U.S. patent application number 17/635044 was filed with the patent office on 2022-09-08 for diagnostic system.
The applicant listed for this patent is TALIS BIOMEDICAL CORPORATION. Invention is credited to Sayeed ANDESHMAND, Thomas H. CAULEY, III, John DIXON, David GLADE, Hedia MAAMAR, Michael John McADAMS, Dzam-Si Jesse NG, David Alexander ROLFE.
Application Number | 20220280081 17/635044 |
Document ID | / |
Family ID | 1000006362507 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220280081 |
Kind Code |
A1 |
ANDESHMAND; Sayeed ; et
al. |
September 8, 2022 |
DIAGNOSTIC SYSTEM
Abstract
Methods and systems are provided for point-of-care nucleic acid
amplification and detection. One embodiment of the point-of-care
molecular diagnostic system includes a cartridge and an instrument.
The cartridge can accept a biological sample, such as a urine or
blood sample. The cartridge, which can comprise one or more of a
loading module, lysis module, purification module and amplification
module, is inserted into the instrument which acts upon the
cartridge to facilitate various sample processing steps that occur
in order to perform a molecular diagnostic test.
Inventors: |
ANDESHMAND; Sayeed; (Dublin,
CA) ; CAULEY, III; Thomas H.; (Redwood City, CA)
; DIXON; John; (Moss Beach, CA) ; GLADE;
David; (San Ramon, CA) ; MAAMAR; Hedia; (El
Dorado Hills, CA) ; McADAMS; Michael John; (Los
Gatos, CA) ; NG; Dzam-Si Jesse; (Fremont, CA)
; ROLFE; David Alexander; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TALIS BIOMEDICAL CORPORATION |
Menlo Park |
CA |
US |
|
|
Family ID: |
1000006362507 |
Appl. No.: |
17/635044 |
Filed: |
August 17, 2020 |
PCT Filed: |
August 17, 2020 |
PCT NO: |
PCT/US20/46721 |
371 Date: |
February 14, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16655007 |
Oct 16, 2019 |
10820847 |
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17635044 |
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16655028 |
Oct 16, 2019 |
11008627 |
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16655007 |
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62887469 |
Aug 15, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/047 20130101;
C12Q 1/6888 20130101; C12Q 1/689 20130101; B01L 2300/0809 20130101;
A61B 5/150221 20130101; A61B 5/150755 20130101; B01L 2200/16
20130101; B01L 3/502761 20130101; A61B 5/150961 20130101; B01L
2300/0681 20130101; C12Q 1/6844 20130101 |
International
Class: |
A61B 5/15 20060101
A61B005/15; B01L 3/00 20060101 B01L003/00; C12Q 1/689 20060101
C12Q001/689 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0004] This invention was made with government support under
contract number HR0011-11-2-0006 awarded by the Department of
Defense (DARPA). The government has certain rights in the
invention.
[0005] This invention was made with government support under
contract number IDSEP160030-02 awarded by the Department of Health
and Human Services (ASPR). The government has certain rights in the
invention.
Claims
1. A method of testing a sample suspected of containing one or more
target pathogens, comprising: accepting a cartridge having a sample
port assembly containing the sample suspected of containing the one
or more target pathogens; advancing the sample suspected of
containing the one or more target pathogens to a lysis chamber
having at least one lysis reagent therein; mixing the sample with
the at least one lysis agent to generate a lysed sample; passing
the lysed sample through a first porous solid support to capture a
nucleic acid on the porous solid support; releasing the captured
nucleic acid from the first porous solid support to generate an
enriched nucleic acid; distributing the enriched nucleic acid to
two or more assay chambers; combining the enriched nucleic acid
with one or more amplification reagents; isolating each one of the
two or more assay chambers from each one of all the other two or
more assay chambers; and performing an isothermal amplification
reaction within each one of the two or more assay chambers while
simultaneously detecting amplification product, wherein presence of
an amplification product is an indication of a presence, an absence
or a quantity of the target pathogen in the sample suspected of
containing the target pathogen.
2. A method of testing a sample according to claim 1, wherein the
sample is a biological sample obtained from a mammal.
3. A method of testing a sample according to claim 2 wherein the
mammal is a person providing a biological sample.
4. The method of testing a sample according to claim 1, wherein the
sample is obtained from a food product, a natural non-growth
hormone crop sample, a crop sample, a water sample, a
non-biological fluid sample or a soil sample.
5. The method of testing a sample according to claim 2, wherein the
step of accepting a cartridge step further comprises reading a bar
code on the cartridge and determining to proceed with the method of
testing.
6. The method of testing a sample according to claim 1, the
accepting a cartridge step further comprising: obtaining and
analyzing an image of a sample window of the sample port assembly
and determining to proceed with the method of testing.
7. The method of testing a sample according to claim 6, wherein the
sample in the sample port assembly is in fluid communication with a
fill chamber, a metering chamber, and an overflow chamber.
8. The method of testing a sample according to claim 6, wherein the
sample window is transparent and formed in at least a portion of a
wall of a metering chamber wherein obtaining an image further
comprises obtaining an image of the transparent viewing window.
9. The method of testing a sample according to claim on 8, wherein
analyzing an image further comprises assessing a height of a sample
liquid in the metering chamber via the transparent viewing
window.
10. The method of testing a sample according to claim 8, wherein
the step of obtaining and analyzing an image further comprises
obtaining an image of the metering chamber comprising a buoyant
ball and analyzing the image comprises identifying a location of
the ball within the metering chamber and determining to proceed
with the method based on the location of the ball.
11. The method of testing a sample according to claim 1, the
accepting a cartridge step further comprising: obtaining and
analyzing an image of a patient ID label and determining to proceed
with the method of testing.
12. The method of testing a sample according to claim 1, the
accepting a cartridge step further comprising: confirming a rotary
valve on the cartridge is in a shipping configuration before
proceeding to the advancing the sample step.
13. The method of testing a sample according to claim 1, the
accepting a cartridge step further comprising: obtaining a reading
from an interference sensor on a valve drive assembly and
confirming based on the reading that a rotary valve on the
cartridge is not in an operational configuration prematurely.
14. The method of testing a sample according to claim 1, the
accepting a cartridge step further comprising: engaging a rotary
valve on the cartridge with a valve drive assembly and rotating the
rotary valve into an operational configuration.
15. The method of testing a sample according to claim 14, wherein
rotating the rotary valve in an operational configuration places a
rotary valve gasket into contact with a stator on the
cartridge.
16. The method of testing a sample according to claim 1, the step
of accepting a cartridge further comprising moving a clamping block
for engaging the cartridge with a door support assembly, a
pneumatic interface assembly, and a thermal clamp assembly.
17. The method of testing a sample according to claim 16, wherein
the moving step is a single continuous movement.
18. The method of testing a sample according to claim 1, the step
of accepting a cartridge further comprising moving a frangible seal
block having a plurality of frangible seal pins into position to
engage one or more frangible seals on the cartridge.
19. The method of testing a sample according to claim 18, wherein
moving the frangible seal block simultaneously engages the
plurality of frangible seal pins with the one or more frangible
seals on the cartridge.
20. The method of testing a sample according to claim 18, wherein
moving the frangible seal block sequentially engages the plurality
of frangible seal pins with the one or more frangible seals on the
cartridge.
21. The method of testing a sample according to claim 18, wherein
the step of moving a frangible seal block is performed after
performing the step of moving a clamp block
22. The method of testing a sample according to claim 18, wherein
the step of moving a frangible seal block is performed initially
with the clamp block and ends in a position separate from the clamp
block.
23. The method of testing a sample according to claim 1, the step
of accepting a cartridge further comprising moving a clamp block
and a frangible seal block together for engaging the cartridge.
24. The method of testing a sample according to claim 23, further
comprising moving the clamp block together with the frangible seal
block until the cartridge is engaged with a door support assembly,
a pneumatic interface assembly, and a thermal clamp assembly.
25. The method of testing a sample according to claim 24, further
comprising: only driving the frangible seal block assembly to
engage one of more frangible seals on the cartridge simultaneously
or sequentially.
26. The method of testing a sample according to claim 1, wherein in
mixing the sample with the at least one lysis agent, the lysis
agent is a mechanical agent.
27. The method of testing a sample according to claim 26, wherein
the mechanical agent is ceramic beads, glass beads or steel beads,
and the mixing the sample step comprises rotating the stir bar at
at least 1000 rpm.
28. The method of testing a sample according to any one of claim 26
or claim 27 further wherein mixing the sample comprises rotating
the stir bar or the ceramic, glass or steel beads along with a
chemical lysis agent.
29. The method of testing a sample according to any one of claim
26, claim 27 or claim 28, wherein the suspected pathogen is a
gram-positive bacterium, a fungus or a plant cell.
30. The method of testing according to claim 1, wherein in the
mixing the sample with the at least one lysis agent step, the at
least one lysis agent is a chemical lysis agent.
31. The method of testing a sample according to claim 30 wherein
the one or more target pathogens is a virus or a gram-negative
bacterium and the lysis reagent is a chaotropic agent.
32. The method of testing a sample according to claim 1, wherein
prior to passing the lysed sample through the porous solid support,
the method further comprises passing the lysed sample through a
size-exclusion filter, wherein nucleic acid passes through the
filter.
33. The method of testing a sample according to claim 1, wherein
the enriched nucleic acid is combined with one or more
amplification reagents before the distributing step.
34. The method of testing a sample according to claim 33, wherein
the one or more amplification reagents are selected from the group
consisting of a DNA polymerase, a reverse transcriptase, a
helicase, nucleotide triphosphates (NTPs), a magnesium salt, a
potassium salt, an ammonium salt, and a buffer.
35. The method of testing a sample according to claim 34, wherein
the one or more amplification reagents further comprise a
primer.
36. The method of testing a sample according to claim 35, wherein
isothermal amplification is initiated prior to distributing the
enriched nucleic acid to the two or more assay chambers.
37. The method of testing a sample according to claim 34, wherein
after the distributing step, but prior to perform the isothermal
amplification reaction, the method further comprises combining the
enriched nucleic acid with a primer set specific to one of the one
or more target pathogens.
38. The method of testing a sample according to claim 1, wherein a
first assay chamber contains a primer set specific to a first
nucleic acid sequence.
39. The method of testing a sample according to claim 38, wherein
the first nucleic acid sequence is present in one of the one or
more target pathogens.
40. The method of testing a sample according to claim 38, wherein
prior to mixing the sample with at least one lysis agent, a process
control is added to the sample and the first nucleic acid sequence
is present in the process control.
41. The method of testing a sample according to claim 38, wherein
prior to passing lysed sample through the porous solid support, a
process control is added to the lysed sample and the first nucleic
acid sequence is present in the process control.
42. The method of testing a sample according to claim 38, wherein a
second assay chamber contains a primer set specific to a second
nucleic acid sequent, wherein the second nucleic acid sequence is
present in one of the one or more target pathogens.
43. The method of testing a sample according to claim 1, wherein
the performing an isothermal amplification reaction step is
completed in less than 20 minutes.
44. The method of testing a sample according to claim 43, wherein
the performing an isothermal amplification reaction step is
completed in less than 15 minutes.
45. The method of testing a sample according to claim 43, wherein
the performing an isothermal amplification reaction step is
completed in less than 10 minutes.
46. The method of testing a sample according to claim 1, further
comprising: providing a result containing a determination made
during the performing step relating to the presence, the absence or
the quantity of the target pathogen in the sample suspected of
containing the target pathogen.
47. The method of testing a sample according to claim 1, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with a chemical reaction.
48. The method of testing a sample according to claim 47, wherein
the sample is sputum and the chemical reaction is incubation with a
mucolytic agent.
49. The method of testing a sample according to claim 48, wherein
the mucolytic agent is dithiothreitol or n-acetylcysteine.
50. The method of testing a sample according to claim 1, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with an enzymatic
reaction.
51. The method of testing a sample according to claim 50, wherein
the enzymatic reaction is incubation of the sample with a nuclease,
a protease, an amylase, a glycosylase, or a lipase.
52. The method of testing a sample according to claim 50, wherein
pretreating comprising incubating the sample with a DNase.
53. The method of testing a sample according to claim 50, wherein
pretreating comprises incubating the sample with a protease.
54. The method of testing a sample according to claim 53, wherein
the protease is selected from pronase, chymotrypsin, trypsin and
pepsin.
55. The method of testing a sample according to claim 1, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with a physical
treatment.
56. The method of testing a sample according to claim 55, wherein
the physical treatment comprises passing the sample through a
size-exclusion filter in a first direction.
57. The method of testing a sample according to claim 56, wherein
the target pathogen passes through the filter.
58. The method of testing a sample according to claim 56, wherein
the target pathogen does not pass through the filter and is thereby
captured on a fill port side of the size-exclusion filter.
59. The method of testing a sample according to claim 58, further
comprising passing a volume of suspension buffer through the
size-exclusion filter in a second direction, wherein second
direction is opposite the first direction, thereby releasing the
target pathogen from the fill port side of the filter.
60. The method of testing a sample according to claim 59, wherein
the volume of suspension buffer is less than the volume of the
sample, and the target pathogen is more concentrated than in the
loaded sample.
61. The method of testing a sample according to claim 58, wherein
the physical treatment comprises exposing the sample to a capture
agent immobilized on a solid substrate.
62. The method of testing a sample according to claim 61, further
comprising, after exposure, separating the solid substrate from the
sample.
63. The method of testing a sample according to claim 61, wherein
the capture agent is a capture antibody.
64. The method of testing a sample according to claim 61, wherein
the capture agent is an antibody with affinity for red blood
cells.
65. The method of testing a sample according to claim 61, wherein
the solid substrate is a magnetic bead, the capture agents has
affinity for a class of cells comprising the one or more target
pathogens and the method further comprises (1) incubating the
magnetic beads with the sample, (2) engaging a magnet to draw the
magnetic beads to a location within the sample loading structure,
(3) washing away unbound sample, (4) releasing the magnet, and (5)
resuspending the magnetic beads and passing the suspension,
including target pathogen bound to the magnetic beads, to the lysis
chamber.
66. The method of testing a sample according to claim 1, wherein
the sample is sputum and the method further comprises, prior to
mixing the sample with the at least one lysis reagent, bead beating
the sputum to liquify the sample.
67. The method of testing a sample according to claim 66, wherein
the bead beating comprises mixing the sputum with ceramic, glass,
or steel beads.
68. The method of testing a sample according to claim 66, wherein
the bead beating comprises mixing the sputum with ceramic, glass,
or steel beads and dithiothreitol.
69. The method of testing a sample according to claim 1, wherein
prior to distributing the enriched nucleic acid to the assay wells,
the method further comprises passing the enriched nucleic acid
through a second porous solid support.
70. The method of testing a sample according to claim 69, wherein
the second porous solid support is the same as the first porous
solid support.
71. The method of testing a sample according to claim 70, wherein
the enriched nucleic acid is mixed with a matrix binding agent
prior to passing through the second solid support.
72. The method of testing a sample according to claim 71, wherein
matrix binding agent is an alcohol or a salt solution.
73. The method of testing a sample according to claim 69, wherein
the second porous solid support is different than the first porous
solid support, and the second solid support has an affinity for
nucleic acid and the method further comprises releasing the
captured nucleic acid from the second solid support to generate a
twice-enriched nucleic acid.
74. The method of testing a sample according to claim 69, wherein
the second porous solid support is different than the first porous
solid support.
75. The method of testing a sample according to claim 1, wherein
prior to passing the lysed sample through a first porous solid
support, the method further comprises passing the lysed sample
through a second porous solid support, wherein the second solid
support does not bind nucleic acid and has affinity for one or more
contaminants, thereby removing contaminant from the lysed
sample.
76. The method of testing a sample according to claim 1, further
comprising releasing the cartridge from engagement with a clamp
block and a frangible seal block after completing the performing an
isothermal amplification reaction step.
77. The method of testing a sample according to claim 1, further
comprising displaying a result produced after the step of
performing an isothermal amplification reaction step.
78. The method of testing a sample according to claim 1, further
comprising storing in a computer memory a result produced after the
step of performing an isothermal amplification reaction step.
79. The method of testing a sample according to claim 1, further
comprising maintaining the cartridge in a vertical orientation
while performing the steps of testing a sample.
80. The method of testing a sample according to claim 79, wherein
the cartridge is inclined no more than 30 degrees while in the
vertical orientation.
81. The method of testing a sample according to claim 79, wherein
the cartridge is inclined no more than 15 degrees while in the
vertical orientation.
82. The method of testing a sample according to claim 1 wherein
during the combining the enriched nucleic acid step in each of the
two or more assay chambers the enriched nucleic acid combines with
a dried reagent contained in each one of the two or more assay
chambers.
83. The method of testing a sample according to claim 82 wherein
the dried reagent is on a surface of a plug in each one of the two
or more assay chambers.
84. The method of testing a sample according to claim 83 wherein
the dried reagent is on a surface of the plug formed from a
material transmissive to excitation wavelengths and emission
wavelengths in at least one of a red spectrum, a blue spectrum and
a green spectrum used during the performing step.
85. The method of testing a sample according to claim 83 wherein
the plug is as in any one of claims 202 to 213 and 214.
86. The method of testing a sample according to claim 1, the
distributing step further comprising distributing the enriched
nucleic acid to two or more assay chambers using a rotary valve on
the cartridge and a pneumatic signal introduced into the rotary
valve further wherein the pneumatic signal continues to be
introduced while the performing step is performed.
87. The method of testing a sample according to claim 1 wherein
performing the isolation step temporarily isolates each one of the
two or more assay chambers are from each one of all the other two
or more assay chambers.
88. The method testing a sample according to claim 87 wherein the
isolation step is performed using a pneumatic signal, a mechanical
system to occlude one or more fluid channels to occlude one or more
passages or channels of a cartridge.
89. The method of testing a sample according to claim 88 wherein
the mechanical system is one of a single pinch valve, a plurality
of pinch valves, and a non-heated staker bar.
90. The method of testing a sample according to claim 1 wherein
performing the isolation step permanently isolates each one of the
two or more assay chambers from each one of all the other two or
more assay chambers.
91. The method of testing a sample according to claim 90 wherein
after performing the isolation step, a portion of the cartridge is
melted or is plastically deformed.
92. The method of testing a sample according to claim 1 wherein
after completing the performing step, each one of the two or more
assay chambers are isolated from each one of all the other two or
more assay chambers.
93. The method of testing a sample according to claim 1, the
distributing step further comprising distributing the enriched
nucleic acid to two or more assay chambers using a rotary valve on
the cartridge and a pneumatic signal introduced into the rotary
valve further wherein the pneumatic signal continues to be
introduced while performing the isolating step by moving a heat
staker into contact with the cartridge to isolate each one of the
two or more assay chambers from each one of all the other two or
more assay chambers.
94. The method of testing a sample according to claim 93 wherein
after performing the isolating step, a single heat stake isolates
each one of the two or more assay chambers from each one of all the
other two or more assay chambers.
95. The method of testing a sample according to claim 94 further
wherein the single heat stake isolates a waste chamber on the
cartridge.
96. The method of testing a sample according to claim 1, the
isolating step further comprising moving a heat staker into contact
with the cartridge to seal each one of the two or more assay
chambers from each one of all the other two or more assay
chambers.
97. The method of testing a sample according to claim 96, further
comprising providing a pneumatic pressure in the cartridge while
moving the heat staker into contact with the cartridge.
98. The method of testing a sample according to claim 1, the
isolating step further comprising forming a heat stake region in
the cartridge to isolate each one of the two or more assay chambers
from each one of all the other two or more assay chambers.
99. The method of testing a sample according to any one of claim
96, claim 97 and claim 98 further comprising: obtaining a first
image of a level of fluid in each of the one or more assay chambers
after the step of distributing the enriched nucleic acid to each
one of the two or more assay chambers.
100. The method of claim 85 further comprising obtaining a second
image of a level of fluid in each of the one or more assay chambers
after the isolating step.
101. The method of testing a sample according to claim 100, further
comprising determining the quality of the heat stake by comparing
the level of fluid in the first image to the level of fluid in the
second image.
102. The method of testing a sample according to claim 1, further
comprising rotating a rotary valve on the cartridge prior to
performing the advancing the sample step.
103. The method of testing a sample according to claim 102, further
comprising advancing the sample to the lysis chamber using a
pneumatic signal introduced into a cartridge pneumatic
interface.
104. The method of testing a sample according to claim 1, further
comprising rotating a rotary valve on the cartridge prior to
performing the step of passing the lysed sample through a first
porous solid support to capture a nucleic acid on the porous solid
support.
105. The method of testing a sample according to claim 104, further
comprising passing the lysed sample through the first porous solid
support using a pneumatic signal introduced into the rotary
valve.
106. The method of testing a sample according to claim 1, further
comprising distributing the enriched nucleic acid to two or more
assay chambers using a rotary valve on the cartridge and a
pneumatic signal introduced into the rotary valve.
107. An apparatus, comprising: an enclosure; a fixed support
bracket within the enclosure; a first imaging system mounted on the
fixed support bracket within the enclosure adjacent to an opening,
the first imaging system configured to collect images from a first
imaging area within the enclosure; a second imaging system mounted
on the fixed support bracket within the enclosure configured to
collect images from a second imaging area within the enclosure
wherein the second imaging area is in non-overlapping relation to
the first imaging area; a moving support bracket within the
enclosure and moveable relative to the fixed support bracket, the
first imaging system and the second imaging system; a drive system
on the fixed support bracket configured to position the moving
support bracket relative to the fixed support bracket; and an
opening positioned in the enclosure to provide access to an
interior portion of the enclosure between the fixed support bracket
and the moving support bracket.
108. The apparatus of claim 107 wherein the moving support bracket
is positioned between the first imaging system and the second
imaging system.
109. The apparatus of claim 107 wherein a rotary connector, a
pneumatic connector and a multiple pin block are connected to and
move with the moving support bracket.
110. The apparatus of claim 109 wherein the multiple pin block is
directly connected to the drive system.
111. The apparatus of claim 109 wherein the multiple pin block is
configured to move together with the rotary connector and the
pneumatic connector and independent of the rotary connector and the
pneumatic connector.
112. The apparatus of claim 107 wherein the opening is a slot,
wherein the slot is aligned to access an upper rail within the
enclosure aligned to an upper portion of the slot and a lower rail
within the enclosure aligned to a lower portion of the slot.
113. The apparatus of claim 112 further comprising a loading and
ejection mechanism within the enclosure in sliding relation to the
lower rail.
114. The apparatus of claim 113 wherein the loading and ejection
mechanism moves between a loading position and a loaded position
wherein when in the loading position the loading and ejection
mechanism is positioned in a forward most position towards the slot
and when in the loaded position the loading and ejection mechanism
is engaged with a load position sensor.
115. The apparatus of claim 114 wherein the load position sensor
provides an electronic indication when the loading and ejection
mechanism has translated into the loaded position.
116. The apparatus of claim 107 further comprising a first heater
and a second heater mounted on the fixed support bracket.
117. The apparatus of claim 116 wherein the first heater is
positioned to heat a portion of the fixed support bracket between
the first imaging area and the second imaging area.
118. The apparatus of claim 116 wherein the second heater is
positioned to heat a portion of the fixed support bracket only
within the second imaging area.
119. The apparatus of claim 107 further comprising a channel in the
fixed support bracket and a heat stake assembly positioned to move
a heating element through the channel.
120. The apparatus of claim 119 wherein the channel is positioned
on the fixed support bracket to allow the heating element to
interact within the enclosure between the first imaging area and
the second imaging area.
121. The apparatus of claim 119 wherein the channel is positioned
within the fixed support bracket such that the heating element may
perform a heat staking operation directly adjacent to but outside
of the second imaging area.
122. The apparatus of claim 107 wherein the moving support bracket
partially blocks the channel when the moving support bracket is
positioned at a closest position to the fixed support bracket.
123. An apparatus, comprising: an enclosure; a fixed support
bracket within the enclosure; a moving support bracket within the
enclosure and moveable relative to the fixed support bracket; a
drive system configured to position the moving support bracket
relative to the fixed support bracket; an opening positioned in the
enclosure to provide access to an interior portion of the enclosure
between the fixed support bracket and the moving support bracket;
and an upper rail and a lower rail in the enclosure positioned
adjacent to the opening wherein a cartridge positioned between the
upper rail and the lower rail remains in a vertical position
between the fixed support bracket and the moving support
bracket.
124. The apparatus of claim 123 further comprising a feature within
the upper rail or the lower rail positioned to interfere with the
movement of a cartridge improperly aligned with respect to the
upper rail and the lower rail.
125. The apparatus of claim 123 further comprising a loading and
ejection assembly within the enclosure positioned to engage with a
cartridge moving along the upper rail and the lower rail.
126. The apparatus of claim 123 further comprising a latch and pin
assembly positioned adjacent to the upper rail adapted to engage a
pin with a cartridge moving along the upper rail.
127. The apparatus of claim 123 further comprising a touch screen
display on an exterior of the enclosure.
128. The apparatus of claim 123 further comprising a cellular
communications module within the enclosure.
129. The apparatus of claim 123 wherein the cellular communication
module is adjacent to the opening.
130. The apparatus of claim 123 further comprising: a cartridge
heater, a driving magnet system, a chemistry heater, a rehydration
motor, a reaction camera and a heat stake assembly coupled to the
fixed support bracket and positioned to interact with a
corresponding portion of a cartridge positioned between the upper
rail and the lower rail.
131. The apparatus of claim 123 further comprising a first imaging
system mounted on the fixed support bracket within the enclosure
adjacent to the opening, the first imaging system configured to
collect images from a first imaging area within the enclosure and a
second imaging system mounted on the fixed support bracket within
the enclosure configured to collect images from a second imaging
area within the enclosure wherein the second imaging area is in
non-overlapping relation to the first imaging area.
132. The apparatus of claim 131 wherein the first imaging area
includes a label of a cartridge positioned within the enclosure
between the upper rail and the lower rail.
133. The apparatus of claim 131 wherein the second imaging area
includes one or more assay chambers of a cartridge positioned
within the enclosure between the upper rail and the lower rail.
134. The apparatus of claim 123 further comprising a clamp block, a
frangible seal block, a valve driver, a pneumatic interface, a
thermal clamp, and a driven magnet system coupled to move along
with the moving support bracket during operation of the drive
system.
135. The apparatus of claim 130 further comprising a plenum
adjacent to the chemistry heater and a fan in fluid communication
with the plenum.
136. The apparatus of claim 130 the heat stake assembly further
comprising a staker blade positioned to move relative to a depth
stop frame, the staker blade coupled to a linear actuator motor and
a spring with pivot washer.
137. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module; a purification module; and a reaction
module; wherein the loading module is in fluidic communication with
the lysis module and the purification module is in fluidic
communication with the reaction module; and further wherein the
loading module, the lysis module, the purification module and the
reaction module are arranged for use while the cartridge is in a
vertical orientation.
138. The integrated diagnostic cartridge of claim 137 further
comprising one or more fluid filling conduits arranged to flow into
an upper portion of a chamber within a fluidic card of the
integrated diagnostics cartridge and one or more fluid outlet
conduits arranged to flow out of a lower portion of the chamber
within the fluidic card of the integrated diagnostics
cartridge.
139. The integrated diagnostic cartridge of claim 138, wherein the
chamber is one or more of a lysis chamber, a metering chamber, a
wash buffer chamber or an elution buffer chambers.
140. The integrated diagnostic cartridge of claim 139, wherein the
chamber further comprises a filter assembly in fluid communication
with a fluid outlet conduit of the chamber.
141. The integrated diagnostic cartridge of claim 137, wherein the
lysis module comprises a mixing assembly having a vertically
oriented lysis chamber containing a lysis agent and a
non-magnetized stir bar.
142. The integrated diagnostic cartridge of claim 141, wherein the
non-magnetized stir bar is made from a metal having a magnetic
permeability to be responsive to a rotating magnetic field induced
between a drive magnetic element and a driven magnetic element of a
magnetic drive system.
143. The integrated diagnostic cartridge according to claim 141,
wherein the non-magnetized stir bar is coated with an impermeable
material to prevent corrosion by a chemical lysis buffer in the
vertically oriented lysis chamber.
144. The integrated diagnostic cartridge of claim 141 wherein, when
in use within a diagnostic instrument, the non-magnetized stir bar
is disposed between a driving magnet system and a driven magnet
system of a magnetic mixing assembly in the diagnostic instrument,
wherein the driving magnet system is configured to rotate the
non-magnetized stir bar within the vertically oriented lysis
chamber at least 1000 rpm.
145. The integrated diagnostic cartridge of claim 141, further
comprising a fluid inlet to the vertically oriented lysis chamber
and a fluid outlet to lysis chamber wherein the vertically oriented
lysis chamber is isolated from the other modules on the cartridge
by a first frangible seal in fluid communication with the fluid
inlet to the vertically oriented lysis chamber and a second
frangible seal in fluid communication with the fluid outlet to the
vertically oriented lysis chamber.
146. The integrated diagnostic cartridge according to claim 137
further comprising a fluidic card and a cover.
147. The integrated diagnostic cartridge of claim 146, wherein the
fluidic card further comprises a first film adhered to a surface of
at least a portion of the fluidic card, wherein the first film
forms one surface of one or more chambers, compartments, or fluid
conduits of the loading module, the lysis module, the purification
module and the reaction module.
148. The integrated diagnostic cartridge of claim 146 further
comprising an interference feature on the cover, wherein the
interference feature is sized and positioned to interact with one
of an upper rail or a lower rail of a loading apparatus of a
diagnostic instrument.
149. The integrated diagnostic cartridge according to claim 148,
wherein a thickness of the fluidic card is selected for sliding
arrangement within an upper rail and a lower rail of a loading
apparatus of the diagnostic instrument.
150. The integrated diagnostic cartridge of claim 146 wherein a
total sample process volume of the integrated diagnostic cartridge
is related to a thickness of the cartridge corresponding to a
spacing between the one or more chambers, compartments, or fluid
conduits of the loading module, the lysis module, the purification
module and the reaction module formed in the fluidic card and the
first film.
151. The integrated diagnostic cartridge of claim 150, wherein a
diagnostic instrument is adapted and configured to accommodate a
variation of the thickness of the cartridge by increasing a width
of an opening of the diagnostic instrument to accommodate the
increased thickness of the cartridge or a displacement range of a
cartridge clamping system of the diagnostic instrument is adapted
to accommodate the increased thickness of the cartridge.
152. The integrated diagnostic cartridge of claim 148 further
comprising a cartridge front face and a cartridge rear face forming
an upper spacing and a lower spacing wherein each of the upper
spacing and the lower spacing is sized and positioned to engage
with the upper rail and lower rail of the diagnostic
instrument.
153. The integrated diagnostic cartridge of claim 152 further
comprising an interference feature within the upper spacing or the
lower spacing positioned to ensure the cartridge engages with the
upper rail and the lower rail in a desired orientation.
154. The integrated diagnostic cartridge of claim 137 further
comprising a plurality of frangible seal chambers in fluid
communication with at least one or more of the loading module, the
lysis module, the purification module or the reaction module.
155. The integrated diagnostic cartridge of claim 137, the
integrated diagnostic cartridge further comprising a
machine-readable code adapted and configured to identify the
cartridge to a diagnostic instrument or an image of a patient
identification marking.
156. An integrated diagnostic cartridge, comprising: a loading
module comprising a sample port assembly having a fill chamber, a
metering chamber, and an overflow chamber arranged in fluid
communication; a lysis module; a purification module; and a
reaction module; wherein the loading module is in fluidic
communication with the lysis module and the purification module is
in fluidic communication with the reaction module.
157. The integrated diagnostic cartridge of claim 156, wherein the
metering chamber comprises a transparent viewing window for
observing the height of a sample within the metering chamber.
158. The integrated diagnostic cartridge of claim 157 further
comprising a ball float in the metering chamber adapted for use
with the transparent viewing window.
159. The integrated diagnostic cartridge of claim 156, wherein the
fill chamber comprises a cap operable to provide access to the fill
chamber.
160. The integrated diagnostic cartridge of claim 159, wherein the
cap is positioned for interaction with a closing apparatus of a
diagnostic instrument.
161. The integrated diagnostic cartridge of claim 156, wherein, the
cartridge is in a vertical orientation when in use within a
diagnostic instrument and a fluid channel connects an outlet at a
lower portion of the fill chamber with an inlet to the metering
chamber located in an upper portion of the metering chamber.
162. The integrated diagnostic cartridge of claim 156, wherein the
metering chamber comprises a transparent viewing window.
163. The integrated diagnostic cartridge of claim 162 further
comprising a buoyant ball within the metering chamber, said buoyant
ball adapted to appear adjacent to the transparent viewing window
permitting an assessment of the height of the sample liquid in the
metering chamber.
164. The integrated diagnostic cartridge of claim 156, wherein the
metering chamber comprises a buoyant ball for assessing a height of
a sample liquid in the metering chamber.
165. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module comprising a mixing assembly having a lysis
chamber containing a lysis agent and a non-magnetized stir bar; a
purification module; and a reaction module; wherein the loading
module is in fluidic communication with the lysis module and the
purification module is in fluidic communication with the reaction
module.
166. The integrated diagnostic cartridge of claim 165, wherein the
non-magnetized stir bar is made from a metal having a magnetic
permeability to be responsive to a rotating magnetic field induced
between a drive magnetic element and a driven magnetic element of a
magnetic drive system.
167. The integrated diagnostic cartridge of claim 166 wherein the
metal comprises a ferritic stainless steel or a duplex stainless
steel.
168. The integrated diagnostic cartridge of claim 166 wherein the
non-magnetized stir bar is made from a metal selected from the
group consisting of a carbon steel, a mild carbon steel, a low
alloy steel, a tool steel, a metal alloy contain nickel, a metal
alloy containing cobalt, a non-austenitic stainless steel, a
ferritic grade of stainless steel including 430 steel, Atlas CR12
steel, 444 steel, F20S steel, a duplex grade of steel including
2205 steel, 2304 steel, 2101 steel, 2507 steel and a martensitic
grade of steel such as 431 steel, 416 steel, 420 steel and 440C
steel wherein the metal has a magnetic permeability to be
responsive to a rotating magnetic field produced within the mixing
chamber.
169. The integrated diagnostic cartridge of claim 168 wherein the
metal has a magnetic permeability between 500-1,000,000.
170. The integrated diagnostic cartridge according to any one of
claims 165-169, wherein the non-magnetized stir bar is coated with
an impermeable material to prevent corrosion by a chemical lysis
buffer in lysis chamber.
171. The integrated diagnostic cartridge of claim 170, wherein the
impermeable material is PTFE, parylene C, parylene D, a
functionalized perfluoropolyether (PFPE), Xylan Fluoropolymer,
epoxy, or urethane.
172. The integrated diagnostic cartridge of claim 165 wherein, when
in use within a diagnostic instrument, the non-magnetized stir bar
is disposed between a driving magnet system and a driven magnet
system of a magnetic mixing assembly in the diagnostic instrument,
wherein the driving magnet system is configured to rotate the
non-magnetized stir bar within the lysis chamber at at least 1000
rpm.
173. The integrated diagnostic cartridge of claim 165, wherein the
lysis agent is a mechanical agent.
174. The integrated diagnostic cartridge of claim 173, wherein the
mechanical agent is ceramic beads, glass beads or steel beads.
175. The integrated diagnostic cartridge of claim 165, wherein the
lysis agent is a chemical agent.
176. The integrated diagnostic cartridge of claim 175, wherein the
chemical agent is an anionic detergent, a cationic detergent, a
non-ionic detergent or a chaotropic agent.
177. The integrated diagnostic cartridge of claim 176, wherein the
cartridge is configured for testing of one or more target pathogens
that is a virus or a gram-negative bacterium.
178. The integrated diagnostic cartridge of claim 165, further
comprising a fluid inlet in fluid communication with the lysis
chamber and a fluid outlet in fluid communication with the lysis
chamber and a filter assembly in fluid communication with the fluid
outlet of the lysis chamber.
179. The integrated diagnostic cartridge of claim 165, further
comprising a fluid inlet to the lysis chamber and a fluid outlet to
lysis chamber wherein the lysis chamber is isolated from the other
modules on the cartridge by a first frangible seal in fluid
communication with the fluid inlet to the lysis chamber and a
second frangible seal in fluid communication with the fluid outlet
of the lysis chamber.
180. The integrated diagnostic cartridge of claim 178 or claim 179
further comprising: a process control chamber having an inlet, an
outlet and a plug comprising a process control wherein the process
control chamber is in fluid communication with the lysis chamber
inlet.
181. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module; a purification module comprising a rotary
valve comprising: a. a stator comprising a stator face and a
plurality of passages, each passage comprising a port at the stator
face; b. a rotor operably connected to the stator and comprising a
rotational axis, a rotor valving face, and a flow channel having an
inlet and an outlet at the rotor valving face, wherein the flow
channel comprises a porous solid support; and c. a retention
element biasing the stator and the rotor together at a rotor-stator
interface to form a fluid tight seal; and a reaction module;
wherein the loading module is in fluidic communication with the
lysis module and the purification module is in fluidic
communication with the reaction module.
182. The integrated diagnostic cartridge of claim 181, wherein the
rotary valve further comprises a gasket between the stator face and
the rotor valving face, and wherein the stator comprises a
displaceable spacer for preventing the gasket from sealing against
at least one of the rotor and stator, and wherein, when the spacer
is displaced the gasket seals the rotor and stator together in a
fluid-tight manner.
183. The integrated diagnostic cartridge of claim 182, wherein,
when the cartridge is positioned within a diagnostic instrument,
engagement with a rotor driver of the diagnostic instrument
displaces the spacer and seals the rotor and stator together in a
fluid-tight manner.
184. The integrated diagnostic cartridge of claim 183, wherein a
rotation movement performed by the rotor driver of the diagnostic
instrument displaces the spacer and seals the rotor and the stator
together in a fluid-tight manner.
185. The integrated diagnostic cartridge of claim 181 further
comprising at least one pair of ridges and spaces on a retention
ring and at least one pair of ridges and spaces on the rotor
wherein while the at least one pair of ridges and spaces of the
retention ring is engaged with the at least one pair of ridges and
spaces of the rotor sealing of the rotor and stator is prevented
wherein, relative movement between the at least one pair of ridges
and spaces on the retention ring and the at least one pair of
ridges and spaces on the rotor seals the rotor and stator together
in a fluid-tight manner.
186. The integrated diagnostic cartridge of claim 185, wherein,
when the cartridge is positioned within a diagnostic instrument,
engagement with a rotor driver of the diagnostic instrument
produces the relative movement between the at least two pairs of
ridges and spaces on the retention ring and the rotor that seals
the rotor and stator together in a fluid-tight manner.
187. The integrated diagnostic cartridge of claim 186, wherein a
rotation movement of less than one full rotation of the rotor
performed by the rotor driver of the diagnostic instrument seals
the rotor and stator together in a fluid-tight manner.
188. The integrated diagnostic cartridge of any one of claims 185,
186 and 187 further comprising a gasket interposed at the
rotor-stator interface.
189. The integrated diagnostic cartridge of claim 181, wherein the
rotary valve is maintained in a storage condition while a threaded
portion of a retention ring is engaged with a threaded portion of
the rotor wherein relative motion between the threaded portion of
the retention ring and the threaded portion of the rotor seals the
rotor and stator together in a fluid-tight manner.
190. The integrated diagnostic cartridge of claim 189 wherein, when
the cartridge is positioned within a diagnostic instrument,
engagement with a rotor driver of the diagnostic instrument
produces the relative movement between the threaded portion of the
retention ring and the threaded portion of the rotor.
191. The integrated diagnostic cartridge of claim 190, wherein a
rotation movement of less than one full rotation of the rotor
performed by the rotor driver of the diagnostic instrument seals
the rotor and stator together in a fluid-tight manner.
192. The integrated diagnostic cartridge of any one of claim 189,
claim 190 and claim 191 further comprising: a gasket interposed at
the rotor-stator interface.
193. The integrated diagnostic cartridge of claim 181, the
purification module further comprising: a waste collection element,
a wash buffer reservoir and an elution buffer reservoir.
194. The integrated diagnostic cartridge of claim 181 further
comprising a pneumatic interface in fluidic communication with at
least the purification module.
195. The integrated diagnostic cartridge of claim 181 wherein the
porous solid support is polymeric.
196. The integrated diagnostic cartridge of claim 181 wherein the
porous solid support is selected from the group consisting of
alumina, silica, celite, ceramics, metal oxides, porous glass,
controlled pore glass, carbohydrate polymers, polysaccharides,
agarose, Sepharose.TM., Sephadex.TM., dextran, cellulose, starch,
chitin, zeolites, synthetic polymers, polyvinyl ether,
polyethylene, polypropylene, polystyrene, nylons, polyacrylates,
polymethacrylates, polyacrylamides, polymaleic anhydride,
membranes, hollow fibers and fibers, and any combination
thereof.
197. The integrated diagnostic cartridge of claim 181 wherein the
rotor valving face comprises a gasket interposed at the
rotor-stator interface.
198. The integrated diagnostic cartridge of claim 197 the gasket
further comprising a fluid connector or a fluid selector comprising
a volume dimensioned to provide an aliquot of liquid when
filled.
199. The integrated diagnostic cartridge of claim 181 wherein the
rotor comprises a plurality of flow channels, each flow channel
comprising an inlet, an outlet, and a porous solid support.
200. The integrated diagnostic cartridge of claim 181 the rotor
valving face further comprising a fluid connector or a fluid
selector comprising a volume dimensioned to provide an aliquot of
liquid when filled.
201. The integrated diagnostic cartridge of any one of claim 181 to
claim 200, the purification module further comprising: a waste
collection element, a wash buffer reservoir and an elution buffer
reservoir.
202. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module; a purification module; and a reaction
module comprising a plurality of individual assay chambers, wherein
at least one wall in each one of the plurality of individual assay
chambers is provided by a plug comprising: a body with a bottom
surface; a central opening in the body; and a dried reagent on the
bottom surface, wherein the body is formed from a material
transmissive to excitation wavelengths and emission wavelengths in
at least one of a red spectrum, a blue spectrum and a green
spectrum; wherein the loading module is in fluidic communication
with the lysis module and the purification module is in fluidic
communication with the reaction module.
203. The integrated diagnostic cartridge of claim 202, wherein the
bottom surface of the plug body comprises a cavity in the bottom
surface with the dried reagent within the cavity, and wherein the
plug has a plug thickness between a central opening bottom and the
plug body bottom, and further wherein a depth of the cavity is less
than 90% of the plug thickness, is less than 70% of the plug
thickness or is less than 50% of the plug thickness.
204. The integrated diagnostic cartridge of claim 202, wherein the
plug has a polished or smooth finish facilitating the
transmissivity of the excitation wavelengths and the emission
wavelengths.
205. The integrated diagnostic cartridge according to any one of
claim 202, claim 203 and claim 204, wherein the dried reagent is
selected from the group consisting of nucleic acid synthesis
reagents, nucleic acids, nucleotides, nucleobases, nucleosides,
monomers, detection reagents, catalysts or combinations
thereof.
206. The integrated diagnostic cartridge of claim 202 further
comprising: a cartridge perimeter, wherein each one of the
plurality of individual assay chambers is in communication with an
air chamber and each air chamber is closer to the cartridge
perimeter than the plug in each one of the plurality of individual
assay chambers.
207. The integrated diagnostic cartridge of claim 202 further
comprising: a reaction area perimeter, wherein each one of the
plurality of individual assay chambers is in communication with an
air chamber and further wherein each plug in each one of the
plurality of individual assay chambers is within the reaction area
perimeter and each air chamber is outside of the reaction area
perimeter.
208. The integrated diagnostic cartridge of claim 202 further
comprising: a cartridge perimeter and a reaction area perimeter
wherein each one of the plurality of individual assay chambers is
in communication with an air chamber and each air chamber is closer
to the cartridge perimeter than the plug in each one of the
plurality of individual assay chambers and is located outside of
the reaction area perimeter and each one of the plurality of
individual assay chambers is within the reaction area
perimeter.
209. The integrated diagnostic cartridge according to claim 202,
wherein the body of the plug protrudes into the monolithic
substrate of the assay chamber at a depth such that the assay
chamber volume can be readily changed by altering the depth at
which the body of the plug protrudes into the monolithic substrate
of the assay chamber.
210. The integrated diagnostic cartridge according to claim 202
further comprising at least one fluid inlet conduit to each one of
the plurality of individual assay chambers of the reaction module
wherein each one of the at least one fluid inlet conduits further
comprises a heat staked region.
211. The integrated diagnostic cartridge according to claim 210
wherein a heat stake in the heat staked region fluidically isolates
the reaction module from the loading module, the lysis module, and
the purification module.
212. The integrated diagnostic cartridge according to any one of
claim 202 to claim 211, wherein the dried reagent is a continuous
film adhered to the plug bottom surface.
213. The integrated diagnostic cartridge according to any one of
claim 202 to claim 212, wherein the dried reagent is a lyophilized
reagent.
214. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module; a purification module; and a reaction
module comprising one or more assay chambers, wherein each assay
chamber comprises: a. a tapered inlet; b. a tapered outlet; c. a
plug comprising a bottom surface and a central opening in the body,
wherein the body is formed from a material transmissive to
excitation wavelengths and emission wavelengths in at least one of
an ultraviolet spectrum, a blue spectrum, a green spectrum and a
red spectrum; d. two curved boundaries, wherein each curved
boundary extends from the tapered inlet to the tapered outlet such
that together, the two curved boundaries and the plug enclose a
volume of the assay chamber; and e. a shoulder extending from each
curved boundary wherein the plug contacts each shoulder such that a
boundary of the assay chamber is provided by the two curved
boundaries, the shoulders extending from each of the curved
boundaries and the plug.
215. The integrated diagnostic cartridge of claim 214, the plug as
in any one of claim 203 to claim 213.
216. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module; a purification module; and a reaction
module comprising: a. a common fluid pathway, and b. a plurality of
independent, continuous fluidic pathways connected to the common
fluid pathway, wherein each independent, continuous fluidic pathway
comprising: i. an assay chamber, and ii. a pneumatic compartment;
1. wherein the assay chamber is connected to the common fluid
pathway, the assay chamber having a fluid volume defined in part by
a plug having a dried reagent thereon; and 2. the pneumatic
compartment, having a pneumatic volume, is connected to the common
fluid pathway via the assay chamber; wherein, each fluidic pathway
of the plurality of independent, continuous fluidic pathways is a
closed system excluding the connection between the assay chamber
and common fluid source, wherein each assay chamber further
comprises: c. a double tapered chamber, the double tapered chamber
comprising: iii. a tapered inlet in fluidic communication with a
terminus of the entry conduit of the fluidic pathway, iv. a tapered
outlet in fluidic communication with a terminus of the pneumatic
compartment, and v. two curved boundaries, wherein each curved
boundary extends from the tapered inlet to the tapered outlet such
that together, the two curved boundaries enclose the volume of the
assay chamber; d. a shoulder extending from each curved boundary
wherein the plug contacts each shoulder such that a boundary of the
assay chamber is provided by the two curved boundaries, the
shoulders extending from each of the curved boundaries and the
plug. wherein the loading module is in fluidic communication with
the lysis module and the purification module is in fluidic
communication with the reaction module.
217. The integrated diagnostic cartridge of claim 216, wherein the
two curved boundaries are formed in a monolithic substrate or a
fluidic card of the cartridge.
218. The integrated diagnostic cartridge according to any one of
claim 202 to claim 217, wherein the body of the plug protrudes into
the monolithic substrate of the assay chamber at a depth such that
the assay chamber volume can be readily changed by altering the
depth at which the body of the plug protrudes into the monolithic
substrate of the assay chamber.
219. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module; a purification module; and a reaction
module comprising a reagent storage component comprising a capsule
capable of holding a liquid or solid sample, said capsule
comprising an opening, a closed end and a wall extending from the
closed end to the opening, wherein the capsule is oval-shaped and
the wall is rounded, and wherein the closed end and wall define an
interior volume having a substantially smooth surface; wherein the
loading module is in fluidic communication with the lysis module
and the purification module is in fluidic communication with the
reaction module.
220. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module; a purification module; and a reaction
module comprising a capsule capable of holding a liquid or a solid
sample, said capsule comprising an inner surface extending from the
bottom of said capsule to an oval-shaped opening at the top of the
capsule, wherein said inner surface is substantially smooth and
comprises a concave shape extending from the bottom of the capsule;
and a planar layer affixed around the oval-shaped opening of said
capsule and oriented in the same plane as the oval-shaped opening
of said capsule, wherein said planar layer comprises a top surface
and a bottom surface, said top surface aligned with the inner
surface of said capsule at said oval-shaped opening to provide a
continuous surface; wherein the loading module is in fluidic
communication with the lysis module and the purification module is
in fluidic communication with the reaction module.
221. An integrated diagnostic cartridge of claim 219 or claim 220
wherein said capsule is capable of holding a volume from
approximately 50 .mu.L to approximately 200 .mu.L or wherein said
oval-shaped opening is contained within an area of 9 mm.times.9
mm.
222. An integrated diagnostic cartridge of claim 219 or claim 220
wherein said capsule comprises a dried reagent according to any one
of claim 205, claim 212 or claim 213.
223. The integrated diagnostic cartridge according to any one of
claim 137 to claim 222 further comprising a fluidic card and a
cover.
224. The integrated diagnostic cartridge of claim 223, wherein at
least two of the loading module, the lysis module, the purification
module and the reaction module are formed in or supported by the
fluidic card.
225. The integrated diagnostic cartridge of claim 223, wherein at
least two of the loading module, the lysis module, the purification
module and the reaction module are formed in or supported by the
cover.
226. The integrated diagnostic cartridge of claim 223, the fluidic
card further comprising a slot positioned to engage with a latch
and pin assembly of a diagnostic instrument to secure the
integrated diagnostic cartridge in a testing position within the
diagnostic instrument.
227. The integrated diagnostic cartridge of claim 223 further
comprising an interference feature on the cover, wherein the
interference feature is sized and positioned to interact with one
of an upper rail or a lower rail of a loading apparatus of a
diagnostic instrument.
228. The integrated diagnostic cartridge according to any one of
claim 223 to claim 227, wherein a thickness of the fluidic card is
selected for sliding arrangement within an upper rail and a lower
rail of a loading apparatus of the diagnostic instrument.
229. The integrated diagnostic cartridge of any one of claim 137 to
claim 228 wherein a total sample process volume of the integrated
diagnostic cartridge is provided by increasing the thickness of the
cartridge.
230. The integrated diagnostic cartridge of claim 229, wherein a
diagnostic instrument is adapted and configured to accommodate the
increased thickness of the cartridge by increasing a width of an
opening of the diagnostic instrument to accommodate the increased
thickness of the cartridge or a displacement range of a cartridge
clamping system of the diagnostic instrument is adapted to
accommodate the increased thickness of the cartridge.
231. The integrated diagnostic cartridge of any one of claim 137 to
claim 230 further comprising a cartridge front face and a cartridge
rear face forming an upper spacing and a lower spacing wherein each
of the upper spacing and the lower spacing is sized and positioned
to engage with an upper rail and a lower rail of the
instrument.
232. The integrated diagnostic cartridge of claim 231 further
comprising an interference feature within the upper spacing or the
lower spacing positioned to ensure the cartridge engages with the
upper rail and the lower rail in a desired orientation.
233. The integrated diagnostic cartridge of any one of claim 137 to
claim 232 further comprising a plurality of frangible seal chambers
in fluid communication with at least one or more of the loading
module, the lysis module, the purification module or the reaction
module.
234. The integrated diagnostic cartridge of any one of claim 137 to
claim 233 further comprising a label section.
235. The integrated diagnostic cartridge of any one of claim 137 to
claim 234 further comprising one or more machine readable marking
indicating the sample type to be used in the cartridge or target
pathogen to be detected.
236. The integrated diagnostic cartridge of any one of claim 137 to
claim 235 further comprising a pneumatic interface.
237. The integrated diagnostic cartridge of any one of claim 137 to
claim 236 wherein prior to loading the cartridge into a diagnostic
instrument a lysis chamber in the cartridge contains a lysis
buffer.
238. The integrated diagnostic cartridge of any one of claim 137 to
claim 237, the integrated diagnostic cartridge further comprising a
machine-readable code adapted and configured to identify the
cartridge to a diagnostic instrument or a patient identification
marking.
239. The integrated diagnostic cartridge of claim 217 or claim 218
further comprising a film adhered to a surface of the monolithic
substrate, wherein the film forms one wall of the assay
chamber.
240. The integrated diagnostic cartridge of any one of claim 137 to
claim 239, further comprising a first film adhered to a surface of
at least a portion of the cartridge, wherein the first film forms
one wall of one or more chambers, compartments, or fluid conduits
of the loading module, the lysis module, the purification module
and the reaction module.
241. The integrated diagnostic cartridge of claim 240, further
comprising a second film adhered to the first film, wherein the
second film has a higher melting temperature than the first
film.
242. The integrated diagnostic cartridge of claim 241 further
comprising a heat staked region formed in each of the fluidic
pathways using the first film or the second film wherein the heat
staked region seals off the common fluid pathway from the assay
chamber and the pneumatic chamber.
243. The integrated diagnostic cartridge of claim 242 further
comprising a raised platform within each of the plurality of
independent, continuous fluidic pathways the raised platform
positioned between an inlet to the assay chamber and the common
fluid pathway wherein the heat staked region is formed using a
portion of the raised platform.
244. An integrated diagnostic cartridge, comprising: a loading
module having a fill chamber within the cartridge having a volume
sufficient to hold a sample, a fluid inlet in fluid communication
with the fill chamber, a fluid outlet in fluid communication with
fill chamber; a lysis module; a purification module; and a reaction
module; wherein the loading module is in fluidic communication with
the lysis module and the purification module is in fluidic
communication with the reaction module; further wherein the loading
module, the lysis module, the purification module and the reaction
module are arranged for use while the cartridge is in a vertical
orientation; and further wherein when the cartridge is in a
horizontal sample loading orientation the fluid inlet accesses the
fill chamber via an upper surface of the cartridge and when the
cartridge is in a vertical sample processing orientation the fluid
inlet is positioned adjacent to an upper portion of the fill
chamber and the fluid outlet is arranged for the sample to flow out
of a lower portion of the fill chamber.
245. The integrated diagnostic cartridge of claim 244 further
comprising one or more fluid filling conduits arranged to flow into
an upper portion of a vertically oriented chamber within a fluidic
card of the integrated diagnostics cartridge and one or more fluid
outlet conduits arranged to flow out of a lower portion of the
vertically oriented chamber within the fluidic card of the
integrated diagnostics cartridge.
246. The integrated diagnostic cartridge of claim 245, wherein the
vertically oriented chamber further comprises a filter assembly in
fluid communication with a fluid outlet conduit of the vertically
oriented chamber.
247. The integrated diagnostic cartridge of claim 244, wherein the
lysis module comprises a mixing assembly having a vertically
oriented lysis chamber containing a lysis agent and a
non-magnetized stir bar.
248. The integrated diagnostic cartridge of claim 247, wherein the
non-magnetized stir bar is made from a metal having a magnetic
permeability to be responsive to a rotating magnetic field induced
between a drive magnetic element and a driven magnetic element of a
magnetic drive system.
249. The integrated diagnostic cartridge according to claim 247,
wherein the non-magnetized stir bar is coated with an impermeable
material to prevent corrosion by a chemical lysis buffer in the
vertically oriented lysis chamber.
250. The integrated diagnostic cartridge of claim 247 wherein, when
in use within a diagnostic instrument, the non-magnetized stir bar
is disposed between a driving magnet system and a driven magnet
system of a magnetic mixing assembly in the diagnostic instrument,
wherein the driving magnet system is configured to rotate the
non-magnetized stir bar within the vertically oriented lysis
chamber at least 1000 rpm.
251. The integrated diagnostic cartridge of claim 247, further
comprising a fluid inlet to the vertically oriented lysis chamber
and a fluid outlet to lysis chamber wherein the vertically oriented
lysis chamber is isolated from the other modules on the cartridge
by a first frangible seal in fluid communication with the fluid
inlet to the vertically oriented lysis chamber and a second
frangible seal in fluid communication with the fluid outlet to the
vertically oriented lysis chamber.
252. The integrated diagnostic cartridge according to claim 244
further comprising a fluidic card and a cover.
253. The integrated diagnostic cartridge of claim 252, wherein the
fluidic card further comprises a first film adhered to a surface of
at least a portion of the fluidic card, wherein the first film
forms one surface of one or more chambers, compartments, or fluid
conduits of the loading module, the lysis module, the purification
module and the reaction module.
254. The integrated diagnostic cartridge of claim 252 further
comprising an interference feature on the cover, wherein the
interference feature is sized and positioned to interact with one
of an upper rail or a lower rail of a loading apparatus of a
diagnostic instrument.
255. The integrated diagnostic cartridge according to claim 254,
wherein a thickness of the fluidic card is selected for sliding
arrangement within an upper rail and a lower rail of a loading
apparatus of the diagnostic instrument.
256. The integrated diagnostic cartridge of claim 252 wherein a
total sample process volume of the integrated diagnostic cartridge
is related to a thickness of the cartridge corresponding to a
spacing between the one or more chambers, compartments, or fluid
conduits of the loading module, the lysis module, the purification
module and the reaction module formed in the fluidic card and the
first film.
257. The integrated diagnostic cartridge of claim 256, wherein a
diagnostic instrument is adapted and configured to accommodate a
variation of the thickness of the cartridge by increasing a width
of a loading slot of the diagnostic instrument to accommodate the
increased thickness of the cartridge or a displacement range of a
cartridge clamping system of the diagnostic instrument is adapted
to accommodate the increased thickness of the cartridge.
258. The integrated diagnostic cartridge of claim 254 further
comprising a cartridge front face and a cartridge rear face forming
an upper spacing and a lower spacing wherein each of the upper
spacing and the lower spacing is sized and positioned to engage
with the upper rail and lower rail of the diagnostic
instrument.
259. The integrated diagnostic cartridge of claim 258 further
comprising an interference feature within the upper spacing or the
lower spacing positioned to ensure the cartridge engages with the
upper rail and the lower rail in a desired orientation.
260. The integrated diagnostic cartridge of claim 244 further
comprising a plurality of frangible seal chambers in fluid
communication with at least one or more of the loading module, the
lysis module, the purification module or the reaction module.
261. The integrated diagnostic cartridge of claim 244, the
integrated diagnostic cartridge further comprising a
machine-readable code adapted and configured to identify the
cartridge to a diagnostic instrument or an image of a patient
identification marking.
262. An integrated diagnostic cartridge, comprising: a loading
module; a lysis module; a purification module comprising a rotary
valve comprising: a. a stator comprising a stator face and a
plurality of passages, each passage comprising a port at the stator
face; b. a rotor operably connected to the stator and comprising a
rotational axis, a rotor valving face, and a flow channel having an
inlet and an outlet at the rotor valving face, wherein the flow
channel comprises a porous solid support; and c. a retention
element biasing the stator and the rotor together at a rotor-stator
interface to form a fluid tight seal; and a reaction module
comprising a plurality of individual assay chambers, wherein at
least one surface in each one of the plurality of individual assay
chambers is provided by a plug comprising: a body with a bottom
surface; a central opening in the body; and a dried reagent on the
bottom surface, wherein the body is formed from a material
transmissive to excitation wavelengths and emission wavelengths in
at least one of a red spectrum, a blue spectrum and a green
spectrum, further wherein the loading module is in fluidic
communication with the lysis module and the purification module is
in fluidic communication with the reaction module; and wherein the
loading module, the lysis module, the purification module and the
reaction module are arranged for use while the cartridge is in a
vertical orientation.
263. The integrated diagnostic cartridge of claim 262, wherein the
bottom surface of the plug body comprises a cavity in the bottom
surface with the dried reagent within the cavity, and wherein the
plug has a plug thickness between a central opening bottom and the
plug body bottom, and further wherein a depth of the cavity is less
than 90% of the plug thickness, is less than 70% of the plug
thickness or is less than 50% of the plug thickness.
264. The integrated diagnostic cartridge of claim 262, wherein the
plug has a polished or smooth finish facilitating the
transmissivity of the excitation wavelengths and the emission
wavelengths.
265. The integrated diagnostic cartridge according to claim 262,
wherein the dried reagent is selected from the group consisting of
nucleic acid synthesis reagents, nucleic acids, nucleotides,
nucleobases, nucleosides, monomers, detection reagents, catalysts
or combinations thereof.
266. The integrated diagnostic cartridge according to claim 262,
wherein the body of the plug protrudes into the monolithic
substrate of the assay chamber at a depth such that the assay
chamber volume can be readily changed by altering the depth at
which the body of the plug protrudes into the monolithic substrate
of the assay chamber.
267. The integrated diagnostic cartridge according to claim 262
further comprising at least one fluid inlet conduit to each one of
the plurality of individual assay chambers of the reaction module
wherein each one of the at least one fluid inlet conduits further
comprises a heat staked region.
268. The integrated diagnostic cartridge according to claim 267
wherein a heat stake in the heat staked region fluidically isolates
the reaction module from the loading module, the lysis module, and
the purification module.
269. The integrated diagnostic cartridge according to claim 244,
the purification module further comprising: a rotary valve
comprising: a. a stator comprising a stator face and a plurality of
passages, each passage comprising a port at the stator face; b. a
rotor operably connected to the stator and comprising a rotational
axis, a rotor valving face, and a flow channel having an inlet and
an outlet at the rotor valving face, wherein the flow channel
comprises a porous solid support; and c. a retention element
biasing the stator and the rotor together at a rotor-stator
interface to form a fluid tight seal.
270. The integrated diagnostic cartridge of claim 269, wherein the
rotary valve further comprises a gasket between the stator face and
the rotor valving face, and wherein the stator comprises a
displaceable spacer for preventing the gasket from sealing against
at least one of the rotor and stator, and wherein, when the spacer
is displaced the gasket seals the rotor and stator together in a
fluid-tight manner.
271. The integrated diagnostic cartridge of claim 270, wherein,
when the cartridge is positioned within a diagnostic instrument,
engagement with a valve drive assembly of the diagnostic instrument
displaces the spacer and seals the rotor and stator together in a
fluid-tight manner.
272. The integrated diagnostic cartridge of claim 269, the
purification module further comprising: a waste collection element,
a wash buffer reservoir and an elution buffer reservoir.
273. The integrated diagnostic cartridge of claim 269 further
comprising a pneumatic interface in fluidic communication with at
least the purification module.
274. A method of testing a sample suspected of containing one or
more target pathogens, comprising: accepting a cartridge having a
sample port assembly containing the sample suspected of containing
the one or more target pathogens; advancing the sample suspected of
containing the one or more target pathogens to a lysis chamber
within the cartridge having at least one lysis reagent therein;
mixing the sample with the at least one lysis agent to generate a
lysed sample; passing the lysed sample through a porous solid
support within the cartridge to capture a nucleic acid on the
porous solid support; releasing the captured nucleic acid from the
first porous solid support to generate an enriched nucleic acid;
introducing the enriched nucleic into a rehydration chamber within
the cartridge containing one or more dried reagents; after
introducing the analyte/reagent solution into a metering channel,
mixing the contents of the rehydration chamber to produce an
analyte/reagent solution; distributing the analyte/reagent solution
to two or more assay chambers within the cartridge after performing
the mixing step; combining the analyte/reagent solution with one or
more amplification reagents after performing the distributing step;
sealing each one of the two or more assay chambers within the
cartridge containing analyte/reagent solution from each one of all
the other two or more assay chambers within the cartridge
containing analyte/reagent solution and a waste chamber; and
performing an isothermal amplification reaction within each one of
the two or more assay chambers in the cartridge while
simultaneously detecting amplification product, wherein presence of
an amplification product is an indication of a presence, an absence
or a quantity of the target pathogen in the sample suspected of
containing the target pathogen.
275. The method of testing a sample according to claim 274, wherein
in mixing the sample with the at least one lysis agent, the lysis
agent is a mechanical agent.
276. The method of testing a sample according to claim 275, wherein
the mechanical agent is ceramic beads, glass beads or steel beads,
and the mixing the sample step comprises rotating a stir bar within
the lysis chamber at at least 1000 rpm.
277. The method of testing a sample according to claim 276, wherein
mixing the sample comprises rotating the stir bar or the ceramic,
glass or steel beads along with a chemical lysis agent.
278. The method of testing according to claim 274, wherein the at
least one lysis agent is a chemical lysis agent.
279. The method of testing a sample according to claim 278, wherein
the one or more target pathogens is a virus or a gram-negative
bacterium and the lysis reagent is a chaotropic agent.
280. The method of testing a sample according to claim 274, wherein
prior to passing the lysed sample through the porous solid support,
the method further comprises passing the lysed sample through a
size-exclusion filter, wherein nucleic acid passes through the
filter.
281. The method of testing a sample according to claim 274, wherein
the enriched nucleic acid is combined with one or more
amplification reagents before the distributing step and further
wherein the one or more amplification reagents comprise a
primer.
282. The method of testing a sample according to claim 281, wherein
the performing of the isothermal amplification reaction step is
initiated prior to the distributing the enriched nucleic acid to
the two or more assay chambers step.
283. The method of testing a sample according to claim 274, wherein
after the distributing step, but prior to performing the isothermal
amplification reaction step, the method further comprises combining
the enriched nucleic acid with a primer set specific to one of the
one or more target pathogens.
284. The method of testing a sample according to claim 274, wherein
a first assay chamber contains a primer set specific to a first
nucleic acid sequence.
285. The method of testing a sample according to claim 284, wherein
the first nucleic acid sequence is present in one of the one or
more target pathogens.
286. The method of testing a sample according to claim 284, wherein
prior to mixing the sample with at least one lysis agent, a process
control is added to the sample and the first nucleic acid sequence
is present in the process control.
287. The method of testing a sample according to claim 284, wherein
prior to passing lysed sample through the porous solid support, a
process control is added to the lysed sample and the first nucleic
acid sequence is present in the process control.
288. The method of testing a sample according to claim 284, wherein
a second assay chamber contains a primer set specific to a second
nucleic acid sequence, wherein the second nucleic acid sequence is
present in one of the one or more target pathogens.
289. The method of testing a sample according to claim 274, wherein
the performing an isothermal amplification reaction step is
completed in less than 15 minutes.
290. The method of testing a sample according to claim 274, further
comprising: providing a result containing a determination made
during the performing step relating to the presence, the absence or
the quantity of the target pathogen in the sample suspected of
containing the target pathogen.
291. The method of testing a sample according to claim 274, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with a chemical reaction.
292. The method of testing a sample according to claim 291, wherein
the sample is sputum and the chemical reaction is incubation with a
mucolytic agent.
293. The method of testing a sample according to claim 274, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with an enzymatic
reaction.
294. The method of testing a sample according to claim 293, wherein
the enzymatic reaction is incubation of the sample with a nuclease,
a protease, an amylase, a glycosylase, or a lipase.
295. The method of testing a sample according to claim 274, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with a physical
treatment.
296. The method of testing a sample according to claim 295, wherein
the physical treatment comprises passing the sample through a
size-exclusion filter in a first direction.
297. The method of testing a sample according to claim 295, wherein
the physical treatment comprises exposing the sample to a capture
agent immobilized on a solid substrate.
298. The method of testing a sample according to claim 297, further
comprising, after exposure, separating the solid substrate from the
sample.
299. The method of testing a sample according to claim 297, wherein
the capture agent is an antibody with affinity for red blood
cells.
300. The method of testing a sample according to claim 274, wherein
the sample is sputum and the method further comprises, prior to
mixing the sample with the at least one lysis reagent, bead beating
the sputum to liquify the sample.
301. The method of testing a sample according to claim 300, wherein
the bead beating comprises mixing the sputum with ceramic, glass,
or steel beads.
302. The method of testing a sample according to claim 274, wherein
prior to distributing the enriched nucleic acid to the assay
chambers, the method further comprises passing the enriched nucleic
acid through a second porous solid support.
303. A method of testing a sample suspected of containing one or
more target pathogens, comprising: accepting a cartridge having a
sample port assembly containing the sample suspected of containing
the one or more target pathogens; advancing the sample suspected of
containing the one or more target pathogens to a lysis chamber
within the cartridge having at least one lysis reagent therein;
mixing the sample with the at least one lysis agent to generate a
lysed sample; passing the lysed sample through a porous solid
support within the cartridge to capture a nucleic acid on the
porous solid support; releasing the captured nucleic acid from the
first porous solid support to generate an enriched nucleic acid;
introducing the enriched nucleic into a rehydration chamber within
the cartridge containing one or more dried reagents to generate an
analyte/reagent solution; after introducing the analyte/reagent
solution into a metering channel, mixing the contents of the
rehydration chamber to homogenize an analyte/reagent solution;
distributing the analyte/reagent solution to two or more assay
chambers within the cartridge after performing the mixing step;
combining the analyte/reagent solution with one or more
amplification reagents after performing the distributing step to
generate an amplification solution; sealing each one of the two or
more assay chambers within the cartridge containing amplification
solution from each one of all the other two or more assay chambers
within the cartridge containing amplification solution and a waste
chamber; and performing an isothermal amplification reaction within
each one of the two or more assay chambers in the cartridge while
simultaneously detecting amplification product, wherein presence of
an amplification product is an indication of a presence, an absence
or a quantity of the target pathogen in the sample suspected of
containing the target pathogen.
304. The method of testing a sample according to claim 303, wherein
in mixing the sample with the at least one lysis agent, the lysis
agent is a mechanical agent.
305. The method of testing a sample according to claim 304, wherein
the mechanical agent is ceramic beads, glass beads or steel beads,
and the mixing the sample step comprises rotating a stir bar within
the lysis chamber at at least 1000 rpm.
306. The method of testing a sample according to claim 305, wherein
mixing the sample comprises rotating the stir bar or the ceramic,
glass or steel beads along with a chemical lysis agent.
307. The method of testing according to claim 303, wherein the at
least one lysis agent is a chemical lysis agent.
308. The method of testing a sample according to claim 307, wherein
the one or more target pathogens is a virus or a gram-negative
bacterium and the lysis reagent is a chaotropic agent.
309. The method of testing a sample according to claim 303, wherein
prior to passing the lysed sample through the porous solid support,
the method further comprises passing the lysed sample through a
size-exclusion filter, wherein nucleic acid passes through the
filter.
310. The method of testing a sample according to claim 303, wherein
a first assay chamber contains a primer set specific to a first
nucleic acid sequence.
311. The method of testing a sample according to claim 310, wherein
the first nucleic acid sequence is present in one of the one or
more target pathogens.
312. The method of testing a sample according to claim 310, wherein
prior to mixing the sample with at least one lysis agent, a process
control is added to the sample and the first nucleic acid sequence
is present in the process control.
313. The method of testing a sample according to claim 310, wherein
prior to passing lysed sample through the porous solid support, a
process control is added to the lysed sample and the first nucleic
acid sequence is present in the process control.
314. The method of testing a sample according to claim 310, wherein
a second assay chamber contains a primer set specific to a second
nucleic acid sequence, wherein the second nucleic acid sequence is
present in one of the one or more target pathogens.
315. The method of testing a sample according to claim 303, wherein
the performing an isothermal amplification reaction step is
completed in less than 15 minutes.
316. The method of testing a sample according to claim 303, further
comprising: providing a result containing a determination made
during the performing step relating to the presence, the absence or
the quantity of the target pathogen in the sample suspected of
containing the target pathogen.
317. The method of testing a sample according to claim 303, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with a chemical reaction.
318. The method of testing a sample according to claim 317, wherein
the sample is sputum and the chemical reaction is incubation with a
mucolytic agent.
319. The method of testing a sample according to claim 303, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with an enzymatic
reaction.
320. The method of testing a sample according to claim 319, wherein
the enzymatic reaction is incubation of the sample with a nuclease,
a protease, an amylase, a glycosylase, or a lipase.
321. The method of testing a sample according to claim 303, wherein
the method further comprises, prior to advancing the sample to a
lysis chamber, pretreating the sample with a physical
treatment.
322. The method of testing a sample according to claim 321, wherein
the physical treatment comprises passing the sample through a
size-exclusion filter in a first direction.
323. The method of testing a sample according to claim 321, wherein
the physical treatment comprises exposing the sample to a capture
agent immobilized on a solid substrate.
324. The method of testing a sample according to claim 323, further
comprising, after exposure, separating the solid substrate from the
sample.
325. The method of testing a sample according to claim 323, wherein
the capture agent is an antibody with affinity for red blood
cells.
326. The method of testing a sample according to claim 303, wherein
the sample is sputum and the method further comprises, prior to
mixing the sample with the at least one lysis reagent, bead beating
the sputum to liquify the sample.
327. The method of testing a sample according to claim 326, wherein
the bead beating comprises mixing the sputum with ceramic, glass,
or steel beads.
328. The method of testing a sample according to claim 303, wherein
prior to distributing the analyte/reagent solution to the two or
more assay chambers, the method further comprises passing the
analyte/reagent solution through a second porous solid support.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 62/887,469, filed Aug. 15, 2019, titled
"DIAGNOSTIC SYSTEM," which is herein incorporated by reference in
its entirety.
[0002] This application also claims priority as a continuation of
U.S. patent application Ser. No. 16/655,007, filed Oct. 16, 2019,
titled "DIAGNOSTIC SYSTEM," which is herein incorporated by
reference in its entirety.
[0003] This application also claims priority as a continuation of
U.S. patent application Ser. No. 16/655,028, filed Oct. 16, 2019,
titled "DIAGNOSTIC SYSTEM," which is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0006] A molecular diagnostic instrument for performing tests on a
sample contained in an integrated diagnostic cartridge.
BACKGROUND OF THE INVENTION
[0007] In the U.S. alone, over one billion infections occur each
year. To combat this, advancements in molecular diagnostic testing
have enabled medical professionals to diagnose infectious diseases
accurately. Nearly all molecular diagnostics testing currently is
performed in centralized laboratories. While such tests performed
in central laboratories are very accurate, results can be delayed
several days or longer and they require expensive, high throughput
instrumentation, regulated infrastructure, and trained personnel.
For example, high throughput instrumentation generally processes
many (e.g. 96 or 384 or more) samples at a time. Samples are
collected during a time period, e.g., a day, and are then processed
in one large batch. Additionally, requiring trained technician,
responsible for operating laboratory equipment, adding reagents,
and overseeing sample processing, e.g., moving samples from step to
step, are too expensive or unavailable to practices in sparsely
populated or economically challenged locations.
[0008] As an alternative to centralized laboratory testing, some
testing can be performed at the point of care (POC) providing near
patient rapid diagnosis outside of a laboratory environment.
However, there are limited POC testing options available and many
known POC tests have poor sensitivity (30-70%), as compared to
highly sensitive central-lab molecular diagnostic tests. Current
POC testing options tend to be single analyte tests with low
analytical quality. These tests are used alongside clinical
algorithms to assist in diagnosis, but are frequently verified by
higher quality, laboratory tests for the definitive diagnosis.
Thus, neither consumers nor physicians achieve a rapid, accurate
test result in the time frame required to "test and treat" a
patient in one visit. As a result, doctors and patients often
determine an empiric course of treatment before they know the
diagnosis. This lack of knowledge has tremendous ramifications:
either antibiotics are not prescribed when needed, leading to
disease progress and/or transmission to another host; or
antibiotics are prescribed when not needed, leading to new
antibiotic-resistant strains in the community.
[0009] In one specific example, Gram-negative Neisseria gonorrhoeae
has progressively developed resistance to the antibiotic drugs
prescribed to treat it and is one of only three organisms on the
CDC's list of urgent threats. Preventing the spread of gonorrhea
relies on prompt diagnosis and treatment of infected persons and
their partners. The turn-around time for centralized lab testing is
1-5 days. Therefore, physicians are faced with one of two choices:
(1) wait days for test results before treating a patient and risk
that a positive patient may continue to spread the infection
through their partners, and their partners' partners or (2) treat
empirically while the patient is in front of them. In a study of
1103 emergency room patients at Johns Hopkins, 440 patients who had
a suspected CT or NG infection were treated with antibiotics though
the vast majority, 323 patients, ultimately turned out to be
negative. As a direct result of the overuse and misuse of
antibiotics through empiric therapy, antibiotic resistance in
gonorrhea is on the verge of becoming a public health crisis. To
prevent the development of future antibiotic resistant strains,
molecular diagnostic testing at the point of care can prevent
unnecessary antibiotics from being prescribed and provide rapid
diagnosis and treatment.
[0010] Highly trained personnel are required to perform molecular
diagnostic tests because these sophisticated assays are powered by
nucleic acid amplification methods, such as PCR, and carried out on
biologic samples, which typically contain a variety of substances
inhibitory to amplification. However, such trained personnel
typically are not available at the locations where patients are
being seen, i.e. at the point of care. Additional challenges
associated with the point of care environment include fulfilling
physician or clinical workflow compatibility coupled with an
unknown skill level for system users. Accordingly, point of care
molecular diagnostic systems must be designed for the ease of use
by system users and be robust in performing sample preparation and
amplification, with minimal user interaction, to generate reliable
diagnostic results
[0011] Thus, despite the existence of some point of care diagnostic
systems, a need exists for improved devices and methods for
molecular diagnostic testing. In particular, an unmet need
continues for an easy-to-use system enabling rapid molecular
diagnostic capabilities in the point of care environment.
SUMMARY
[0012] In general, in one embodiment, a method of testing a sample
suspected of containing one or more target pathogens includes: (1)
accepting a cartridge having a sample port assembly containing the
sample suspected of containing the one or more target pathogens;
(2) advancing the sample suspected of containing the one or more
target pathogens to a lysis chamber having at least one lysis
reagent therein; (3) mixing the sample with the at least one lysis
agent to generate a lysed sample; (4) passing the lysed sample
through a first porous solid support to capture a nucleic acid on
the porous solid support; (5) releasing the captured nucleic acid
from the first porous solid support to generate an enriched nucleic
acid; (6) distributing the enriched nucleic acid to two or more
assay chambers; (7) combining the enriched nucleic acid with one or
more amplification reagents; (8) isolating each one of the two or
more assay chambers from each one of all the other two or more
assay chambers; and (9) performing an isothermal amplification
reaction within each one of the two or more assay chambers while
simultaneously detecting amplification product, wherein presence of
an amplification product is an indication of a presence, an absence
or a quantity of the target pathogen in the sample suspected of
containing the target pathogen.
[0013] This and other embodiments can include one or more of the
following features. The sample can be a biological sample obtained
from a mammal. The mammal can be a person providing a biological
sample. The sample can be obtained from a food product, a natural
non-growth hormone crop sample, a crop sample, a water sample, a
non-biological fluid sample or a soil sample. The step of accepting
a cartridge step can further include reading a bar code on the
cartridge and determining to proceed with the method of testing.
The method can further include obtaining and analyzing an image of
a sample window of the sample port assembly and determining to
proceed with the method of testing. The sample in the sample port
assembly can be in fluid communication with a fill chamber, a
metering chamber, and an overflow chamber. The sample window can be
transparent and formed in at least a portion of a wall of a
metering chamber. Obtaining an image can further include obtaining
an image of the transparent viewing window. Analyzing an image can
further include assessing a height of a sample liquid in the
metering chamber via the transparent viewing window. The step of
obtaining and analyzing an image can further include obtaining an
image of the metering chamber including a buoyant ball and
analyzing the image can include identifying a location of the ball
within the metering chamber and determining to proceed with the
method based on the location of the ball. The method can further
include obtaining and analyzing an image of a patient ID label and
determining to proceed with the method of testing. The method can
further include confirming a rotary valve on the cartridge is in a
shipping configuration before proceeding to the advancing the
sample step. The method can further include obtaining a reading
from an interference sensor on a valve drive assembly and
confirming based on the reading that a rotary valve on the
cartridge is not in an operational configuration prematurely. The
method can further include engaging a rotary valve on the cartridge
with a valve drive assembly and rotating the rotary valve into an
operational configuration. Rotating the rotary valve in an
operational configuration can place a rotary valve gasket into
contact with a stator on the cartridge. The method can further
include moving a clamping block for engaging the cartridge with a
door support assembly, a pneumatic interface assembly, and a
thermal clamp assembly. The moving step can be a single continuous
movement. The method can further include moving a frangible seal
block having a plurality of frangible seal pins into position to
engage one or more frangible seals on the cartridge. Moving the
frangible seal block simultaneously can engage the plurality of
frangible seal pins with the one or more frangible seals on the
cartridge. Moving the frangible seal block sequentially can engage
the plurality of frangible seal pins with the one or more frangible
seals on the cartridge. The step of moving a frangible seal block
can be performed after performing the step of moving a clamp block.
The step of moving a frangible seal block can be performed
initially with the clamp block and ends in a position separate from
the clamp block. The method can further include moving a clamp
block and a frangible seal block together for engaging the
cartridge. The method can further include moving the clamp block
together with the frangible seal block until the cartridge is
engaged with a door support assembly, a pneumatic interface
assembly, and a thermal clamp assembly. The method can further
include only driving the frangible seal block assembly to engage
one of more frangible seals on the cartridge simultaneously or
sequentially. In mixing the sample with the at least one lysis
agent, the lysis agent can be a mechanical agent. The mechanical
agent can be ceramic beads, glass beads or steel beads, and the
mixing the sample step can include rotating the stir bar at at
least 1000 rpm. Mixing the sample can include rotating the stir bar
or the ceramic, glass or steel beads along with a chemical lysis
agent. The suspected pathogen can be a gram-positive bacterium, a
fungus or a plant cell. In the mixing the sample with the at least
one lysis agent step, the at least one lysis agent can be a
chemical lysis agent. The one or more target pathogens can be a
virus or a gram-negative bacterium and the lysis reagent can be a
chaotropic agent. Prior to passing the lysed sample through the
porous solid support, the method can further include passing the
lysed sample through a size-exclusion filter, wherein nucleic acid
can pass through the filter. The enriched nucleic acid can be
combined with one or more amplification reagents before the
distributing step. The one or more amplification reagents can be
selected from the group consisting of a DNA polymerase, a reverse
transcriptase, a helicase, nucleotide triphosphates (NTPs), a
magnesium salt, a potassium salt, an ammonium salt, and a buffer.
The one or more amplification reagents can further include a
primer. Isothermal amplification can be initiated prior to
distributing the enriched nucleic acid to the two or more assay
chambers. After the distributing step, but prior to perform the
isothermal amplification reaction, the method can further include
combining the enriched nucleic acid with a primer set specific to
one of the one or more target pathogens. A first assay chamber can
contain a primer set specific to a first nucleic acid sequence. The
first nucleic acid sequence can be present in one of the one or
more target pathogens. Prior to mixing the sample with at least one
lysis agent, a process control can be added to the sample and the
first nucleic acid sequence is present in the process control.
Prior to passing lysed sample through the porous solid support, a
process control can be added to the lysed sample and the first
nucleic acid sequence can be present in the process control. A
second assay chamber can contain a primer set specific to a second
nucleic acid sequent. The second nucleic acid sequence can be
present in one of the one or more target pathogens. The performing
an isothermal amplification reaction step can be completed in less
than 20 minutes. The performing an isothermal amplification
reaction step can be completed in less than 15 minutes. The
performing an isothermal amplification reaction step can be
completed in less than 10 minutes. The method of testing a sample
can further include providing a result containing a determination
made during the performing step relating to the presence, the
absence or the quantity of the target pathogen in the sample
suspected of containing the target pathogen. The method can further
include, prior to advancing the sample to a lysis chamber,
pretreating the sample with a chemical reaction. The sample can be
sputum and the chemical reaction can be incubation with a mucolytic
agent. The mucolytic agent can be dithiothreitol or
n-acetylcysteine. The method can further include, prior to
advancing the sample to a lysis chamber, pretreating the sample
with an enzymatic reaction. The enzymatic reaction can be
incubation of the sample with a nuclease, a protease, an amylase, a
glycosylase, or a lipase. Pretreating can include incubating the
sample with a DNase. Pretreating can include incubating the sample
with a protease. The protease can be selected from pronase,
chymotrypsin, trypsin and pepsin. The method can further include,
prior to advancing the sample to a lysis chamber, pretreating the
sample with a physical treatment. The physical treatment can
include passing the sample through a size-exclusion filter in a
first direction. The target pathogen can pass through the filter.
The target pathogen may or may not pass through the filter and can
thereby capture on a fill port side of the size-exclusion filter.
The method can further include passing a volume of suspension
buffer through the size-exclusion filter in a second direction,
wherein second direction can be opposite the first direction,
thereby releasing the target pathogen from the fill port side of
the filter. The volume of suspension buffer can be less than the
volume of the sample, and the target pathogen can be more
concentrated than in the loaded sample. The physical treatment can
include exposing the sample to a capture agent immobilized on a
solid substrate. The method can further include, after exposure,
separating the solid substrate from the sample. The capture agent
can be a capture antibody. The capture agent can be an antibody
with affinity for red blood cells. The solid substrate can be a
magnetic bead, the capture agents can have affinity for a class of
cells including the one or more target pathogens and the method can
further include: (1) incubating the magnetic beads with the sample;
(2) engaging a magnet to draw the magnetic beads to a location
within the sample loading structure; (3) washing away unbound
sample; (4) releasing the magnet; and (5) resuspending the magnetic
beads and passing the suspension, including target pathogen bound
to the magnetic beads, to the lysis chamber. The sample can be
sputum and the method can further include, prior to mixing the
sample with the at least one lysis reagent, bead beating the sputum
to liquify the sample. The bead beating can include mixing the
sputum with ceramic, glass, or steel beads. The bead beating can
include mixing the sputum with ceramic, glass, or steel beads and
dithiothreitol. Prior to distributing the enriched nucleic acid to
the assay wells, the method can further include passing the
enriched nucleic acid through a second porous solid support. The
second porous solid support can be the same as the first porous
solid support. The enriched nucleic acid can be mixed with a matrix
binding agent prior to passing through the second solid support.
Matrix binding agent can be an alcohol or a salt solution. The
second porous solid support can be different than the first porous
solid support, and the second solid support can have an affinity
for nucleic acid and the method can further include releasing the
captured nucleic acid from the second solid support to generate a
twice-enriched nucleic acid. The second porous solid support can be
different than the first porous solid support. Prior to passing the
lysed sample through a first porous solid support, the method can
further include passing the lysed sample through a second porous
solid support, wherein the second solid support may or may not bind
nucleic acid and can have affinity for one or more contaminants,
thereby removing contaminant from the lysed sample.
[0014] The method can further include releasing the cartridge from
engagement with a clamp block and a frangible seal block after
completing the performing an isothermal amplification reaction
step. The method can further include displaying a result produced
after the step of performing an isothermal amplification reaction
step. The method can further include storing in a computer memory a
result produced after the step of performing an isothermal
amplification reaction step. The method can further include
maintaining the cartridge in a vertical orientation while
performing the steps of testing a sample. The cartridge can be
inclined no more than 30 degrees while in the vertical orientation.
The cartridge can be inclined no more than 15 degrees while in the
vertical orientation.
[0015] In some embodiments, during the combining the enriched
nucleic acid step in each of the two or more assay chambers, the
enriched nucleic acid can combine with a dried reagent contained in
each one of the two or more assay chambers. The dried reagent can
be on a surface of a plug in each one of the two or more assay
chambers. The dried reagent can be on a surface of the plug formed
from a material transmissive to excitation wavelengths and emission
wavelengths in at least one of a red spectrum, a blue spectrum and
a green spectrum used during the performing step.
[0016] The method can further include distributing the enriched
nucleic acid to two or more assay chambers using a rotary valve on
the cartridge and a pneumatic signal introduced into the rotary
valve further wherein the pneumatic signal continues to be
introduced while the performing step is performed. Performing the
isolation step can temporarily isolate each one of the two or more
assay chambers are from each one of all the other two or more assay
chambers. The isolation step can be performed using a pneumatic
signal, a mechanical system to occlude one or more fluid channels
to occlude one or more passages or channels of a cartridge. The
mechanical system can be one of a single pinch valve, a plurality
of pinch valves, and a non-heated staker bar. Performing the
isolation step can permanently isolate each one of the two or more
assay chambers from each one of all the other two or more assay
chambers. After performing the isolation step, a portion of the
cartridge can be melted or can be plastically deformed. After
completing the performing step, each one of the two or more assay
chambers can be isolated from each one of all the other two or more
assay chambers. The method can further include distributing the
enriched nucleic acid to two or more assay chambers using a rotary
valve on the cartridge and a pneumatic signal introduced into the
rotary valve. The pneumatic signal can continue to be introduced
while performing the isolating step by moving a heat staker into
contact with the cartridge to isolate each one of the two or more
assay chambers from each one of all the other two or more assay
chambers. After performing the isolating step, a single heat stake
can isolate each one of the two or more assay chambers from each
one of all the other two or more assay chambers. The single heat
stake can isolate a waste chamber on the cartridge. The method can
further include moving a heat staker into contact with the
cartridge to seal each one of the two or more assay chambers from
each one of all the other two or more assay chambers. The method
can further include providing a pneumatic pressure in the cartridge
while moving the heat staker into contact with the cartridge. The
method can further include forming a heat stake region in the
cartridge to isolate each one of the two or more assay chambers
from each one of all the other two or more assay chambers. The
method can further include obtaining a first image of a level of
fluid in each of the one or more assay chambers after the step of
distributing the enriched nucleic acid to each one of the two or
more assay chambers. The method can further include obtaining a
second image of a level of fluid in each of the one or more assay
chambers after the isolating step. The method can further include
determining the quality of the heat stake by comparing the level of
fluid in the first image to the level of fluid in the second image.
The method can further include rotating a rotary valve on the
cartridge prior to performing the advancing the sample step. The
method can further include advancing the sample to the lysis
chamber using a pneumatic signal introduced into a cartridge
pneumatic interface. The method of testing a sample can further
include rotating a rotary valve on the cartridge prior to
performing the step of passing the lysed sample through a first
porous solid support to capture a nucleic acid on the porous solid
support. The method of testing a sample can further include passing
the lysed sample through the first porous solid support using a
pneumatic signal introduced into the rotary valve. The method of
testing a sample can further include distributing the enriched
nucleic acid to two or more assay chambers using a rotary valve on
the cartridge and a pneumatic signal introduced into the rotary
valve.
[0017] In general, in one embodiment, an apparatus includes an
enclosure, a fixed support bracket within the enclosure, a first
imaging system mounted on the fixed support bracket within the
enclosure adjacent to an opening, a second imaging system mounted
on the fixed support bracket within the enclosure configured to
collect images from a second imaging area within the enclosure, a
moving support bracket within the enclosure and moveable relative
to the fixed support bracket, the first imaging system and the
second imaging system, a drive system on the fixed support bracket
configured to position the moving support bracket relative to the
fixed support bracket, and an opening positioned in the enclosure
to provide access to an interior portion of the enclosure between
the fixed support bracket and the moving support bracket. The first
imaging system is configured to collect images from a first imaging
area within the enclosure. The second imaging area is in
non-overlapping relation to the first imaging area.
[0018] This and other embodiments can include one or more of the
following features. The moving support bracket can be positioned
between the first imaging system and the second imaging system. A
rotary connector, a pneumatic connector and a multiple pin block
can be connected to and move with the moving support bracket. The
multiple pin block can be directly connected to the drive system.
The multiple pin block can be configured to move together with the
rotary connector and the pneumatic connector and independent of the
rotary connector and the pneumatic connector. The opening can be a
slot. The slot can be aligned to access an upper rail within the
enclosure aligned to an upper portion of the slot and a lower rail
within the enclosure aligned to a lower portion of the slot. The
apparatus can further include a loading and ejection mechanism
within the enclosure in sliding relation to the lower rail. The
loading and ejection mechanism can move between a loading position
and a loaded position. When in the loading position, the loading
and ejection mechanism can be positioned in a forward most position
towards the slot and when in the loaded position the loading and
ejection mechanism can be engaged with a load position sensor. The
load position sensor can provide an electronic indication when the
loading and ejection mechanism has translated into the loaded
position. The apparatus can further include a first heater and a
second heater mounted on the fixed support bracket. The first
heater can be positioned to heat a portion of the fixed support
bracket between the first imaging area and the second imaging area.
The second heater can be positioned to heat a portion of the fixed
support bracket only within the second imaging area. The apparatus
can further include a channel in the fixed support bracket and a
heat stake assembly positioned to move a heating element through
the channel. The channel can be positioned on the fixed support
bracket to allow the heating element to interact within the
enclosure between the first imaging area and the second imaging
area. The channel can be positioned within the fixed support
bracket such that the heating element may perform a heat staking
operation directly adjacent to but outside of the second imaging
area. The moving support bracket can partially block the channel
when the moving support bracket is positioned at a closest position
to the fixed support bracket.
[0019] In general, in one embodiment, an apparatus includes an
enclosure, a fixed support bracket within the enclosure, a moving
support bracket within the enclosure and moveable relative to the
fixed support bracket, a drive system configured to position the
moving support bracket relative to the fixed support bracket, an
opening positioned in the enclosure to provide access to an
interior portion of the enclosure between the fixed support bracket
and the moving support bracket; and an upper rail and a lower rail
in the enclosure positioned adjacent to the opening wherein a
cartridge positioned between the upper rail and the lower rail
remains in a vertical position between the fixed support bracket
and the moving support bracket.
[0020] This and other embodiments can include one or more of the
following features. The apparatus can further include a feature
within the upper rail or the lower rail positioned to interfere
with the movement of a cartridge improperly aligned with respect to
the upper rail and the lower rail. The apparatus can further
include a loading and ejection assembly within the enclosure
positioned to engage with a cartridge moving along the upper rail
and the lower rail. The apparatus can further include a latch and
pin assembly positioned adjacent to the upper rail adapted to
engage a pin with a cartridge moving along the upper rail. The
apparatus can further include a touch screen display on an exterior
of the enclosure. The apparatus can further include a cellular
communications module within the enclosure. The cellular
communication module can be adjacent to the opening. The apparatus
can further include a cartridge heater, a driving magnet system, a
chemistry heater, a rehydration motor, a reaction camera and a heat
stake assembly coupled to the fixed support bracket and positioned
to interact with a corresponding portion of a cartridge positioned
between the upper rail and the lower rail. The apparatus can
further include a first imaging system mounted on the fixed support
bracket within the enclosure adjacent to the opening. The first
imaging system can be configured to collect images from a first
imaging area within the enclosure and a second imaging system can
be mounted on the fixed support bracket within the enclosure
configured to collect images from a second imaging area within the
enclosure. The second imaging area may be in non-overlapping
relation to the first imaging area. The first imaging area can
include a label of a cartridge positioned within the enclosure
between the upper rail and the lower rail. The second imaging area
can include one or more assay chambers of a cartridge positioned
within the enclosure between the upper rail and the lower rail. The
apparatus can further include a clamp block, a frangible seal
block, a valve driver, a pneumatic interface, a thermal clamp, and
a driven magnet system coupled to move along with the moving
support bracket during operation of the drive system. The apparatus
can further include a plenum adjacent to the chemistry heater and a
fan in fluid communication with the plenum. The apparatus can
further include a staker blade positioned to move relative to a
depth stop frame. The staker blade can be coupled to a linear
actuator motor and a spring with pivot washer.
[0021] In general, in one embodiment, an integrated diagnostic
cartridge includes a loading module, a lysis module, a purification
module, and a reaction module. The loading module is in fluidic
communication with the lysis module and the purification module is
in fluidic communication with the reaction module. The loading
module, the lysis module, the purification module and the reaction
module are arranged for use while the cartridge is in a vertical
orientation.
[0022] This and other embodiments can include one or more of the
following features. The integrated diagnostic cartridge can further
include one or more fluid filling conduits arranged to flow into an
upper portion of a chamber within a fluidic card of the integrated
diagnostics cartridge and one or more fluid outlet conduits
arranged to flow out of a lower portion of the chamber within the
fluidic card of the integrated diagnostics cartridge. The chamber
can be one or more of a lysis chamber, a metering chamber, a wash
buffer chamber or an elution buffer chambers. The chamber can
further include a filter assembly in fluid communication with a
fluid outlet conduit of the chamber. The lysis module can include a
mixing assembly having a vertically oriented lysis chamber
containing a lysis agent and a non-magnetized stir bar. The
non-magnetized stir bar can be made from a metal having a magnetic
permeability to be responsive to a rotating magnetic field induced
between a drive magnetic element and a driven magnetic element of a
magnetic drive system. The non-magnetized stir bar can be coated
with an impermeable material to prevent corrosion by a chemical
lysis buffer in the vertically oriented lysis chamber. When in use
within a diagnostic instrument, the non-magnetized stir bar can be
disposed between a driving magnet system and a driven magnet system
of a magnetic mixing assembly in the diagnostic instrument. The
driving magnet system can be configured to rotate the
non-magnetized stir bar within the vertically oriented lysis
chamber at least 1000 rpm. The integrated diagnostic cartridge can
further include a fluid inlet to the vertically oriented lysis
chamber and a fluid outlet to lysis chamber wherein the vertically
oriented lysis chamber can be isolated from the other modules on
the cartridge by a first frangible seal in fluid communication with
the fluid inlet to the vertically oriented lysis chamber and a
second frangible seal in fluid communication with the fluid outlet
to the vertically oriented lysis chamber. The integrated diagnostic
cartridge can further include a fluidic card and a cover. The
fluidic card can further include a first film adhered to a surface
of at least a portion of the fluidic card. The first film can form
one surface of one or more chambers, compartments, or fluid
conduits of the loading module, the lysis module, the purification
module and the reaction module. The integrated diagnostic cartridge
can further include an interference feature on the cover. The
interference feature can be sized and positioned to interact with
one of an upper rail or a lower rail of a loading apparatus of a
diagnostic instrument. A thickness of the fluidic card can be
selected for sliding arrangement within an upper rail and a lower
rail of a loading apparatus of the diagnostic instrument. A total
sample process volume of the integrated diagnostic cartridge can be
related to a thickness of the cartridge corresponding to a spacing
between the one or more chambers, compartments, or fluid conduits
of the loading module, the lysis module, the purification module
and the reaction module formed in the fluidic card and the first
film. A diagnostic instrument can be adapted and configured to
accommodate a variation of the thickness of the cartridge by
increasing a width of an opening of the diagnostic instrument to
accommodate the increased thickness of the cartridge or a
displacement range of a cartridge clamping system of the diagnostic
instrument is adapted to accommodate the increased thickness of the
cartridge. The integrated diagnostic cartridge can further include
a cartridge front face and a cartridge rear face forming an upper
spacing and a lower spacing. Each of the upper spacing and the
lower spacing can be sized and positioned to engage with the upper
rail and lower rail of the diagnostic instrument. The integrated
diagnostic cartridge can further include an interference feature
within the upper spacing or the lower spacing positioned to ensure
the cartridge engages with the upper rail and the lower rail in a
desired orientation. The integrated diagnostic cartridge can
further include a plurality of frangible seal chambers in fluid
communication with at least one or more of the loading module, the
lysis module, the purification module or the reaction module. The
integrated diagnostic cartridge can further include a
machine-readable code adapted and configured to identify the
cartridge to a diagnostic instrument or an image of a patient
identification marking.
[0023] In general, in one embodiment, an integrated diagnostic
cartridge includes a loading module including a sample port
assembly having a fill chamber, a metering chamber, and an overflow
chamber arranged in fluid communication, a lysis module, a
purification module, and a reaction module. The loading module is
in fluidic communication with the lysis module and the purification
module is in fluidic communication with the reaction module.
[0024] This and other embodiments can include one or more of the
following features. The metering chamber can include a transparent
viewing window for observing the height of a sample within the
metering chamber. The integrated diagnostic cartridge can further
include a ball float in the metering chamber adapted for use with
the transparent viewing window. The fill chamber can include a cap
operable to provide access to the fill chamber. The cap can be
positioned for interaction with a closing apparatus of a diagnostic
instrument. The cartridge can be in a vertical orientation when in
use within a diagnostic instrument and a fluid channel connects an
outlet at a lower portion of the fill chamber with an inlet to the
metering chamber located in an upper portion of the metering
chamber. The metering chamber can include a transparent viewing
window. The integrated diagnostic cartridge can further include a
buoyant ball within the metering chamber. Said buoyant ball can
adapt to appear adjacent to the transparent viewing window
permitting an assessment of the height of the sample liquid in the
metering chamber. The metering chamber can include a buoyant ball
for assessing a height of a sample liquid in the metering
chamber.
[0025] In general, in one embodiment, an integrated diagnostic
cartridge includes a loading module, a lysis module including a
mixing assembly having a lysis chamber containing a lysis agent and
a non-magnetized stir bar, a purification module, and a reaction
module. The loading module is in fluidic communication with the
lysis module and the purification module is in fluidic
communication with the reaction module.
[0026] This and other embodiments can include one or more of the
following features. The non-magnetized stir bar can be made from a
metal having a magnetic permeability to be responsive to a rotating
magnetic field induced between a drive magnetic element and a
driven magnetic element of a magnetic drive system. The metal can
include a ferritic stainless steel or a duplex stainless steel. The
non-magnetized stir bar can be made from a metal selected from the
group consisting of a carbon steel, a mild carbon steel, a low
alloy steel, a tool steel, a metal alloy contain nickel, a metal
alloy containing cobalt, a non-austenitic stainless steel, a
ferritic grade of stainless steel including 430 steel, Atlas CR12
steel, 444 steel, F20S steel, a duplex grade of steel including
2205 steel, 2304 steel, 2101 steel, 2507 steel and a martensitic
grade of steel such as 431 steel, 416 steel, 420 steel and 440C
steel. The metal can have a magnetic permeability to be responsive
to a rotating magnetic field produced within the mixing chamber.
The metal can have a magnetic permeability between 500-1,000,000.
The non-magnetized stir bar can be coated with an impermeable
material to prevent corrosion by a chemical lysis buffer in lysis
chamber. The impermeable material can be PTFE, parylene C, parylene
D, a functionalized perfluoropolyether (PFPE), Xylan Fluoropolymer,
epoxy, or urethane. When in use within a diagnostic instrument, the
non-magnetized stir bar can be disposed between a driving magnet
system and a driven magnet system of a magnetic mixing assembly in
the diagnostic instrument. The driving magnet system can be
configured to rotate the non-magnetized stir bar within the lysis
chamber at at least 1000 rpm. The lysis agent can be a mechanical
agent. The mechanical agent can be ceramic beads, glass beads or
steel beads. The lysis agent can be a chemical agent. The chemical
agent can be an anionic detergent, a cationic detergent, a
non-ionic detergent or a chaotropic agent. The cartridge can be
configured for testing of one or more target pathogens that is a
virus or a gram-negative bacterium. The integrated diagnostic
cartridge can further include a fluid inlet in fluid communication
with the lysis chamber and a fluid outlet in fluid communication
with the lysis chamber and a filter assembly in fluid communication
with the fluid outlet of the lysis chamber. The integrated
diagnostic cartridge can further include a fluid inlet to the lysis
chamber and a fluid outlet to lysis chamber wherein the lysis
chamber can be isolated from the other modules on the cartridge by
a first frangible seal in fluid communication with the fluid inlet
to the lysis chamber and a second frangible seal in fluid
communication with the fluid outlet of the lysis chamber. The
integrated diagnostic cartridge can further include a process
control chamber having an inlet, an outlet and a plug including a
process control wherein the process control chamber is in fluid
communication with the lysis chamber inlet.
[0027] In general, an integrated diagnostic cartridge includes a
loading module, a lysis module, a purification module including a
rotary valve including (a.) a stator including a stator face and a
plurality of passages, each passage including a port at the stator
face; (b.) a rotor operably connected to the stator and including a
rotational axis, a rotor valving face, and a flow channel having an
inlet and an outlet at the rotor valving face, wherein the flow
channel includes a porous solid support; and (c.) a retention
element biasing the stator and the rotor together at a rotor-stator
interface to form a fluid tight seal, and a reaction module. The
loading module is in fluidic communication with the lysis module
and the purification module is in fluidic communication with the
reaction module.
[0028] This and other embodiments can include one or more of the
following features. The rotary valve can further include a gasket
between the stator face and the rotor valving face. The stator can
include a displaceable spacer for preventing the gasket from
sealing against at least one of the rotor and stator. When the
spacer is displaced, the gasket can seal the rotor and stator
together in a fluid-tight manner. When the cartridge is positioned
within a diagnostic instrument, engagement with a rotor driver of
the diagnostic instrument can displace the spacer and can seal the
rotor and stator together in a fluid-tight manner. A rotation
movement performed by the rotor driver of the diagnostic instrument
can displace the spacer and can seal the rotor and the stator
together in a fluid-tight manner. The integrated diagnostic
cartridge can further include at least one pair of ridges and
spaces on a retention ring and at least one pair of ridges and
spaces on the rotor. While the at least one pair of ridges and
spaces of the retention ring is engaged with the at least one pair
of ridges and spaces of the rotor sealing of the rotor and stator
can be prevented. Relative movement between the at least one pair
of ridges and spaces on the retention ring and the at least one
pair of ridges and spaces on the rotor can seal the rotor and
stator together in a fluid-tight manner. When the cartridge is
positioned within a diagnostic instrument, engagement with a rotor
driver of the diagnostic instrument can produce the relative
movement between the at least two pairs of ridges and spaces on the
retention ring and the rotor that can seal the rotor and stator
together in a fluid-tight manner. A rotation movement of less than
one full rotation of the rotor performed by the rotor driver of the
diagnostic instrument can seal the rotor and stator together in a
fluid-tight manner. The integrated diagnostic cartridge can further
include a gasket interposed at the rotor-stator interface. The
rotary valve can be maintained in a storage condition while a
threaded portion of a retention ring is engaged with a threaded
portion of the rotor. Relative motion between the threaded portion
of the retention ring and the threaded portion of the rotor can
seal the rotor and stator together in a fluid-tight manner. When
the cartridge is positioned within a diagnostic instrument,
engagement with a rotor driver of the diagnostic instrument can
produce the relative movement between the threaded portion of the
retention ring and the threaded portion of the rotor. A rotation
movement of less than one full rotation of the rotor performed by
the rotor driver of the diagnostic instrument can seal the rotor
and stator together in a fluid-tight manner. The integrated
diagnostic cartridge can further include a gasket interposed at the
rotor-stator interface. The integrated diagnostic cartridge can
further include a waste collection element, a wash buffer reservoir
and an elution buffer reservoir. The integrated diagnostic
cartridge can further include a pneumatic interface in fluidic
communication with at least the purification module. The porous
solid support can be polymeric. The porous solid support can be
selected from the group consisting of alumina, silica, celite,
ceramics, metal oxides, porous glass, controlled pore glass,
carbohydrate polymers, polysaccharides, agarose, Sepharose.TM.,
Sephadex.TM., dextran, cellulose, starch, chitin, zeolites,
synthetic polymers, polyvinyl ether, polyethylene, polypropylene,
polystyrene, nylons, polyacrylates, polymethacrylates,
polyacrylamides, polymaleic anhydride, membranes, hollow fibers and
fibers, and any combination thereof. The rotor valving face can
include a gasket interposed at the rotor-stator interface. The
integrated diagnostic cartridge can further include a fluid
connector or a fluid selector including a volume dimensioned to
provide an aliquot of liquid when filled. The rotor can include a
plurality of flow channels, each flow channel can include an inlet,
an outlet, and a porous solid support. The integrated diagnostic
cartridge can further include a fluid connector or a fluid selector
including a volume dimensioned to provide an aliquot of liquid when
filled. The integrated diagnostic cartridge can further include a
waste collection element, a wash buffer reservoir and an elution
buffer reservoir.
[0029] In general, in one embodiment, an integrated diagnostic
cartridge includes a loading module, a lysis module, a purification
module, and a reaction module including a plurality of individual
assay chambers. At least one wall in each one of the plurality of
individual assay chambers is provided by a plug include a body with
a bottom surface; a central opening in the body; and a dried
reagent on the bottom surface, wherein the body is formed from a
material transmissive to excitation wavelengths and emission
wavelengths in at least one of a red spectrum, a blue spectrum and
a green spectrum. The loading module is in fluidic communication
with the lysis module and the purification module is in fluidic
communication with the reaction module.
[0030] This and other embodiments of an assay chamber of an
integrated diagnostic cartridge can include a plug having one or
more or a combination of the following features. The bottom surface
of the plug body can include a cavity in the bottom surface with
the dried reagent within the cavity. The plug can have a plug
thickness between a central opening bottom and the plug body
bottom, and further wherein a depth of the cavity is less than 90%
of the plug thickness, is less than 70% of the plug thickness or is
less than 50% of the plug thickness. The plug can have a polished
or smooth finish facilitating the transmissivity of the excitation
wavelengths and the emission wavelengths. The plug may have a dried
reagent that can be selected from the group consisting of nucleic
acid synthesis reagents, nucleic acids, nucleotides, nucleobases,
nucleosides, monomers, detection reagents, catalysts or
combinations thereof. The dried reagent can be a continuous film
adhered to the plug bottom surface. The dried reagent can be a
lyophilized reagent. The body of the plug can protrude into the
monolithic substrate of the assay chamber at a depth such that the
assay chamber volume can be readily changed by altering the depth
at which the body of the plug protrudes into the monolithic
substrate of the assay chamber. In some embodiments, during the
combining the enriched nucleic acid step in each of the two or more
assay chambers, the enriched nucleic acid can combine with a dried
reagent contained in each one of the two or more assay chambers.
The dried reagent can be on a surface of a plug in each one of the
two or more assay chambers. The dried reagent can be on a surface
of the plug formed from a material transmissive to excitation
wavelengths and emission wavelengths in at least one of a red
spectrum, a blue spectrum and a green spectrum used during the
performing step. In one aspect, the surface of the plug having the
dried reagent is also used during the performing an isothermal
amplification reaction step. Images collected through the plug
surface that contained the dried reagent are processed as part of
the detection of an amplification product within an assay
chamber.
[0031] In still additional embodiments, the integrated diagnostic
cartridge can further include a cartridge perimeter. Each one of
the plurality of individual assay chambers can be in communication
with an air chamber and each air chamber is closer to the cartridge
perimeter than the plug in each one of the plurality of individual
assay chambers. The integrated diagnostic cartridge can further
include a reaction area perimeter. Each one of the plurality of
individual assay chambers can be in communication with an air
chamber and further wherein each plug in each one of the plurality
of individual assay chambers can be within the reaction area
perimeter and each air chamber is outside of the reaction area
perimeter. The integrated diagnostic cartridge can further include
a cartridge perimeter and a reaction area perimeter wherein each
one of the plurality of individual assay chambers can be in
communication with an air chamber and each air chamber is closer to
the cartridge perimeter than the plug in each one of the plurality
of individual assay chambers and is located outside of the reaction
area perimeter and each one of the plurality of individual assay
chambers is within the reaction area perimeter. The integrated
diagnostic cartridge can further include at least one fluid inlet
conduit to each one of the plurality of individual assay chambers
of the reaction module. Each one of the at least one fluid inlet
conduits can further include a heat staked region. A heat stake in
the heat staked region can fluidically isolate the reaction module
from the loading module, the lysis module, and the purification
module.
[0032] In general, in one embodiment, an integrated diagnostic
cartridge includes a loading module, a lysis module, a purification
module, and a reaction module including one or more assay chambers.
Each assay chamber includes: (1) a tapered inlet; (2) a tapered
outlet; (3) a plug including a bottom surface and a central opening
in the body, wherein the body is formed from a material
transmissive to excitation wavelengths and emission wavelengths in
at least one of an ultraviolet spectrum, a blue spectrum, a green
spectrum and a red spectrum; (4) two curved boundaries, wherein
each curved boundary extends from the tapered inlet to the tapered
outlet such that together, the two curved boundaries and the plug
enclose a volume of the assay chamber; and (5) a shoulder extending
from each curved boundary wherein the plug contacts each shoulder
such that a boundary of the assay chamber is provided by the two
curved boundaries, the shoulders extending from each of the curved
boundaries and the plug.
[0033] In general, in one embodiment, an integrated diagnostic
cartridge, includes a loading module; a lysis module; a
purification module; and a reaction module. Additionally or
optionally, the reaction module may also include a common fluid
pathway, and a plurality of independent, continuous fluidic
pathways connected to the common fluid pathway. Still further, each
independent, continuous fluidic pathway also is in fluidic
communication with an assay chamber, and a pneumatic compartment,
and wherein the assay chamber is connected to the common fluid
pathway, the assay chamber having a fluid volume defined in part by
a plug having a dried reagent. In additional aspects, the pneumatic
compartment, including a pneumatic volume, is connected to the
common fluid pathway via the assay chamber. Still further, each
fluidic pathway of the plurality of independent, continuous fluidic
pathways is a closed system excluding the connection between the
assay chamber and common fluid source. In additional aspects, each
assay chamber includes a double tapered chamber that includes a
tapered inlet in fluidic communication with a terminus of the entry
conduit of the fluidic pathway, a tapered outlet in fluidic
communication with a terminus of the pneumatic compartment, and two
curved boundaries, wherein each curved boundary extends from the
tapered inlet to the tapered outlet such that together, the two
curved boundaries enclose the volume of the assay chamber. There is
also a shoulder extending from each curved boundary wherein the
plug contacts each shoulder such that a boundary of the assay
chamber is provided by the two curved boundaries, the shoulders
extending from each of the curved boundaries and the plug.
Additionally, the loading module is in fluidic communication with
the lysis module and the purification module is in fluidic
communication with the reaction module.
[0034] This and other embodiments can include one or more of the
following features. The two curved boundaries can be formed in a
monolithic substrate or a fluidic card of the cartridge. The body
of the plug can protrude into the monolithic substrate of the assay
chamber at a depth such that the assay chamber volume can be
readily changed by altering the depth at which the body of the plug
protrudes into the monolithic substrate of the assay chamber.
[0035] In general, in one embodiment, an integrated diagnostic
cartridge includes a loading module, a lysis module, a purification
module, and a reaction module including a reagent storage component
including a capsule capable of holding a liquid or solid sample,
said capsule including an opening, a closed end and a wall
extending from the closed end to the opening, wherein the capsule
is oval-shaped and the wall is rounded, and wherein the closed end
and wall define an interior volume having a substantially smooth
surface. The loading module is in fluidic communication with the
lysis module and the purification module is in fluidic
communication with the reaction module.
[0036] In general, in one embodiment, an integrated diagnostic
cartridge includes a loading module, a lysis module, a purification
module, and a reaction module including a capsule capable of
holding a liquid or a solid sample. Said capsule includes an inner
surface extending from the bottom of said capsule to an oval-shaped
opening at the top of the capsule and a planar layer affixed around
the oval-shaped opening of said capsule and oriented in the same
plane as the oval-shaped opening of said capsule. Said inner
surface is substantially smooth and includes a concave shape
extending from the bottom of the capsule. Said planar layer
includes a top surface and a bottom surface. Said top surface
aligned with the inner surface of said capsule at said oval-shaped
opening to provide a continuous surface. The loading module is in
fluidic communication with the lysis module and the purification
module is in fluidic communication with the reaction module.
[0037] These and other embodiments can include one or more of the
following features. Said capsule can be capable of holding a volume
from approximately 50 .mu.L to approximately 200 .mu.L or said
oval-shaped opening can contained within an area of 9 mm.times.9
mm. Said capsule can include a dried reagent. The integrated
diagnostic cartridge can further include a fluidic card and a
cover. At least two of the loading module, the lysis module, the
purification module and the reaction module can be formed in or
supported by the fluidic card. At least two of the loading module,
the lysis module, the purification module and the reaction module
can be formed in or supported by the cover. The integrated
diagnostic cartridge can further include a slot positioned to
engage with a latch and pin assembly of a diagnostic instrument to
secure the integrated diagnostic cartridge in a testing position
within the diagnostic instrument. The integrated diagnostic
cartridge can further include an interference feature on the cover.
The interference feature can be sized and positioned to interact
with one of an upper rail or a lower rail of a loading apparatus of
a diagnostic instrument. A thickness of the fluidic card can be
selected for sliding arrangement within an upper rail and a lower
rail of a loading apparatus of the diagnostic instrument. A total
sample process volume of the integrated diagnostic cartridge can be
provided by increasing the thickness of the cartridge. A diagnostic
instrument can be adapted and configured to accommodate the
increased thickness of the cartridge by increasing a width of an
opening of the diagnostic instrument to accommodate the increased
thickness of the cartridge or a displacement range of a cartridge
clamping system of the diagnostic instrument is adapted to
accommodate the increased thickness of the cartridge. The
integrated diagnostic cartridge can further include a cartridge
front face and a cartridge rear face forming an upper spacing and a
lower spacing. Each of the upper spacing and the lower spacing can
be sized and positioned to engage with an upper rail and a lower
rail of the instrument. The integrated diagnostic cartridge can
further include an interference feature within the upper spacing or
the lower spacing positioned to ensure the cartridge engages with
the upper rail and the lower rail in a desired orientation. The
integrated diagnostic cartridge can further include a plurality of
frangible seal chambers in fluid communication with at least one or
more of the loading module, the lysis module, the purification
module or the reaction module. The integrated diagnostic cartridge
can further include a label section. The integrated diagnostic
cartridge can further include one or more machine readable marking
indicating the sample type to be used in the cartridge or target
pathogen to be detected. The integrated diagnostic cartridge can
further include a pneumatic interface. Prior to loading the
cartridge into a diagnostic instrument, a lysis chamber in the
cartridge can contain a lysis buffer. The integrated diagnostic
cartridge can further include a machine-readable code adapted and
configured to identify the cartridge to a diagnostic instrument or
a patient identification marking. The integrated diagnostic
cartridge can further include a film adhered to a surface of the
monolithic substrate, wherein the film forms one wall of the assay
chamber. The integrated diagnostic cartridge can further include a
first film adhered to a surface of at least a portion of the
cartridge. The first film can form one wall of one or more
chambers, compartments, or fluid conduits of the loading module,
the lysis module, the purification module and the reaction module.
The integrated diagnostic cartridge can further include a second
film adhered to the first film. The second film can have a higher
melting temperature than the first film. The integrated diagnostic
cartridge can further include a heat staked region formed in each
of the fluidic pathways using the first film or the second film
wherein the heat staked region seals off the common fluid pathway
from the assay chamber and the pneumatic chamber. The integrated
diagnostic cartridge can further include a raised platform within
each of the plurality of independent, continuous fluidic pathways
the raised platform positioned between an inlet to the assay
chamber and the common fluid pathway wherein the heat staked region
is formed using a portion of the raised platform.
[0038] In general, in one embodiment, an integrated diagnostic
cartridge includes a loading module having a fill chamber within
the cartridge having a volume sufficient to hold a sample, a fluid
inlet in fluid communication with the fill chamber, a fluid outlet
in fluid communication with fill chamber; a lysis module; a
purification module, and a reaction module. The loading module is
in fluidic communication with the lysis module and the purification
module is in fluidic communication with the reaction module.
Further, the loading module, the lysis module, the purification
module and the reaction module are arranged for use while the
cartridge is in a vertical orientation. Further, when the cartridge
is in a horizontal sample loading orientation the fluid inlet
accesses the fill chamber via an upper surface of the cartridge and
when the cartridge is in a vertical sample processing orientation
the fluid inlet is positioned adjacent to an upper portion of the
fill chamber and the fluid outlet is arranged for the sample to
flow out of a lower portion of the fill chamber.
[0039] This and other embodiments can include one or more of the
following features. The integrated diagnostic cartridge can further
include one or more fluid filling conduits arranged to flow into an
upper portion of a vertically oriented chamber within a fluidic
card of the integrated diagnostics cartridge and one or more fluid
outlet conduits arranged to flow out of a lower portion of the
vertically oriented chamber within the fluidic card of the
integrated diagnostics cartridge. The vertically oriented chamber
can further include a filter assembly in fluid communication with a
fluid outlet conduit of the vertically oriented chamber. The lysis
module can include a mixing assembly having a vertically oriented
lysis chamber containing a lysis agent and a non-magnetized stir
bar. The non-magnetized stir bar can be made from a metal having a
magnetic permeability to be responsive to a rotating magnetic field
induced between a drive magnetic element and a driven magnetic
element of a magnetic drive system. The non-magnetized stir bar can
be coated with an impermeable material to prevent corrosion by a
chemical lysis buffer in the vertically oriented lysis chamber.
When in use within a diagnostic instrument, the non-magnetized stir
bar can be disposed between a driving magnet system and a driven
magnet system of a magnetic mixing assembly in the diagnostic
instrument. The driving magnet system can be configured to rotate
the non-magnetized stir bar within the vertically oriented lysis
chamber at least 1000 rpm. The integrated diagnostic cartridge can
further include a fluid inlet to the vertically oriented lysis
chamber and a fluid outlet to lysis chamber. The vertically
oriented lysis chamber can be isolated from the other modules on
the cartridge by a first frangible seal in fluid communication with
the fluid inlet to the vertically oriented lysis chamber and a
second frangible seal in fluid communication with the fluid outlet
to the vertically oriented lysis chamber. The integrated diagnostic
cartridge can further include a fluidic card and a cover. The
fluidic card can further include a first film adhered to a surface
of at least a portion of the fluidic card, wherein the first film
forms one surface of one or more chambers, compartments, or fluid
conduits of the loading module, the lysis module, the purification
module and the reaction module. The integrated diagnostic cartridge
can further include an interference feature on the cover. The
interference feature can be sized and positioned to interact with
one of an upper rail or a lower rail of a loading apparatus of a
diagnostic instrument. A thickness of the fluidic card can be
selected for sliding arrangement within an upper rail and a lower
rail of a loading apparatus of the diagnostic instrument. A total
sample process volume of the integrated diagnostic cartridge can be
related to a thickness of the cartridge corresponding to a spacing
between the one or more chambers, compartments, or fluid conduits
of the loading module, the lysis module, the purification module
and the reaction module formed in the fluidic card and the first
film. A diagnostic instrument is adapted and configured to
accommodate a variation of the thickness of the cartridge by
increasing a width of a loading slot of the diagnostic instrument
to accommodate the increased thickness of the cartridge or a
displacement range of a cartridge clamping system of the diagnostic
instrument can be adapted to accommodate the increased thickness of
the cartridge. The integrated diagnostic cartridge can further
include a cartridge front face and a cartridge rear face forming an
upper spacing and a lower spacing. Each of the upper spacing and
the lower spacing can be sized and positioned to engage with the
upper rail and lower rail of the diagnostic instrument. The
integrated diagnostic cartridge can further include an interference
feature within the upper spacing or the lower spacing positioned to
ensure the cartridge engages with the upper rail and the lower rail
in a desired orientation. The integrated diagnostic cartridge can
further include a plurality of frangible seal chambers in fluid
communication with at least one or more of the loading module, the
lysis module, the purification module or the reaction module. The
integrated diagnostic cartridge can further include a
machine-readable code adapted and configured to identify the
cartridge to a diagnostic instrument or an image of a patient
identification marking.
[0040] In still another alternative implementation, there is
integrated diagnostic cartridge with a loading module, a lysis
module, and a purification module. The purification module also
includes a purification module comprising a rotary valve. The
rotary valve may also include a stator comprising a stator face and
a plurality of passages, each passage comprising a port at the
stator face; a rotor operably connected to the stator and
comprising a rotational axis, a rotor valving face, and a flow
channel having an inlet and an outlet at the rotor valving face,
wherein the flow channel comprises a porous solid support, and a
retention element biasing the stator and the rotor together at a
rotor-stator interface to form a fluid tight seal. The integrated
cartridge also includes a reaction module. The reaction module
includes a plurality of individual assay chambers, wherein at least
one surface in each one of the plurality of individual assay
chambers is provided by a plug. Each plug includes, for example, a
body with a bottom surface, a central opening in the body and a
dried reagent on the bottom surface. Still further, the body is
formed from a material transmissive to excitation wavelengths and
emission wavelengths in at least one of a red spectrum, a blue
spectrum and a green spectrum. Additionally, the loading module is
in fluidic communication with the lysis module and the purification
module is in fluidic communication with the reaction module. Still
further, the loading module, the lysis module, the purification
module and the reaction module are arranged for use while the
cartridge is in a vertical orientation.
[0041] This and other embodiments can include one or more of the
following features. The bottom surface of the plug body can include
a cavity in the bottom surface with the dried reagent within the
cavity. The plug can have a plug thickness between a central
opening bottom and the plug body bottom, and further, a depth of
the cavity can be less than 90% of the plug thickness, can be less
than 70% of the plug thickness or can be less than 50% of the plug
thickness. The plug can have a polished or smooth finish
facilitating the transmissivity of the excitation wavelengths and
the emission wavelengths. The dried reagent can be selected from
the group consisting of nucleic acid synthesis reagents, nucleic
acids, nucleotides, nucleobases, nucleosides, monomers, detection
reagents, catalysts or combinations thereof. The body of the plug
can protrude into the monolithic substrate of the assay chamber at
a depth such that the assay chamber volume can be readily changed
by altering the depth at which the body of the plug protrudes into
the monolithic substrate of the assay chamber. The integrated
diagnostic cartridge can further include at least one fluid inlet
conduit to each one of the plurality of individual assay chambers
of the reaction module. Each one of the at least one fluid inlet
conduits can further include a heat staked region. A heat stake in
the heat staked region can fluidically isolate the reaction module
from the loading module, the lysis module, and the purification
module. The purification module can further include a rotary valve
including: (a) a stator including a stator face and a plurality of
passages, each passage comprising a port at the stator face; (b) a
rotor operably connected to the stator and including a rotational
axis, a rotor valving face, and a flow channel having an inlet and
an outlet at the rotor valving face, wherein the flow channel can
include a porous solid support; and (c.) a retention element
biasing the stator and the rotor together at a rotor-stator
interface to form a fluid tight seal. The rotary valve can further
include a gasket between the stator face and the rotor valving
face. The stator can include a displaceable spacer for preventing
the gasket from sealing against at least one of the rotor and
stator, and when the spacer is displaced the gasket can seal the
rotor and stator together in a fluid-tight manner. When the
cartridge is positioned within a diagnostic instrument, engagement
with a valve drive assembly of the diagnostic instrument can
displace the spacer and seal the rotor and stator together in a
fluid-tight manner. The purification module can further include a
waste collection element, a wash buffer reservoir and an elution
buffer reservoir. The integrated diagnostic cartridge can further
include a pneumatic interface in fluidic communication with at
least the purification module. The loading module can further
include a dried antifoam agent. The method of testing a sample can
further include combining the sample with a dried antifoam agent in
the sample port assembly before the accepting step.
[0042] In general, in one embodiment, a method of testing a sample
suspected of containing one or more target pathogens includes: (1)
accepting a cartridge having a sample port assembly containing the
sample suspected of containing the one or more target pathogens;
(2) advancing the sample suspected of containing the one or more
target pathogens to a lysis chamber within the cartridge having at
least one lysis reagent therein; (3) mixing the sample with the at
least one lysis agent to generate a lysed sample; (4) passing the
lysed sample through a porous solid support within the cartridge to
capture a nucleic acid on the porous solid support; (5) releasing
the captured nucleic acid from the first porous solid support to
generate an enriched nucleic acid; (6) introducing the enriched
nucleic into a rehydration chamber within the cartridge containing
one or more dried reagents; (7) after introducing the
analyte/reagent solution into a metering channel, mixing the
contents of the rehydration chamber to produce an analyte/reagent
solution; (8) distributing the analyte/reagent solution to two or
more assay chambers within the cartridge after performing the
mixing step; (9) combining the analyte/reagent solution with one or
more amplification reagents after performing the distributing step;
(10) sealing each one of the two or more assay chambers within the
cartridge containing analyte/reagent solution from each one of all
the other two or more assay chambers within the cartridge
containing analyte/reagent solution and a waste chamber; and (11)
performing an isothermal amplification reaction within each one of
the two or more assay chambers in the cartridge while
simultaneously detecting amplification product, wherein presence of
an amplification product is an indication of a presence, an absence
or a quantity of the target pathogen in the sample suspected of
containing the target pathogen.
[0043] This and other embodiments can include one or more of the
following features. In mixing the sample with the at least one
lysis agent, the lysis agent can be a mechanical agent. The
mechanical agent can be ceramic beads, glass beads or steel beads,
and the mixing the sample step can include rotating a stir bar
within the lysis chamber at at least 1000 rpm. Mixing the sample
can include rotating the stir bar or the ceramic, glass or steel
beads along with a chemical lysis agent. The at least one lysis
agent can be a chemical lysis agent. The one or more target
pathogens can be a virus or a gram-negative bacterium and the lysis
reagent is a chaotropic agent. Prior to passing the lysed sample
through the porous solid support, the method can further include
passing the lysed sample through a size-exclusion filter, wherein
nucleic acid can pass through the filter. The enriched nucleic acid
can be combined with one or more amplification reagents before the
distributing step and the one or more amplification reagents can
include a primer. The performing of the isothermal amplification
reaction step can be initiated prior to the distributing the
enriched nucleic acid to the two or more assay chambers step. After
the distributing step, but prior to performing the isothermal
amplification reaction step, the method can further include
combining the enriched nucleic acid with a primer set specific to
one of the one or more target pathogens. A first assay chamber can
contain a primer set specific to a first nucleic acid sequence. The
first nucleic acid sequence can be present in one of the one or
more target pathogens. Prior to mixing the sample with at least one
lysis agent, a process control can be added to the sample and the
first nucleic acid sequence can be present in the process control.
Prior to passing lysed sample through the porous solid support, a
process control can be added to the lysed sample and the first
nucleic acid sequence can be present in the process control. A
second assay chamber can contain a primer set specific to a second
nucleic acid sequence. The second nucleic acid sequence can be
present in one of the one or more target pathogens. The performing
an isothermal amplification reaction step can be completed in less
than 15 minutes. The method of testing a sample can further include
providing a result containing a determination made during the
performing step relating to the presence, the absence or the
quantity of the target pathogen in the sample suspected of
containing the target pathogen. The method can further include
prior to advancing the sample to a lysis chamber, pretreating the
sample with a chemical reaction. The sample can be sputum and the
chemical reaction can be incubation with a mucolytic agent. The
method can further include, prior to advancing the sample to a
lysis chamber, pretreating the sample with an enzymatic reaction.
The enzymatic reaction can be incubation of the sample with a
nuclease, a protease, an amylase, a glycosylase, or a lipase. The
method can further include, prior to advancing the sample to a
lysis chamber, pretreating the sample with a physical treatment.
The physical treatment can include passing the sample through a
size-exclusion filter in a first direction. The physical treatment
can include exposing the sample to a capture agent immobilized on a
solid substrate. The method of testing a sample can further
include, after exposure, separating the solid substrate from the
sample. The capture agent can be an antibody with affinity for red
blood cells. The sample can be sputum and the method can further
include, prior to mixing the sample with the at least one lysis
reagent, bead beating the sputum to liquify the sample. The bead
beating can include mixing the sputum with ceramic, glass, or steel
beads. Prior to distributing the enriched nucleic acid to the assay
chambers, the method can further include passing the enriched
nucleic acid through a second porous solid support.
[0044] In general, in one embodiment, a method of testing a sample
suspected of containing one or more target pathogens include: (1)
accepting a cartridge having a sample port assembly containing the
sample suspected of containing the one or more target pathogens;
(2) advancing the sample suspected of containing the one or more
target pathogens to a lysis chamber within the cartridge having at
least one lysis reagent therein; (3) mixing the sample with the at
least one lysis agent to generate a lysed sample; (4) passing the
lysed sample through a porous solid support within the cartridge to
capture a nucleic acid on the porous solid support; (5) releasing
the captured nucleic acid from the first porous solid support to
generate an enriched nucleic acid; (6) introducing the enriched
nucleic into a rehydration chamber within the cartridge containing
one or more dried reagents to generate an analyte/reagent solution;
(7) after introducing the analyte/reagent solution into a metering
channel, mixing the contents of the rehydration chamber to
homogenize an analyte/reagent solution; (8) distributing the
analyte/reagent solution to two or more assay chambers within the
cartridge after performing the mixing step; (9) combining the
analyte/reagent solution with one or more amplification reagents
after performing the distributing step to generate an amplification
solution; (10) sealing each one of the two or more assay chambers
within the cartridge containing amplification solution from each
one of all the other two or more assay chambers within the
cartridge containing amplification solution and a waste chamber;
and (11) performing an isothermal amplification reaction within
each one of the two or more assay chambers in the cartridge while
simultaneously detecting amplification product, wherein presence of
an amplification product is an indication of a presence, an absence
or a quantity of the target pathogen in the sample suspected of
containing the target pathogen.
[0045] This and other embodiments can include one or more of the
following features. In mixing the sample with the at least one
lysis agent, the lysis agent can be a mechanical agent. The
mechanical agent can be ceramic beads, glass beads or steel beads,
and the mixing the sample step can include rotating a stir bar
within the lysis chamber at at least 1000 rpm. Mixing the sample
can include rotating the stir bar or the ceramic, glass or steel
beads along with a chemical lysis agent. The at least one lysis
agent can be a chemical lysis agent. The one or more target
pathogens can be a virus or a gram-negative bacterium and the lysis
reagent can be a chaotropic agent. Prior to passing the lysed
sample through the porous solid support, the method can further
include passing the lysed sample through a size-exclusion filter,
wherein nucleic acid can pass through the filter. A first assay
chamber can contain a primer set specific to a first nucleic acid
sequence. The first nucleic acid sequence can be present in one of
the one or more target pathogens. Prior to mixing the sample with
at least one lysis agent, a process control can be added to the
sample and the first nucleic acid sequence can be present in the
process control. Prior to passing lysed sample through the porous
solid support, a process control can be added to the lysed sample
and the first nucleic acid sequence can be present in the process
control. A second assay chamber can contain a primer set specific
to a second nucleic acid sequence. The second nucleic acid sequence
can be present in one of the one or more target pathogens. The
performing an isothermal amplification reaction step can be
completed in less than 15 minutes. The method of testing a sample
can further include providing a result containing a determination
made during the performing step relating to the presence, the
absence or the quantity of the target pathogen in the sample
suspected of containing the target pathogen. The method can further
include, prior to advancing the sample to a lysis chamber,
pretreating the sample with a chemical reaction. The sample can be
sputum and the chemical reaction can be incubation with a mucolytic
agent. The method can further include, prior to advancing the
sample to a lysis chamber, pretreating the sample with an enzymatic
reaction. The enzymatic reaction can be incubation of the sample
with a nuclease, a protease, an amylase, a glycosylase, or a
lipase. The method can further include, prior to advancing the
sample to a lysis chamber, pretreating the sample with a physical
treatment. The physical treatment can include passing the sample
through a size-exclusion filter in a first direction. The physical
treatment can include exposing the sample to a capture agent
immobilized on a solid substrate. The method of testing a sample
can further include, after exposure, separating the solid substrate
from the sample. The capture agent can be an antibody with affinity
for red blood cells. The sample can be sputum and the method can
further include, prior to mixing the sample with the at least one
lysis reagent, bead beating the sputum to liquify the sample. The
bead beating can include mixing the sputum with ceramic, glass, or
steel beads. Prior to distributing the analyte/reagent solution to
the two or more assay chambers, the method can further include
passing the analyte/reagent solution through a second porous solid
support.
DESCRIPTION OF THE DRAWINGS
[0046] The foregoing and other objects, features and advantages
will be apparent from the following description of particular
embodiments of the invention, as illustrated in the accompanying
drawings in which like reference characters refer to the same parts
throughout the different views. The drawings are not necessarily to
scale, emphasis instead placed upon illustrating the principles of
various embodiments of the invention.
[0047] FIG. 1 is an illustration of a diagnostic instrument for
conducting a molecular diagnostic test, in accordance to with an
embodiment.
[0048] FIG. 2A and FIG. 2B depict an integrated diagnostic
cartridge, configured to be used in conjunction with a diagnostic
instrument, during filling by a user, in accordance with an
embodiment.
[0049] FIG. 2C depicts an integrated diagnostic cartridge with a
loading module sealed after filling is completed and prior to being
inserted into a diagnostic instrument, in accordance with an
embodiment.
[0050] FIG. 3 depicts an integrated diagnostic cartridge being
inserted into a diagnostic instrument to perform a diagnostic test,
in accordance with an embodiment.
[0051] FIG. 4A depicts a diagnostic instrument after an integrated
diagnostic cartridge is inserted during initialization of a
diagnostic test. The integrated diagnostic instrument is shown
having a display configured to show information associated with a
diagnostic test run, in accordance with an embodiment.
[0052] FIG. 4B depicts a diagnostic instrument when running a
diagnostic test on an integrated diagnostic cartridge, in
accordance with an embodiment.
[0053] FIG. 5 depicts a diagnostic instrument ejecting an
integrated diagnostic cartridge upon completion of a diagnostic
test, in accordance with an embodiment.
[0054] FIG. 6 depicts a frontal exploded illustration of a
diagnostic instrument, in accordance with an embodiment.
[0055] FIG. 7 depicts a rear exploded illustration of a diagnostic
instrument, in accordance with an embodiment.
[0056] FIG. 8 and FIG. 9 are frontal perspective views of a
diagnostic instrument clamping subsystem during clamping of an
integrated diagnostic cartridge.
[0057] FIG. 10 and FIG. 11 are rear perspective views of a
diagnostic instrument clamping subsystem during clamping of an
integrated diagnostic cartridge.
[0058] FIG. 12 is a frontal exploded view of a diagnostic
instrument clamping subsystem with an integrated diagnostic
cartridge disposed between a fixed bracket assembly and a moving
bracket assembly.
[0059] FIG. 13 is a rear exploded view of a diagnostic instrument
clamping subsystem with an integrated diagnostic cartridge disposed
between a fixed bracket assembly and a moving bracket assembly.
[0060] FIG. 14 is a perspective view of a moving bracket assembly
of a clamping subsystem. The view of the moving bracket assembly is
shown from a first surface of a clamp block.
[0061] FIG. 15A is a frontal exploded view of a moving bracket
assembly of a clamping subsystem.
[0062] FIG. 15B is a rear exploded view of a moving bracket
assembly of a clamping subsystem.
[0063] FIG. 16A is a view of a clamping subsystem with an
integrated diagnostic cartridge inserted between a fixed bracket
assembly and moving bracket assembly taken from the front of a
diagnostic instrument, as seen in FIG. 4A. The clamping subsystem
is in a zero clamping position.
[0064] FIG. 16B is a view of a clamping subsystem with an
integrated diagnostic cartridge inserted between a fixed bracket
assembly and a moving bracket assembly taken from the front of a
diagnostic instrument, as seen in FIG. 4A. The clamping subsystem
is in a first clamping position with a valve drive assembly and a
thermal clamp assembly of the moving bracket assembly contacting
the integrated diagnostic cartridge.
[0065] FIG. 16C is a view of a clamping subsystem with an
integrated diagnostic cartridge inserted between a fixed bracket
assembly and a moving bracket assembly taken from the front of a
diagnostic instrument, as seen in FIG. 4A. The clamping subsystem
is in a second clamping position to clamp the integrated diagnostic
cartridge. A valve drive assembly, a thermal clamp assembly, a door
support assembly, and a pneumatic interface of the moving bracket
assembly contact the integrated diagnostic cartridge.
[0066] FIG. 16D is a view of a clamping subsystem with an
integrated diagnostic cartridge inserted between a fixed bracket
assembly and a moving bracket assembly taken from the front of a
diagnostic instrument, as seen in FIG. 4A. The clamping subsystem
is in a third clamping position to render the integrated diagnostic
cartridge fluidically active with a frangible seal block.
[0067] FIG. 16E is a view of a clamping subsystem with an
integrated diagnostic cartridge inserted between a fixed bracket
assembly and a moving bracket assembly taken from the front of a
diagnostic instrument, as seen in FIG. 4A. The clamping subsystem
is in a fourth clamping position to unclamp the integrated
diagnostic cartridge and eject the integrated diagnostic cartridge
from the clamping subsystem.
[0068] FIG. 17A is a frontal perspective view of a fixed support
bracket of a clamping subsystem. The fixed support bracket is shown
with a loading assembly for accepting and ejecting an integrated
diagnostic cartridge. An integrated diagnostic cartridge is seen in
a loading position.
[0069] FIG. 17B is an enlarged view of a loading assembly, as shown
in FIG. 17A, in a loading position.
[0070] FIG. 17C is an enlarged partial view of a loading assembly,
as shown in FIGS. 17A and 17B, depicting a spring which provides a
motive force for ejecting an integrated diagnostic cartridge.
[0071] FIG. 18A is another frontal perspective view of a fixed
support bracket of a clamping subsystem. The fixed support bracket
is shown with a loading assembly from FIG. 17A for accepting and
ejecting an integrated diagnostic cartridge. The loading assembly
is now shown in a loaded position.
[0072] FIG. 18B is an enlarged view of a loading assembly in a
loaded position. A load position sensor on the loading assembly is
triggered by a flag.
[0073] FIG. 19A is a perspective frontal view of a fixed support
bracket of a clamping subsystem with an integrated diagnostic
cartridge inserted into a loading assembly from FIGS. 17A and 18A.
The integrated diagnostic cartridge is in a loaded position.
[0074] FIG. 19B is an additional enlarged view of a loading
assembly in a loaded position similar to FIG. 18B. A load position
sensor on the loading assembly is triggered by a flag.
[0075] FIG. 19C is an additional frontal view of FIG. 19A with a
fixed bracket assembly of a clamping subsystem and an integrated
diagnostic cartridge inserted into a loading assembly. The
integrated diagnostic cartridge and loaded assembly is shown in a
loaded position.
[0076] FIG. 20 is a perspective view of loading assembly rails
showing guide features extending along the rails.
[0077] FIG. 21 is an illustration of a loading assembly with rails
viewed from the front of a diagnostic instrument, as seen in FIGS.
4A and 16A-16E.
[0078] FIG. 22A is a top view of an integrated diagnostic cartridge
prior to being loaded into a loading assembly. The integrated
diagnostic cartridge is shown with a gap formed between a fluidics
card and a cover configured to align with a guide feature on a top
rail, as shown in FIGS. 20 and 21.
[0079] FIG. 22B is a top view of an integrated diagnostic cartridge
during loading into a loading assembly. A guide feature on a top
rail is shown inserted between a gap formed between a fluidics card
and a cover.
[0080] FIG. 23A is a bottom view of an integrated diagnostic
cartridge prior to being loaded into a loading assembly. The
integrated diagnostic cartridge is shown with a gap formed between
a fluidics card and a cover configured to align with a guide
feature on a bottom rail, as shown in FIGS. 20 and 21.
[0081] FIG. 23B is a bottom view of an integrated diagnostic
cartridge during loading into a loading assembly. A guide feature
on a bottom rail is shown inserted between a gap formed between a
fluidics card and a cover.
[0082] FIG. 24 is a rear perspective view of a latch and pin
assembly of a clamping subsystem. An integrated diagnostic
cartridge is inserted between a fixed bracket assembly and a moving
bracket assembly of the clamping subsystem as shown in FIGS. 10 and
11.
[0083] FIG. 25A is a frontal perspective view of a latch and pin
assembly of FIG. 24. An integrated diagnostic cartridge is seen
inserted and latched. A latch from the latch and pin assembly is
shown disposed within a notch of the integrated diagnostic
cartridge to prevent the integrated diagnostic cartridge from being
ejected.
[0084] FIG. 25B is an enlarged view of the latch and pin assembly
of FIG. 24.
[0085] FIG. 25C is an additional view of a latch and pin assembly
with a pin positioned within a narrow portion of a latch release
arm. A latch from the latch and pin assembly is shown dropped into
a notch of an integrated diagnostic cartridge to prevent the
integrated diagnostic cartridge from being ejected.
[0086] FIG. 25D is an illustration of a latch and pin assembly
after an integrated diagnostic cartridge is latched to prevent the
integrated diagnostic cartridge from ejection. The integrated
diagnostic cartridge is seen in an unclamped position and is viewed
from a front of a diagnostic instrument shown in FIG. 4A.
[0087] FIG. 26A is an illustration of a latch and pin assembly
after an integrated diagnostic cartridge is latched and clamped.
The integrated diagnostic cartridge is seen in a clamped position
and is viewed from a front of a diagnostic instrument shown in FIG.
4A.
[0088] FIG. 26B is a top view of a latch and pin assembly from FIG.
26A. A pin is shown in a wide portion of a latch arm slot when an
integrated diagnostic cartridge is in a clamped position.
[0089] FIG. 27 is an illustration of a latch and pin assembly when
an integrated diagnostic cartridge is ejected. The end of a latch
release arm is shown contacting the end of a pin to lift the latch
and is viewed from a front of a diagnostic instrument shown in FIG.
4A.
[0090] FIG. 28 is an illustration of a latch and pin assembly after
an integrated diagnostic cartridge is ejected. The end of a latch
release arm is shown not in contact with an end of a pin and is
viewed from a front of a diagnostic instrument shown in FIG.
4A.
[0091] FIG. 29 is a perspective view of a valve drive assembly
engaging with a rotary valve on an integrated diagnostic cartridge.
The integrated diagnostic cartridge is shown inserted into a
loading assembly and is in a loaded position, as demonstrated by
FIGS. 18A and 19A.
[0092] FIG. 30 is an enlarged view of a valve drive assembly from
FIG. 29. A valve drive and valve drive pins engage with a rotary
valve on an integrated diagnostic cartridge.
[0093] FIG. 31 is an isometric view of a frangible seal block from
a moving bracket assembly shown in FIGS. 14, 15A, and 15B, in
accordance with an embodiment.
[0094] FIG. 32 is an isometric view of a pocket formed within a
fixed support bracket of a fixed bracket assembly. The pocket
configured to receive portions of a frangible seal block.
[0095] FIG. 33 is a frontal view moving bracket assembly, in
accordance with an alternative embodiment. A first frangible seal
pin on a frangible seal block is shown to be longer than the
remainder of a plurality of frangible seal pins.
[0096] FIG. 34 is an isolated perspective view of a frangible seal
block, shown in FIG. 31, engaging with frangible seals on an
integrated diagnostic cartridge. The integrated diagnostic
cartridge is shown inserted into a loading assembly and is in a
loaded position, similarly shown in FIGS. 18A-18B, 19A-19C, and
29.
[0097] FIG. 35 is a perspective view of a diagnostic instrument
pneumatic interface engaging with an integrated diagnostic
cartridge pneumatic interface. The integrated diagnostic cartridge
is shown inserted into a loading assembly and is in a loaded
position, similarly shown in FIGS. 18A-18B, 19A-19C, 29, and FIG.
34.
[0098] FIG. 36A is a frontal perspective view of a diagnostic
instrument pneumatic interface, according to one embodiment. The
pneumatic interface is shown having a flat plunger surface.
[0099] FIG. 36B is a cross-sectional view of FIG. 35. A diagnostic
instrument pneumatic interface with a flat plunger surface is
engaged with an integrated diagnostic cartridge pneumatic interface
cover adaptor. The pneumatic interface is shown with a gimbaling
mechanism active.
[0100] FIG. 36C is a cross-sectional view of a diagnostic
instrument pneumatic interface of with a flat plunger surface
retracted from an integrated diagnostic cartridge pneumatic
interface cover adaptor during unclamping. The pneumatic interface
is shown with a gimbaling mechanism locked.
[0101] FIG. 37A is a frontal perspective view of a diagnostic
instrument pneumatic interface, according to another embodiment.
The pneumatic interface is shown having an angled plunger
surface.
[0102] FIG. 37B is an additional cross-sectional view of FIG. 35. A
diagnostic instrument pneumatic interface with an angled plunger
surface is engaged with an integrated diagnostic cartridge
pneumatic interface cover adaptor. The pneumatic interface is shown
with a gimbaling mechanism active.
[0103] FIG. 37C is a cross-sectional view of a diagnostic
instrument pneumatic interface with an angled plunger surface
retracted from an integrated diagnostic cartridge pneumatic
interface cover adaptor during unclamping. The pneumatic interface
is shown with a gimbaling mechanism locked.
[0104] FIG. 38 is a top down view of a thermal clamp assembly in a
zero clamping position.
[0105] FIG. 39 is a top down view of a thermal clamp assembly in a
first clamping position.
[0106] FIG. 40 is a top down view of a thermal clamp assembly in a
second clamping position.
[0107] FIG. 41 is a top down view of a thermal clamp assembly in a
fourth clamping position.
[0108] FIG. 42 is a perspective view of a thermal clamp assembly
from FIGS. 38-41, engaged with a reaction area of an integrated
diagnostic cartridge. An optical block of a reaction imaging
assembly is shown enclosing the thermal clamp assembly and reaction
area.
[0109] FIG. 43 is an enlarged perspective view of a clamping
subsystem from and a reaction imaging assembly of a diagnostic
instrument optical subsystem. A thermal clamp assembly from FIGS.
38-42 is shown disposed within the reaction imaging assembly. The
clamping subsystem is viewed in a similar perspective in FIGS. 8
and 9.
[0110] FIG. 44 is a broadened perspective view of FIG. 43. A
reaction imaging assembly of a diagnostic instrument optical
assembly is shown attached to a fixed support bracket of the
clamping subsystem. A clamping subsystem clamps an integrated
diagnostic cartridge, as shown in FIGS. 8-13, 16A-16E, 26A, and
38-41. Furthermore, A frangible seal block is shown contained
within a clamp block of a moving bracket assembly.
[0111] FIG. 45 is an isometric view of a diagnostic instrument
optical subsystem comprising a label imaging assembly and a
reaction imaging assembly. An integrated diagnostic cartridge is in
a loading position, as shown in FIGS. 17A and 17B. A reaction area
of the integrated diagnostic cartridge is outside of an optical
block from a reaction imaging assembly in a loading position.
[0112] FIG. 46 is an additional isometric view of a diagnostic
instrument optical subsystem of FIG. 45. An integrated diagnostic
cartridge is in a loaded position, as shown by FIGS. 18A-18B,
19A-19C, 29, 34, and 35. A reaction area of the integrated
diagnostic cartridge is disposed below an optical block from a
reaction imaging assembly in a loaded position.
[0113] FIG. 47A is an exploded view of a magnetic mixing assembly
of a clamping subsystem.
[0114] FIG. 47B is a perspective view of a driving magnet system
and a driven magnet system of a magnetic mixing assembly.
[0115] FIG. 48 is a perspective view of a diagnostic instrument
pneumatic subsystem shown in FIGS. 6 and 7.
[0116] FIG. 49 is an enlarged perspective internal view of FIGS. 6
and 7. An arrangement of a clamping subsystem, an optical
subsystem, and a pneumatic subsystem is readily apparent in this
view. A valve drive assembly is removed from a moving bracket
assembly to show a pneumatic interface, shown in FIGS. 35-37C, is
connected to the pneumatic subsystem, shown in FIG. 48, via
tubing.
[0117] FIG. 50 is a perspective frontal view of a cartridge heating
area of a cartridge heater zone and a reaction well zone of a
diagnostic instrument thermal subsystem.
[0118] FIG. 51 is an isometric enlarged view of FIG. 50 showing a
reaction well zone comprising grooves to form a machined pocket
geometry.
[0119] FIG. 52 is a perspective rear view of a cartridge heater
assembly of a diagnostic instrument thermal subsystem.
[0120] FIG. 53 is an exploded view of FIG. 52. A cartridge heater
assembly is shown comprising a chemistry heater, an insulator, a
plurality of perforations and a plurality of cutouts.
[0121] FIG. 54 is a cross-sectional view of a chemistry heater
assembly of a diagnostic instrument thermal subsystem.
[0122] FIG. 55 is an exploded rear view of FIG. 54. A chemistry
heater assembly of a diagnostic instrument thermal subsystem is
shown comprising a chemistry heater, a chemistry heater plate, a
chemistry heater fan, a fan plenum, a flow vane, a flow guide
frame, and a heater plenum.
[0123] FIG. 56 is a perspective view of a heat staking assembly of
a diagnostic instrument thermal subsystem.
[0124] FIG. 57A is an isometric view of a heat staker bar assembly
within a heat staking assembly of FIG. 56.
[0125] FIG. 57B is a cross-sectional view of FIG. 57A showing a
heat staker bar assembly within a heat staking assembly.
[0126] FIG. 58 is a rear perspective view of FIG. 49. An integrated
diagnostic cartridge is clamped, as shown in FIGS. 8-11, and is in
a loaded position, as described regarding FIGS. 18A-B and 19A-C. A
cellular antenna is shown mounted to an antenna ground plate,
wherein the ground plate is attached to a fixed support bracket of
the fixed bracket assembly.
[0127] FIG. 59 is an enlarged view of a cellular antenna and label
imaging assembly of FIG. 58. The label imaging assembly of a
diagnostic instrument is shown fixed to an antenna ground plate. A
patient label area of the cartridge label and a loading module are
disposed within the field of view of a label imaging assembly
camera.
[0128] FIG. 60 is a perspective view of a label imaging assembly
from FIG. 59. The field of view of the label imaging assembly
includes a patient label area of a cartridge label and a loading
module of an integrated diagnostic cartridge.
[0129] FIG. 61 is a cross-sectional view of a label imaging
assembly from FIGS. 59 and 60.
[0130] FIG. 62 is a top down cross-sectional view of a reaction
imaging assembly of a diagnostic instrument optical subsystem.
Excitation wavelengths are shown contacting an image plane of an
integrated diagnostic cartridge reaction area. Emission paths are
shown emanating from an image plane of an integrated diagnostic
cartridge reaction area to a reaction camera.
[0131] FIG. 63 is a cross-sectional view of an excitation lens cell
of a reaction imaging assembly from FIG. 62.
[0132] FIG. 64 is an additional enlarged cross-sectional view of a
bottom of an excitation lens cell.
[0133] FIG. 65 is an enlarged top down cross-sectional view FIG.
62. A reaction imaging assembly of a diagnostic instrument optical
subsystem is shown with emission wavelengths reflected off a fold
mirror, through a dichroic beam splitter and into a reaction
camera.
[0134] FIG. 66 is an isometric view of a diagnostic instrument
optical subsystem, as shown by FIGS. 45 and 46. A label imaging
assembly and a reaction imaging assembly of the optical subsystem
are attached to a fixed support bracket. An integrated diagnostic
cartridge is inserted into a loading assembly and is in a loaded
position, as described with regard to FIGS. 18A-18B and
19A-19C.
[0135] FIG. 67A is a schematic diagram of an exemplary instrument
computer control system
[0136] FIG. 67B is a schematic diagram of the optical cartridge
label subsystem of the exemplary computer control system of FIG.
67A.
[0137] FIG. 67C is a schematic diagram of the optical reaction or
assay well subsystem of the exemplary computer control system of
FIG. 67A.
[0138] FIG. 67D is a schematic diagram of the thermal subsystem of
the exemplary computer control system of FIG. 67A.
[0139] FIG. 67E is a schematic diagram of the lysing drive
subsystem of the exemplary computer control system of FIG. 67A.
[0140] FIG. 67F is a schematic diagram of the loading cartridge
subsystem of the exemplary computer control system of FIG. 67A.
[0141] FIG. 67G is a schematic diagram of the pneumatic subsystem
of the exemplary computer control system of FIG. 67A.
[0142] FIG. 67H is a schematic diagram of the valve drive subsystem
of the exemplary computer control system of FIG. 67A.
[0143] FIG. 67I is a schematic diagram of the rehydration mixing
subsystem of the exemplary computer control system of FIG. 67A.
[0144] FIG. 68 is a schematic layout of an integrated diagnostic
cartridge according to an embodiment described herein.
[0145] FIG. 69A is an illustration of an integrated diagnostic
cartridge, according to an embodiment described herein, viewed from
a feature side.
[0146] FIG. 69B is an illustration of an exemplary cartridge label
for supplying a user and a diagnostic instrument with information
associated with a given diagnostic test for use with a cartridge of
FIG. 69A.
[0147] FIG. 70A is an illustration of an integrated diagnostic
cartridge, according to an embodiment described herein, viewed from
a fluidics side.
[0148] FIG. 70B is an enlarged view of the waste collection element
1470 of FIG. 70A.
[0149] FIG. 70C is an enlarged view of the upper proximal portion
and lower proximal portion of the cartridge of FIG. 70A.
[0150] FIG. 70D is an exemplary chamber with a chamber reference
line used to indicate an upper chamber portion and a lower chamber
portion.
[0151] FIG. 70E is the exemplary chamber of FIG. 70E with chamber
reference line with an inlet a top most position in the upper
chamber portion and an outlet in a lower chamber portion in a lower
most position.
[0152] FIG. 70F is the exemplary chamber of FIG. 70D with chamber
reference line indicating a top zone for locating an inlet in the
upper chamber portion and a bottom zone for locating an outlet.
[0153] FIG. 71 is an isometric view of a loading module in
accordance with an embodiment shown in FIGS. 69A and 70. A fill
chamber, a metering chamber, and an overflow chamber are shown in
fluidic communication.
[0154] FIG. 72 is a view of an integrated diagnostic cartridge and
a cartridge heating zone provided by a diagnostic instrument
thermal subsystem.
[0155] FIG. 73 is a top view of a filter assembly on an integrated
diagnostic cartridge, as described herein.
[0156] FIG. 74 is a cross-sectional view of a filter assembly shown
in FIG. 73.
[0157] FIG. 75A is a cross-sectional view of an integrated
diagnostic cartridge pneumatic interface and a filter assembly
illustrated in FIGS. 73 and 74.
[0158] FIG. 75B is an enlarged cross section view of a portion of
the filter in FIG. 75A when under pressure.
[0159] FIG. 76A is a cross-sectional perspective view of a rotary
valve illustrating an interface between a rotor and a stator,
according to an embodiment of the invention.
[0160] FIGS. 76B and 76C are bottom of views of exemplary gaskets
for use with a rotor as in FIG. 76A.
[0161] FIG. 77 is a perspective drawing of a rotor comprising a
plurality of flow channels. A magnified view of a single solid
support chamber within one of the flow channels is shown.
[0162] FIGS. 78 and 79 are perspective cross-sectional views of a
rotary valve with a threaded rotor in a shipping configuration.
[0163] FIGS. 80 and 81 are perspective cross-sectional views of the
rotary valve of FIGS. 78 and 79 with a threaded rotor in an
operational configuration with a gasket forming a fluid tight seal
with the stator.
[0164] FIG. 82 is a three-dimensional, cross-sectional illustration
of a rehydration chamber, in accordance with an embodiment.
[0165] FIG. 83A is a cross-sectional view of an assay chamber taken
through an inlet and an outlet.
[0166] FIG. 83B is a cross-sectional view of an assay chamber taken
through the midpoint of an assay chamber.
[0167] FIG. 84 is a top down illustration of a reaction area with a
plurality of assay chambers of FIGS. 83A and 83B showing a signal
indicative of the presence of target nucleic acids from a target
pathogen viewed through a transparent plug.
[0168] FIG. 85 is a cross-sectional illustration of a raised
platform within each of the loading channels used to form a portion
of a heat staked region. FIG. 85 additionally shows an illustration
of a reaction area with a main loading channel configured with a
u-bend.
[0169] FIG. 86 is a cross-sectional illustration of a raised
platform within a main loading channel used to form a portion of a
heat staked region.
[0170] FIG. 87 is a cross-sectional illustration of an assay
chamber taken through an inlet and a loading channel with a raised
platform of FIGS. 85 and 86 within.
[0171] FIG. 88 is an illustration of a waste collection element of
an integrated diagnostic cartridge. A channel for filling the waste
chamber and a vent channel are shown in proximity to loading
channels forming a shared heat staking portion of the integrated
diagnostic cartridge.
[0172] FIG. 89 is an exploded view of a cartridge, according to an
exemplified embodiment described herein with regard to FIGS. 69A
and 70, comprising a loading module, a lysing module, a
purification module, and an amplification module.
[0173] FIG. 90 is an illustration of an exemplary cartridge label
for supplying a user and a diagnostic instrument with information
associated with a given diagnostic test.
[0174] FIG. 91 is an illustration of a cartridge label with one or
more perforated areas configured to break when a diagnostic
instrument contacts an integrated diagnostic cartridge.
[0175] FIG. 92 is an illustration of an alternate cartridge,
according to an embodiment, comprising a loading module, a lysing
module, and a purification module.
[0176] FIG. 93 illustrates the state of an integrated diagnostic
cartridge after a biological sample is loaded into the sample port
assembly, prior to insertion into a diagnostic instrument and/or
prior to actuation of any cartridge features by the diagnostic
instrument.
[0177] FIG. 94 illustrates the status of the integrated diagnostic
cartridge features after cartridge preparation steps are completed
and frangible seals are broken. All of the fluids remain in their
original positions, as no motive force has yet been applied to the
cartridge features.
[0178] FIG. 95 illustrates the status of the integrated diagnostic
cartridge features after the lysis steps are performed.
[0179] FIG. 96 illustrates the status of the integrated diagnostic
cartridge features after the filtration and binding steps--the
lysis chamber is empty, and fluid has passed to a waste collection
element.
[0180] FIG. 97 illustrates the status of the integrated diagnostic
cartridge features after completion of the wash step.
[0181] FIG. 98 illustrates the status of the integrated diagnostic
cartridge features after completion of the air dry step.
[0182] FIG. 99 illustrates the status of the integrated diagnostic
cartridge features after the elution and metering step.
[0183] FIG. 100 illustrates the status of the integrated diagnostic
cartridge features after loading the assay chambers.
[0184] FIG. 101 illustrates the status of the integrated diagnostic
cartridge features after heat staking.
[0185] FIG. 102 illustrates the status of the integrated diagnostic
cartridge features after release of pressure and during the assay
step.
[0186] FIG. 103-105 depict a table of reference numbers used
herein.
[0187] FIG. 106A-106E depict an exemplary sequence of operations
executed by a diagnostic instrument to perform a molecular
diagnostic test on an integrated diagnostic cartridge, as described
in FIGS. 93-102.
[0188] FIG. 107 depicts a workflow diagram of an assay method of
testing a sample suspected of containing a target pathogen.
[0189] FIG. 108 depicts a workflow diagram of a minimal assay
method of testing a sample suspected of containing a target
pathogen.
[0190] FIG. 109 depicts a workflow diagram of a blood assay method
of testing a sample suspected of containing a target pathogen.
[0191] FIG. 110 depicts a workflow diagram of a vaginitis assay
method of testing a sample suspected of containing a target
pathogen.
[0192] FIG. 111 depicts a workflow diagram of a sputum assay method
of testing a sample suspected of containing a target pathogen.
[0193] FIG. 112 depicts a workflow diagram of a stool assay method
of testing a sample suspected of containing a target pathogen.
[0194] FIG. 113 depicts a workflow diagram of a solid tissue assay
method of testing a sample suspected of containing a target
pathogen.
DETAILED DESCRIPTION
[0195] Described herein is a diagnostic system for performing rapid
molecular diagnostic testing at the point of care. The diagnostic
system comprises a diagnostic instrument and an integrated
diagnostic cartridge as described in greater detail below. FIGS.
1-5 depict an exemplary workflow of using an integrated diagnostic
cartridge in conjunction with a diagnostic instrument to conduct a
molecular diagnostic test at the point of care. FIG. 1 illustrates
an exemplary instrument configured to be used with this diagnostic
system. As seen in FIGS. 2A and 2B, the first step of the workflow
is depicted. A user is shown loading an integrated diagnostic
cartridge with a sample loader, such as a bulb, syringe or pipette
1060. FIG. 2C illustrates the integrated diagnostic after sample
loading is completed and the user seals the cartridge by closing a
cap.
[0196] FIG. 3 illustrates the step of inserting a diagnostic
cartridge 1000 into an opening, i.e. front slot 2072, of the front
2073 of instrument 2000. The instrument includes features to ensure
that a cartridge is loaded into the instrument only in the
preferred orientation. Further description of the loading sequence
is detailed below with reference to FIGS. 17A-23B.
[0197] Once a cartridge is properly loaded and verified by the
instrument, the cartridge remains within the instrument slot as
shown in FIGS. 4A and 4B. Regarding FIG. 4A, as part of the
cartridge verification process, the display 2820 provides
information regarding the patient information from the cartridge
label and the type of test to be performed by the instrument.
Additionally, the display 2820 may be configured to provide touch
screen/GUI interactions with the instrument computer operating
system. While running the diagnostic test, the instrument display
may further provide information regarding the remaining time left
for the diagnostic test. Once the automated testing sequence is
completed, the cartridge is ejected from the instrument as shown in
FIG. 5. Additional details and exemplary workflow for the use of
embodiments of instruments and cartridges described herein may be
appreciated with reference to commonly assigned U.S. patent
application Ser. No. 16/928,994, filed Jul. 14, 2020 entitled,
"Point-of-Care Diagnostic Instrument Workflow" (incorporated herein
by reference for all purposes in its entirety).
[0198] By way of introduction, the diagnostic system will be
described according instrument embodiments and cartridge
embodiments presented herein. The diagnostic instrument 2000 will
be described according to several subsystems and assemblies shown
in FIGS. 6-66. The various subsystems and assemblies, as described
herein, may operate under the control of a computer system shown in
FIGS. 67A-671. In one aspect, the instrument 2000 is configured to
accept an integrated diagnostic cartridge of different
configurations. The large number of different cartridge
configurations are detailed below with regard to FIGS. 68-92. An
exemplary method of using one embodiment of an integrated
diagnostic cartridge 1000 is described in FIGS. 93-102. The
exemplary method describes how a cartridge can be used to prepare a
biological sample to amplify nucleic acid and detect the presence
of a suspected pathogen in a diagnostic test. As a result of the
modular and highly configurable design of the cartridge, a wide
array of sample types may be analyzed by the instrument as
described with regard to FIGS. 107-113.
A. Instrument General Overview
[0199] FIG. 1 is a front isometric view of a diagnostic instrument
2000 to be used with the diagnostic system described herein. The
various embodiments of the instrument 2000 described herein are
adapted and configured to accept and process samples using any of a
wide array of different testing methodologies and sample types. The
instrument 2000 includes a clamping subsystem, a pneumatic
subsystem, a thermal subsystem and an optical subsystem. The
various relationship between the various subsystems may be
appreciated with reference to the exploded isometric views of
instrument 2000 provided in FIGS. 6 and 7. The clamping subsystem
is described with reference to FIGS. 8-47B. The pneumatic subsystem
is described with regard to FIGS. 48 and 49. The thermal subsystem
is described with reference to FIGS. 50-57B. Additionally, the
optical subsystem is described with regard to FIGS. 58-66.
[0200] Returning to FIGS. 6 and 7, in these views, the subsystems
are shown outside of the instrument enclosure 2070 with pneumatic
subsystem 2130 shown in its position within the instrument
enclosure. The major assemblies of the fixed bracket assembly 2010
and the moving bracket assembly 2040 are shown in these views. In
FIG. 6, subsystems and assemblies of the diagnostic instrument are
shown in a right side exploded view or from a first surface of a
fixed support bracket. A reaction imaging assembly 2700 of the
optical subsystem is viewed as detached from the fixed bracket
assembly 2010 and valve drive assembly 2400 is similarly detached
from the moving bracket assembly 2040. Furthermore, a cellular
assembly 2800, which provides communication to and from an
instrument, and a label imaging assembly 2770 are readily apparent
in this view. In FIG. 7, subsystems and assemblies of the
diagnostic instrument are shown in a left side exploded view or
from a second surface of a fixed support bracket. The fixed bracket
assembly 2010 shows multiple components and assemblies supported
from the second surface of the fixed support bracket 2013.
Additionally, moving bracket assembly 2040 is viewed from a first
surface of a clamp block 2042 and holds the remaining assemblies
and components configured to interface with an integrated
diagnostic cartridge to perform various processing steps.
[0201] Throughout the disclosure that follows, the term "vertical"
position refers to the relationship of a testing cartridge to a
vertical plane and a horizontal plane orientation provided by the
design characteristics of a specific instrument embodiment. The
vertical plane orientation is one allowing for the use of gravity
for fluid movement for processing and handling steps performed
during system operations. As such, terms of orientation such as
higher and lower, upper and lower are understood in the context of
gravitational flows of a generally vertical system orientation. In
use, an instrument may be placed on a table or shelf that induces a
tilt or incline to the instrument during use. Even though the
instrument and cartridge are tilted during use this tilting up to
and including +/-30 degrees is considered vertical as used herein.
Moreover, tilting may be within the range of +/-15 degrees and also
be considered vertical as used herein. Tilting within the above
mentioned ranges would retain sufficient desired vertical
orientation so as to maintain desired and expected gravity flow and
characteristics.
[0202] The single use biologic test cartridge is received into and
maintained within the instrument enclosure in a single orientation.
This orientation is readily identified by the orientation of an
opening of the instrument enclosure and along with the vertical and
horizontal planes of the instrument. The instrument is adapted and
configured to operate with cartridges configured to operate in such
an orientation. Accordingly, the instrument receives a cartridge
via an opening within the instrument enclosure when the cartridge
is oriented with proper alignment. In various embodiments, the
opening within the enclosure is a hole, gap, space, slot, window,
drawer, cabinet or any other aperture for permitting limited access
to the interior of the instrument. In one embodiment, the opening
within the enclosure is a slot. In a preferred embodiment, the
opening within the enclosure is a vertically oriented slot. As
such, the meaning of upright is that positioning of the cartridge
relative to the components of the instrument while maintaining an
orientation of the cartridge so as to operate the cartridge within
the designed cartridge orientation principals. In one embodiment,
upright refers to an orientation of the cartridge within the
instrument to being vertical within the instrument. This is the
orientation that is illustrated in the several views of the
instrument. In the views of FIGS. 68-72, and 89-92 an arrow 1900
indicates the vertical orientation and points towards UP. However,
the operation and configurations of the instrument is not so
limited. Based on variations in fluid flow characterizations of a
specific single use cartridge, the orientation of the cartridge to
the components of the instrument may be modified while still
enabling the upright fluid flow principals implemented in a
specific cartridge design. As a result, in other configurations,
upright may include a slightly inclined orientation where the
cartridge may be inclined relative to a vertical plane of the
instrument while still providing the needed discrete actions of
having an up and a down within the cartridge fluid schemes.
B. Clamping Subsystem
[0203] The clamping subsystem disposed within the instrument
orchestrates the various physical interactions between the
instrument 2000 and cartridge 1000 to perform a molecular
diagnostic test run on the cartridge. The coordinated operation of
the clamping subsystem is under control of the instrument computer
controller (see FIGS. 67A-671). The clamping subsystem is
configured to accept and align a cartridge once inserted into the
instrument and maintain the cartridge in an operational orientation
within the instrument until the completion of a testing protocol.
The clamping process is used to sequentially initiate one or more
interfaces between the instrument and specific cartridge
components. Once diagnostic testing of a cartridge sample is
completed, the clamping subsystem unclamps the cartridge and is
ejected from the instrument. In one embodiment, the clamping
subsystem includes a mechanism to break frangible seals within
cartridge 1000, thus allowing fluid flow. In another embodiment, a
magnetic mixing assembly 2300 is coupled to the clamping subsystem
to provide mixing capabilities performed by the cartridge. In one
implementation, a valve drive assembly 2400 actuates a rotary valve
1400 on the cartridge to move fluids and includes various sensors
to monitor valving positions. In yet another implementation, the
clamping subsystem supports an additional magnetic mixing motor to
dissolve and rehydrate reagents within a cartridge to perform a
diagnostic test.
1. Overview
[0204] As will be described further herein, a combination of
instrument assemblies, subsystems, and an appropriate computer
control system can be used to automate a plurality of steps in a
testing protocol to perform rapid molecular diagnostic testing at
the point of care. Upon cartridge insertion, an instrument computer
control system may cause the instrument to automatically engage a
clamping subsystem to immobilize the cartridge within the
instrument enclosure in an operational orientation for conducting
the testing sequence. Once engaged by the clamping subsystem, a
cartridge is immobilized within the instrument during testing. The
design of the clamping subsystem may vary based on the specific
cartridge arrangement. Accordingly, instrument 2000 and the
clamping subsystem may be configured with corresponding
instrument-to-cartridge interfaces to accept and clamp an
integrated diagnostic cartridge of varying different
configurations. It is to be appreciated that the following
embodiments and configurations are solely for the purposes of
understanding and changes and modifications can be made thereto
without departing from the spirit or scope of the appended
claims.
[0205] In various aspects of the invention, the clamping subsystem
is configured to clamp the integrated diagnostic cartridge in an
operational orientation for performing the molecular diagnostic
test. In one implementation, the clamping subsystem clamps the
cartridge in a vertical orientation. Such orientation may be
directed and maintained by a clamping subsystem that comprises a
fixed-bracket assembly 2010 and moving bracket assembly 2040 which
provide the foundation from which all other subsystems and
assemblies are mounted from. FIGS. 8 and 9 are two frontal views of
the clamping subsystem at two angles with cartridge 1000
inserted.
[0206] Additionally, FIGS. 10 and 11 are two rear views of the
clamping subsystem at two angles with cartridge 1000 inserted. Door
support assembly 2280 is seen pressing against a sample port
assembly 1100 on cartridge 1000. Linear actuator 2014, with lead
screw 2016, mates with lead nut 2044 of the frangible seal block
within the moving bracket assembly 2040. In FIG. 9 the valve drive
assembly 2400 is readily visible. Furthermore, loading assembly
2230 is in a loaded cartridge position while thermal clamp assembly
2680 presses against the distal end of the cartridge. FIGS. 10 and
11 illustrate the clamping subsystem, with cartridge 1000 inserted,
from two angles of the second surface of the fixed support bracket
2013. Linear actuator 2014, latch and pin assembly 2210, drive
motor 2330 of the driving magnet system 2310, rehydration motor
2510 and thermal subsystem are illustrated in these views.
[0207] An exploded view of the clamping subsystem with cartridge
1000 is seen from two angles in FIGS. 12 and 13. Notch 2015 on the
bottom of the first surface of fixed support bracket 2012 and
linear slide 2043 on the moving bracket assembly 2040 define the
direction the clamp block can move, such that the moving block
assembly is configured to move toward the first surface of the
fixed support bracket 2012 in a positive direction and away from
the first surface of the fixed bracket support in a negative
direction. In one embodiment, the linear actuator 2014 uses lead
screw 2016 coupled to lead nut 2044 to move the moving bracket
assembly 2040 to clamp and unclamp a cartridge. Lead nut 2044 is
bolted to frangible seal block 2260 to drive the moving bracket
assembly 2040 along linear slide 2043. Further detail of the
assemblies and operation of the fixed bracket assembly 2010 in
conjunction with a moving bracket assembly 2040 is described in the
following sections.
2. Fixed Bracket Assembly
[0208] Fixed bracket assembly 2010 is the stationary component of
the clamping subsystem and is composed of the loading assembly
2230, pin and latch assembly 2210, a driving magnet system 2310,
and rehydration motor 2510. Various views of the fixed bracket
assembly are provided in FIGS. 9, 10, 11, 12 and 13. In one
embodiment, the fixed bracket assembly 2010 further supports the
thermal subsystem responsible for generating the thermal
requirements for executing a molecular diagnostic test and the
optical subsystem for imaging separate distinctive areas of a
cartridge. The optical subsystem comprises two assemblies: the
label imaging assembly and the reaction imaging assembly. The label
imaging assembly 2770 is attached to the bottom proximal end of the
fixed support bracket, while the reaction imaging assembly 2700 is
fixed to the distal end of the fixed support bracket. A frontal
view of the fixed support bracket is viewed from a first surface
2012 or cartridge side in FIG. 12. Loading assembly 2230, which
accepts and detects a loaded cartridge within the instrument and
ejects the cartridge upon completion of a diagnostic test, is
attached to the first surface of the fixed support bracket. Notch
2015 on the bottom of the first surface of the fixed support
bracket provides the area in which linear slide 2043 within moving
bracket assembly 2040 resides. In some embodiments, a sensor 2019
is mounted to the fixed bracket assembly 2010 to detect when the
cartridge is successfully clamped between the fixed bracket
assembly 2010 and the moving bracket assembly 2040. The sensor 2019
can be viewed in FIGS. 12, 13, and 15A-15E.
[0209] A rear view or a view from a second surface 2013 of fixed
bracket assembly 2010 is depicted in FIGS. 10, 11 and 13. The fixed
bracket assembly 2010 further comprises a linear actuator 2014
attached to a second surface of the fixed support bracket 2013. The
linear actuator 2014 uses a lead nut 2016, coupled to lead nut 2044
on the frangible seal block 2260, to pull the moving bracket
assembly toward the first surface of the fixed support bracket 2012
during clamping and push the frangible seal block 2260 and clamp
block 2041 away from the fixed support bracket during unclamping.
Further description of the clamping mechanism between the fixed
bracket assembly 2010 and moving bracket assembly 2040 is discussed
in greater detail with regard to the clamp block 2041 and frangible
seal block 2260. The second surface of the fixed support bracket
2013 additionally serves as the surface responsible for carrying a
driving magnet system 2310, rehydration motor 2510, and thermal
subsystem of the instrument.
3. Moving Bracket Assembly
[0210] A front perspective view of the moving bracket assembly 2040
is viewed in FIG. 14. Exploded views of the moving bracket assembly
2040 from two different angles is viewed in FIGS. 15A and 15B. The
moving bracket assembly is the dynamic component of the clamping
subsystem and is configured to move linearly toward the fixed
support bracket 2011 to clamp and contact the cartridge at numerous
locations. The clamp block 2041 supports various systems
interfacing with the cartridge and is configured to enable each
system to perform respective tasks when running a diagnostic test.
In one embodiment, assemblies supported by the clamp block 2041
include a frangible seal block 2260, a door support assembly 2280,
a valve drive assembly 2400, a pneumatic interface 2100, a driven
magnet system 2350, and a thermal clamp assembly 2680. As described
in greater detail in the following sections, it is advantageous to
separate the clamp block 2041 and the frangible seal block 2260 to
separate the clamping action from the frangible seal actuation. In
one implementation, the frangible seal block 2260 is configured to
initially move with the moving bracket assembly 2040 and is capable
of moving independently of the clamp block 2041. Additionally, the
thermal clamp assembly 2680 is configured to move independently
from the clamp block 2041. The door support assembly 2280, valve
drive assembly 2400, pneumatic interface 2100, and driven magnet
system 2350 are fixedly mounted to the clamp block 2041, such that
movement of these assemblies entirely depends on the position of
the clamp block 2041. The clamp block 2041 further comprises a
first surface 2042 from which all cartridge interfacing features
extend out of. The first surface of the clamp block 2042 is seen in
FIGS. 14 and 15A.
[0211] The clamp block sits along linear slide 2043 which
corresponds to notch 2015 on the bottom of the first surface of the
fixed support bracket 2012 to connect the fixed bracket assembly
2010 to the moving bracket assembly 2040. As described above, the
linear actuator 2014 is coupled to lead nut 2044 on the frangible
seal block 2260. The linear actuator 2014 rotates lead screw 2016
in a first direction within lead nut 2044 of the frangible seal
block 2260 to pull the moving bracket assembly toward the first
surface of the fixed bracket assembly 2010 during clamping.
Clamping force applied to the cartridge by the moving bracket
assembly is not a result of the clamp block contacting the fixed
bracket. In one implementation, extension springs 2045, seen in
FIGS. 18A and 24, provide the force needed to clamp all assemblies
supported by the clamp block to interface with a cartridge. During
unclamping the moving bracket assembly is driven away from the
fixed support bracket as the lead screw 2016 of the linear actuator
2014 is rotated in a second direction, opposite to the first
rotational direction.
[0212] The largest displacement the clamp block is configured to
move, in the positive direction toward the fixed bracket, is
constrained by hard stops 2211 at the top of the clamp block. This
configuration separates the clamping action from the frangible seal
action, allowing the clamp block to clamp and interface with the
cartridge without actuating frangible seals and allowing fluid
flow.
[0213] The moving bracket assembly includes a door support assembly
2280 comprising a door support 2281 and spring 2282. During
clamping, spring 2282 contact pushes door support 2281 against a
top of a cap 1181 on a cartridge. Door support 2281 ensures the cap
remains closed and sealed during pressurization of cartridge
1000.
4. Frangible Seal Block (Clamp Block)
[0214] Frangible seals keep fluids contained within cartridge 1000
and fluidic components isolated when the cartridge is not in use,
such as during shipping and storage conditions. Accordingly, the
diagnostic instrument includes a puncture mechanism for actuating
frangible seals and allowing fluids within the cartridge to flow.
The frangible seal block 2260 operates to break the frangible seals
of the cartridge and is disposed within clamp block 2041 as a part
of the moving bracket assembly 2040. The frangible seal block is a
separate component from the clamp block 2041, wherein the frangible
seal block and clamp block are coupled by linear slide 2264 to
allow the frangible seal block 2260 to move independently from the
clamp block 2041 during clamping. FIG. 34 illustrates the frangible
seal block 2260 separated from the remaining moving bracket
assembly 2040. This configuration disconnects the clamping action
from the actuation of frangible seals to enable a cartridge to be
clamped but not fluidically active until commanded. Frangible seal
block 2260 can be viewed in FIGS. 14, 15A, 15B, 31, 33, 34 and 44.
The basic structure of the frangible seal block includes frangible
seal pins 2261 and hard stop 2263. Lead nut 2044 is bolted to the
front of the frangible seal block and is used to pull the frangible
seal block 2260 and moving bracket assembly 2040 toward the fixed
bracket assembly 2010 in the positive direction during clamping and
drive the frangible seal block 2260 and moving bracket assembly
2040 away from the fixed bracket assembly 2010 in the negative
direction during unclamping. The lead nut 2044 is coupled to the
lead screw 2016 of linear actuator 2014 mounted on the second
surface of the fixed support bracket 2013. The linear actuator 2014
rotates lead screw 2016 in a first rotational direction to pull the
frangible seal block in a positive direction towards the fixed
bracket assembly 2010. Extension springs 2045 housed within the top
of the moving bracket assembly 2040 provide tension to pull the
clamp block 2041 against the frangible seal block 2260 to move the
frangible seal block and clamp block together along linear slide
2043 during clamping movement in the positive direction. In one
implementation, extension springs 2045 are attached to pins 2018
which are fixed to portions of the fixed support bracket 2011 and
clamp block 2041.
[0215] The moving bracket assembly 2040 is configured such that the
frangible seal block 2260 and clamp block 2041 initially move
together due to extension springs 2045 until hard stop 2211 on
clamp block 2041 contacts the first surface of the fixed support
bracket 2012. Hard stop 2211 prevents the clamp block 2041 from
being displaced a further distance in the positive direction toward
the fixed bracket assembly 2010. However, the separation between
the frangible seal block 2260 and the clamp block 2041 enables the
frangible seal block to be further displaced in the positive
direction along linear slide 2264 toward the fixed support bracket
to actuate frangible seals and render the cartridge fluidically
active. To actuate frangible seals 1201-1207 on the cartridge,
linear actuator 2014 continues to rotate the lead screw 2016 in a
first rotational direction after the cartridge is clamped. While
the clamp block 2041, remains stationary due to the contact between
hard stop 2211 and the first surface of the fixed support bracket
2012, the frangible seal block 2260 is pulled along linear slide
2264, seen in FIG. 31. Frangible seal pins 2261 on the frangible
seal block 2260 press against frangible seals 1201-1207 and into
pocket 2262, shown in FIG. 32, formed in the first surface of the
fixed support bracket 2012 to actuate the seals. Movement of the
frangible seal block 2260 is configured to move in the positive
direction until hard stop 2263 contacts upper rail 2231a of the
loading assembly 2230. Hard stop 2263 prevents the frangible seal
pins from over puncturing the frangible seals, which may result in
the breaking of the one or more backing films on the cartridge and
produce a leak. Additionally, hard stop 2263 prevents damaging the
pins.
[0216] In one implementation, frangible seal pins 2261 are
cylindrical in shape. Other pin shapes are possible included
rounded tips or other shapes suited to produce the desired opening
in a frangible seal or to have a complementary shape with a
preferred seal rupture pattern or design.
[0217] One aspect of the invention provides the frangible seal pins
2261 of substantially equivalent length. When frangible seals are
of substantially equal length, frangible seal pins on the frangible
seal block 2260 will actuate all frangible seals on cartridge 1000
with one linear motion in a positive direction. Furthermore, when
the frangible seal block 2260 is moved in a negative direction, all
frangible seal pins are retracted from pocket 2262 in the first
surface of the fixed support bracket 2012 to release the cartridge
during unclamping and ejection. In an alternative embodiment, one
or more frangible seal pins 2261 may be of varying lengths, such
that different frangible seals on the cartridge may be actuated at
different times. In this configuration, the frangible seal block
may actuate frangible seals in a sequence to convert one or more
frangible seals fluidically active while one or more frangible
seals remain fluidically inactive. Sequential actuation of one or
more frangible seals depends on the position of the frangible seal
block 2260, such that in a first actuation position frangible seal
pins 2261 longer in length will actuate frangible seals before
frangible seal pins smaller in length. Subsequently, the frangible
seal block must be moved in the positive direction to a second
actuation position to actuate frangible seals with smaller
frangible seal pins to render respective seals fluidically active.
As described in the previous sections, the clamp block 2041 remains
stationary, clamping the cartridge, due to hard stop 2211 as
frangible seal pins are actuated either all at once or in a
sequence. This alternative embodiment is illustrated in FIG. 33
with first frangible seal pin 2261a being shown longer than
remaining pins 2261b-g.
[0218] When a diagnostic test is complete and a cartridge is ready
to be unclamped and ejected, linear actuator 2014 rotates the lead
screw 2016 in a second rotational direction. Rotating the lead
screw 2016 in a second rotational direction initially pushes the
frangible seal block in the negative direction along linear slide
2264 away from frangible seals 1201-1207. As an integrated part of
the moving bracket assembly 2040, the frangible seal block
continues to move in a negative direction along linear slide 2264
until the frangible seal block contacts ledge 2046 of clamp block
2041. The frangible seal block presses against ledge 2046 to
subsequently move the entire moving bracket assembly 2040 in the
negative direction away from the fixed bracket assembly 2010 to
unclamp the cartridge.
5. Clamping Sequence
[0219] As described herein, the clamping subsystem can clamp a
cartridge 1000 using a sequence of clamping positions to engage
different interfaces of the moving bracket assembly 2040 with the
cartridge either simultaneously or sequentially. A representative
clamping sequence will be described with reference to FIGS. 16A-16E
for clamping an integrated diagnostic cartridge in a preferred
vertical orientation. In the preferred orientation, the instrument
is configured to maintain the cartridge in the vertical orientation
during the duration of the testing sequence to determine the
presence of a target pathogen The exemplary clamping sequence
begins at FIG. 16A with the moving bracket assembly 2040 described
above in a zero clamping position. Specifically, hard stop 2211
located at the top of the clamp block 2041 does not contact the
first surface of the fixed support bracket 2012 and the moving
bracket assembly 2040 is spaced apart from the fixed bracket
assembly 2010 to allow a cartridge 1000 to be inserted into the
instrument 2000. Engagement between each interface, e.g., a
frangible seal block 2260, a door support assembly 2280, a valve
drive assembly 2400, a pneumatic interface 2100, a driven magnet
system 2350, and a thermal clamp assembly 2680, on the moving
bracket assembly 2040 and the cartridge has yet to be
established.
[0220] FIG. 16B shows the moving bracket assembly 2040 after it is
moved in the positive direction from the zero clamping position to
a first clamping position when linear actuator 2014 rotates lead
screw 2016 in a first rotational direction. In the first position,
the valve drive assembly 2400, mounted within clamp block 2041, is
engaged with rotary valve 1400 on the cartridge. Hard stop 2211 has
yet to contact the first surface of the fixed bracket 2012 and
sensor 2019 is untriggered. Additionally, the thermal clamp
assembly contacts the distal end of the cartridge but is not
engaged to seal the reaction area 1600. This position enables the
instrument to execute multiple rotary valve verification tests on
rotary valve 1400 of the cartridge, described in the sections
below, before executing the remainder of the clamping sequence.
Rotary valve verification tests ensure a cartridge rotary valve
1400 is in a shipping configuration to ensure an inserted cartridge
is unused and can perform a diagnostic test.
[0221] FIG. 16C shows the moving bracket assembly 2040 after it is
moved in the positive direction from the first clamping position to
a second clamping position when linear actuator 2014 rotates lead
screw 2016 again in a first rotational direction. In the second
position, hard stops 2211 contact the first surface of the fixed
support bracket 2012 and sensor 2019, which now hidden from view,
is triggered. The door support assembly 2280, pneumatic interface
2100, valve drive assembly 2400, and thermal clamp assembly 2680
are actively engaged with each respective location on the
cartridge. In this view, the cartridge is clamped, but not
fluidically active. Furthermore, this position is the greatest
distance the clamp block 2041 and all assemblies fixedly attached
to the clamp block (i.e. door support assembly 2280, valve drive
assembly 2400, pneumatic interface 2100, thermal clamp assembly
2680, and driven magnet system 2350) are permitted to move in the
direction of the fixed support bracket 2011.
[0222] FIG. 16D shows the frangible seal block 2260 after it moved
in the positive direction from the second clamping position to a
third clamping position. In the third position, the clamp block
2041 remains in the second clamping position and is prevented from
moving as hard stops 2211 contact the first surface of the fixed
support bracket 2012. All assemblies fixedly to the clamp block
2041 including door support assembly 2280, pneumatic interface
2100, valve drive assembly 2400, and driven magnet system 2350
remain in the second clamping position. Note, while the thermal
clamp assembly 2680 is configured to move independently from the
clamp block 2041, the thermal clamp assembly also remains in the
second clamping position due to being in sealing contact with the
distal end of the cartridge. As described herein, frangible seal
block 2260 is configured to move independently of the clamp block
2041 along linear slide 2264. The frangible seal block 2260 moves
to the third clamping position in the positive direction when
linear actuator 2014 rotates lead screw 2016 in a first rotational
direction, thus actuating frangible seals on a cartridge. This
independent movement is observed by gap 2265 between the frangible
seal block 2260 and clamp block 2041. The separation between the
clamp block 2041 and frangible seal block 2260 isolates the
clamping action of the cartridge from the actuation of frangible
seals on the cartridge. In the third clamping position, the
cartridge is clamped, fluidically active, and ready to run a
diagnostic test in the third position.
[0223] FIG. 16E shows the moving bracket assembly 2040 when it is
moved in the negative direction away from the fixed bracket
assembly 2010 to a fourth clamping position when linear actuator
2014 rotates lead screw 2016 in a second rotational direction. In
the fourth position, the moving bracket assembly 2040 is located at
a negative distance measured from the zero clamping position to
unclamp a cartridge when the diagnostic test is completed. During
unclamping, the frangible seal block 2260 is first driven away from
the fixed support bracket until the frangible seal block contacts
ledge 2046 of the clamp block 2041, thus eliminating gap 2265 seen
in FIG. 16D. As the frangible seal block 2260 continues to move in
a negative direction, the frangible seal block pushes against ledge
2046 to drive the entire moving bracket assembly 2040 away from the
cartridge 1000 and fixed bracket assembly 2010.
6. Loading Assembly (Fixed Support Bracket)
a) Loading
[0224] In one aspect, the invention provides a loading assembly
2230 configured to accept a cartridge inserted into instrument 2000
and eject the cartridge upon completion of a diagnostic test. FIGS.
17A-17C, 18A-18B and 19A-19C illustrate various views of the
operation of the loading assembly 2230 within instrument 2000.
FIGS. 17A-17B illustrate the loading assembly 2230 in a loading
position. FIG. 18A-18B illustrate the loading assembly 2230 in a
loaded position. FIGS. 19A-19C illustrate a cartridge inserted into
the loading assembly 2230 in a loaded position. The loading
assembly comprises rails 2231, rack 2232, pinion 2233, pusher
carriage 2234, spring 2235 and a load position sensor 2236.
[0225] A cartridge inserted into the loading assembly 2230 is
viewed in a loading position in FIG. 17A. The cartridge is inserted
along upper and lower rails 2231 until the distal end of the
cartridge contacts pusher carriage 2234. In a loading position,
pusher carriage 2234 is in a forward most position toward the front
slot 2072 of the instrument such that load position sensor 2236 is
not triggered by flag 2237 located on the pusher carriage. Further
description of the load position sensor 2236 and flag 2237 is
discussed in reference to the cartridge in a loaded position. An
enlarged view of the pusher carriage 2234, rack 2232, and pinion
2233 is viewed in FIG. 17B when a cartridge is in a forward most
loading position. FIG. 17C shows an enlarged view of spring 2235
which is fixed between post 2239 and pusher carriage 2234, such
that when the cartridge and loading assembly is in a forward most
loading position, spring 2235 is in a resting equilibrium
position.
[0226] FIGS. 18A-18B illustrate the loading assembly 2230 in a
loaded position without a cartridge. In the loaded position, pusher
carriage 2234 is in a backward most position away from the front
slot 2071 of the instrument. As viewed in FIGS. 18B and 19B, load
position sensor 2236 is triggered by flag 2237 on the pusher
carriage. FIGS. 19A and 19C are perspective views of a cartridge
inserted into the loading assembly 2230 while in a loaded position.
The cartridge transitions from the loading position, viewed in FIG.
17A, to a loaded position when the cartridge continues to move
along rails 2231, with the distal end of the cartridge pushing
against the pusher carriage. The cartridge is permitted to move
along rails 2231 until pinion 2233 reaches the end of rack 2232 and
flag 2237 triggers load position sensor 2236, thus confirming the
cartridge is inserted into the instrument. The latch and pin
assembly 2210, described in the next section, obstructs the
cartridge while in a loaded position to prevent the cartridge from
being ejected by spring 2235 prior to the cartridge being clamped
by the moving bracket assembly 2040. The cartridge remains in the
loaded position for the duration of the diagnostic test until the
cartridge is ejected upon completion of the test. A view of the
cartridge in a loaded position from the outside of the instrument
is seen in FIGS. 4A and 4B.
[0227] In one aspect of the invention, the loading assembly 2230
allows an inserted cartridge 1000 to ride along two rails 2231
until the distal end of the cartridge contacts pusher carriage
2234. Interaction between a cartridge and the upper and lower rails
is shown in the various views of FIGS. 20-23B. Proper cartridge
insertion orientation is ensured through the use of complementary
features on both the cartridge and the rails. FIGS. 20 and 21
illustrate an upper rail 2231a and lower rail 2231b, of the loading
assembly 2230, both comprising guide features 2240. A properly
aligned cartridge is configured to align with guide features 2240
to maintain proper vertical orientation as described herein. In one
embodiment, the width of the rail gap corresponds to the width or
edge thickness of the fluidic card. It is to be appreciated that
features used to ensure proper cartridge orientation may be used to
interfere with one or both of the fluidic card, the cover or any
designed gap or spacing formed or partially formed between the
fluidic card and cover. In some embodiments, interference features
may be included in one or both of a cartridge component or an upper
rail or a lower rail to ensure proper cartridge insertion
orientation.
[0228] A cartridge inserted with proper alignment is shown in a top
down view in FIGS. 22A-B and shown in a bottom up view in 23A-B.
FIG. 22A illustrates the distal end of the cartridge prior to being
inserted into the loading assembly 2230 and prior to interacting
with upper guide feature 2240. FIG. 22B shows a cartridge during
loading with upper guide feature 2240 in alignment with the
cartridge gap or spacing formed or partially formed between the
fluidic card and cover. The gap or spacing formed between the
fluidic card and cover is configured to interface with the upper
guide feature 2240 to direct the cartridge along the upper rail
2231a. Additionally notch 1021, used to obstruct the cartridge from
being ejected, is further viewed in FIGS. 22A and 22B. In one
implementation, an interference feature 1022 is formed within the
cartridge cover, as shown in FIGS. 23A and 23B. FIG. 23A
illustrates the distal end of the cartridge, in a bottom up view,
prior to being inserted into the loading assembly 2230 and prior to
lower guide feature 2240 interacting with interference feature
1022. FIG. 23B shows a cartridge during loading with a lower guide
feature 2240 in alignment with interference feature 1022. Alignment
between the lower guide 2240 and interference feature 1022 prevents
a user from inserting the cartridge with an incorrect
orientation.
b) Ejection
[0229] When the diagnostic test is complete, the cartridge is
unclamped by the moving bracket assembly 2240 and unlatched by the
latch and pin assembly 2210. The loading assembly 2230 uses spring
2235, as shown in FIG. 17C, along the bottom rail 2231 to provide
the force to eject the cartridge upon completion of the diagnostic
test. Spring 2235 is fixed between post 2239 and pusher carriage
2234 such that when the cartridge is in a backward most loaded
position (i.e. the load position sensor is triggered), spring 2235
is stretched out of equilibrium. During ejection, spring 2235
returns to its resting equilibrium position and pulls the pusher
carriage and cartridge back to a forward most position toward the
front slot 2072. The cartridge is returned to the loading position,
as viewed in FIG. 17A, to eject the cartridge. An ejected cartridge
is viewed from the outside of the instrument in FIG. 5.
7. Latch and Pin Assembly (Fixed Support Bracket)
[0230] In one embodiment, the instrument of the present invention
comprises a latch and pin assembly 2210 to prevent a cartridge from
being ejected by spring 2235. The latch and pin assembly 2210 keeps
the cartridge stationary in the loaded position while the moving
bracket assembly 2240 moves in a positive direction toward the
first surface of the fixed support bracket 2011 to clamp the
cartridge. Specifically, the latch and pin assembly 2210 is fixed
to the second surface of the fixed support bracket 2013 and
comprises latch 2212, spring 2213, latch arm 2214, arm slot 2215,
and pin 2216. The latch and pin assembly 2210 illustrated in FIG.
24 is discussed in greater detail with regard to FIGS. 25A-28.
[0231] FIG. 25A is a perspective frontal view of the latch and pin
assembly 2210 with cartridge 1000 fully inserted. In some
embodiments, a latch release arm 2214 is attached to the moving
bracket assembly 2010 and extends to the second surface of the
fixed support bracket 2013 to interact with pin 2216, described in
further detail with regards to FIG. 27. Latch 2212 is seen within
notch 1021 at the top of the cartridge to prevent the cartridge
from being ejected by the loading assembly 2230. It is to be
appreciated features used to obstruct the cartridge from being
ejected may be formed or partially formed in the fluidic card, such
as the one depicted in FIG. 25A, and optionally extent through to
the cover. FIGS. 25B and 25C are additionally views illustrating
the pin and latch assembly 2210 at two angles with spring 2213
configured to provide a downward force to drop the latch 2212 into
notch 1021 when the cartridge is inserted into the loading assembly
2230 of the instrument. In FIGS. 25A-25C, pin 2216 resides within a
narrow portion of a slot 2215 formed within the latch release arm
2214.
[0232] As described herein, the cartridge travels along upper and
lower rails 2231 of the loading assembly 2230 when a user inserts a
cartridge with proper alignment and orientation into the
instrument. When implemented, the rounded distal end of the
cartridge lifts latch 2212 up and spring 2213 drops latch 2212 into
notch 2210. When the latch is trapped within the notch, the
cartridge is obstructed and remains in a loaded position (i.e. with
load position sensor 2236 triggered). FIG. 25D shows the latch and
pin assembly 2210 in a side view with cartridge 1000 in a loaded
position and latched by the latch and pin assembly. However, the
cartridge remains unclamped. Hard stops 2211 of the moving bracket
assembly 2040 do not contact the first surface of the fixed support
bracket 2012 and hard stop 2263 of the frangible seal block 2260
has yet to contact upper rail 2231 of the loading assembly 2230.
Pin 2216 is constrained between the narrow portion of slot 2215
formed within the latch release arm and latch release arm 2214 does
not contact the bottom of pin 2216.
[0233] FIG. 26A illustrates when a cartridge is in a loaded and
latched position. Additionally, the cartridge is clamped and
rendered fluidically active as denoted by hard stops 2211
contacting the first surface of the fixed support bracket 2012 and
hard stop 2263 contacting upper rail 2231. As shown in FIG. 26B,
pin 2216 resides in a widened section of slot 2215 formed within
the latch release arm 2214 when a cartridge is clamped by the
moving bracket assembly 2040. In some embodiments, latch and pin
assembly 2210 uses latch release arm slot 2215 to constrain the
movement of pin 2216 within the slot opening. The position of the
pin, in relation to slot 2215, is free to gimbal during the
clamping of the cartridge due to the widening of the slot opening
at the vertical bend of the latch release arm. This feature
addresses mild variation tolerances generated from the interaction
between cartridge and the various interface features of the moving
bracket assembly 2240 when the cartridge is clamped and ensures
latch 2212 catches notch 1021 to prevent the cartridge from being
ejected.
[0234] When the cartridge is ready to be ejected the moving bracket
assembly 2240 travels in a negative direction away from the first
surface of the fixed support bracket 2012, thus causing the latch
release arm, fixedly attached to the moving bracket assembly, to
simultaneously move in the negative direction. The unclamping
motion of the moving bracket assembly causes tab 2217 on the latch
release arm to contact the bottom of pin 2216 and urge latch 2212
upward, shown in FIG. 27. In this configuration, the cartridge is
no longer obstructed by the latch 2212 and the cartridge 1000 is
free to be ejected by the loading assembly 2230. FIG. 28
illustrates the latch and pin assembly 2210 when the used cartridge
is removed from the instrument. Latch 2212 is returned to its
resting position and the moving bracket assembly 2040 is separated
from the first surface of the fixed support bracket 2012.
8. Valve Drive Assembly (Clamp Block)
[0235] As described herein, the moving bracket assembly 2040
comprises a valve drive assembly 2400 to facilitate the delivery
and redirection of a sample and any of the necessary reagents
through rotary valve 1400 on a cartridge 1000. FIGS. 29 and 30
provide an enlarged view and a perspective view of the details and
operation of a valve drive assembly. The valve drive assembly is
configured to index the rotary valve 1400 to different valving
positions in a sequence of steps for performing a diagnostic test.
The valve drive assembly includes a valve drive 2401, a valve drive
shaft 2408, a motor 2403, a pulley 2406, and various sensors to
detect the valve drive position. As seen in FIG. 29, valve drive
2401 is connected to the valve drive shaft 2408 wherein the end of
the valve drive shaft 2408 is coupled to pulley 2406. Motor 2430
supplies the motive force to rotate the valve drive to index the
rotary valve to different valving positions. The motor is
mechanically coupled to the valve drive using valve drive shaft
2408 and pulley 2406. Specifically, as the motor rotates, belt 2407
translates rotational motion to the pulley thereby causing the
valve drive shaft 2408 to rotate the valve drive. In some
embodiments, the valve drive assembly may incorporate the use of
various sensors to perform multiple verification checks on the
rotary valve to ensure an inserted cartridge is suitable to run a
diagnostic test (i.e. the cartridge is unused and untampered). In
one implementation, the valve drive assembly 2400 uses an
interference sensor 2404 to track the linear displacement of the
valve drive assembly. In a further implementation, valve drive
assembly 2400 includes a homing sensor 2409 to monitor the
rotational position of the valve drive 2401.
[0236] Valve drive 2401 defines the operational coupling between
the valve drive assembly and rotary valve 1400 on a cartridge. In
some implementations, the valve drive may further include a
plurality, e.g., two, three, four, or more, valve drive pins 4202,
shown in FIG. 30, which extend from an outermost peripheral wall or
edge of a rotary valve. Valve drive pins 2402 are associated with
engagement openings on the rotary valve to interface between the
valve drive assembly and rotary valve 1400 when indexing. In
various embodiments, the configuration is reversed and the valve
drive may include a series of receptacles for receiving
projections. In some versions, a rotor portion forms a gear that
interlocks with a propulsion element, or a portion thereof and the
gear interaction drives the indexing of the rotor. Typically, valve
drive pins are arranged concentrically about the rotational axis of
the rotary valve. In one embodiment, the valve drive pins may be of
cylindrical shape. In a further embodiment, the valve drive pins
include a chamfered edge to guide the valve drive pins into
engagement openings when the valve drive engages with the rotary
valve.
[0237] The valve drive assembly 2400 is configured to move linearly
in the positive and negative directions, depending on the moving
bracket assembly position during clamping and unclamping. In this
manner, the valve drive 2401, valve drive shaft 2408, pulley 2406,
and belt 2407 are capable of both linear and rotational motion.
Interference sensor 2404 tracks the linear position of the valve
drive with respect to the cartridge while homing sensor 2405
monitors the rotational position of the valve drive shaft. Both
sensors are used to ascertain information about rotary valve 1400
and enable the instrument to perform a series of verification
checks to ensure the rotary valve is satisfactory for running the
diagnostic test.
[0238] Cartridge 1000 is configured for long-term storage and
includes a rotary valve 1400 configured for a shipping
configuration and an operational configuration when actuated on
command. Accordingly, the valve drive assembly is configured to
perform a series of verification tests on rotary valve 1400 to
verify cartridge 1000 can support a diagnostic test and
subsequently actuate the rotary valve into an operational
configuration to deliver and direct fluids. In one implementation,
the shipping configuration of a rotary valve is determined using
interference sensor 2404. When the moving bracket assembly is moved
to the first position, the valve drive assembly is the first
interface to contact the cartridge. In this position, valve drive
pins 2404 are inserted into engagement openings of the rotary
valve. The engagement between the valve drive and the rotary valve
causes the valve drive shaft to be located at a linear distance
away from the cartridge. The interference sensor uses the end of
the valve drive shaft to determine the status of the rotary valve.
For example, when the valve drive 2401 correctly engages with the
rotary valve 1400 the interference sensor 2404 is triggered by the
end of the valve drive shaft 2405, thus confirming the rotary valve
is in a shipping configuration. Alternatively, the rotary valve
1400 may be defected and not be in a shipping configuration. In
such case, the valve drive must move a larger distance in the
positive direction to mate valve drive pins 2404, seen in FIG. 30,
with the rotary valve. This results in the end of the valve drive
shaft being located at a different linear distance from the
cartridge. The interference sensor is not triggered by the
interference sensor and notifies the instrument that the rotary
valve in not in a shipping configuration and is unfit to run a
diagnostic test. Upon successful confirmation of rotary valve 1400
in a shipping configuration, the valve drive assembly rotates to
transition the rotary valve from a shipping configuration and into
an operational configuration, as described herein with in greater
detail with regard to the cartridge.
[0239] In a further embodiment, the valve drive assembly 2400 is
configured to conduct a second rotary valve verification check
prior to the moving bracket assembly 2010 moving in the positive
direction to a second clamping position. The second rotary valve
verification check confirms a valve drop into an operational
configuration is successful. In a similar manner as the first
rotary valve verification check, the valve drive assembly uses the
interference sensor 2404 and end of the valve drive shaft 2405 to
verify the operational configuration. In one implementation, the
valve drive shaft will not trigger the interference sensor,
indicating a successful rotary valve drop and proceed onto
commanding the moving bracket assembly to a second clamping
position. When the interference sensor is triggered, the valve
drive assembly 2400 detects a failed valve drop and ejects the
cartridge due to an unusable rotary valve. After a successful valve
drop into operational configuration, the instrument proceeds to
clamp the cartridge to a second clamping position. When all
subsequent verification checks are performed and the cartridge is
rendered fluidically active, the valve drive assembly may begin the
valving sequence to direct the sample and reagents throughout the
cartridge to different processing modules. In one embodiment, the
valve drive assembly 2400 uses a sensor (i.e. homing sensor 2405)
to monitor the valve drive rotational position during rotation.
9. Pneumatic Interface (Clamp Block)
a) General Description
[0240] In one embodiment of the present invention, fluids (i.e., a
sample, reagents, air) are advanced through the cartridge using a
pneumatic source. A pneumatic interface 2100 is included within the
moving bracket assembly 2040 and is appreciated with respect to the
various views in FIGS. 14, 15A-B, 33, and 35-37C. The pneumatic
interface is configured to provide pressurized air from the
pneumatic subsystem 2130 to the cartridge to motivate fluids though
various locations of a cartridge for different sample processing
steps. Shown in FIGS. 14, 15A, and 15B, the pneumatic interface is
fixed to the clamp block 2041 such that movement of the pneumatic
interface is dictated by the movement of the moving bracket
assembly 2040. The pneumatic interface 2100 engages with the
cartridge to form a pneumatic seal when the moving bracket assembly
is moved in the positive direction to the second clamping position.
Plunger 2104 breaks the pneumatic interface perforations on the
cartridge label and the plunger surface grips the pneumatic
interface cover adaptor. Spring 2102 is urges plunger 2104 into the
pneumatic interface cover adaptor by pushing against shim 2105 and
housing 2106. FIG. 35 illustrates the pneumatic interface 2100
engaged with the cartridge pneumatic interface.
[0241] Referring to FIGS. 36A and 37A, the basic design employs a
spring 2102 loaded plunger 2101, with a plunger surface 2104, a
shim 2105, and housing 2106. In one implementation, the housing
2106 is fixed to the clamp block and includes plunger 2101 further
configured to be moveable within the inner surface of the housing
2108. In one embodiment, housing 2108 has a central opening wherein
the central opening has a smaller portion of the central opening
and a larger portion of the central opening. Plunger 2101 has a
long cylindrical shape and includes a proximal end with a plunger
surface 2104, a central portion housed within the smaller portion
of the central opening, and a distal end housed within the larger
portion of the central opening. Additionally, the plunger further
comprises an outer plunger surface 2107. The body of the plunger
can be made from any material with appropriate rigidity such as
plastics or metals, but is preferentially made from steel. The
central portion of the plunger is substantially equivalent in
diameter to the proximal end of the plunger, such that the central
portion and the proximal end of the plunger are both smaller in
diameter than the diameter of the distal end of the plunger. A
step-up feature 2109 links the central portion to the distal end of
the plunger. The central portion of the plunger is disposed in the
smaller portion of the housing central opening. Furthermore, the
distal end of the plunger disposed within the larger portion of the
housing central opening forms a gap between the outer surface of
the plunger 2107 and inner surface of the housing 2108. The space
formed between the outer surface of the plunger and inner surface
of the housing constrains the movement of the plunger for properly
engaging the pneumatic interface 2100 with the pneumatic interface
adaptor 1172 on a cartridge. The proximal end of the steel plunger
comprises a plunger surface 2104 that is responsible for gripping
the pneumatic interface adaptor to form a pneumatic seal. In one
embodiment the shape of the plunger surface 2104 is designed with
an angled surface, shown in FIG. 37A, to minimize potential
pneumatic leaks. In an alternative embodiment shown in FIG. 36A,
the plunger surface flat. Engagement with a pneumatic cover
interface 1172 on the cartridge is shown in FIGS. 36B, 36C, 37B and
37C and further discussed in greater detail with regard to a
gimbaling mechanism described below.
[0242] In one embodiment, the pneumatic interface includes a
gimbaling mechanism to account for any potential parallelism issues
arising during engagement between the moving bracket assembly 2040
and cartridge 1000. FIGS. 36B and 36C depict the gimbaling
mechanism of the pneumatic interface with a flat plunger surface
seen in FIG. 36A. FIG. 36B illustrates a pneumatic interface with
the gimbaling mechanism active when the pneumatic interface engages
with the pneumatic interface cover adaptor 1172. Housing 2106 is
fixed to the clamp block 2041, such that when plunger 2101 contacts
the pneumatic interface cover adaptor, the housing 2106 remains
stationary while plunger 2101 is pushed back into the housing
central opening to cause spring 2102 to compress between shim 2105
and housing 2106. The position of the plunger within the housing
central opening creates a gap between the plunger step-up feature
2109 and the inner surface of the housing 2108. In this
configuration, the plunger is permitted to pivot within the housing
central opening to ensure a secure pneumatic seal is established
when the plunger surface 2104 contacts the pneumatic interface
cover adaptor 1172. The degree of pivoting is constrained by the
inner surface of housing 2106, where the central portion of the
plunger, step up feature, and distal end of the plunger can pivot
until any one part of the plunger contacts the inner surface of the
housing.
[0243] FIG. 36C shows the pneumatic interface with a flat plunger
surface 2104 and the gimbaling mechanism locked when the moving
bracket assembly 2040 moves to unclamp a cartridge. As the moving
bracket assembly 2040 moves in the negative direction away from the
pneumatic interface cover adaptor 1172, housing 2106 retracts the
plunger 2101 from the pneumatic interface cover adaptor 1172. The
larger portion of the central opening contacts the corner of the
distal end of the plunger, adjacent to step-up feature 2109, to
pull the plunger back as the moving bracket assembly 2040 is moved
in the negative direction. The contact between the inner surface of
the housing 2108 and the corner of the distal end of the plunger
eliminates the gap seen in FIG. 36B when the gimbaling mechanism is
active. In this configuration, the plunger is prevented from
pivoting while the larger portion of the central opening remains in
contact with the corner of the distal end of the plunger. FIG. 37B
illustrates the pneumatic interface gimbaling mechanism active when
the pneumatic interface 2100 contacts the pneumatic interface cover
adaptor 1172 with an angled plunger surface 2104. FIG. 37C
illustrates the pneumatic interface with an angled surface when the
gimbaling mechanism is locked.
10. Thermal Clamp Assembly (Clamp Block)
[0244] The thermal clamp assembly 2680 is a component of the moving
bracket assembly 2040 and is connected to the clamp block 2041 (see
the various views of FIGS. 38-43). In some embodiments, the thermal
clamp assembly is configured to move independently of the clamp
block 2041 and is not fixedly attached to clamp block 2040, such
that the position of the thermal clamp assembly 2680 does not
solely depend on the position of clamp block 2041. The thermal
clamp assembly 2680 comprises a clamp plate 2681, a light frame
2686, and a plurality of clamp posts 2682, wherein each clamp post
2682 further comprises a shoulder screw 2684, spring 2683, and
bushing 2685. The thermal clamp assembly 2680 is configured to
presses against a cartridge 1000 to ensure the cartridge remains
flat against the fixed support bracket 2011 during the heat staking
process, as described herein, and additionally produces a light
seal around the reaction area 1600 of the cartridge during imaging
and detection by the reaction imaging assembly 2700. In
implementations where the thermal clamp assembly 2680 is configured
to move independently of the clamp block, the thermal clamp
assembly is connected to the clamp block using a bushing 2685 for
each of the plurality of clamp posts 2682, wherein the each bushing
is operably coupled to a shoulder screw 2684 thus permitting
independent movement of the thermal clamp assembly 2680 along
shoulder screws 2684. In one embodiment, each of the plurality of
clamp posts, 2682 comprises one or more springs 2683 along shoulder
screws 2684 for constraining the maximum movement of the clamp
block 2041, with respect to the thermal clamp assembly 2680, in the
positive and negative direction during clamping and unclamping.
Such configuration allows clamp block 2041 to move in the positive
direction toward the fixed bracket assembly until contacted by
spring 2683a and allows clamp block 2041 to move in the negative
direction until contacted by spring 2683b. In one implementation, a
clamp plate 2681 is fixed to the plurality of clamp posts 2682.
Furthermore, as shown by FIG. 38, a light frame 2686 is housed
within clamp plate 2681, wherein the light frame 2686 is configured
to contact the distal end of the cartridge during the clamping
sequence, as described herein below. The light frame 2686 is shaped
to correspond to a perimeter about the assay chambers within a
specific reaction module configuration of a diagnostic cartridge
embodiment.
[0245] The thermal clamp assembly 2680 is arranged between the
optical block 2710 and cartridge as seen in FIG. 42, wherein the
optical block is shown with dashed lines. As illustrated in FIGS.
38-41, the clamp plate 2681 resides in the space between the moving
block assembly 2040 and a cartridge in a loaded position defined by
loading assembly 2230. In one implementation illustrated in these
views, the light frame 2686 is disposed within a pocket 2710 formed
within optical block 2710. Additional views of the optical block is
further shown in FIGS. 45, 46, 62 and 66. The position of the light
frame within the optical block enables the thermal clamp assembly
2680 to form a light seal around reaction area 1600 of a cartridge,
viewed in FIG. 45. The light seal around the reaction area, as
provided by the light frame 2686, helps ensure the darkest possible
background is achieved for a reaction camera 2701 of a reaction
imaging assembly 2700 to capture fluorescent images of assay
chambers within the reaction area. When a cartridge is in a loaded
position, the movement of clamp plate 2681 is constrained between
the distal end of the cartridge and optical block 2710, such that
the thermal clamp is permitted from moving in the negative
direction until clamp block 2041 contacts spring 2685b and light
frame 2686 contacts the edge of pocket 2710 within the optical
block. By way of example, further description of the movement of
the thermal clamp assembly 2680 is depicted in a top down view of
the thermal clamp assembly 2680 and optical block 2710 according to
the clamping sequence in FIGS. 38-41.
[0246] FIG. 38 illustrates the thermal clamp assembly 2680 when the
moving bracket assembly 2040 is in the zero clamping position.
Cartridge 1000 is in a loaded position given by loading assembly
2230 and latched by the latch and pin assembly 2210. Note light
frame 2686 is not in contact with cartridge 1000.
[0247] The moving bracket assembly 2040 is moved in the positive
direction from the zero clamping position to a first clamping
position when linear actuator 2014 rotates lead screw 2016 in a
first rotational direction. The clamp block 2041 slides along
shoulder screws 2684 and causes light frame 2686 to contact the
cartridge. However, a seal between the light frame 2686 and the
reaction area 1600 is not established in the first position. As
described herein, the first clamping position only establishes an
operational coupling between the valve drive assembly 2400 and
cartridge rotary valve 1400. FIG. 39 illustrates the thermal clamp
assembly 2680 after the moving bracket assembly 2040 is in the
first clamping position with light frame 2686 lightly contacting
the distal end of the cartridge. In the first clamping position,
the movement of the moving bracket assembly 2040 and thermal clamp
assembly 2680 causes light frame 2686 to move in the positive
direction toward the cartridge and away from pocket 2711 of optical
block 2710.
[0248] After rotary valve verification checks are performed on the
rotary valve in the first clamping position, the moving bracket
assembly 2040 is moved in the positive direction to a second
clamping position when linear actuator 2014 rotates lead screw 2016
in a first rotational direction. Clamp block 2041 slides along
shoulder screws 2684 until the clamp block 2041 compresses spring
2683a to exert a force against the clamp plate 2681. Accordingly,
the clamp plate 2681 urges the light frame 2686 into the cartridge
and establishes a light seal around the cartridge imaging area
1600. FIG. 40 illustrates the thermal clamp assembly 2680 after the
moving bracket assembly 2040 is in the second clamping position to
clamp the cartridge. In the second position, the thermal clamp
assembly is prevented from moving any further in the positive
direction, such that when the frangible seal block 2260 is moved to
the third clamping position to actuate frangible seals the position
of the thermal clamp assembly 2680 remains unchanged due to light
frame 2686 contacting the cartridge and clamp block 2041 contacting
spring 2683a. The thermal clamp assembly 2680 will remain in the
second clamping position until a diagnostic test run is completed
on the cartridge.
[0249] The moving bracket assembly 2040 is moved in the negative
direction to a fourth clamping position when linear actuator 2014
rotates lead screw 2016 in a second rotational direction to unclamp
the cartridge. Clamp block 2041 is driven away from the cartridge
by the coupling between the lead screw 2016 and frangible seal
block 2260 contacting ledge 2046 of the clamp block, as described
herein. This action causes clamp block 2041 to slide along shoulder
screws 2684 until the clamp block 2041 contacts spring 2683b.
Pocket 2711 formed within optical block 2710 of the reaction
imaging assembly 2700 allows the light frame 2686 of the thermal
clamp assembly to retract away from the cartridge to establish a
clearance between the clamp plate 2681 and cartridge for ejection.
FIG. 41 illustrates the thermal clamp assembly 2680 after the
moving bracket assembly 2040 is in a fourth clamping position.
11. Magnetic Mixing (Fixed Support Bracket & Clamp Block)
a) Magnetic Mixing Assembly
[0250] The clamping subsystem supports two magnetic mixing systems
that interface with elements within a cartridge to perform
respective functions. The first magnetic mixing system of
instrument 2000 is magnetic mixing assembly 2300, illustrated in an
exploded view in FIG. 47A and a perspective assembly view in FIG.
47B. The various views illustrate the arrangement, spacing,
orientation and operation of the magnetic mixing assembly for use
with various vertically oriented diagnostic cartridge and
instrument embodiments described herein. Magnetic mixing assembly
2300 provides the means to mix a sample in a vertically oriented
lysis chamber using a stir bar alone or in combination with other
lysis agents, while minimizing the amount of contact of the stir
bar with the walls of said vertical lysis chamber. The driving
magnet system 2310 and driven magnet system 2350, as seen in FIGS.
47A, 47B, are arranged to effectuate a magnetic coupling between
the one or more driving magnets and the one or more driven
magnets.
[0251] Specifically, each driving magnet and driven magnet are
arranged with respect to one another such that an alignment of the
driving magnet magnetic axis and an alignment of the driven magnet
magnetic axis effectuate a magnetic coupling between the driving
magnetic and the driven magnet. Still further, the arrangement and
operation of the magnetic mixing assembly is adapted for rotation
of a stir bar within the magnetic field produced between the
driving and driven magnets. In certain embodiments, to effectuate
magnetic coupling between a driving magnet and driven magnet, the
driven magnet magnetic axis is parallel to the driving magnet
magnetic axis. In further, preferred embodiments, the driven magnet
magnetic axis is substantially collinear with the corresponding
driving magnet magnetic axis. As used herein, "substantially
collinear" encompasses deviations from absolute collinearity of up
to 10.degree. and/or 3 mm at a plane bisecting the gap between the
driving and driven magnet system.
[0252] The magnetic coupling between the driving magnet system and
the driven magnet system comprises an attractive magnetic coupling.
In such embodiments, the one or more driving magnets and the one or
more driven magnets are arranged with respect to one another such
that the alignment of the each driving magnet magnetic axis and the
alignment of the each driven magnet magnetic axis effectuate an
attractive magnetic coupling between the one or more driving
magnets and the one or more driven magnets. In general, to
effectuate an attractive magnetic coupling between a driving magnet
and a driven magnet, the driving magnet magnetic axis and the
driven magnet magnetic axis are aligned such that opposing poles of
the driving magnet magnetic axis and the driven magnet magnetic
axis are located in proximity to one another.
[0253] In certain embodiments, a strength of the magnetic coupling
between the driving magnet system and driven magnet system is based
on a distance of the gap located between the one or more driving
magnets and the corresponding one or more driven magnets.
Additionally, the magnetic coupling is based on a magnet strength
of the one or more driving magnets, as well as a magnet strength of
the one or more driven magnets. In some embodiments, the gap
separating the driving magnet system from a driven magnet system is
between about 10 mm and about 30 mm. Furthermore, in a preferred
embodiment, the magnetic strength of one or more driving magnets is
the same as the strength of the one or more driven magnets.
[0254] FIG. 47A is an illustration of an exploded view of the
magnetic mixing assembly 2300 of instrument 2000, in accordance
with an embodiment. The exemplary mixing assembly shows a driving
magnet system 2310 comprising a first driving magnet 2311 and a
second driving magnet 2316 separated by a distance, and a driven
magnet system 2350 comprising a first driven magnet 2353 and a
second driven magnet 2356 separated by a distance. As shown in both
FIGS. 47A, 47B, the drive motor is operably/mechanically coupled to
the drive belt 2332, wherein the drive belt is
operably/mechanically coupled to the driving magnet spindle 2361,
and further wherein the spindle is operably coupled to the driving
magnet holder 2325. In some embodiments, the driving magnet holder
is configured to house one or more driving magnets. The illustrated
embodiment shows the driving magnet holder contains the first
driving magnet 2311 and a second driving magnet 2316. In a
preferred embodiment, the driving magnet holder 2325 is positioned
in proximity and aligned to a first face, e.g. fluidic side 1006,
of a cartridge containing a vertically oriented lysis chamber
1371.
[0255] Analogous to the driving magnet system is a driven magnet
system 2350. In some embodiments, the driven magnet system
comprises at least one driven magnet, a driven magnet holder, and a
driven magnet spindle. In some embodiments, the driven magnet
holder is configured to house one or more driven magnets. FIGS. 47A
and 47B show an embodiment where the driven magnet holder 2365
contains the first driven magnet 2351 and the second driven magnet
2356, further wherein the driven magnet holder is operably coupled
to a driven magnet spindle 2361. In a preferred embodiment, the
driven magnet holder 2365 is similarly positioned in proximity and
aligned to a second face, e.g. feature side 1007, of a cartridge
containing a vertically oriented lysis chamber 1371. FIGS. 12 and
13 illustrate the complementary arrangement of the driving magnet
system 2310 and driven magnet system 2350 with respect to a
vertically oriented cartridge containing a vertically oriented
lysis chamber position therebetween.
[0256] As further detailed in the sections below, in embodiments
where a magnetic stir bar is provided within the lysis chamber, the
operation of the magnetic mixing assembly induces a magnetic field
to rotate the stir bar substantially within the vertical plane of
the diagnostic cartridge, i.e. rotating within a plane collinear
with the cartridge width axis 1025, when clamped in an operational
orientation within the instrument. Further, in certain
implementations, a first driving magnet field focuser 2312 can be
coupled to the first driving magnet 2311 and/or a first driven
magnet field focuser 2352 can be coupled to the first driven magnet
2351 to concentrate magnetic fields generated toward the center of
the vertically oriented lysis chamber.
[0257] In certain embodiments, the magnetic mixing assembly can
further comprise an acoustic mechanism for detecting magnetic
decoupling of the stir bar 1390 from one or more of the driving
magnet system 2310 and the driven magnet system 2350. In such
embodiments, the acoustic mechanism is configured to detect a
change in one or more of an amplitude and a frequency of vibrations
produced by the stir bar during rotation of the driving magnet
system, the change indicating the magnetic decoupling of the stir
bar. In some embodiments, the change comprises a sudden decrease in
one or more of the amplitude and the frequency of the vibrations
produced by the stir bar. In some embodiments, the acoustic
mechanism comprises a microphone 2380 (see FIG. 11).
b) Rehydration
[0258] The second magnetic mixing system supported by the clamping
subsystem is the mechanism for rehydrating dried reagents contained
within a cartridge. In one implementation, motor 2500 contains a
magnet to gyrate a magnetic element contained within a reservoir of
a cartridge. The motor is mounted to the fixed support bracket and
is best seen in the views of FIGS. 10 and 11. In one embodiment,
the cartridge reservoir containing a magnetic element holds dried
reagents, such that gyration of the magnetic element facilitates
rehydration and mixing of dried reagents with fluids.
C. Pneumatic Subsystem
1. Overview
[0259] In one embodiment, the instrument includes a pneumatic
subsystem that is configured to generate pneumatic pressure to
advance fluids to various locations within the cartridge that are
responsible for sample preparation, nucleic acid amplification, and
detection. FIGS. 48 and 49 illustrate a pneumatic subsystem 2130 in
isolation and in position within the instrument enclosure,
respectfully. The pneumatic subsystem comprises at least a pump
2131, a pressure regulator 2132, a proportional valve 2133, an
accumulator 2135, and a pressure sensor 2134. In some
implementations, the pneumatic subsystem includes an output
selector valve 2136. The pneumatic pump compresses air to convey
fluids through the cartridge, wherein pump 2131 is connected to
pressure regulator 2132 to down regulate the pressure to a desired
value. An accumulator 2135, in line with a proportional valve 2133,
acts as a pressure storage reservoir until pressure is needed on
demand.
[0260] In one aspect, the pneumatic subsystem includes
environmental sensors and additional hardware contained within the
instrument to monitor various instrument measurements including
internal temperature, atmospheric pressure, and humidity of the
instrument. As described herein, the firmware of the pneumatic
subsystem allows the instrument to control the time spent varying
increasing or decreasing pressure set points and control steady
state pressure with varying flow resistances from the cartridge. In
some embodiments, the pneumatic subsystem includes a flow sensor to
monitor the flow rates of various fluids within the cartridge for
sample preparation and amplification. In a preferred embodiment,
the pneumatic subsystem contains no flow sensors to monitor flow
rates of fluids in the cartridge. In a further preferred
embodiment, indirect measurements are used to determine when the
pneumatic subsystem completes the act of pushing a fluid or
substance through the porous solid support chamber prior to moving
on to the next processing step. A feedback control system uses a
pressure feedback sensor 2134 and a proportional valve 2133 to push
finite amounts of fluid through the porous solid support of the
cartridge and indicate when all the fluid has exited the channel.
The feedback system, as described herein, replaces a flow sensor by
using an actuation signal to indicate when the system is ready for
the next fluid sequence.
[0261] In one embodiment, the pneumatic subsystem provides
pressurized atmospheric air to the cartridge via the pneumatic
interface 2100 shown in FIGS. 35, 36A-36C and 37A-37C. The
pneumatic interface 2100 punctures a perforated area 1052 on the
label of the cartridge to access the cartridge pneumatic interface
1170 located on the cartridge. As described herein, spring 2102
establishes a connection between the cartridge pneumatic interface
and pneumatic interface 2100 to deliver the pressurized atmospheric
air. In yet another aspect of the invention as described herein, a
gimbaling mechanism is used to account for small degrees of
misalignment between the cartridge and instrument. FIG. 49 is a
perspective view of the clamping subsystem and optical system
engaged with a cartridge in a loaded position with the valve drive
assembly 2400 removed from the moving block assembly 2040 to
demonstrate the connection between the pneumatic subsystem 2130 and
the pneumatic interface 2100. In this view, the instrument
pneumatic interface 2100 is shown connected to the pneumatic
subsystem via tubing 2190. Furthermore, FIG. 49 demonstrates the
relationship of the pneumatic subsystem position in reference to
the clamping subsystem and the reaction imaging assembly and label
imaging assembly of the instrument optical subsystem. In one
implementation, the pneumatic subsystem is fixed to the bottom of
the instrument, unlike all other subsystems and assemblies which
are fixed to either the fixed bracket assembly 2010 or the moving
bracket assembly 2040. As a result, the pneumatic subsystem remains
stationary during the clamping and unclamping sequence in a similar
manner to the fixed bracket assembly.
[0262] In one implementation, each pneumatic pressure control
component or aspects thereof, such as the pump 2131, pressure
regulator 2132, proportional valve 2133, accumulator 2135, output
selector valve 2136, and various sensors are mounted in the
manifold block 2137. In a further implementation, a control board
2138 contains the proportional valve 2133, pressure sensor 2134,
and various environmental sensors, wherein the control board 2138
is mounted within manifold block 2137, as shown in FIG. 48. The
manifold block can, in various aspects, be made of one or more
rigid materials, such as a polymeric material, like plastic. In
some implementations, the manifold block is machined from acrylic.
In a further embodiment, the acrylic manifold block is vapor
polished. In one aspect, pneumatic routing channels and mounting
ports are fabricated in the manifold block for all components of
the pneumatic subsystem. Additionally, due to the thermodynamics of
the compression of air in the pump, humidity in the air can be
condensed. In one embodiment, the instrument manages condensation
control with the regulator's manifold entry geometry.
Advantageously, the implemented geometry vents
moisture/condensation within the instrument enclosure through the
use of one or more bleed orifices 2191 so that it does not enter
the regulator inlet.
[0263] Given the pressurization of the pneumatic subsystem, in one
implementation filters are installed on the pump's intake, inlet,
and outlet to eliminate the possibility of external particulates
from reaching the manifold or cartridge to control the risk of
contamination within the instrument. In an exemplified
implementation, the pneumatic subsystem is shown in FIG. 48
comprising a pump filter 2160 and outlet filter 2162.
[0264] In some embodiments, the pneumatic subsystem optionally
comprises several components to minimize noise due to vibration. In
one implementation, the assembly uses pump isolation mounts. In
another implementation the assembly includes silicone foam damping
pads reduce noise of pump components vibrating against manifold. In
an alternate implementation, the assembly uses isolation grommets
2194 to reduce the vibration of the pneumatic subsystem against the
instrument's enclosure. In a preferred embodiment, the pneumatic
subsystem uses pump isolation mounts, silicone foam damping pads,
and isolation grommets to provide noise damping.
D. Thermal Subsystem
[0265] In one aspect, a cartridge configured to perform sample
preparation or both sample preparation and amplification requires
the use of one or more heaters supported by the instrument 2000. In
one implementation, the thermal subsystem is configured to provide
a controlled steady state temperature to areas of the cartridge
used to conduct sample preparation and enable controlled heating
and cooling of assay chambers to permit isothermal amplification
and detection of target nucleic acids during a diagnostic test. In
implementations where amplification is performed, avoiding
cross-contamination between assay chambers as well as isolating the
reaction from the outside environment is imperative to prevent
amplicon contamination. Containing amplified nucleic acids ensures
false positive results are not obtained on all cartridge runs
performed on the instrument thereafter. The diagnostic instrument
includes one or more mechanisms to provide the desired containment
by either temporary or permanent isolation between one or more
components, modules or chambers within a cartridge. Temporary
isolation refers to a mode of isolation present in the cartridge so
long as the cartridge is clamped within the instrument. Once,
unclamped and ejected from the instrument, isolation is not
maintained. Examples of temporary isolation include the use of
pneumatic pressure, or a mechanical system such as one or a number
of pinch valves or non-heated stakes to occlude one or more
passages or channels of a cartridge. In contrast, permanent
isolation refers to a mode of isolation that once formed is present
in the cartridge even after the cartridge is ejected from the
instrument. Permanent isolation includes any suitable form of
modification to produce a suitable fluid tight constriction or
occlusion, region of plastic deformation, or sealing between one or
more components, modules or chambers within a cartridge.
[0266] In one specific implementation, the thermal subsystem
further includes a heat staker assembly 2640 used as an isolation
mechanism for permanent isolation by sealing a portion of a
cartridge containing amplified nucleic acids. Additionally, the
thermal subsystem includes a cartridge heater assembly 2550 and a
chemistry heater assembly 2600 for providing the thermal
requirements of sample preparation and amplification when
performing a diagnostic test. The various views of the components
of the thermal subsystem are provided in FIGS. 50-58. The various
components of the thermal subsystem operate under the control of
the instrument computer control system as described in FIGS.
67A-671. The thermal subsystem, as described herein, contains
various embodiments used to precisely control the temperature of
specific areas of the cartridge to prepare a sample and, if
desired, amplify and detect target nucleic acids and prevent
amplicon from escaping the cartridge.
1. Overview
[0267] A thermal subsystem of the present invention comprises a
chemistry heater assembly 2600, a cartridge heater assembly 2550,
and a heat staker assembly 2640, wherein the heat staker assembly
further comprises a staker bar assembly 2641. All assemblies and
components of the thermal subsystem are supported by the fixed
bracket assembly 2010. In one implementation, more than one heater
e.g., two or more, heaters are used to provide multiple controlled
temperatures to different areas of the cartridge responsible for
conducting sample preparation and amplification. In one embodiment,
the cartridge heater assembly 2550 is configured to maintain an
operational temperature within a cartridge heating zone 2552 which
include portions of the integrated cartridge containing the wash
buffer reservoir 1475, elution buffer reservoir 1500, rehydration
chamber 1520, and lysis chamber 1371. In another embodiment, the
chemistry heater 2601 is configured to maintain a reaction
temperature to the reaction area 1600 of a cartridge to enable the
amplification of target nucleic acids within assay chambers. In yet
another embodiment, a third heater is used to seal a cartridge
according to an embodiment described herein.
2. Chemistry Heater Assembly
[0268] In one embodiment, a chemistry heater assembly 2600 is
configured to provide a reaction temperature for amplifying nucleic
acids contained within a plurality of assay chambers in a
cartridge. A cross sectional view of the chemistry heater assembly
2600 is illustrated in FIG. 54 and an exploded view of the
chemistry heater assembly 2600 is seen in FIG. 55. In one
embodiment, the chemistry heater assembly 2600 comprises a
chemistry heater 2601, a flow guide frame 2606, a chemistry heater
plate 2602, a chemistry heater fan 2603, a heater plenum 2607, and
a fan plenum 2604 with a flow vane 2605. The chemistry heater 2601
can be of any suitable design but is most preferably a resistance
heater (e.g. a Kapton heater). In certain aspects of the invention,
the chemistry heater assembly further consists a thermistor
integrated with the chemistry heater 2601.
[0269] In one embodiment, shown in FIGS. 54 and 55, the chemistry
heater 2601 is in thermal contact and bonded to a second surface
2622 of chemistry heater plate 2602 using a pressure sensitive
adhesive or other adhesive appropriate to the operating temperature
range. When assembled, there is a reaction well zone 2620 formed in
a first surface of the chemistry heater plate 2621, wherein the
first surface of the chemistry heater plate is in thermal contact
with the film side of a cartridge. The chemistry heater zone 2620
is viewed from the first surface of the chemistry heater plate 2621
in FIGS. 50 and 51. The cartridge heater plate 2602 is susceptible
to thermal effects from the ambient environment. Thus, in one
embodiment, the chemistry heater assembly addresses the thermal
effects on the thermal gradient of the chemistry heater plate by
bonding chemistry heater plate 2602 to a flow guide frame 2606. In
a further embodiment, the flow guide frame 2606 is flush with the
second surface of the fixed support bracket 2013. In another
implementation the chemistry heater plate 2602 is bonded to the
flow guide frame 2606 to ensure proper thermal contact is
maintained between the first surface of the chemistry heater plate
2621 and the film side of the cartridge regardless of mechanical
tolerances.
[0270] In some implementations, a chemistry heater fan 2603 is
fluidically coupled to a fan plenum 2604 with flow vane 2605 and a
heater plenum 2607 to direct cooled air through a cutout disposed
within the flow guide frame 2606 and directly over chemistry heater
2601. As shown in FIG. 54, arrows demonstrate the flow path of air
from the chemistry heater fan 2603 and through the opening formed
within the flow guide frame 2606 and heater plenum 2607. This
configuration is advantageous when optionally thermally fluctuating
the chemistry heater between two or more temperatures rapidly prior
to setting the chemistry heater to a reaction temperature. As
described according to an embodiment herein, thermally fluctuating
the chemistry heater generates convection of the fluids within the
assay chambers. Specifically, the convection generated within a
plurality of assay chambers facilitates mixing of a sample with
dried reagents within the assay chambers prior to beginning
amplification. Chemistry heater fan 2603, fan plenum 2604, flow
vane 2605, and heater plenum 2607 are fluidically coupled to
facilitate a faster cooling ramp rate of the chemistry heater 2601
to a low temperature during the sequence of thermal fluctuations.
In one implementation, the chemistry heater fan 2603 may be turned
off after the sequence of thermally fluctuations and remains off
for the remainder of the diagnostic test while the chemistry heater
is set to the reaction temperature.
[0271] In various aspects, a flow guide frame 2606 is composed,
e.g., entirely composed, of one or more polymeric materials (e.g.,
materials having one or more polymers including, for example,
plastic). A flow guide frame 2606 can be composed of any of the
elastic materials provided herein. Materials of interest for the
flow guide frame include, but are not limited to, polymeric
materials, e.g., plastics. In a preferred embodiment, the flow
guide frame is polyether ether ketone (PEEK).
[0272] Thermal boundary conditions affect the temperature gradient
of the chemistry heater plate 2601 in contact with the film side of
a cartridge and can result in undesired temperature variation
across assay chambers. Uniformity among assay chambers is critical
to amplifying nucleic acids for accurate detection. According to
various embodiments, the chemistry heater assembly 2600 includes a
chemistry heater plate 2602 comprising a machine pocket geometry
for thermal gradient reduction.
[0273] Returning to FIGS. 50 and 51, the chemistry heater plate
2601 is viewed from a first surface 2621. In one embodiment, the
reaction well zone includes a machined pocket geometry 2623
comprising grooves 2624. The machined pocket geometry 2623 is a
series of grooves arranged in a pattern to decrease heat flux
through the center of the reaction well zone 2620 to compensate for
edge heat loss due to the environment. The configuration, as
described herein, provides precise isothermal control of the
reaction well zone 2620 of the chemistry heater plate 2602 to
supply a uniform temperature to the plurality of assay chambers
conducing amplification.
[0274] The term "grooves," is used herein, refer to any hole,
cutout, orifice, aperture, gap, or space machined into the
chemistry heater plate 2602 to reduce heat flux variation between
the chemistry heater 2601 and chemistry heater plate 2602 to supply
a consistent temperature to the reaction well zone for
amplification. In some embodiments, grooves extend entirely through
the chemistry heater plate from a first surface of the chemistry
heater plate 2621 to a second surface 2622. In other
implementations, grooves extend partially at a depth measured from
the first surface of the chemistry heater plate. Examples of
geometries for cutouts include, but are not limited to, circles,
rectangles, rounded rectangles, ovals, ellipses, or any
combinations thereof.
3. Cartridge Heater Assembly
[0275] In one embodiment, a cartridge heater assembly 2550 provides
controlled heating of sample preparation areas of the cartridge,
i.e., a cartridge heater zone. FIG. 50 provides a perspective view
of cartridge heater zone 2552 from the first surface of the fixed
support bracket 2012, wherein the first surface of the fixed
support bracket is in contact with the film side of the cartridge.
In one embodiment, the cartridge heater zone 2552 is in thermal
contact with a wash buffer reservoir 1475, elution buffer reservoir
1500, rehydration chamber 1520, and lysis chamber 1371 housed
within an integrated diagnostic cartridge to provide a controlled
steady state temperature to areas of the cartridge performing
sample preparation. In one aspect, the cartridge heater assembly
comprises a cartridge heater 2551 and insulator 2553. FIG. 52
illustrates the cartridge heater assembly and FIG. 53 illustrates
the cartridge heater assembly in an exploded view. The cartridge
heater 2551 can be of any suitable design but is most preferably a
resistance heater (e.g. a Kapton heater). In one embodiment, the
cartridge heater 2551 is in thermal contact and bonded to a second
surface of the fixed support bracket 2013 with a pressure sensitive
adhesive. The thermal contact between cartridge heater 2551 and the
second surface of the fixed support bracket forms the cartridge
heater zone 2552 in the first surface of the fixed support bracket
2012 viewed in FIG. 50. In another embodiment, an insulator 2553 is
in thermal contact with the cartridge heater 2551 to prevent
thermal energy from escaping into the ambient environment.
[0276] Thermal boundary conditions affect the uniformity of heat
transfer between the cartridge heater assembly zone 2552 and areas
of the cartridge housing the wash buffer reservoir 1475, elution
buffer reservoir 1500, rehydration chamber 1520, and lysis chamber
1371. In one implementation, as shown by FIG. 50, the cartridge
heater assembly 2550 further comprises a series of cutouts 2554
around the perimeter of cartridge heater zone 2552 for thermal
gradient reduction to control heat loss.
[0277] The term "cutouts," as used herein, refer to any hole,
groove, orifice, aperture, gap, or space machined into the fixed
support bracket 2011 to reduce heat flux variation between the
cartridge heater 2551 and the cartridge heater zone 2552 to provide
a consistent temperature to areas of the cartridge responsible for
sample preparation. In some implementations, cutouts may extend
through from the first surface of the fixed support bracket 2012 to
the second surface of the fixed support bracket 2013. In other
implementations, the cutouts may partially extend a depth measured
from the first surface of the fixed support bracket 2012. Examples
of geometries for cutouts include, but are not limited to, circles,
rectangles, rounded rectangles, ovals, ellipses, or any
combinations thereof.
[0278] In a further embodiment, the cartridge heater zone may
comprise a plurality of perforations 2377. In embodiments of the
magnetic mixing assembly of the instrument, the driving magnet
system 2310 and driven magnet system 2350 rotate in a circular
pattern. As such, eddy currents are induced in the fixed support
bracket 2012 radially from a center of the circular pattern. To
limit the induction of eddy currents in the fixed support bracket,
the plurality of perforations can be arranged in a concentric
pattern around the center of the circular pattern of the magnetic
mixing assembly 2300, as described herein. This concentric
arrangement of the plurality of perforations causes the eddy
currents induced in the fixed support bracket to follow a
convoluted path along their radial induction pathways. This
convoluted pathway limits the formation of the eddy currents in the
fixed support bracket.
4. Heat Staker Assembly
[0279] The high sensitivity of nucleic acid amplification methods,
in particular isothermal amplification methods, pose the threat of
amplicon contamination. A cartridge unable to successfully contain
amplified nucleic acids may cause cross contamination between assay
chambers or in the case of leakage may contaminate the instrument.
Cross contamination between assay chambers produces erroneous
results in the cartridges while cartridge leakage within the
instrument will lead to subsequent false positive results on all
cartridges run thereafter. The instrument's thermal subsystem
provides a heat staker assembly 2640 as appreciated with reference
to the various views of FIGS. 51, 52, 55, 56, 57A, 57B and 58. The
operation of the heat staker assembly forms a heat stake across a
number of individual fluid channels on the integrated diagnostic
cartridge to seal off the channels one from another and prevent
sample contamination. Advantageously, the heat stake is performed
under sufficient pressure across the main loading channel leading
to the assay chambers to prevent amplified nucleic acids from
escaping the cartridge and mitigate the risk of amplicon
contamination. In addition, the heat staker assembly is configured
to heat stake the channel leading to and exiting from the waste
collection element to stop fluids from exiting the waste chamber
when the cartridge is removed from the instrument. In a preferred
embodiment, the integrated diagnostic cartridge has a portion of
the fluid channels arranged with planar portions intended to
support this integrated heat stake operation. Still further, the
fluid pathways of the integrated diagnostic cartridge are arranged
so that they are adjacent in a spacing which permits the use of a
single linear heat stake element.
[0280] The term "heat stake," as used herein, refers to an
exemplary permanent isolation technique for performing the process
of melting and rapidly cooling a portion of the cartridge to form a
seal and prevent fluids from leaving the cartridge. In
implementations where the cartridge comprises one or more polymeric
films, the heat staker assembly 2640 provides the means to melt and
fuse the stack of polymeric films attached to the fluidics side of
the cartridge, wherein melting the one or more films into the
cartridge forms a barrier across selected fluid channels to retain
liquids within. The term "heat stake" may also refer to the seal or
barrier formed as a result of the heat staking process. According
to an embodiment described herein, the one or more thermoplastic
films may be placed on the fluidic card or used as part of the
cartridge as part of a heat stake compatible design. In one
specific embodiment, there are two thermoplastic films used each
having a different melting temperature wherein the first film has a
substantially similar melting temperature as the cartridge and the
second film has a higher melting temperature than the first film
such that only the second film will melt during a heat stake
operation to form the barrier. The two thermoplastic film approach
described herein has the added benefit of protecting other
components or the integrity of the fluidic card or cartridge during
heat staking.
[0281] As previously described herein, the fixed bracket assembly
2010 is configured to support the thermal subsystem responsible for
generating the thermal requirements for executing a molecular
diagnostic test. In one embodiment, the thermal subsystem comprises
a heat staker assembly 2640 for providing one means of permanently
sealing each one of the cartridge assay chambers. In such
implementation, the fixed support bracket 2011 may contain contains
a channel 2020 integrally formed therein to accommodate such heat
staker assembly. Channel 2020 permits a staker bar assembly to
directly contact the cartridge to perform the sealing action and is
most readily apparent in FIG. 51. In FIG. 56 depicts a heat staker
assembly 2640 comprises a linear actuation motor 2642, a spring
2643, a staker bar assembly 2641, heat staker fan 2644, and an
inductive linear sensor 2645. In one embodiment, a spring 2643
provides the force needed to perform heat staking. As used herein,
the linear actuation motor is configured to move the heat staker
bar assembly 2641 to make contact with the film side of a
cartridge. The linear actuator motor does not, however, provide the
force or depth control necessary for heat staking. The linear
actuation motor 2642 releases spring 2643, which supplies the force
necessary to heat stake, to push the staker bar assembly 2641 into
the film side of the cartridge. In one implementation, an inductive
linear sensor 2645 enables the measurement of linear displacement
heat staker assembly 2640 and provides a means for heat staking
error detection.
[0282] FIGS. 57A and 57B provide perspective and cross section
views of the staker bar assembly 2641. As shown in FIGS. 57A and
57B the staker bar assembly 2461 comprises a heater 2661, a staker
blade 2660, and a depth stop 2662. The heater can be of any
suitable design but is most preferably a resistance heater (e.g. a
wire heater) and is in thermal contact with the staker blade 2660.
In one implementation, the staker blade 2660 has a draft angle to
form the heat stake when the blade contacts the polymeric films of
the cartridge without tearing. In a further implementation, the
draft angle of the staker blade is surrounded by a depth stop 2662
to control the depth of the heat stake to a desired displacement
range. Linear actuation motor 2642 moves the staker bar assembly
2641 to the cartridge and then releases spring 2643 to apply the
force needed to press the heated staker blade 2660 into the film
side of the cartridge. The staker blade is permitted to melt into
the cartridge until depth stop 2662 contacts the cartridge, thus
preventing the staker blade from traveling further.
[0283] In various aspects, a depth stop is composed, e.g., entirely
composed, of one or more polymeric materials (e.g., materials
having one or more polymers including, for example, plastic). The
polymeric depth stop can be composed of any of the elastic
materials provided herein. Materials of interest include, but are
not limited to, polymeric materials, e.g., plastics. In a preferred
embodiment, the depth stop is polyether ether ketone (PEEK) suited
to the operational temperature range of the heat stake
assembly.
[0284] According to the subject embodiments, the staker blade can
be composed of a variety of materials and can be composed of the
same or different materials. Materials that the staker blade
described herein can be composed of include, but are not limited
to, metals, such as aluminum. In a preferred embodiment the staker
blade is aluminum.
E. Optical Subsystem
[0285] Instrument 2000 includes an optical subsystem comprising two
assemblies which separately interact with cartridge 1000. FIGS. 58,
59, 60 and 61 provide various views of a label imaging assembly
2770. The label imaging subsystem illuminates and captures an image
of the cartridge label area. The label imaging assembly may further
be configured to illuminate and capture a series of images of the
loading module to aid in monitoring and verification that an
adequate sample is loaded into the cartridge prior to running a
diagnostic test. The reaction imaging assembly 2700 is illustrated
in the various views of FIGS. 62-66. In implementations where one
or more assay chambers are configured to produce a fluorescent
signal indicative of the presence of a target pathogen, the
reaction imaging assembly 2700 provides excitation wavelength
illumination to the cartridge reaction area 1600 and captures
images of fluorescence resulting from the amplification of target
nucleic acids. Both optical assemblies are supported by the fixed
support bracket and remain stationary during the clamping and
unclamping of a cartridge.
1. Label Imaging Assembly
[0286] Label imaging assembly 2770 is configured to illuminate and
capture images of the patient label and loading module. As shown in
FIGS. 58 and 59, the label imaging assembly is mounted to the
antenna ground plate 2810 and comprises a camera 2771, LED 2772,
aperture 2773 and diffuser 2774. The label imaging assembly 2770
will include at least one, but preferably more than one (e.g., two
or three), LEDs 2772 for illuminating the patient label area 1040
and the loading module while minimizing shadows cast in the patient
label area. The aperture 2773 defines an opening to transmit and
reshape illumination by LEDs to reduce off axis light and stray
light from affecting the patient label image quality. Once
illumination from the LEDs passes through each respective aperture
2773, light travels through diffuser 2774 which generates a more
uniform illumination intensity on the patient label and loading
module. In one implementation, seen in FIGS. 60 and 61, the LEDs
may be arranged in an oblique configuration to illuminate the
patient label. This arrangement can be advantageous for increasing
the contrast of images and improving the overall image quality of
the cartridge.
[0287] In a preferred implementation, the label imaging assembly
2770 is further configured to image the sample port assembly 1100
to verify adequate sample is loaded into a cartridge prior to
running a diagnostic test. Given the low concentrations of target
pathogens in some samples, it is advantageous to determine a
sufficient sample volume is present in the loading module. In a
preferred implementation, the label imaging assembly is configured
to capture an image of the sample port assembly 1100 and detect a
mechanism (e.g., a ball disposed within the loading module) to
determine the sample volume. Alternatively, the label imaging
assembly may detect the meniscus of the sample fluid. Furthermore,
the label imaging assembly may be configured to read the sample
volume through a sample window 1050 provided by a cut out in the
cartridge label.
2. Reaction Imaging Assembly
[0288] In some implementations of the present invention, a visual
signal, e.g., fluorescent signal, is used to indicate the presence
of nucleic acids of a target pathogen within a sample.
Specifically, a plurality of target nucleic acids may be detected
using one or more distinct excitation and emission wavelengths. A
wide variety of fluorophores, with varying emission spectra, are
known in the art and one of ordinary skill would be able to select
an appropriate fluorophore for a given assay performance. A
reaction imaging assembly 2100 permits the instrument 2000 to
simultaneously detect nucleic acids from one or more target
pathogens. The reaction imaging assembly 2100 may be configured to
provide excitation wavelengths to excite one or more fluorophores.
Additionally, various elements to filter and capture emitted
wavelengths are described herein to determine the presence or
absence of target nucleic acids. The arrangement and operation of
the reaction imaging assembly are shown in FIGS. 62-66.
[0289] In a preferred implementation, the reaction imaging assembly
2700 shown in FIG. 62 is designed with an epifluorescence
arrangement, such that illumination and emitted wavelengths travel
through the same objective lens. Unlike oblique illumination, the
epifluorescence arrangement illuminates uniformly within the plug
structures, described herein with regard to a cartridge
amplification module, of the assay chambers to minimize or prevent
shadows. Shadows casted on assay chambers hinder the likelihood of
detecting a positive sample for an infectious disease. In one
implementation, the reaction imaging assembly 2700 comprises a
camera 2701, dichroic beam splitter 2702, excitation lens cell
2730, emission lens cell 2750, objective lens 2706, and a fold
mirror 2704. In a preferred embodiment, all components of the
reaction imaging assembly are either contained within or fixedly
attached to an optical block 2710 or a beam splitter block 2707. In
one embodiment, the optical block and beam splitter block are
joined to form the reaction imaging assembly. The optical block
2710 may be configured with a pocket 2711 which is an opening
therein to permit the transmission of excitation wavelengths from
the excitation lens cell to the cartridge imaging plane 2760 and
emission wavelengths from the plurality of assay chambers to the
reaction camera. Further the pocket 2711 surrounding the cartridge
reaction area 1600 may prevent any potential stray light within the
instrument enclosure from interfering with the detection of
fluorophore emission spectra by generating the darkest reference
background when capturing images of the reaction area. In a further
preferred embodiment, the reaction imaging assembly is fixed to the
first side of the fixed support bracket 2012 and therefore remains
stationary during the clamping and unclamping of a cartridge.
[0290] In various embodiments, reaction camera 2701 captures images
of the assay chambers within a cartridge reaction area 1600 for an
instrument image processing to determine the presence of target
nucleic acids and generate a result of the diagnostic test. In some
implementations, the reaction camera 2701 is monochromatic. In such
implementations, the reaction camera may be optically coupled to a
corresponding cartridge configuration adapted to perform with said
monochromatic reaction camera. For example, a cartridge may
comprise a plurality of assay chambers, wherein each assay chamber
contains a distinct primer set and fluorescent probe. When the
reaction camera captures an image of the cartridge reaction area
containing a plurality of assay chambers, image processing within
the instrument computer system may determine the presence of target
nucleic acids based on the visual signal and corresponding chamber
position to determine a diagnostic result. In an alternative
embodiment, the reaction camera 2701 is a multicolor camera. In
such implementations, a cartridge configured to perform with said
multicolor reaction camera may comprise assay chambers where
multiple primer sets and probes are used. For example, the
multicolor reaction camera may capture images a cartridge reaction
area with assay chambers comprising a plurality of primer/probe
sets within a single assay chamber. Further, the multicolor
reaction camera may capture images of a cartridge reaction area
with assay chambers comprising a plurality of primer/probe sets
within multiple assay chambers. Appropriate optical components,
e.g. LEDS, filters, lenses, and sensors, may be selected such that
imaging processing within the instrument computer system may
determine the presence of target nucleic acids based one the
plurality of emission wavelengths.
[0291] In a further embodiment, additionally included in the
reaction imaging assembly is a dichroic beam splitter 2702, which
separates excitation light from emitted light by reflecting shorter
wavelengths of light from the excitation lens cell 2703 and passing
longer wavelengths emitted from the fluorophore. in another
embodiment, a fold mirror 2704 directs excitation wavelengths to
the reaction well image plane 2760 and redirects emitted
wavelengths from the reaction well image plane to the reaction
camera 2701.
[0292] In one embodiment, an excitation lens cell generates
excitation wavelengths for a fluorophore to absorb and is comprised
of at least one or more excitation LED 2731, a plano-convex lens
2733, an aspheric lens 2734, an aperture 2732, and a bandpass
filter 2735. The excitation lens cell is shown in FIGS. 63 and 64
as section views. In one embodiment, the at least one or more
excitation LED 2731 illuminates the plurality of assay chambers. A
person of ordinary skill may select the appropriate number and type
of LEDs such that emission spectra corresponds to the excitation
wavelengths of a chosen fluorophore. The optical path of the
excitation light travels through aspheric lens 2734 to correct for
spherical aberration, an optical effect commonly observed with
plano-convex lenses, where incident light rays focus at different
points resulting in a blurry image. Aspheric lens 2734 focuses
incident light from the excitation LED 2731 to a small point, thus
improving the image quality. In one implementation, an aperture is
used to reshape excitation illumination. Focused excitation light
is transmitted through the aspheric lens 2734 and enters aperture
2732 such that, aperture 2732 alters the illumination shape from
the excitation LED to minimize off axis light and stray light from
hindering fluorescent imaging. In one embodiment, one or more
bandpass filters may be used within the excitation lens cell to
selectively transmit light of specific wavelengths. Excitation
light passes through bandpass filter 2735 to filter wavelengths
outside the fluorophore excitation bandwidth and transmit
wavelengths within the excitation bandwidth. Furthermore, the
excitation bandpass filter substantially prevents light in the
fluorophore emission band from entering the reaction well imaging
plane due to the epifluorescence arrangement. Filtered excitation
light travels through a plano-convex lens 2733 to diffuse the light
prior to reaching dichroic beam splitter 2702. Filtered excitation
light strikes the dichroic beam splitter 2702 and reflects shorter
excitation wavelengths to the objective lens 2706 while longer
wavelengths are transmitted through the dichroic beam splitter
2702. In one implementation, transmitted longer wavelengths from
the dichroic beam splitter are directed to a light trap 2703. When
implementing a light trap, the light trap prevents excitation light
from entering the camera by reflecting the light off multiple
angled surfaces substantially away from the camera. Excitation
wavelengths reflected by the dichroic beam splitter 2702 are
transmitted through the objected lens 2706 where fold mirror 2704
redirects the light to the image plane 2760 of the reaction well
area 1600 of the cartridge.
[0293] The excitation LED peak wavelength and intensity can vary
with temperature, thus requiring precise thermal control of the LED
temperature. The excitation lens cell further includes various
elements to ensure the excitation cell 2730 functions properly. As
shown in FIG. 64, a temperature sensor 2738 provides temperature
feedback control while photodiode 2739 monitors the LED output to
ensure the LED is on. A thermal isolation spacer 2737 isolates the
excitation system from short ambient thermal transients and heat
sink 2736 provides cooling.
[0294] In another embodiment, the reaction imaging assembly 2700
includes emission lens cell 2750, shown in FIG. 65, comprising an
image lens 2751, long pass filter 2752, and objection lens 2706.
The fluorophore absorbs excitation light from the excitation lens
cell 2730 and almost instantaneously emits emission wavelengths to
fold mirror 2704. The bent emitted light travels through objective
lens 2706 where the longer emitted wavelengths are subsequently
transmitted through the dichroic beam splitter 2702. In one
implementation, the emission lens cell 2750 comprises one or more
longpass filters to transmit emitted wavelengths from the
fluorophore. A longpass filter 2752 ensures light in the emission
band enters the reaction camera 2701 and substantially eliminates
interfering wavelengths, outside of the emission band, from the
excitation LED.
[0295] FIGS. 45, 46 and 66 illustrate the relationship between the
label imaging assembly 2700 and the reaction imaging assembly 2700
of the instrument optical subsystem. Regarding FIG. 66, the label
imaging assembly is shown fixed to the antenna ground plate 2810 at
the proximal end of the instrument near front slot 2072, while the
reaction imaging assembly is fixed to the distal end of the
instrument in close proximity to the loading assembly 2230. In such
a configuration, the label imaging assembly 2770 is advantageously
separated from the reaction imaging assembly 2700. In this way, the
label imaging system 2770 may be used initially to detect an
insufficient sample volume error and eject a cartridge prior to
executing sample preparation steps for an assay to be imaged by the
reaction imaging assembly 2700.
[0296] With regard to FIGS. 45 and 46, a cartridge 1000 is shown
with respect to the label imaging assembly 2770 and reaction
imaging assembly 2700 in a loading and loaded position, as
described herein. FIG. 45 depicts the cartridge in a forward most
loading position within the loading assembly 2230. Patient label
area 1040 is not within the field of view of the label imaging
assembly 2770, as observed in FIGS. 59, 60, and 61. Furthermore, in
the forward most loading position the reaction area 1600 of a
cartridge containing a plurality of assay chambers 1621 is adjacent
to the reaction imaging assembly 2700 and outside of pocket 2711
within optical block 2710. Note, the loading position of the
loading assembly 2230 in FIG. 45 reiterates the loading position
shown in FIGS. 17A and 17B. The cartridge is shown in a loaded
position in FIG. 46. Patient label area 1040 is now hidden by the
label imaging assembly 2770 and is within the field of view as
shown in FIGS. 59, 60, and 61. Furthermore, the reaction area of
the cartridge 1600 is disposed within pocket 2711 of the optical
block and is hidden from view. The loaded position of the loading
assembly 2230 similarly reflects the loaded position in FIGS. 18A,
18B, 19A, 19B, and 19C. Additional details of the positioning of
the cartridge and the movement of the thermal clamp assembly 2680
with respect to the reaction imaging system 2700 may be appreciated
with reference to FIGS. 38-42. The position of the reaction imaging
assembly relative to other components of the instrument, (e.g., the
moving bracket assembly 2040) may be appreciated with reference to
the various views provided in FIGS. 43 and 44.
F. Exemplary Computer System
[0297] FIGS. 67A-671 represent various schematic views of a
representative computer control system for use with a diagnostic
instrument described herein. Generally, the instrument computer
control system includes instructions in computer readable code used
to coordinate the synchronous performance of the one or more of the
operations described herein related to receiving, handling,
processing and analyzing a suspected sample in a cartridge.
Additional details of the various steps performed related to
receiving, handling, processing and analyzing a suspected sample in
a cartridge are provided with regard to FIGS. 93-102 and 106A-113.
The computer system may comprise an exemplary client or server
computer system. Computer system includes a number of communication
channels or busses for communicating control signals, sensor
information, or other information from a component or system within
the instrument to a processor. These various communication pathways
are indicated by the lines connecting each of the various
components, systems and subsystems. The host processor 2900 is used
for processing information and generating signals according to one
or a number of programmed control sequences. Processor 2900 may be
any suitable computer controller, processor with co-processor,
microprocessor or suitable combination thereof.
[0298] Additionally or optionally, the instrument computer control
system may include one or more of a random access memory (RAM), or
other dynamic storage device (referred to as main memory) coupled
to bus for storing information and instructions to be executed by
processor. Main memory also may be used for storing temporary
variables or other intermediate information during execution of
instructions by processor.
[0299] Instrument computer system also includes a read only memory
(ROM) and/or other static storage device coupled to bus for storing
static information and instructions for processor, and a data
storage device, such as a magnetic disk or optical disk and its
corresponding disk drive. Data storage device is coupled to bus for
storing information and instructions.
[0300] With reference to FIG. 67A, the host processor 2900 is in
communication with a communications module 2905 which includes a
cellular antenna 2800 located in the front panel 2073 of the
instrument 2000 along with associated firmware and software.
Additionally, the host processor 2900 is in communication with a
USB and Ethernet port 2903 as well as any other external
communication port. There is access provided to data storage
including encrypted data 2901 along with calibration, firmware
upgrade and test results data. There is also provided appropriate
storage for de-identified patient results data. The host processor
2900 is also in communication with a display or graphical user
interface 2902 such as the one on the instrument front panel 2073.
The host processor 2900 is also in communication with various
instrument application software 2904. This software and firmware
corresponds, by way of example, to particular testing routines to
be implemented by the diagnostic instrument 2000 based on the type
of sample/integrated diagnostic cartridge 1000 that is loaded into
and detected by the instrument 2000. Additionally, the instrument
software and firmware 2904 includes computer readable instructions
for an instrument operating system along with the various
appropriate computer drivers for instrument components. The host
processor 2900 is also configured to access and execute the camera
operation and imaging firmware 2915 responsible for executing the
specific imaging routines performed by the label imaging camera
2771 and the reaction chemistry or assay chamber camera 2701.
[0301] FIG. 67A also illustrates the communication busses to each
of the different computer subsystems utilized in the instrument.
Each one includes appropriate software, firmware and communication
components adapted and configured to the functional and operational
requirements of that specific instrument subsystem. As such, each
instrument subsystem is provided appropriate communication channels
for transmission and receipt of computer readable instructions from
the one or more processors, co-processors or suitable
microprocessor(s). Additionally, a number of specifically
configured subsystem of the instrument control system are
configured to deliver, receive or monitor signals from one or more
actuators, components, switches or sensors as will now be
described.
[0302] Advantageously, the instrument computer system may include a
host processor and a co-processor 2900 in coordinated operation. In
one configuration, the host processor 2900 includes instrument
operating system and device drivers, specific instrument
application software and firmware 2915 for operation of the label
camera 2771 and reaction well camera 2701. A second processor may
be configured as a slave processor to handle other commands such as
the operation of various motors and actuator in the diagnostic
instrument 2000. Additionally, the co-processor would be
responsible for prioritization and execution of various control
signals throughout the various instrument subsystems. The
instrument computer system memory or computer readable storage may
include stored or accessible computer records of various test
methods, scripts, parameters, completed records storage, instrument
calibration readings and results based on specific operations
performed by the instrument 2000 for a specific cartridge
diagnostic test or sample type.
[0303] In general, the instrument computer system includes the
following functional subsystems adapted and configured to
correspond to the steps performed in a wide variety of functions
corresponding to a desired preprogrammed testing sequence. As shown
in FIG. 67A, the functional subsystems are optical cartridge label
subsystem 2910, the optical reaction well subsystem 2990, the
thermal subsystem 2970, the lysing drive subsystem 2950, the
loading cartridge subsystem 2920, the cartridge seal rupturing
subsystem 2930, the pneumatic-interface subsystem 2960, the valve
drive subsystem 2940 and the rehydration mixing subsystem 2980. In
one aspect, these functional groups may be functionally grouped
more generally into an optical subsystem, a thermal subsystem and a
clamping subsystem. One or more of these functional groups may be
assigned to the co-processor.
[0304] The optical subsystem includes an optical cartridge label
subsystem 2910 (FIG. 67B) and an optical reaction well subsystem
2990 (FIG. 67C).
[0305] As shown in FIG. 67B, the optical cartridge label subsystem
2910 includes software, firmware and communication components
adapted and configured to for use with a cartridge label imaging
camera 2771, a label illumination LED 2792 and a sample
illumination LED 2775. Under control of instructions from the one
or more processors 2900, the optical cartridge label subsystem 2910
interacts with the patient label area 1040 and sample port assembly
1100 of a cartridge 1000 undergoing processing within the
instrument 2000.
[0306] As shown in FIG. 67C, the optical reaction well or assay
imaging subsystem 2990 includes software, firmware and
communication components 2980 adapted and configured for use with a
reaction camera 2701. Additionally, the optical reaction well
subsystem 2990 controls a bright field LED 2753, an excitation LED
heater 2741, an excitation LED 2791, an LED excitation intensity
sensor 2740, an assay well reaction camera 2701, and an LED
excitation temperature sensor 2738. Under control of instructions
from the one or more processors 2900, optical reaction well
subsystem 2990 interacts with the assay chamber 1621 in the
reaction area 1600 of a cartridge 1000 undergoing processing within
the instrument 2000.
[0307] As shown in FIG. 67D, the thermal subsystem 2970 includes
software, firmware and communication components 2960 adapted and
configured to for use with an heat stake cooling fan 2644, a
chemistry heater 2601, chemistry heater sensor 2608, heat staker
heater 2661, cartridge heater temperature sensor 2555, heat staker
motor 2642, cartridge heater 2551, heat stake temperature sensor
2646, staker linear displacement sensor 2645, and chemistry heater
cooling fan 2603. Under control of instructions from the one or
more processors 2900, the thermal subsystem 2970 interacts with the
central cartridge portion, assay wells and heat stake zone portions
of a cartridge undergoing processing within the instrument.
[0308] As shown in FIG. 67E, the lysing drive subsystem 2950
includes software, firmware and communication components adapted
and configured for use with a lysing drive motor 2330 and an
audible sensor/microphone 2380. Under control of instructions from
the one or more processors 2900, the lysing drive subsystem 2950
interacts with the stir bar or other lysing agents within the
lysis-lysis chamber 1371 of the cartridge while being monitored for
magnetic uncoupling via the audible sensor 2380.
[0309] As shown in FIG. 67F, the loading cartridge subsystem 2920
includes software, firmware and communication components adapted
and configured for coordinated operation of a linear actuator 2014,
hardstop clamping sensor 2019, cartridge door support assembly
2280, homing clamping sensor 2017, cartridge loading sensor 2236,
and frangible seal switch 2266. Under control of instructions from
the one or more processors 2900, the loading cartridge subsystem
2920 provides coordinated interactions with cartridge to ensure
proper loading, positioning and clamping of the cartridge with
respect to the instrument interior.
[0310] As shown in FIG. 67G, the pneumatic subsystem 2960 includes
software, pneumatic control firmware and communication components
adapted and configured to for use with a pneumatic pump 2131,
proportional valve 2133, output selector valve 2136, altitude
sensor 2140, pressure regulator 2132, and a humidity sensor 2142.
Under control of instructions from the one or more processors 2900,
the pneumatic subsystem 2960 interacts with a pneumatic interface
2100 on the cartridge to deliver pneumatic drive signals to the
cartridge undergoing processing within the instrument.
[0311] As shown in FIG. 67H, the valve drive subsystem 2940
includes software, firmware and communication components adapted
and configured to for use with a valve drive motor 2403, an
interference sensor 2404 and a valve drive homing sensor 2409.
Under control of instructions from the one or more processors 2900,
the valve drive subsystem 2940 interacts with the rotary valve on
the cartridge to index the rotary valve for alignment of a desired
flow channel on the cartridge undergoing processing within the
instrument.
[0312] As shown in FIG. 67I, the rehydration mixing subsystem 2980
includes software, firmware and communication components adapted
and configured for use with a rehydration motor 2510 and
rehydration motor rotation sensor 2530. Under control of
instructions from the one or more processors 2900, the rehydration
motor 2510 interacts with a stir ball or other component within the
master mixing rehydration chamber of the cartridge while being
monitored for rotation using the motor rotation sensor 2530.
[0313] Additional alternative computing environments and
modifications to both user experience and user interaction are
possible and within the scope of the various embodiments described
herein, The instrument computer control system may further be
coupled to a display device, such as a liquid crystal display (LCD)
including touch screen or other functionality by direct connection
or wirelessly. The display is also coupled to bus for displaying
information to an instrument user. An alphanumeric input device,
including alphanumeric and other keys, may also be provided via the
touch display or coupled to bus for communicating information and
command selections to processor. An additional user input device is
cursor control, such as a mouse, trackball, trackpad, stylus, or
cursor direction keys, voice or touch controllers coupled to bus
for communicating direction information and command selections to
processor, and/or for controlling cursor movement on display.
[0314] Another device that may be coupled to bus is hard copy
device, which may be used for marking information on a medium such
as paper, film, or similar types of media. Additionally, the
computer system may include wired and wireless communication
capabilities depending on configuration. Remote communications
using the communications module described above with the instrument
computer system may be utilized for transferring information,
calibration, service, maintenance or other system or patient
information collected or produced by the instrument computer
system.
[0315] Note that any or all of the components of system and
associated hardware may be used in the present invention. However,
it can be appreciated that other configurations of the instrument
computer system may include some or all of the devices. Certain
variations of system may include peripherals or components not
illustrated in these various exemplary figures. Additional such
components may be included and configured to receive different
types of user input, such as audible input, or a touch sensor such
as a touch screen.
[0316] Certain embodiments may be implemented as a computer program
product that may include instructions stored on a machine-readable
medium. These instructions may be used to program a general-purpose
or special-purpose processor to perform the described operations. A
machine-readable medium includes any mechanism for storing or
transmitting information in a form (e.g., software, processing
application) readable by a machine (e.g., a computer). The
machine-readable medium may include, but is not limited to,
magnetic storage medium (e.g., floppy diskette); optical storage
medium (e.g., CD-ROM); magneto-optical storage medium; read-only
memory (ROM); random-access memory (RAM); erasable programmable
memory (e.g., EPROM and EEPROM); flash memory; electrical, optical,
acoustical, or other form of propagated signal (e.g., carrier
waves, infrared signals, digital signals, etc.); or another type of
medium suitable for storing electronic instructions. The label
imaging camera firmware or the optical cartridge label subsystem
may be adapted and configured to recognize machine readable
markings as part of a cartridge verification protocol as well as to
aid in the identification of a particular sample type and/or
diagnostic testing routine to be performed with that
sample/cartridge.
[0317] Additionally, some embodiments may be practiced in
distributed computing environments where the machine-readable
medium is stored on and/or executed by more than one computer
system. In addition, the information transferred between computer
systems may either be pulled or pushed across the communication
medium connecting the computer systems.
[0318] The digital processing device(s) described herein may
include one or more general-purpose processing devices such as a
microprocessor or central processing unit, a controller, or the
like. Alternatively, the digital processing device may include one
or more special-purpose processing devices such as a digital signal
processor (DSP), an application specific integrated circuit (ASIC),
a field programmable gate array (FPGA), or the like. In an
alternative embodiment, for example, the digital processing device
may be a network processor having multiple processors including a
core unit and multiple micro engines. Additionally, the digital
processing device may include any combination of general-purpose
processing device(s) and special-purpose processing device(s).
G. Integrated Diagnostic Cartridge
[0319] The embodiments described herein relate to a disposable
single use device (a "cartridge") used for molecular diagnostic
testing. The cartridge can contain a plurality of modules for
performing a variety of functions in order to effect the diagnostic
test including, but not limited to, a loading module, a lysis
module, a purification module, and an amplification module. The
loading module is configured to receive a sample, minimize the
spilling of the sample, and prepare the sample for lysis. The lysis
module is configured to disrupt cells walls and cell membranes in
order to release inter-cellular materials such as nucleic acids
(DNA, RNA), protein or organelles from a cell, and, in some cases,
clear debris from the lysate. The purification module is configured
to isolate and/or enrich nucleic acid from a lysed sample. The
amplification module is configured to generate and detect a signal
from target amplicon, indicative of the presence of target pathogen
in the sample.
[0320] Generally, cartridge dimensions are defined by its length,
width, and height. Accordingly, each dimension has a respective
associated axis, e.g. a cartridge length axis, a cartridge width
axis, and a cartridge height axis. FIGS. 68-70, 89, and 92 are
exemplary embodiments of an integrated diagnostic cartridge 1000.
In FIGS. 68-70 and 92 the dimensions of the integrated diagnostic
cartridge are arranged such that the cartridge length axis 1035 and
cartridge width axis 1025 lie within the plane of the page.
Further, the cartridge height axis, i.e. axis defining the
thickness of the cartridge, is represented by a circle 1030 which
is normal to the plane of the page. Additionally, FIGS. 68-72 and
89-92 depict an arrow 1900 adjacent to the illustrated cartridge
embodiments. Arrow 1900 corresponds to one preferred embodiment of
an operational cartridge orientation maintained within the
instrument during the performance of a diagnostic test. As
described herein and further detailed below, the preferred
orientation is a vertical cartridge orientation.
[0321] FIGS. 3-5, 8, 11-13, and 16A-16E depict various views of the
cartridge positioned between the clamping subsystem in the vertical
orientation from outside and within the instrument enclosure.
Accordingly, arrow 1900, which indicates the preferred cartridge
orientation, is collinear with the cartridge width axis 1025 and
slot 2072 such that the cartridge width axis 1025 is substantially
normal to a base of the instrument and the cartridge length axis
1035 is substantially normal to a rear wall of the instrument when
inserted and clamped by the instrument.
[0322] In many embodiments, one or more portions of the cartridge
and/or instrument may comprise alignment features, e.g. rails,
protrusions, indents, or keys, for inserting the cartridge into the
instrument in the acceptable orientation. The following cartridge
embodiments describing various modules for performing and housing
the molecular diagnostic test will be described according to this
orientation. A discussion of several vertical cartridge advantages
will be made readily apparent throughout the remaining disclosure.
However, it is to be appreciated that a person of ordinary skill
may design a cartridge based on alternative orientations while
achieving the same desired objective of detecting nucleic
acids.
[0323] FIG. 68 is a side view of one cartridge interface schematic
of an integrated diagnostic cartridge 1000. In this illustrative
embodiment, the integrated cartridge 1000 includes a loading
module, a lysis module, a purification module and an amplification
module. In alternative embodiments, the diagnostic cartridge may
optionally and/or additionally include a pre-amplification module,
a distinct detection module, a plurality of any of the
aforementioned modules, e.g. a second purification module, or any
other module designed for the effect of performing a molecular
assay, In the embodiment shown in FIG. 68, the interface region
allocated to the loading module is on a proximal end of the
cartridge 1920 adjacent to the patient label area 1040. Further,
the interface region for the amplification module, containing
reaction area 1600, is on a distal end of the cartridge 1915.
Further to the compact and modular design aspects of the various
cartridge embodiments, the interstitial portion of the cartridge
between respective interfaces for the loading module and
amplification module can be occupied by remaining module
interfaces. As shown in FIG. 68, the interface regions for the
lysis module and the purification modules are arranged between the
indicated proximal and distal modules . . . As such, the additional
interface regions of the lysis and purification modules are
occupied by the frangible seal area 1200, the rotary valve 1400,
the rehydration chamber 1520, the cartridge pneumatic interface
1170 and the lysis chamber 1371. The placement of these interfaces,
and others described herein, are in advantageous arrangement not
only as to the loading module and the amplification module but also
to take advantage of vertical orientation within the instrument for
sample processing.
[0324] The inventive modular design of the cartridge embodiments
described herein leverage the ability to easily modify cartridge
configurations, modules, and/or their respective interfaces to
process samples and detect specific target pathogens with a desired
diagnostic assay. One or more distinct modules may be simply
altered, redesigned, or substituted in whole, without significantly
impacting the remaining cartridge modules, to change the diagnostic
assay performed by the instrument. Such modifications may enable
the processing of a different sample type, the lysing of various
target pathogens, the purification of different analytes of
interest, and/or the amplification and detection of one or more
pathogens. Accordingly, the modular design of the cartridge
embodiments presented herein advantageously permit simple
substitution of individual elements within respective modules. In
one specific example, described in greater detail below, assay
chambers for amplifying and detecting purified analyte may be
modified with reagent plugs comprising dried down reagents. The
dried down reagents may comprise one or more primer sets and probes
for the specific detection of a target pathogen, such that one may
replace one or more reagent plugs in the amplification module to
detect a different pathogen. Specifically, cartridge modifications
driven by altering the sample type to be processed and target
pathogen can be made with little to no impact on the design and
functionality of analogous instrument subsystems. However, in any
case, it is most advantageous and preferable to modify the
cartridge configuration, modules, and/or interfaces within one or
more fixed parameters of instrument-to-cartridge interfaces
established to minimize the redesign of the diagnostic instrument.
Additionally or optionally, the plugs may be modified by shape,
size or placement to provide for a range of assay chamber volumes
as described herein.
[0325] In many embodiments, the instrument is configured to
recognize and interact with the cartridge interfaces to perform the
diagnostic assay. Accordingly, the instrument interfaces which
interact with the cartridge may be one or more of those which are
physically coupled or non-physically coupled. The aforementioned
cartridge configuration of FIG. 68 illustrates a combination of
physically and non-physically coupled interfaces. Physically
coupled interfaces may include elements that directly contact the
cartridge, such as interfaces which are in thermal contact or
direct physical contact. For example, the door support assembly
2280 presses upon the fill port cap 1181, the valve drive assembly
2400 inserts a valve drive into engagement openings in the rotary
valve 1400, and the instrument pneumatic interface 2100 presses
against the cartridge pneumatic interface 1170. Alternatively,
non-physically coupled interfaces may include elements that still
interact with the cartridge, but otherwise do not physically
contact the cartridge. Such non-physical interfaces include, but
are not limited to, magnetic, optic, acoustic, ultrasonic, and
electromagnetic. For example, the magnetic mixing assembly 2300
acts upon the contents of the lysis chamber 1371 and a magnet
associated with the rehydration motor 2510 acts upon a magnetic
ball 1524 with the rehydration chamber 1520. The illustrated
embodiment is further configured to interact with a camera 2771
within the label imaging assembly 2770 to capture an image of the
patient label area 1040. Further, a reaction camera 2701 of the
reaction imaging assembly 2700 may capture and image of the
reaction area 1600 of the cartridge.
[0326] In certain implementations, the cartridge is comprised of a
fluidics card, which comprises most of the functional structures of
the cartridge, and a cover 1004, which protects the active areas of
the cartridge. FIG. 69A illustrates a cartridge from the feature
side 1007 of the fluidics card 1001. The cover is substantially
removed to permit visualization of the fluidics features hidden
behind the cover. Similarly, FIG. 70A illustrates the cartridge
from the fluidics side 1006, which provides the fluidic network for
transporting a sample and various substances to different modules
of a cartridge. Typically the fluidics side comprises a plurality
of fluidic channels, ducts, and pathways formed within the surface
of the cartridge. In many embodiments, the channels, ducts and
pathways are enclosed with a film applied against the fluidics side
of the cartridge. In a preferred implementation, the channels,
ducts and pathways are microfluidic features having a smallest
dimension of 750 .mu.m or less. In other implementations, the
smallest dimension can be 600 .mu.m or less, 500 .mu.m or less, 400
.mu.m or less, 200 .mu.m or less, or 100 .mu.m or less. In another
aspect, the fluidics side 1006 can include multiple vias, e.g.
openings, passages or ports configured for passing fluids
therethrough from one side of the fluidics card to the other, e.g.
from the fluidics side 1006 to a structure on the features side
1007. In another aspect, the fluidics side 1006 can include
multiple vias, e.g. openings configured for passing fluids
therethrough from one side of the fluidics card to the other, e.g.
from the fluidics side 1006 to a structure on the features side
1007. A via can have any of the dimensions, such as the
cross-sectional diameter of any of the channels provided herein. In
another embodiment, the feature side 1007 of a fluidics card 1001
defines the various structures to enable the loading, lysing,
purifying, and amplification of a sample.
[0327] In certain implementations, the cartridge is comprised of a
fluidics card, which comprises most of the functional structures of
the cartridge, and a cover 1004, which protects the active areas of
the cartridge. FIG. 69A illustrates a cartridge from the feature
side 1007 of the fluidics card 1001. The cover is substantially
removed to permit visualization of the fluidics features hidden
behind the cover. Similarly, FIG. 70A illustrates the cartridge
from the fluidics side 1006, which provides the fluidic network for
transporting a sample and various substances to different modules
of a cartridge. Typically the fluidics side comprises a plurality
of fluidic channels, ducts, and pathways formed within the surface
of the cartridge. In many embodiments, the channels, ducts and
pathways are enclosed with a film applied against the fluidics side
of the cartridge. In a preferred implementation, the channels,
ducts and pathways are microfluidic features having a smallest
dimension of 750 .mu.m or less. In other implementations, the
smallest dimension can be 600 .mu.m or less, 500 .mu.m or less, 400
.mu.m or less, 200 .mu.m or less, or 100 .mu.m or less. In another
aspect, the fluidics side 1006 can include multiple vias, e.g.
openings, passages or ports configured for passing fluids
therethrough from one side of the fluidics card to the other, e.g.
from the fluidics side 1006 to a structure on the features side
1007. In another aspect, the fluidics side 1006 can include
multiple vias, e.g. openings configured for passing fluids
therethrough from one side of the fluidics card to the other, e.g.
from the fluidics side 1006 to a structure on the features side
1007. A via can have any of the dimensions, such as the
cross-sectional diameter of any of the channels provided herein. In
another embodiment, the feature side 1007 of a fluidics card 1001
defines the various structures to enable the loading, lysing,
purifying, and amplification of a sample.
[0328] In some implementations, one or more fluidic channels may
specifically be pneumatic channels, wherein only pressurized air or
gas is permitted to flow. The diameter of the pneumatic channels
may be of similar dimensions of fluidic channels as described
herein. Such pneumatic channels may be configured for venting and
rerouting air or gas within the cartridge when a sample is
loaded.
[0329] The terms "fluidic communication" as used herein, refers to
any duct, channel, tube, pipe, or pathway through which a
substance, such as a liquid, gas, or solid may pass substantially
unrestricted when the pathway is open. When the pathway is closed,
the substance is substantially restricted from passing through.
[0330] It is noted that, as used herein, the term "input" refers to
vias or channels of a cartridge where active pressurization is
applied to motivate a liquid (i.e. a sample or reagent) or a gas
(i.e. air) residing within a channel. As used herein, the term
"output" refers to the leading front of said motivated liquid or
gas which is displaced as a result of active pressurization and
which terminates at a via or channel for venting. In one aspect,
the input and/or output may comprise one or more filter plugs for
filtering a fluid. In one embodiment, a filter plug is configured
to capture pollutants and particles from pressurized air. In
another embodiment, a filter plug is configured to be hydrophobic
to vent gasses while retaining liquids.
[0331] As described above, fluids are motivated throughout the
fluidic network of a cartridge using pressurized air. Thus, the
cartridge is configured to receive pressurized air through one or
more pneumatic vias. In the cartridge exemplified in FIG. 69A, main
pneumatic via 1193 is present on the feature side of the cartridge.
Each pneumatic via is in fluidic communication with a pneumatic
channel, such that pneumatic channels enable the motive force to
transport a sample and liquids through various modules within the
cartridge.
[0332] Given the pressurization of the device, as will become
apparent in the sections to follow, in some embodiments, the
cartridge, optionally, includes a liquid trap configured to capture
liquids to prevent contamination of various structures of the
cartridge. The liquid trap, preferably, is formed by a widening or
depression in a pneumatic channel, wherein liquid droplets fall to
bottom of the depression thereby captured outside the main
pneumatic flow within the cartridge. Alternatively, the liquid trap
can be a physical structure, such as a sintered vent plug placed
within the pneumatic channel.
[0333] In some embodiments, the cartridge cover can further include
a cartridge label to supply the user and instrument with
information associated with a given diagnostic test. FIGS. 69B and
90 illustrate an exemplary cartridge labels 1005. In some
embodiments, the cartridge label includes a cut out to provide
visual access to a sample window 1050 formed within a metering
chamber 1110, enabling the user and/or a system, such as instrument
2000 as described herein, to view and detect the sample volume
loaded into the device. Additionally, one cut out within the
cartridge label may be configured to exclude the reaction area 1600
enabling the amplification and detection of target nucleic acids
from optically transparent plugs, as described herein. A portion of
the cartridge label, i.e., the patient label area 1040, in some
embodiments, is configured to be written on to enable a user to
provide patient information relating to the diagnostic test. Such
information can include, for example, the name of the patient, the
date of birth of a patient, and the sample type gathered from the
patient. In some embodiments, the cartridge label may include
computer readable information. In some embodiments, the cartridge
label provides a computer readable visual code 1053 to store
computer readable information. Such information can include, for
example, the type of test the cartridge is configured to run and
general manufacturing information, e.g., a lot number, expiration
date, and/or recalls associated with the cartridge. In some
implementations, the computer readable information is configured to
be encrypted. The computer readable information may be configured
to be read by a system or instrument, such as instrument 2000, as
described herein. In a further implementation, illustrated in FIG.
91, the cartridge label 1005 can include one or more perforated
areas within said cartridge label configured to be broken when
contacted. In one implementation, a perforated area 1051 exists
around the frangible seal area. In another implementation, a
perforated area 1052 is located around the cartridge pneumatic
interface to enable a pneumatic interface, for example like
pneumatic interface 2100 of instrument 2000 to break the perforated
area to make contact with the device.
[0334] Returning to FIGS. 69A and 69B, there is provided a
cartridge orientation reference. In these views the cartridges are
in a vertical orientation as in indicated by the arrow which
corresponds to the orientation during use when processing a sample
while within the instrument 2000. Treating the rotary valve as an
origin, there is one dashed line extending along the longitudinal
axis of the cartridge and another extending along the vertical axis
of the cartridge. As a result, any cartridge embodiment may be
described by reference to the cartridge distal end 1915, the
cartridge proximal end 1920, cartridge upper portion 1905 and
cartridge lower portion 1910. Thus, by using the dashed lines and
this convention, the cartridge may be described according to an
upper proximal portion 1914, upper distal portion 1907, lower
proximal portion 1917 and lower distal portion 1912. By way of
illustrative example, FIG. 69A is a top down view of another
cartridge embodiment. In this embodiment there is a reaction area
1600 in the cartridge upper distal region 1907, a waste collection
element 1470 in the cartridge lower distal region 1912, a cap for
the sample port assembly 1181 is in the cartridge upper proximal
region 1914 and exemplary manufacturing bar codes are located in
the cartridge lower proximal region 1917, but may be provided in
other locations depending upon various configurations.
[0335] Additionally, in an aspect which aids in the mitigation or
elimination of bubble formation during process, many of the various
chambers in the integrated cartridge have been designed so that in
a general way once the cartridge is in a vertical orientation
fluids will flow from the top of a chamber of enclosure to the
bottom of the chamber or enclosure. Even if a chamber or enclosure
employs reversible flows for mixing or other purposes, there may
still be advantages to this general design guideline of upper inlet
and lower out placement. FIG. 70A provides a number of examples of
this design guidance. Fill/sample chamber 1101 has a pneumatic
inlet 1176 in an upper portion and a fill chamber outlet 1102 in a
lower portion. Wash buffer reservoir 1475 has a wash inlet 1476 in
an upper portion and a wash outlet 1477 in a lower portion. Lysis
chamber 1371 has a lysis chamber inlet 1371 located in an upper
portion of the lysis chamber while the lysis outlet/bead filter
channels 1387 are provided in a lower portion of the lysis
chamber.
[0336] Also shown in FIG. 70A are dashed lines for the enlarged
views of FIG. 70B and FIG. 70C. FIG. 70B is an enlarged view of the
waste collection element 1470 of FIG. 70A. A chamber reference line
1310 has been added to the waste collection element to divide it
into an upper portion and a lower portion. The waste collection
element provides an exception to the general design rule of in from
the top and out from the bottom described above. Because of the
special handling of the various waste products generated by
cartridge operations as well as for venting, the waste collection
element includes the inlet 1471 and as illustrated a number of
waste outlets 1471. Both the inlet 1471 and the several outlets
1474 are in the upper chamber portion--that is above the chamber
reference line 1310.
[0337] FIG. 70C is an enlarged view of the upper proximal portion
1914 and lower proximal portion 1917 of the cartridge of FIG. 70A.
In this view, a chamber reference line 1310 has been added to each
of fill/sample chamber 1101, wash buffer reservoir 1475 and the
metering chamber 1120. As is clear from a chamber reference line in
the approximal mid vertical point in the fill/sample chamber 1101,
it is clear that the pneumatic inlet 1176 in an upper chamber
portion that is above the chamber reference line. Still further,
the fill chamber outlet 1102 is below the chamber reference line
1310 and is such in a lower chamber portion. Similarly, the
reference chamber line 1310 placed on wash buffer reservoir 1475
makes clear that the wash inlet 1476 is in an upper chamber portion
above the chamber reference line. The wash outlet 1477 is below the
chamber reference line and is in a lower chamber portion. A similar
result is found when considering the chamber reference line 1310
with respect to the metering chamber 1120. The inlet 1111 is above
the reference line 1310 and is therefore in an upper chamber
portion. The outlet 1115 is below the chamber reference line 1310
and is therefore considered in a lower chamber portion.
[0338] This general design guidance is summarized in the example
chamber views of FIGS. 70D, 70E and 70F. FIG. 70D is a front view
of an exemplary chamber as positioned in a vertical processing
orientation as in FIGS. 69A and 70A. FIG. 70D is an exemplary
chamber with a chamber reference line 1310 used to indicate an
upper chamber portion and a lower chamber portion. To the general
case of FIG. 70D, FIG. 70E illustrates the exemplary chamber of
FIG. 70D having an inlet in a top middle or upper most portion of
the upper portion. Along the same lines, the outlet is shown in the
middle bottom or bottom most portion of the chamber bottom portion.
FIG. 70F illustrates that the inlet and the outlet may be provided
in a more general way but still within the chamber upper portion
and the chamber lower portion and still within the design guidance
of in from the top and out from the bottom. By way of illustrative
example, if one were to apply a clock face to the exemplary
chamber, the (i) the chamber reference line 1310 would run from the
9 o'clock position to the 3 o'clock position (FIG. 70D); (ii) the
top most and bottom most positions would be positioned,
respectively, at the 12 o'clock position and the six o'clock
position (FIG. 70E); and the top zone and bottom zones would be
positioned, respectively, between the 10 o'clock to 2 o'clock
position and between the 4 o'clock and the 8 o'clock position.
[0339] Another advantageous design guide of several embodiment of
the integrated diagnostic cartridge are also shown with reference
to FIGS. 69A and 69B. Each cartridge embodiment includes a
cartridge perimeter 1011 within which are arranged the various
components of a specific cartridge embodiment. Additionally, there
is a reaction area perimeter 1601 which is shown in relation to the
reaction area 1600. The reaction area includes plugs 1770 (FIG.
69A) and assay chambers 1621 (FIG. 70A). As described herein, with
reference to FIGS. 69A and 70A, each one of the plurality of
individual assay chambers 1621 is in communication with an air
chamber 1631 (FIGS. 69A and 70A). In one embodiment, each air
chamber 1631 is closer to the cartridge perimeter 1601 than the
plug 1770 in each one of the plurality of individual assay
chambers. 1621. In another aspect, each one of the plurality of
individual assay chambers 1621 is in communication with an air
chamber 1631. Additionally, each plug 1770 in each one of the
plurality of individual assay chambers 1621 is within the reaction
area perimeter 1601 and each air chamber 1631 is outside of the
reaction area perimeter 1601. In another aspect, there is an
integrated diagnostic cartridge having a perimeter 1011 and a
reaction area perimeter 1601. Each one of the plurality of
individual assay chambers 1621 of the cartridge is in communication
with an air chamber 1631. Still further, each air chamber 1631 is
closer to the cartridge perimeter 1011 than the plug 1770 in each
one of the plurality of individual assay chambers 1621. Each air
chamber 1631 is located outside of the reaction area perimeter 1601
and each one of the plurality of individual assay chambers 1621
(and plug 1770) is within the reaction area perimeter 1601.
1. Loading Module
[0340] In one embodiment, a cartridge of the present invention
comprises a loading module configured to, e.g., accept a sample,
prevent the sample from spilling liquids outside the cartridge, and
optionally prepare the sample for lysis. The loading module defines
a sample volume used to perform a diagnostic test. In some
implementations, the loading module includes a metering chamber and
an overflow chamber to produce a metered sample volume. The loading
module may further comprise a mechanism for detecting a sufficient
sample volume is present in the device. A window may be included to
allow a user or an instrument to detect the mechanism indicative of
a sufficient sample volume. In another implementation, the loaded
sample is drawn into the cartridge using a converging channel.
[0341] In some embodiments the loading module comprises a sample
port assembly 1100 disposed within the cartridge. Optionally, the
sample port assembly 1100 is configured to produce a metered sample
of predetermined volume. Specifically, as discussed in greater
detail below regarding FIG. 71, the sample port assembly comprises
an entry port 1140, a fill chamber 1101, a metering chamber 1110,
metering channel 1113, an overflow chamber 1120, overflow channel
1122, vent 1165, and gas conduit 1150. The entry port 1140 of the
assembly defines an opening of the fill chamber to receive a
sample, wherein the fill chamber 1101 is in fluidic communication
with metering chamber 1110. A sample loader, such as a bulb,
syringe or pipette 1060, can be useful for loading a sample into
the cartridge.
[0342] The fill chamber has dimensions including a volume, said
volume being between 100 .mu.l and 15 ml, between 200 .mu.l and 7.5
ml, between 0.5 ml and 5 ml, between 0.5 ml and 3 ml, between 5 ml
and 10 ml, between 1 ml and 3 ml, between 0.5 and 1.5 ml. While the
fill chamber illustrated in the FIGS. 69-71 is configured to hold
up to 2.4 ml of fluid, cartridges of the invention may accommodate
larger sample volume by increasing the depth of the fill chamber.
Increasing the depth of the fill chamber results in an overall
thickness increase of the cartridge as a function of the depth of
the fill chamber. Advantageously, increasing the thickness of the
cartridge can allow for increased volume of chambers holding liquid
reagents and the waste chamber as discussed in greater detail
below. Thicker cartridges can be accommodated by the instrument
simply by changing the clamping position of the moving bracket
assembly 2040 as described herein.
[0343] When implemented, the metering chamber 1110 is in fluidic
communication with the fill chamber 1101 via a metering channel
1113. In some embodiments, the metering chamber includes a
mechanism for detecting a sample volume present within the metering
chamber, e.g., a buoyant ball 1114. The ball may be detected
through a sample window 1050 by a user or instrument 2000 for
indicating an adequate sample volume is in the metering chamber
1110 prior to executing a diagnostic test. Alternatively, the
meniscus of the fluid sample can be detected through the sample
window by a user or instrument. Referring to FIG. 68, the label
imaging system 2770, that captures an image of the patient label
area, can also capture an image of the meniscus or buoyant ball
1114 through the sample window 1050. The metering chamber has
dimensions including a volume, which can range from 0.1 to 10 ml,
from 0.5 to 5 ml, from or 1 to 3 ml.
[0344] When implementing a metering chamber, the cartridge
typically further comprises an overflow chamber 1120 configured to
capture excess sample that was loaded into the fill chamber 1101
and cannot be accommodated by fully filled metering chamber. The
overflow chamber is in fluidic communication with the metering
chamber 1110 through an overflow channel 1122, such that excess
sample flows though the overflow channel and is retained in the
overflow chamber. Taking advantage of the vertical orientation of
the cartridge with the instrument, the sample flows from the fill
chamber 1101 through the metering channel 1113 into the top of the
metering chamber 1110. Once the metering chamber 1110 is filled,
any excess fluid remaining in the fill chamber passes from the
metering channel 1113 to the overflow channel 1122 and then to the
overflow chamber 1120 without substantially entering the metering
chamber. This geometry can be advantageous for instances in which
the metering chamber comprises a chemical or enzymatic agent to
pretreat the sample prior to passing the sample on to the lysis
module. After metering, the meter fluid within the metering channel
can be withdrawn from the metering chamber though a channel at the
bottom of the metering chamber. In a preferred embodiment, the
lower bound of the metering chamber is angled toward the exit such
that gravity will assist in emptying the chamber.
[0345] The sample port assembly 1100 further comprises a structure
for separating the sample from the outside environment, e.g. a cap
1181 configured to be opened to permit addition of a sample and
then resealed prior to the sample being loaded to the device. Given
the pressurization inherent to the devices described herein, the
closure, i.e. the cap, preferably is air tight. The configuration,
as described herein, creates an airlock within the loading module
to prevent sample within fill chamber 1001 from passing to the
metering chamber 1110 until actuated by pressurization. Such an
airlock further prevents liquid from entering the metering chamber
when the device is tilted vertically.
[0346] In some embodiments, the loading module is configured to be
emptied using pneumatic force. Specifically, a sample is
transferred from the fill chamber 1001 to the metering chamber 1114
when pneumatic line 1171 to the fill chamber is pressurized. In one
implementation, the port is pressurized using constant pressure. In
another implementation the port is pressurized using a series of
applied pressure pulses each followed by a period of zero applied
pressure. In the instance where an excess sample volume is present,
excess sample enters overflow channel 1121 and is retained in the
overflow chamber 1120.
[0347] In an additional specific aspect implemented to mitigate or
eliminate bubble formation during sample processing, embodiments of
the integrated diagnostic cartridge may incorporate an antifoaming
agent that is mixed with the sample during sample processing. The
use of an antifoaming agent may be advantageous for reducing the
bubbling of protein-rich and/or surfactant-rich mixtures, e.g. a
sample combined with lysis reagents, in implementations where
pneumatic pressure drives fluid movement. Surfactant- and
protein-rich mixtures can easily generate bubbles and/or become
foamy. The resulting bubbles increase the difficultly of directing
fluids by means of gravity in a vertically oriented cartridge and
may interfere with downstream optical visualization of reactions
within assay chambers. In one embodiment, an antifoaming agent is
including within the loading module. The antifoaming agent may be
in liquid or dried form. Depending on the properties of a selected
antifoam agent, the antifoam agent may be used in full strength or
in any suitable concentration based on properties of the antifoam
agent, sample type, specific cartridge designs, cartridge volume
and other factors. In one embodiment, the antifoaming agent may be
diluted by combination with another a fluid such as an engineered
fluid or a solvent. In one exemplary embodiment, the antifoam agent
is the silicone fluid antifoam compound available commercially from
The Dow chemical company under the trade name XIAMETER.TM.
ACP-0001. In one exemplary embodiment, the antifoam agent is
diluted with an engineered fluid solvent available under the trade
name Novec.TM. 7000 commercially available from Sigma Aldrich, Inc.
In one specific implementation, 2 microliters of the antifoam agent
is combined with 100 microliters of the engineered fluid solvent
and dried to produce a dried antifoam agent. During cartridge
manufacturing, the dried antifoam agent is included in the sample
fill chamber. As a result, for integrated cartridge embodiments
that include a dried antifoam agent, when a user introduces a
sample into the fill chamber as part of the sample loading process
(see FIGS. 2A-2C), the sample is combined with the dried antifoam
agent. Thereafter, the cartridge is accepted as explained with
regard to FIGS. 3, 4A and 4B and will proceed to process the sample
combined with the antifoam agent.
[0348] In an alternative embodiment shown in FIG. 72, the loading
module may comprise an entry port in fluidic communication with a
reservoir containing a converging channel and one or more diverging
channels. Such a configuration enables a loaded sample to be drawn
to the distal end of the converging channel, wherein the sample
exits the converging channel and fills the one or more diverging
channels. Further description of a sample port configured to wick a
sample can be found in U.S. patent application titled "Vented
Converging Capillary Sample Port and Reservoir," filed 13 Sep.
2018, and assigned application Ser. No. 16/130,927, which is
incorporated by reference herein.
[0349] Lysis Module
[0350] The cartridge further comprises a lysis module configured to
disrupt cell walls and/or cells membrane to release inter-cellular
materials such as nucleic acids (DNA, RNA), protein or organelles
from a cell. In a one implementation, the lysis module comprises a
lysis chamber 1371 and a stir bar 1390. In one aspect, the lysis
module may further include a filter assembly to rid the sample of
cell debris after lysis to minimize the potential for clogging
downstream features in the cartridge.
[0351] In a preferred implementation, the lysis module comprises a
mixing assembly for combining the sample with one or more lysis
agents. In one embodiment, illustrated in FIG. 70A, the cartridge
includes a mixing assembly comprising a lysis chamber 1371 and a
stir bar 1390. The lysis chamber 1371 is configured to receive the
sample from a sample transfer channel 1386 through inlet 1373 that
is preferably located at or near the top of the lysis chamber 1371.
In some implementations, the cartridge comprises a chemical lysis
reagent prior to loading the sample. When the chemical lysis
reagent is a liquid, the reagent preferably is sealed into the
lysis chamber prior to use of the cartridge. In one such
embodiment, the channel leading to the lysis chamber 1386 and the
channel for draining the lysis chamber 1388 are both closed with a
frangible seal prior to use. When the cartridge is inserted into
the instrument and readied for use, the frangible seals are broken,
thus permitting pressurized air to transfer the sample into the
lysis chamber for lysis. The magnetic mixing assembly, as described
herein with regard to instrument 2000, is activated to rotate the
stir bar 1390 and thereby mix the sample with the one or more lysis
reagents.
[0352] When used with the balanced magnet mixing assembly 2300
described above, the stir bar 1390 need not be a permanent magnet.
In a preferred implementation, the stir bar is a composed of a
ferromagnetic material, which is not magnetized in the absence of
an external magnetic field. In some embodiments, the ferromagnetic
material of the stir bar is ferritic stainless steel or duplex
stainless steel. In additional embodiments, a relative magnetic
permeability of the stir bar can be between 500-1,000,000. The stir
bar can comprise any shape and/or volume. For example, the shape of
the stir bar can be selected from a group consisting of
cylindrical, spherical, and triangular-prism-shaped. Further
description of stir bar, lysis chamber and balanced magnetic mixing
assembly can be found in US patent publication 2019/0160443 A1,
titled "Magnetic Mixing Apparatus," which is incorporated by
reference herein.
[0353] In embodiments where one of the one or more lysis reagents
is a chemical agent, the ferromagnetic material is preferably
coated with an inert material to protect the stir bar from
corrosion and to deter release of iron, a suspected inhibitor of
amplification, from being released into the lysate. One of ordinary
skill in the art would be able to select an appropriate impermeable
material that would not interfere with magnetic flux through the
stir bar. Example materials include, but are not limited to PTFE,
parylene C, parylene D, functionalized perfluoropolyethers (PFPEs),
FEP, Xylan Fluoropolymer, epoxy, and urethane. Similarly, the
impermeable material can be applied to the stir bar by any method
known in the art, such as by tumble coating. In one implementation,
the ferromagnetic material of the stir bar is passivated prior to
coating. In a preferred implementation, the stir bar is
tumble-coated with a layer of parylene C between 20 .mu.m and 200
.mu.m thick.
[0354] By placing a ferromagnetic stir bar within the lysis chamber
1371 located in a gap between a driving magnet system 2310 and a
driven magnet system 2350 of instrument 2000, a magnetic dipole can
be induced across and within the stir bar. This dipole of the stir
bar effectuates a magnetic coupling between the stir bar 1390, the
one or more driving magnets of the driving magnet system 2310, and
the one or more driven magnets of the driven magnet system 2350.
Specifically, the introduction of the stir bar 1390 into the
magnetic field causes the stir bar to be attracted to the one or
more driving magnets and the one or more driven magnets. In
preferred embodiments in which a magnetic strength of the
corresponding driving magnet equals a magnetic strength of the
driven magnet, and the driving magnet magnetic axis is
substantially collinear with the driven magnet magnetic axis,
attraction of the stir bar to the driving magnet and driven magnet
causes the stir bar to be located roughly equidistant from driving
magnet and driven magnet. In an even further preferred embodiment
in which a center of the lysis chamber 1371 is located an equal
distance from the driving magnet system and the driven magnet
system, as a result of the attractive forces between the stir bar
and the one or more driving magnets and the stir bar and the one or
more driven magnets, the stir bar can be centered within the lysis
chamber thereby minimizing the amount of contact between the stir
bar and the bounding surface.
[0355] In some embodiments, the lysis chamber 1371 further
comprises beads. In such embodiments, mixing the fluid sample with
the beads promotes lysis of the one or more cells. Preferably, the
sample and beads, plus optionally one or more additional lysis
reagents, are stirred at least 500 rpm, at least 1000 rpm, at least
2000 rpm or at least 3000 rpm for at least 15 seconds 30 seconds,
60 seconds or 2 minutes to generate a lysed sample, or lysate.
Following mixing of the fluid sample with the beads, the lysate is
removed from the lysis chamber. In a preferred embodiment, the
beads are separated from the fluid sample in conjunction with the
sample being removed from the lysis chamber. To separate the beads
from the fluid sample, in some embodiments, bead filter channels
1387 are appended to the lysis chamber. The bead filter channels
are located along an edge of the lysis chamber and are configured
to retain the beads in the lysis chamber while allowing the fluid
sample to exit. Preferably the bead filter channels are located at
the bottom of the lysis chamber to take advantage of gravitation
forces to move the lysate from the lysis chamber without generating
bubbles or foam in the lysate. In a preferred implementation, a
cross sectional area of each bead filter channel comprises a first
dimension such that the beads are too large to enter the bead
filter channels, and a second dimension such that the beads are
unable to block fluid flow. In this way use of the bead filter
channels enables fluid to be drawn from the lysis chamber without
beads.
[0356] In some implementations, the lysis module further comprises
a process control. A process control establishes a factor of
confidence in a test result when executing a diagnostic test.
Controls are treated and tested in parallel with target pathogen
and are used to generate a predetermined expected result. When the
expected result is reported, one or more aspects of the diagnostic
test are confirmed to be working as intended, enabling the user of
to verify the diagnostic test as valid. However, when the
predetermined result is not obtained, one or more aspects of the
test does not meet the expected performance and would invalidate
the test results obtained from a cartridge. In one embodiment, a
cartridge can include a process control chamber 1130 comprising an
inlet 1131, an outlet 1132, and a control plug 1133 for doping a
sample with a process control. In one aspect, sample within the
metering chamber is flowed through the process control chamber to
dope a sample with a process control. In a further embodiment, the
process control is a positive control. Prior to mixing the sample
with at least one lysis agent, a process control can be added to
the sample. In such an implementation, one of the assay chambers
will comprise a primer set specific to a nucleic acid sequence
found in the process control. The process control chamber is
exemplified in FIGS. 69-71.
[0357] Preferably, the process control can function as a positive
control for lysis, purification and amplification within the
cartridges described herein. One exemplified process control is a
bacterial spore, such as a spore of a Bacillus species. Bacterial
spores typically are more difficult to lyse than any other target
cell and can therefore serve as a universal control for cell lysis.
Suitable spores can be comprised of any species of Bacillus,
including, e.g., Bacillus globgii, Bacillus atrophaeus, Bacillus
subtilis, and Bacillus stearothermophilus. Alternatively, a process
control can be added to the lysed sample prior to passing the lysed
sample through the porous solid support. Such a process control
would act as a positive control for purification and amplification,
but not lysis.
[0358] In some implementations, the cartridge further comprises one
or more filter assemblies 1330 to remove undesired cellular
material and debris from a sample by passing the sample through
filter assembly 1330. A filter assembly comprises at least a
filter, an inlet, and an outlet. In one implementation, the lysis
module comprises a filter assembly located before the lysis chamber
to filter a sample before lysing. In another embodiment, the lysis
module comprises a filter assembly placed after the lysis chamber
to filter a lysed sample. Specifically, the lysis module can
comprise one or more filter assemblies located downstream of the
lysis chamber.
[0359] FIGS. 73-75B illustrate a filter assembly according to one
embodiment described herein. FIGS. 74 and 75A provide section views
through the cartridge depicting a filter assembly 1330. FIG. 75B
illustrates an enlarged in view of the exemplary filter assembly
during operation when pressurized. In one embodiment, the filter
assembly 1330 comprises a filter 1331, an inlet via 1332, an outlet
via 1333, flow directors 1334, a filter plug 1336, and a pneumatic
interface cover adaptor 1172. Filter 1331 can be configured to
capture one substance, e.g., larger cells, more effectively, e.g.,
substantially more effectively, than another substance, e.g., a
liquid, such as a sample suspected on contained a target pathogen,
when the substances are exposed to the filter and at least one of
them is moved substantially therethrough. For example, a filter
1331 can enable the solid components, such as, e.g., cells, debris
or contaminant, to be separated from the liquid components of the
solution. Alternatively, a filter can enable larger solid
components, such as, e.g., proteinaceous aggregates, aggregated
cell debris, or larger cell, to be separated from smaller
components, e.g. virus, bacterial cells or nucleic acid, from a
solution. In aspects of this embodiment, a filter useful for
separating components contained in a solution can be, e.g., a
size-exclusion filter, a plasma filter, an ion-exclusion filter, a
magnetic filter, or an affinity filter. In other aspects of this
embodiment, a filter useful for separating components contained in
a solution can have a pore size of, e.g., 0.1 .mu.m, 0.2 .mu.m, 0.5
.mu.m, 1.0 .mu.m, 2.0 .mu.m, 5.0 .mu.m, 10.0 .mu.m, 20.0 .mu.m, 30
.mu.m, 40 .mu.m, 50 .mu.m, 60 .mu.m, 70 .mu.m, 80 .mu.m, 90 .mu.m,
100 .mu.m, or more. In yet other aspects of this embodiment, a
filter useful for separating components contained in a solution can
have a pore size of, e.g., at least 0.2 .mu.m, at least 0.5 .mu.m,
at least 1.0 .mu.m, at least 2.0 .mu.m, at least 5.0 .mu.m, at
least 10.0 .mu.m, at least 20.0 .mu.m, at least 30.0 .mu.m, at
least 40.0 .mu.m, at least 50.0 .mu.m, at least 60.0 .mu.m, at
least 70.0 .mu.m, at least 80.0 .mu.m, at least 90.0 .mu.m, or at
least 100.0 .mu.m. In still other aspects of this embodiment, a
filter useful for separating components contained in a solution can
have a pore size of, e.g., at most 0.1 .mu.m, at most 0.2 .mu.m, at
most 0.5 .mu.m, at most 1.0 .mu.m, at most 2.0 .mu.m, at most 5.0
.mu.m, at most 10.0 .mu.m, at most 20.0 .mu.m, at most 30.0 .mu.m,
at most 40.0 .mu.m, at most 50.0 .mu.m, at most 60.0 .mu.m, at most
70.0 .mu.m, at most 80.0 .mu.m, at most 90.0 .mu.m, or at most
100.0 .mu.m. In other aspects of this embodiment, a filter useful
for separating components contained in a solution can have a pore
size between, e.g., about 0.2 .mu.m to about 0.5 .mu.m, about 0.2
.mu.m to about 1.0 .mu.m, about 0.2 .mu.m to about 2.0 .mu.m, about
0.2 .mu.m to about 5.0 .mu.m, about 0.2 .mu.m to about 10.0 .mu.m,
about 0.2 .mu.m to about 20.0 .mu.m, about 0.2 .mu.m to about 30.0
.mu.m, about 0.2 .mu.m to about 40.0 .mu.m, about 0.2 .mu.m to
about 50.0 .mu.m, about 0.5 .mu.m to about 1.0 .mu.m, about 0.5
.mu.m to about 2.0 .mu.m, about 0.5 .mu.m to about 5.0 .mu.m, about
0.5 .mu.m to about 10.0 .mu.m, about 0.5 .mu.m to about 20.0 .mu.m,
about 0.5 .mu.m to about 30.0 .mu.m, about 0.5 .mu.m to about 40.0
.mu.m, about 0.5 .mu.m to about 50.0 .mu.m, about 1.0 .mu.m to
about 2.0 .mu.m, about 1.0 .mu.m to about 5.0 .mu.m, about 1.0
.mu.m to about 10.0 .mu.m, about 1.0 .mu.m to about 20.0 .mu.m,
about 1.0 .mu.m to about 30.0 .mu.m, about 1.0 .mu.m to about 40.0
.mu.m, about 1.0 .mu.m to about 50.0 .mu.m, about 2.0 .mu.m to
about 5.0 .mu.m, about 2.0 .mu.m to about 10.0 .mu.m, about 2.0
.mu.m to about 20.0 .mu.m, about 2.0 .mu.m to about 30.0 .mu.m,
about 2.0 .mu.m to about 40.0 .mu.m, about 2.0 .mu.m to about 50.0
.mu.m, about 5.0 .mu.m to about 10.0 .mu.m, about 5.0 .mu.m to
about 20.0 .mu.m, about 5.0 .mu.m to about 30.0 .mu.m, about 5.0
.mu.m to about 40.0 .mu.m, about 5.0 .mu.m to about 50.0 .mu.m,
about 10.0 .mu.m to about 20.0 .mu.m, about 10.0 .mu.m to about
30.0 .mu.m, about 10.0 .mu.m to about 40.0 .mu.m, about 10.0 .mu.m
to about 50.0 .mu.m, about 10.0 .mu.m to about 60.0 .mu.m, about
10.0 .mu.m to about 70.0 .mu.m, about 20.0 .mu.m to about 30.0
.mu.m, about 20.0 .mu.m to about 40.0 .mu.m, about 20.0 .mu.m to
about 50.0 .mu.m, about 20.0 .mu.m to about 60.0 .mu.m, about 20.0
.mu.m to about 70.0 .mu.m, about 20.0 .mu.m to about 80.0 .mu.m,
about 20.0 .mu.m to about 90.0 .mu.m, about 20.0 .mu.m to about
100.0 .mu.m, about 30.0 .mu.m to about 40.0 .mu.m, about 30.0 .mu.m
to about 50.0 .mu.m, about 30.0 .mu.m to about 60.0 .mu.m, about
30.0 .mu.m to about 70.0 .mu.m, about 30.0 .mu.m to about 80.0
.mu.m, about 30.0 .mu.m to about 90.0 .mu.m, about 30.0 .mu.m to
about 100.0 .mu.m, about 40.0 .mu.m to about 50.0 .mu.m, about 40.0
.mu.m to about 60.0 .mu.m, about 40.0 .mu.m to about 70.0 .mu.m,
about 40.0 .mu.m to about 80.0 .mu.m, about 40.0 .mu.m to about
90.0 .mu.m, about 40.0 .mu.m to about 100.0 .mu.m, about 50.0 .mu.m
to about 60.0 .mu.m, about 50.0 .mu.m to about 70.0 .mu.m, about
50.0 .mu.m to about 80.0 .mu.m, about 50.0 .mu.m to about 90.0
.mu.m, or about 50.0 .mu.m to about 100.0 .mu.m. An artisan of
ordinary skill may select an appropriate filter based on
considerations such as sample type and the target pathogen of
interest.
[0360] In certain implementations, the filter can be a depth
filter. Depth filters consist of a matrix of randomly oriented,
bonded fibers that capture particulates within the depth of the
filter, as opposed to on the surface. The fibers in the depth
filter can be comprised of glass, cotton or any of a variety of
polymers. Exemplified depth filter materials may include, type
GF/F, GF/C and GMF150 (glass fiber, Whatman), Metrigard.RTM. (glass
fiber, Pall-Gelman), APIS (glass fiber, Millipore), as well as a
variety of cellulose, polyester, polypropylene or other fiber or
particulate filters, so long as the filter media can retain a
sufficient contaminant to allow further processing of the
sample.
[0361] In alternate implementations, the size-exclusion filter can
be a membrane filter, or mesh filter. Membrane filters typically
performs separations by retaining particles larger than its pore
size on the upstream surface of the filter. Particles with a
diameter below the rated pore size may either pass through the
membrane or be captured by other mechanisms within the membrane
structure. Membrane filters can support smaller pore sizes,
including small enough to exclude bacterial cells. Membrane filters
can be used to concentrate solutions, e.g. bacterial cell
suspensions, by filtering a first larger volume through the
membrane filter, thereby holding the bacterial cells to the
upstream surface of the membrane filter (or suspended in residual
fluid retained on the upstream side of the filter). The bacterial
cells can then be resuspended in a second small volume of fluid by
either passing the suspension fluid in the reverse direction to
float the bacterial cells off the membrane surface or by washing
the suspension fluid across the upstream surface of the filter to
wash the bacterial cells off the filter. Exemplified membranes may
include, polyethersulfone (PES) membranes (e.g., Supor.RTM. 200,
Supor.RTM. 450, Supor.RTM. MachV (Pall-Gelman, Port Washington,
N.Y.), Millipore Express PLUS.RTM. (Millipore)). Other possible
filter materials may include, HT Tuffryn.RTM. (polysulfone), GN
Metricel.RTM. (mixed cellulose ester), Nylaflo.RTM. (Nylon), FP
Verticel (PVDF), all from Pall-Gelman (Port Washington, N.Y.), and
Nuclepore (polycarbonate) from Whatman (Kent, UK).
[0362] In various embodiments, the filter can be enclosed by a
frame, a chamber, or any other housing for containing the filter
material. In some embodiments, the frame holding the filter can be
integrated into another cartridge structure adjacent to the filter
assembly. In one implementation, the filter assembly comprises a
filter fixedly attached, e.g., laser welded, to a feature side 1007
of a fluidics card 1001, as shown in FIGS. 74, 75A, and 75B.
Further, the illustrated embodiment depicts that the frame
enclosing filter 1331 is provided by pneumatic interface cover
adaptor 1172. Such arrangement produces deformation space 1335
which is formed between the filter 1331 and the pneumatic interface
cover adaptor 1172. The deformation space is most readily apparent
in FIG. 75A, wherein the filter 1331 flush against fluidic card
1001 prior to pressurizing the cartridge during operation. In
another implementation, the filter frame, i.e. the pneumatic
interface cover adaptor 1172, is configured with a plurality of
flow directors 1334 integrally formed within the body of the
pneumatic interface cover adaptor for directing a filtered liquid
to an outlet via.
[0363] In many implementations, a filter assembly is configured to
filter a liquid, e.g. a sample or lysate, when pressurized by the
instrument pneumatic subsystem. FIG. 73 shows a top view of the
exemplary filter assembly 1330 described herein. An inlet via 1332
provides the opening for a fluid to enter the filter assembly. As
best shown in the cross section view of FIG. 74, inlet 1332 permits
a liquid to advance from fluidic side 1006 to the feature side
1007. In the exemplary embodiment where the filter is fixedly
attached to the fluidic card, fluid pressure is generated as a
result of the liquid entering the filter assembly 1330 and causes
filter 1331 to expand, i.e. deflect away from feature side of
fluidic card 1101. FIG. 75B is an enlarged section view of the
filter assembly shown in FIG. 75A when pressurized during cartridge
operation. Expansion of the filter is accommodated by deformation
space 1335 (FIG. 75A), such that the filter 1331 is permitted to
expand until contacted by flow directors 1334. The contacting of
filter 1331 against flow directors 1334 transforms deformation
space 1335 into a plurality of defined channels, wherein three
surfaces are formed by the pneumatic interface cover adaptor 1172
(specifically, two surfaces are formed by flow directors 1334), and
one surface of each channel is formed by the porous filter 1331.
Accordingly, the active pressurization advances the liquid through
the filter. Substances, e.g., a sample, such as a lysed sample, is
transmitted through the filter while other substances, e.g.,
particles, such as larger cells or cell debris, is prevented from
passing therethrough to produce a filtered sample. The resulting
filtered liquid is collected in the plurality of channels formed by
the flow directors 1134 and is directed toward outlet 1333 shown at
the bottom of the filter assembly in FIG. 73. Further, filter 1331
includes a cutout around outlet via 1333 to permit the filtered
sample to enter the outlet via and travel from the feature side
1007 to the fluidic side 1006 of fluidic card 1001.
[0364] In other aspects, the pneumatic interface cover adaptor can
be a structure which receives pressurized air from the instrument
pneumatic interface 2100. In various embodiments, the pneumatic
interface cover adaptor is configured to hold a filter plug 1336
for filtering the pressurized air input. As illustrated in FIG.
75A, pressurized air enters input via 1195 of the cartridge
pneumatic interface 1170 and is filtered by filter plug 1336 before
exiting the main pneumatic via 1193 and pneumatic via 1194, such
that main pneumatic via 1193 is fluidically coupled to the main
pneumatic line 1171 and pneumatic via 1194 is fluidically coupled
to pneumatic line 1178.
2. Purification Module
[0365] The cartridges of the invention further comprise a
purification module for capturing nucleic acids from a lysed
sample. In one aspect the purification module is configured to
purify a lysed sample using a rotary valve, wherein the rotary
valve comprises a porous solid support. The porous solid support
captures nucleic acid while allowing the remainder of the sample
and liquid waste to be directed to a waste collection element. In
such an embodiment, the device additionally includes reagent
reservoirs to store on-board reagents necessary for sample
purification.
[0366] In one aspect, the purification module comprises one or more
rotary valves comprising an integrated flow channel containing a
porous solid support for filtering, binding and/or purifying
analytes within a fluid stream. In one implementation, the rotary
valve comprises a stator 1450 comprising a stator face and a
plurality of passages 1454, each passage comprising a port 1453 at
the stator face; a rotor 1410 operably connected to the stator and
comprising a rotational axis, a rotor valving face, and a flow
channel having an inlet 1441 and an outlet 1442 at the rotor
valving face, wherein the flow channel comprises a porous solid
support 1445; and a retention element 1490 biasing the stator and
the rotor together at a rotor-stator interface to form a fluid
tight seal.
[0367] The rotor usable in the devices and methods described herein
typically include a first face, e.g., a valving face 1412, and a
second face, e.g., outer face 1413 (not shown), opposite the first
face. The valving face and/or outer face can each be planar or have
a planar portion. In such circumstances, the rotational axis of the
rotor is perpendicular or substantially perpendicular to the
valving face and/or the outer face. Also, in a cylindrical rotor, a
rotational axis can be defined by and/or be a portion of the rotor
located equidistant or substantially equidistant from all points on
an outermost radial edge of the rotor or on an outermost radial
edge of the rotor and/or outer face. The rotor valve face 1412
optionally comprises a gasket 80. The valving face typically also
will comprise one or more fluid handling features, such as an inlet
and/or outlet to a flow channel, a fluidic connector or a fluidic
selector. In some embodiments, it may be advantageous to use
fluidic handling features, e.g. a connector or selector further
described herein, integrally formed within the rotor valving face
or a gasket to deliver exact volumes of fluid to selected portions
of the cartridge. In one exemplary embodiment, in operation, a
rotor may be indexed to a position allowing fluidic communication
between one stator port and a fluidic connector comprising a known
connector volume. A fluid may be introduced into the fluidic
connector within the rotor valving face, via the stator port, thus
filling the fluidic connector volume with fluid against a hard
stop. Subsequently, the rotor may be indexed to a second position
to transfer the volume of fluid within the connector to a desired
cartridge location when fluidic communication is established
between an inlet and outlet stator port. Such embodiment is
advantageous in methods where delivery of exact fluid volumes is
desired, e.g. aliquoting and performing dilutions.
[0368] In some aspects, a rotary valve includes a gasket between
the stator face and the rotor valving face. A gasket is a
mechanical seal that fills a space between two or more mating
surfaces of objects, generally to prevent leakage from or into the
joined objects while the gasket is under compression. In various
aspects, the gasket is composed, e.g., entirely composed, of an
elastic and/or compressible material. In some versions, the rotor
comprises the gasket and in other versions, the stator comprises
the gasket. In embodiments wherein the rotor comprises a gasket, is
fixedly, e.g., adhesively, attached to a rotor and forms a sliding
interface along the stator. Also, in those embodiment where the
stator comprises the gasket, the gasket is fixedly, e.g.,
adhesively, attached to a stator and forms a sliding interface
along the rotor.
[0369] One embodiment of a rotary valve gasket is shown in FIGS.
76B and 76C. Specifically, FIGS. 76B and 76C illustrate a gasket
1480 configured to slidably engage a stator. The gasket 1480 also
includes a first inlet 1484 aligned with the inlet 1441 of the
rotor's flow channel, and a outlet 1485 aligned with the outlet
1442.
[0370] As used herein, a fluid handling feature is a physical
structure in the rotor or gasket that, when aligned with two stator
ports, fluidically connect the two ports and associated passages to
form a continuous fluidic path. In some embodiments, the fluid
handling feature is a fluidic connector 1486. A fluidic connector
is configured to fluidically connect a first stator port to a
second stator port. In implementations, such as illustrated in
FIGS. 76B and 76C, the fluidic connector is an elongated groove in
the rotor or gasket with the longest dimension along a line
radiating from the center of the rotor. Such a radially aligned
fluidic connector is capable of sequentially connecting a plurality
of pairs of stator ports, wherein each of the plurality of pairs
has one proximal port and one distal port, wherein all proximal
ports are one distance from the rotational axis and all distal
ports are a second, larger, distance from the axis. Alternatively,
a fluidic connector may be configured to fluidically connect a
first stator port to a second stator port along the same arc, i.e.
stator ports along different points around the rotational axis with
an equal radial distance. In some embodiments, the fluid handling
feature is a flow channel, wherein when the flow channel inlet is
aligned with one stator port and the flow channel outlet is aligned
with a second stator port, the full volume of the flow channel
fluidically connects the two stator ports. Accordingly, the flow
channel can act as a fluidic connector. In some embodiments, the
fluid handling feature is a fluidic selector 77 having a first
portion that is an arc with all points along the first portion
being equidistant from the rotational axis, and a second portion
extending radially toward or away from the center of the rotor.
[0371] One aspect of the invention provides a rotary valve having a
rotor wherein the rotor valving face comprises a first fluidic
connector, wherein in a first rotor position a first port of the
stator is fluidically connected to a second port of the stator via
the first connector. In a second rotor position, a third port is
fluidically connected to a fourth port via the first fluidic
connector. Optionally, in a third rotor position, a fifth port is
fluidically connected to a sixth port via the first fluidic
connector. In one implementation, the fluidic connector is an
elongate groove. In another implementation, the fluidic connector
is a flow channel in the rotor.
[0372] In various aspects, a gasket is substantially cylindrical
and/or disk-shaped wherein the distance between the axis of
rotation and the outer circumference of the gasket is greater than
the distance between the axis of rotation to the most distant port
on the stator. In some embodiments, such as illustrated in FIGS.
76B and 76C, the gasket is annular having an outer circumference
beyond the most distant stator port as described above and wherein
the distance between the axis of rotation and inner circumference
of the annulus is less than the distance between the axis and the
most proximal stator port. A gasket can have an outer
cross-sectional diameter such as any of the rotor diameters
provided herein. A gasket can have an out cross-sectional diameter,
for example, of 100 mm or less, such as 45 mm or less, such as 50
mm or less, such as 40 mm or less, such as 20 mm or less, such as
10 mm or less. The inner and outer gasket diameters can range, for
example, from to 1 mm to 100 mm, 3 mm to 50 mm, 3 mm to 25 mm or 5
mm to 35 mm. A gasket can also have a thickness such as any of the
thicknesses of device components provided herein, such as 10 mm or
less, such as 5 mm or less, such as 1 mm or less or 1 mm or more, 5
mm or more, or 10 mm or more.
[0373] In various aspects, a gasket is composed, e.g., entirely
composed, of one or more polymeric materials (e.g., materials
having one or more polymers including, for example, plastic and/or
rubber and/or foam). A gasket can also be composed of a silicone
material. A gasket can be composed of any of the elastic materials
provided herein. Gasket materials of interest include, but are not
limited to: polymeric materials, e.g., plastics and/or rubbers,
such as polytetrafluoroethene or polytetrafluoroethylene (PTFE),
including expanded polytetrafluoroethylene (e-PTFE), polyester
(Dacron.TM.), nylon, polypropylene, polyethylene, polyurethane,
etc., or any combinations thereof. In some embodiments, the gasket
comprises Neoprene (polychloroprene), a polysiloxane, a
polydimethylsiloxane, a fluoropolymer elastomer (e.g. VITON.TM.), a
polyurethane, a thermoplastic vulcanizate (TPV, such as
Santoprene.TM.), butyl, or a styrenic block copolymer
(TES/SEBS).
[0374] According to some embodiments, a gasket includes one or more
apertures fully penetrating the thickness of the gasket. In those
implementations, wherein the gasket is affixed to the stator, the
gasket comprises an aperture corresponding to and aligned with each
stator port, to permit fluid flow therethrough. In implementations
wherein the gasket is affixed to the rotor, the gasket comprises an
aperture corresponding to and aligned with each of the flow channel
inlet and outlet, if present, to permit flow across the
rotor-stator interface. In alternative embodiments, the gasket may
include one or more apertures, structures, or geometries partially
formed therethrough. Such embodiment may be useful for delivering
small precise volumes of fluid to various locations within the
cartridge. FIGS. 12, 17A, 19A, 19C, and 29 illustrate an exemplary
embodiment of a rotary valve 1400 in an operational position within
a vertically oriented diagnostic cartridge. For the purposes of
understanding, the cross section view of FIG. 76A illustrates an
orientation of the rotor and the stator not in an operational
position. However, associated cartridge axis are depicted alongside
the rotor cross section to demonstrate the operational orientation
and is further described below. The rotor may be indexed
rotationally, with respect to to the stator, e.g. a fluidic card,
such that a fluid pathway is established through a plurality of
features formed within a stator, a rotor and optionally a gasket.
As a result, a flow channel 1440 within the rotor body 1411
provides fluid communication with the porous solid support 1445
within the solid support chamber 1446 to purify a lysed sample and
produce an enriched nucleic acid when introduced into the solid
support chamber. In many embodiments, fluid communication to the
flow channel 1440 is accessed when stator ports within the stator
face align with fluid conduits integrally formed within the rotor.
In a further embodiment, a gasket, may interpose the rotor and
stator at a rotor-stator interface to facilitate a fluid-tight
seal.
[0375] Specifically, the exemplary fluid pathway in FIG. 76A begins
at the stator 1450. A lysed sample first enters at a first stator
fluid passage 1454a and stator port 1453a and advances through the
gasket 1480 via the gasket inlet 1484. The fluid enters the rotor
body 1411 via inlet 1441 and traverses through the first fluid
conduit 1443. The outlet of the first conduit 1443 leads to a fluid
pathway defined by a spacing between the rotor upper surface and a
bottom surface of the cap cover 1430. The upper surface of the
rotor body in this region is shaped to include a short channel to
provide a portion of desired flow path between the first fluid
conduit 1443 and the solid support chamber 1446. The partial flow
path is completed when the cap cover 1430 is secured to the rotor
top surface, thus allowing a filtered sample exiting the first
fluid conduit to travel in a direction along the cartridge width
axis 1025 (additionally see FIGS. 68-72) when oriented in an
operational position as shown in FIG. 29. Next, the fluid enters
the solid support chamber 1446 containing the porous solid support
1445. Fluid passes through the porous solid support 1445 to
generate a purified or enriched nucleic acid and is directed to the
bottom of solid support chamber to the second conduit 1444 and
exits via a rotor outlet 1442. Fluid exits the rotor, passes
through the gasket 1480 via outlet 1485 and enters the stator, i.e.
fluidic card 1001, through stator opening 1453b and fluid passage
1454b.
[0376] The rotary valve for use in the cartridges of the invention
are described in greater detail in U.S. patent application titled
"Rotary Valve," filed 15 Feb. 2018, and assigned application Ser.
No. 15/898,064, and in international patent application, also
titled "Rotary Valve," filed 15 Feb. 2019 and assigned application
no. PCT/US2019/018351, each of which is incorporated by reference
herein.
[0377] As an integral part of the rotor, a flow channel is
configured for rotational motion, rotating with the other portions
of the rotor with respect to other valve aspects, such as a stator.
In a preferred implementation, the flow channel is not concentric
with the rotational axis of the rotor. As illustrated in FIG. 76A,
a flow channel can include one or more inlets 1441 and one or more
outlets 1442 and provide fluidic communication between the inlet
and the outlet. In a preferred implementation, each flow channel
will comprise a single inlet and a single outlet. The inlet and
outlet typically, but not necessarily, will adopt the same form as
a cross-section of the flow channel immediately adjacent to that
inlet or outlet. The inlet and/or outlet can be circular,
rectangular or any other appropriate shape consistent with forming
fluid-tight fluidic connections within the valve interface.
[0378] The rotor can be configured to hold one or more porous solid
supports. As shown in FIG. 77, each support chamber 1446a-1446d may
vary in shape, size, dimension, volume or by the content of the
solid support contained in a specific support chamber 1446.
[0379] Optionally, the flow channel also includes a flow channel
spacer 1449 for spacing a porous solid support from a surface,
e.g., a bottom surface, of a porous solid support chamber 1446. In
various embodiments, a flow channel spacer can be crescent shaped
and extend in an arcuate manner along its length. The flow channel
spacer can facilitate fluid flow through the outlet by preventing
the porous solid support, e.g., beads or fibers, from physically
blocking the exit from the solid support chamber. Illustrious flow
channel spacer variations include: (a) a flow channel spacer may be
segmented rather than a continuous structure; (b) a flow channel
spacer may include more than one structure along a surface of the
solid support chamber such as a sidewall or bottom; (c) a flow
channel spacer may be spaced apart from the chamber exit or
terminate at the edge of the exit; and (d) a flow channel spacer
may be raised above a chamber interior surface such as a bottom or
a sidewall, recessed into a chamber interior surface such as a
bottom or a sidewall.
[0380] Porous solid supports can be configured to capture and
thereby concentrate analyte, e.g., concentrate analyte from a first
concentration to a second concentration, from a sample flowed
therethrough by an amount of analyte concentration, such as
1000.times. or more in any of the time amounts described herein,
such as in 30 min or less, such as 1 hour or less. In various
embodiments, a porous solid support is bounded, such as bounded at
an upstream face and/or a downstream face by a frit.
[0381] In some aspects, a porous solid support can be a selective
membrane or a selective matrix. As used herein, the terms
"selective membrane" or "selective matrix" as referred to herein is
a membrane or matrix which retains one substance, e.g., an analyte,
more effectively, e.g., substantially more effectively, than
another substance, e.g., a liquid, such as portions of a sample
other than the analyte and/or water and/or buffer, when the
substances are exposed to the porous solid support and at least one
of them is moved at least partially therethrough. For example, a
porous solid support, such as a selective matrix, having a
biological sample flowed therethrough can retain an analyte, e.g.,
nucleic acids, while the remainder of the sample passes through the
porous solid support.
[0382] Examples of porous solid supports include, but are not
limited to: alumina, silica, celite, ceramics, metal oxides, porous
glass, controlled pore glass, carbohydrate polymers,
polysaccharides, agarose, Sepharose.TM., Sephadex.TM., dextran,
cellulose, starch, chitin, zeolites, synthetic polymers, polyvinyl
ether, polyethylene, polypropylene, polystyrene, nylons,
polyacrylates, polymethacrylates, polyacrylamides, polymaleic
anhydride, membranes, hollow fibers and fibers, or any combinations
thereof. The choice of matrix material is based on such
considerations as the chemical nature of the affinity ligand pair,
how readily the matrix can be adapted for the desired specific
binding.
[0383] In some embodiments, a porous solid support is a polymeric
solid support and includes a polymer selected from polyvinylether,
polyvinylalcohol, polymethacrylate, polyacrylate, polystyrene,
polyacrylamide, polymethacrylamide, polycarbonate, or any
combinations thereof. In one embodiment, the solid support is a
glass-fiber based solid support and includes glass fibers that
optionally can be functionalized. In some embodiments, the solid
support is a gel and/or matrix. In some embodiments, the solid
support is in bead, particle or nanoparticle form.
[0384] A myriad of functional groups can be employed with the
subject embodiments to facilitate attachment of a sample analyte or
ligand to a porous solid support. Non-limiting examples of such
functional groups which can be on the porous solid support include:
amine, thiol, furan, maleimide, epoxy, aldehyde, alkene, alkyne,
azide, azlactone, carboxyl, activated esters, triazine, and
sulfonyl chloride. In one embodiment, an amine group is used as a
functional group. A porous solid support can also be modified
and/or activated to include one or more of the functional groups
provided that facilitate immobilization of a suitable ligand or
ligands to the support.
[0385] In some embodiments, a porous solid support has a surface
which includes a reactive chemical group that is capable of
reacting with a surface modifying agent which attaches a surface
moiety, such as a surface moiety of an analyte or ligand of a
sample, to the solid support. A surface modifying agent can be
applied to attach the surface moiety to the solid support. Any
surface modifying agent that can attach the desired surface moiety
to the solid support may be used in the practice of the present
invention. A discussion of the reaction a surface modifying agent
with a solid support is provided in: "An Introduction to Modern
Liquid Chromatography," L. R. Snyder and Kirkland, J. J., Chapter
7, John Wiley and Sons, New York, N.Y. (1979), the entire
disclosure of which is incorporated herein by reference for all
purposes. The reaction of a surface modifying agent with a porous
solid support is described in "Porous Silica," K. K. Unger, page
108, Elsevier Scientific Publishing Co., New York, N.Y. (1979), the
entire disclosure of which is incorporated herein by reference for
all purposes. A description of the reaction of a surface modifying
agent with a variety of solid support materials is provided in
"Chemistry and Technology of Silicones," W. Noll, Academic Press,
New York, N.Y. (1968), the entire disclosure of which is
incorporated herein by reference for all purposes.
[0386] As described above, in some versions of the rotary valve,
the valve includes a gasket between the stator face and the rotor
valving face, and a structure for maintaining the valve in a
storage configuration wherein the rotor and stator are spaced apart
such that the gasket is not compressed at the rotor-stator
interface. Gaskets, typically formed of compressible, elastomeric
materials, are susceptible to compression-set and adhesion to
adjacent surfaces if stored under compression for extended periods
of time. Accordingly, described herein is a preferred
implementation of a rotary valve that includes a threaded rotor and
a threaded retention ring for maintaining a gap between the gasket
and at least one of the rotor and stator, thereby preventing the
gasket from sealing against at least one of the rotor and stator,
wherein when threaded rotor is rotated, the gasket seals the rotor
and stator together in a fluid tight manner. In a preferred
embodiment, the mechanism for sealing the rotor and stator together
in a fluid tight manner is irreversible.
[0387] As best seen in FIGS. 78 and 79, a retention ring 1491
includes a threaded portion 1492. In the illustrated embodiment,
the threaded portion 1492 includes threads. A rotor includes an
outer wall having a threaded portion 1411. In the illustrated
embodiment, the threaded portion includes grooves 1411 that
correspond to the threads 1492 of the retention ring. In the
shipping configuration shown in FIGS. 78 and 79, a biasing element
1496 maintains engagement between threads 1492 and grooves 1411
aiding in maintaining the desired gap between the rotor sealing
surface (gasket 1480) and the stator valving face 1452. As best
seen in FIG. 79, the top of rotor cap 1430 is substantially flush
with an upper surface of retention ring 1491 maintaining a
low-profile rotary valve design factor. Rotation of the rotor
relative to the retention ring 1491 moves the rotor towards the
stator and into the operational configuration shown in FIGS. 80 and
81. The transition out of the storage configuration is clear in
this view, as the rotor cap is recessed below the top surface of
the retention ring 1491 and the gasket 1480 provides a fluidic seal
between the rotor and the stator. Also visible in FIG. 81 it is
that the rotor is detached from the threaded portion 1492 of the
retention ring 1491. Movement of the threaded rotor into this
position ensures that the rotor is free to be indexed relative to
the stator as described herein.
[0388] In consideration of FIGS. 79-81, there is provided a rotary
valve comprising a rotor 1410 having a rotational axis, a rotor
valving face, an outer face opposite the rotor valving face.
Additionally, there is a stator 1450 having a stator valving face
positioned opposite the rotor valving face. The rotary valve also
includes a retention element 1490 biasing the rotor and stator
towards one another comprising a retention ring 1491 and a biasing
element 1496. The rotary valve is maintained in a shipping
configuration while a threaded portion of the retention ring is
engaged with a threaded portion of the rotor. In one configuration,
a relative motion between the rotor and the stator produces a fluid
tight arrangement between the rotor valving surface and the stator
valving surface or the relative motion between the rotor and the
stator is rotation of the rotor so as to move the rotor along the
threaded portion of the retention ring until released to seal
against the stator. As such, a rotary valve having a threaded rotor
used for engagement in a shipping configuration may be configured
to transition to provide a fluid tight seal within the rotary valve
with a rotation of less than one revolution, half a revolution, a
quarter of a revolution or one-eighth of a revolution of the
threaded rotor. Still further, it is to be appreciated that while
the threaded components of a threaded rotor rotary valve are
engaged a gasket disposed between the rotor valving face and the
stator valving face does not form a fluid tight seal with the
stator valving surface.
[0389] In an alternative embodiment, one or more displaceable
spacers may configured for preventing the gasket from sealing
against at least one of the rotor and the stator. When the spacers
are displaced, e.g., displaced from a pre-activated configuration
to an activated configuration, the gasket seals the rotor and
stator together in a fluid-tight manner. According to the subject
embodiments, displaceable spacers can be part of and/or integral
with a stator or rotor.
[0390] In one implementation, in the storage configuration, a
displaceable spacer comprises a plurality of tabs that contact a
lip on the rotor to hold the rotor away from the stator. Each of
the plurality of tabs is displaceable to disengage from the lip in
the operational configuration. In one embodiment, the displaceable
spacer comprises a plurality of tabs displaceable from a first tab
configuration, i.e. storage configuration, to a second tab
configuration, i.e. operational configuration. In a further
embodiment, the stator comprises the plurality of tabs.
Displaceable spacers, such as tabs, can be shaped substantially as
a three-dimensional box or rectangular shape so as to contact a lip
of a rotor during a storage configuration. To facilitate
displacement of the spacer when transitioning from a storage
configuration to an operational configuration, the rotor can
comprise one or more ramping portions adjacent to the lip on the
rotor to interact with each of the spacers. Specifically, the
ramping portions exert a force in an outward or substantially
outward direction, such as a direction away from a rotational axis
of a rotor, on the displaceable spacers, thus allowing the
retention element to bias the rotor and stator to form a
fluid-tight seal in the operational configuration.
[0391] In one aspect of the invention, the purification module
stores on-board liquid reagents in reagent reservoirs for easy
delivery of reagents used herein to prepare a sample suspected of
containing a target pathogen. Such reagent reservoirs can be of any
structure formed in the fluidics card 1001 configured to contain
liquid therein, such that the fluidics card forms a first bounding
surface. In one embodiment reagent reservoirs may comprise a second
bounding surface provided by one or more sealing films fixedly
attached, e.g. welded, to the fluidics side 1006 of the fluidics
card (see, e.g. FIG. 89, discussed in greater detail below). In one
implementation, reagent reservoirs are sealed by frangible seals to
define a receptacle for long-term storage of the liquid reagents
contained therein. Reagent reservoirs are rendered fluidically
active when actuated to break the frangible seal allowing fluid to
be emptied from the reagent reservoir and redirected throughout the
cartridge. In one implementation, reagent reservoirs are in direct
fluidic communication with main pneumatic line 1171 to deliver
pressurized air to a reagent reservoir inlet to empty the contents
of the reagent reservoir. The reagent reservoir additionally
includes a reagent reservoir outlet to transfer the contents of the
reagent reservoir from its holding receptacle to appropriate sample
processing locations on the cartridge.
[0392] In one implementation, the device comprises one or more
frangible seals configured to seal the device and allow on board
liquids to be stored therein. In some implementations, breaking the
one or more frangible seals renders the device fluidically active
to allow liquid substances contained therein, e.g., lysis buffer or
wash buffer, to be directed through the fluidic network of
channels. A variety of configurations of frangible seals for
closing microfluidic channels are well known in the art, any of
which can be used in conjunction with the cartridges disclosed
herein. For example, a description of frangible seals can be found
in U.S. Pat. Nos. 10,183,293, 10,173,215, 9,309,879, 9,108,192,
U.S. Patent Application publication 2017/0157611, and published
European Patent Application 3406340 A1, all of which are
incorporated by reference herein.
[0393] In one implementation, a reagent reservoir is configured to
store a wash buffer to form a wash buffer reservoir 1475 (see, e.g.
FIG. 70A). Wash buffer removes unbound or loosely bound
contaminants from a porous solid support while target analyte, e.g.
nucleic acids, remain bound to the porous solid support. In one
embodiment, the wash buffer reservoir is in direct fluidic
communication with the main pneumatic line 1171 such that
pressurized air can be sent to the wash buffer reservoir through a
wash reservoir inlet 1476 to empty the wash buffer reservoir. The
wash buffer exits the wash buffer reservoir through wash outlet
1477 and is transferred to the porous solid support chamber within
the rotary valve, thus washing the porous solid support of
contaminants. Wash buffer exits the porous solid support chamber,
and the wash buffer containing cell debris is then conveyed to the
waste collection element 1470.
[0394] In another one implementation, a reagent reservoir is
configured to store an elution buffer to form an elution buffer
reservoir 1500 (see, e.g. FIG. 70A). Elution buffer enables the
release of target nucleic acids bound to a porous solid support in
a sample to form a purified sample. The elution buffer reservoir
further includes an elution reservoir inlet 1501 from which
pressurized air can enter the elution buffer reservoir to empty the
contents. The inlet of the elution buffer reservoir can be in
direct fluidic communication with main pneumatic line 1171 to
deliver the pressurized air. The elution buffer is emptied from the
elution buffer reservoir through an elution reservoir outlet 1502
and flowed over the porous solid support in the rotary valve to
release target nucleic acid to produce an eluate, or eluted nucleic
acid. In another embodiment, the eluate may subsequently be
directed to a rehydration chamber, described in further detail
below.
a) Waste Collection Element
[0395] The waste collection element 1470 is configured for
receiving and storing liquid waste in a secure manner. In some
embodiments, the waste collection element 1470 comprises a waste
inlet 1471, a vent channel 1472, vent 1473, at least one waste
outlet 1474, and outlet filter plug 1478. Accordingly, liquid waste
is directed to the waste collection element from channel 1362
through a waste inlet 1472. The waste collection element will
include at least one, but preferably more than one, waste outlets
in fluidic communication with a vent channel 1472. Multiple waste
outlets coupled to the vent channel allow continuous venting in the
instance that one or more waste outlets become clogged or
obstructed by fluid. In one embodiment, the vent channel terminates
at a vent 1473. The vent 1473, optionally, can include an outlet
filter plug 1478 configured to capture aerosolized liquid particles
that may travel through the waste collection element given the
pressurization applied to the device.
[0396] In one aspect of the invention, the cartridge uses the force
of gravity for retaining fluids within the waste collection element
during a diagnostic test run. Active pressurization applied to the
cartridge motivates fluids, e.g., the sample, reagents, and air,
through the fluidic network of the cartridge. Specifically, liquid
waste is directed through channels and enters the to the waste
collection element through channel 1362. The vertical orientation
of the cartridge within the instrument 2000 allows the waste
collection element to be configured as a liquid trap until all
incoming and outgoing channels in fluidic communication with the
waste collection element are either temporarily or permanently
isolated from other portions of the cartridge. In one
implementation of permanent isolation all incoming and outgoing
channels in fluidic communication with the waste collection are
sealed. In one specific implementation, the channels are heat
staked separately or simultaneously as part of isolating one or
more assay chambers. Sealing the channels to and from the waste
collection element forms a closed system to prevent liquid waste
contained therein to escape the waste collection element regardless
of the cartridge orientation.
[0397] As mentioned above, sealing the channels leading to and
exiting from the waste collection element to retain fluids therein,
regardless of cartridge orientation, can be achieved by selectively
heat staking a portion of the device. In one embodiment, the
cartridge prevents liquid waste from exiting the waste collection
element to mitigate contamination control by heat staking channel
1362 leading to the waste collection element and vent channel 1472
in a process described herein. Heat staking the channel 1362 and
channel 1472 seals all access channels leading to the waste
collection element. In some embodiments, a portion of the cartridge
may be configured to include a raised platform 1605 to facilitate
heat staking. Further description of the raised platform in context
to sealing the cartridge by heat staking is discussed in the
sections to follow. In another implementation, the waste collection
element can contain a bibulous pad for absorbing liquid waste
captured by the waste collection element.
3. Amplification Module
[0398] In an additional embodiment, a device comprises an
amplification module configured to supply amplification reagents
required to perform an assay, amplify nucleic acid from a purified
sample and detect a signal indicative of the presence of a target
pathogen. The amplification module has a reaction area comprising a
plurality of assay chambers of defined volume, each configured to
receive nucleic acids, where the said nucleic acids are amplified
to yield a greater copy number of the nucleic acid sequence for
detection. One or more nucleic acid targets can be read on a
chamber-by-chamber basis to permit multiplex amplification and
detection. The large number of amplicons generated in nucleic acid
amplification poses a threat for contamination to laboratory work
surfaces. In some implementations, the amplification module
includes a mechanism for amplicon containment.
[0399] In various aspects, the amplification module includes one or
more rehydration chambers for rehydrating dried reagents with a
substance, e.g., a liquid, such as a purified sample. As
illustrated in FIG. 70A, the cartridge can comprise a rehydration
chamber 1520 that accepts nucleic acid solution eluted from the
porous solid support of the rotary valve. Referring to FIG. 82, one
exemplified rehydration chamber comprises a double tapered chamber
which in turn comprises tapered inlet 1521, a tapered outlet 1522,
two curved boundaries 1525, and a reagent plug 1523. In certain
implementations, a first bounding surface is formed by the fluidics
card 1001, and a second bounding surface is formed by a plug. The
plug comprises a body and a cap. The body of the plug protrudes
into the fluidics card 1001 of the rehydration chamber 1520 to form
the second bounding surface of the rehydration chamber. In further
embodiments, one or more films form a third bounding surface of the
rehydration chamber such that the first bounding surface, the
second bounding surface, and the third bounding surface together
enclose the rehydration chamber volume. In some embodiments, the
plug cap comprises an internal cavity 1774 configured to hold one
or more dried amplification reagents for use in an assay to take
place in the assay chambers, described in greater detail in the
following section. Additionally, a magnetic mixing element may be
located in the rehydration chamber to facilitate actuation of an
assay in the assay chamber. In one implementation, the magnetic
mixing element is a magnetic ball 1524.
[0400] In various embodiments, the amplification module of the
cartridge comprises one or more assay chambers 1621 configured to
detect a signal indicative of target amplicon generated from the
nucleic acid. Referring to FIG. 70A, the assay chambers are located
within the reaction area 1600 and are visible to the reaction
camera 2701 of the reaction imaging assembly 2700.
[0401] In one implementation, the assay chambers 1621 comprise a
double tapered chamber which in turn comprises tapered inlet 1641,
a tapered outlet 1642, two curved boundaries, and a reagent plug
1770. In certain embodiments, the assay chamber comprises a first
bounding surface formed in a monolithic substrate (i.e., fluidics
card 1001), and a second bounding surface formed by a plug. The
plug comprises a body and a reagent surface. The body of the plug
protrudes into the monolithic substrate of the assay chamber at a
depth such that the assay chamber volume can be readily changed by
altering the depth at which the body of the plug protrudes into the
monolithic substrate of the assay chamber. In particular, the
reagent surface of the plug forms the second bounding surface of
the assay chamber. In further embodiments, a film may form a third
bounding surface of the assay chamber such that the first bounding
surface, the second bounding surface, and the third bounding
surface together enclose the assay chamber volume. In some
embodiments, the plug reagent surface comprises an internal cavity
1774 configured to hold one or more dried reagents for use in an
assay for a diagnostic test to take place in the assay chamber.
[0402] In one implementation of an assay chamber of an integrated
diagnostic cartridge can include a plug 1770 having one or more or
a combination of the following features. The bottom surface of the
plug body can include a cavity in the bottom surface with the dried
reagent within the cavity. The plug can have a plug thickness
between a central opening bottom and the plug body bottom, and
further wherein a depth of the cavity is less than 90% of the plug
thickness, is less than 70% of the plug thickness or is less than
50% of the plug thickness. The plug can have a polished or smooth
finish facilitating the transmissivity of the excitation
wavelengths and the emission wavelengths. The plug may have a dried
reagent that can be selected from the group consisting of nucleic
acid synthesis reagents, nucleic acids, nucleotides, nucleobases,
nucleosides, monomers, detection reagents, catalysts or
combinations thereof. The dried reagent can be a continuous film
adhered to the plug bottom surface. The dried reagent can be a
lyophilized reagent. The body of the plug can protrude into the
monolithic substrate of the assay chamber at a depth such that the
assay chamber volume can be readily changed by altering the depth
at which the body of the plug protrudes into the monolithic
substrate of the assay chamber. In some embodiments, during the
combining the enriched nucleic acid step in each of the two or more
assay chambers, the enriched nucleic acid can combine with a dried
reagent contained in each one of the two or more assay chambers.
The dried reagent can be on a surface of a plug in each one of the
two or more assay chambers. The dried reagent can be on a surface
of the plug formed from a material transmissive to excitation
wavelengths and emission wavelengths in at least one of a red
spectrum, a blue spectrum and a green spectrum used during the
performing step. In one aspect, the surface of the plug having the
dried reagent is also used during the performing an isothermal
amplification reaction step. Images collected through the plug
surface that contained the dried reagent are processed as part of
the detection of an amplification product within an assay
chamber.
[0403] With regard to FIGS. 83A and 83B, in some embodiments, the
plug further comprises a flange 1773 that can be welded and/or
adhered to a surface of the assay chamber to stabilize the position
of the plug body within the opening of the fluidic card of the
assay chamber. The plug body further includes a central opening
1777 with a side wall 1778 and a bottom surface 1776. The plug
protrudes into the monolithic substrate at a depth such that the
component of the plug that is visible on the exterior of the assay
chamber is the surfaces of the central opening of the plug. In
embodiments in which the plug cap includes a flange, the flange is
also visible on the exterior of the assay chamber as shown in FIGS.
83A and 83B. FIG. 83A is a cross section view of an assay chamber
taken through the tapered inlet 1622 and the tapered outlet 1632
which shows the plug flange 1753 supported by a raised annulus 1797
integrally formed within a fluidics card 1001. FIG. 83B is a cross
section view of an assay chamber taken through the midpoint of the
assay chamber showing the flange supporting the plug and the double
tapered sidewalls towards the inlet.
[0404] In some embodiments, such as embodiments in which the assay
chamber is used to contain an assay, the plug is transparent such
that the assay within the assay chamber is optically detectable
from outside of the assay chamber. FIG. 84 shows a signal
indicative of the presence of target nucleic acids from a target
pathogen viewed through a transparent plug as described herein. In
a preferred embodiment, the signal visible through the transparent
plug is a fluorescent signal. Alternatively, the signal visible
through the transparent plug is a colorimetric (i.e. color change)
signal.
[0405] The one or more dried reagents, used in combination with a
plurality of assay chambers, generates an amplification solution
and enables multiplexing to test a sample for the presence of more
than one target nucleic acids. The cartridge exemplified herein can
achieve multiplexing through several methods. First, the cartridge
can comprise a plurality of assay chambers, with each chamber
comprising primers and probes specific to a different target
pathogen or process control. Additionally, a single assay chamber
can comprise multiple primer/probe sets, each set specific to a
different target pathogen or process control. Alternatively, a
single assay chamber can comprise multiple prime sets with the same
probe, where each set is specific to the same target pathogen or
process control. The probe for each different target can be
differentiated by the signal generated by the probe. For example a
single assay chamber can contain a first primer/probe set in which
the probe comprises a Texas Red fluorophore and a second
primer/probe set in which the probe comprises a fluorescein (green)
fluorophore. A wide variety of fluorophores are known in the art,
as well as the mechanisms and filters one can use to differentiate
signals from multiple fluorophores in the same assay chamber. In
one implementation, the plurality of assay chambers can detect the
presence of up to 3 target nucleic acids. In one implementation,
the plurality of assay chambers can detect the presence of up to 5
target nucleic acids. Similarly stated, in some embodiments, the
assay chambers can produce a visible signal, wherein the visible
signal is associated with the presence of the target amplicon
and/or target pathogen.
[0406] In an alternative embodiment, the cartridge can comprise a
plurality of assay chambers with two or more chambers comprising
primers and probes specific to a single target pathogen. For
example, a single assay chamber can contain a first primer/probe
set for detecting a specific target pathogen and a second assay
chamber can contain a second primer//probe set for detecting the
same target pathogen. In yet another alternative embodiment,
multiple assay chambers can comprise the same primer/probe set such
that identical amplification solutions are generated within at
least two or more assay chambers.
[0407] In some implementations, it may be desirable to fill the
assay chambers simultaneously regardless of the assay chamber fluid
volume. In such implementations, one or more air chambers 1631 are
included in a cartridge to balance a ratio of the volume of the
assay chamber to the volume of the air chamber to fill
simultaneously (FIG. 70A). For example, air chambers may be
described in U.S. Pat. No. 10,046,322, titled "Reaction Well for
Assay Device" and assigned application no. PCT/US19/23764, all of
which are incorporated herein by reference. The invention
contemplates cartridges having assay chambers that differ in
volume, see for example, the assay chambers 1621 illustrated in
FIG. 70A. In such embodiments, each assay chamber will be
associated with its own air chamber. In order to achieve concurrent
filling of each assay chambers that differ in volume, the ratio of
the assay chamber volume to its associated air spring volume will
be approximately the same for each assay chamber/air spring pair on
the cartridge.
[0408] In an alternative embodiment, the above described features,
characteristics and functionality of the various the reagent plug
1523, plug cap or plug 1770 embodiments may be provided by an plug
that similarly forms part of an associated assay chamber without
extending into the diagnostic cartridge as in FIGS. 82, 83A, 83B,
84, 88 and 89. In contrast, these alternative reagent plugs
embodiments may be positioned in a planar or raised aspect to the
assay chamber or other associated component. In one variation the
plug functionality is provided by a capsule design that is raised
above surface of the diagnostic cartridge. Additionally or
optionally, the capsule style plug may be mounted to the surface of
diagnostic cartridge with appropriately shaped raised or recessed
support elements to aid in readily mounting the capsule plug in
position. The capsule plug mounts may provide appropriately sized
and shaped raised or recessed mounting features similar to the plug
cap flange 1773. Appropriate capsule plug flanges or mounting
features may be incorporated which ensure placement of the capsule
relative to the assay chamber or other chamber while ensuring
appropriate fluidic communication relative to inlets, outlets or
other conduits associated with the chamber.
[0409] As a result, in general, in one embodiment, an integrated
diagnostic cartridge includes a loading module, a lysis module, a
purification module, and a reaction module. The reaction module
includes a reagent storage component including a capsule capable of
holding a liquid or solid sample. In one embodiment the capsule
includes an opening, a closed end and a wall extending from the
closed end to the opening. The capsule is oval-shaped and the wall
is rounded, and the closed end and wall define an interior volume
having a substantially smooth surface.
[0410] In still another alterative capsule style plug embodiment,
there is an integrated diagnostic cartridge includes a loading
module, a lysis module, a purification module, and a reaction
module. The reaction module includes a capsule capable of holding a
liquid or a solid sample. The capsule includes an inner surface
extending from the bottom of said capsule to an oval-shaped opening
at the top of the capsule, wherein said inner surface is
substantially smooth and includes a concave shape extending from
the bottom of the capsule, and a planar layer affixed around the
oval-shaped opening of said capsule and oriented in the same plane
as the oval-shaped opening of said capsule. The planar layer
includes a top surface and a bottom surface. The top surface is
aligned with the inner surface of said capsule at said oval-shaped
opening to provide a continuous surface.
[0411] This and other capsule style plug embodiments may include
one or more of the following features. The capsule can be capable
of holding a volume from approximately 50 .mu.L to approximately
200 .mu.L. Still other embodiments provide for an oval-shaped
opening contained within an area of 9 mm.times.9 mm. Still further,
the capsule can include a dried reagent as described elsewhere in
this specification. Additional details of these and additional
embodiments are provided in Published International Patent
Application WO 2018/111728 entitled "Capsule Containment of Dried
Reagents" having International Application Number PCT/US2017/065444
filed on Dec. 8, 2017, incorporated herein by reference. In
particular, the details of the embodiment of the capsule plug
configuration illustrated and described with regard to FIG. 6 as
well as the contents of paragraphs [0149-0152] are incorporated
herein specifically.
[0412] The cartridge of the invention can be configured to be
provide isolation between cartridge elements either temporarily or
permanently. In one specific implementation of a form of permanent
isolation, one or more heat stake regions are used to seal off and
maintain sample within each assay chamber. In one implementation,
the configuration of the main loading channel 1671 may consist of a
u-bend 1607 (FIG. 85). By sealing off a connection between a main
channel 1671 and any loading channels 1672, the loading channel,
assay chamber 1621 and air chambers 1631 form a completely closed
system from which matter cannot travel in or out, and for which,
internal pressure within the assay chamber, loading channel, and
air chamber remains constant, unless the environment is
substantially changed, e.g. by heating the cartridge. One
acceptable method of isolating the loading channels 1672 is heat
staking with a heated element such that the loading channels are
sealed off from the main channel. In one implementation of the
method, the heated element is heat staker assembly 2640 of
instrument 2000. Note that the supply pressure of the fluid sample
is maintained during the heat staking process.
[0413] In some embodiments, as described herein, a first film is
adhered to the fluidics side 1006 of a fluidics card 1001, such
that the first film forms one wall of the main channel and loading
channels. In one implementation the first film has a similar
melting point as the substrate of the device. In further
embodiments, a second film is adhered to the first film. In such
embodiments, the second film has a higher melting point than the
first film and the surface of the device such that when heat is
applied to the device via the heat staker assembly 2640 to heat
stake the loading channel, the first film and the surface of the
device melt prior to the second film. This higher melting point of
the second film prevents the pressurized sample from escaping from
the loading channels, thus emptying assay chambers, as the first
film and the surface of the device re melted. The result of this
heat staking process is a melted first film, which forms a heat
stake 1603 seen in FIGS. 101 and 102.
[0414] In some embodiments, the fluidics card 1001 can further
include a raised platform 1605 within each of the loading channels
1672 such that, the raised feature is positioned between an inlet
to the assay chamber and the main channel. The heat staked region
can be formed using a portion of the raised platform, as depicted
in FIGS. 85, 86, and 87. In various implementations, the raised
platform may further extend throughout a fluidics card 1001 to
include one or more channels from different modules. For example,
the raised platform may extend to include channel 1362 leading to
the waste collection element and vent channel 1472 exiting the
waste collection element as seen in FIG. 88. In such a
configuration, the heat staker assembly 2640 contacts the main
channel 1671, each of the plurality of loading channels 1672,
u-bend 1607, channel 1362, and vent channel 1472 to selectively
melt these areas of the cartridge to form closed systems.
4. Example Cartridge
a) 4 Module Cartridge--Sample Prep+Amp
[0415] FIG. 89 is an exploded view of the exemplary cartridge
illustrated in FIGS. 69A and 70A, configured for a disposable,
single use diagnostic test. The cartridge, according to the
exemplified embodiment, comprises a loading module, a lysing
module, a purification module, and an amplification module.
Cartridge 1000 comprises a fluidics card 1001, wherein the fluidics
card further comprises a fluidic side 1006 and a feature side 1007,
a first film 1002, a second film 1003, and a cartridge cover 1004.
The loading module, lysing module, purification module, and
amplification module are integrally formed, e.g., molded, within
the fluidics card 1001 to provide the structures necessary to
perform each sample processing step for a diagnostic test. In some
embodiments, the cartridge is between 150 and 200 mm long, 75 mm to
100 mm wide and 10 to 30 mm tall. The cartridge can be 175 to 200
mm long, 80 to 90 mm wide and 10 to 20 mm tall. In a particularly
preferred embodiment, as illustrated in FIG. 70A, the cartridge is
approximately 180 mm long, about 90 mm and about 12 mm tall.
[0416] The loading module is configured to accept and seal a
sample. As described herein, the loading module is configured to
define a metered sample and comprises an entry port 1140, a fill
chamber 1101, a metering chamber 1110 and an overflow chamber 1120.
Such a configuration defines the volume of the sample and can
accommodate for excess sample loaded into the fill chamber by
directing the excess sample to the overflow chamber.
[0417] The lysis module is configured to lyse a metered sample
generated by the loading module. The lysing module produces a lysed
sample upon mixing a sample in the lysis chamber 1371 with one or
more lysis reagents and subsequently produces a filtered lysate
after flowing the lysed sample through a filter assembly 1330. The
lysis chamber 1371 formed within the fluidics card 1001 is
configured to hold a stir bar 1390 to mix the metered sample with a
substance contained therein, e.g., lysis buffer, to disrupt the
cell wall and/or outer membrane of cells. Lysing a sample releases
the contents of cells including various organelles, proteins, and
nucleic acids. As exemplified, the lysing module includes a filter
assembly 1330 through which the lysed sample flows. The filter
assembly is fluidically downstream from the lysis chamber 1371 to
filter a lysed sample. An inlet via 1332 allows the lysed sample to
enter the filter assembly where filter 1331 is configured to filter
the lysed sample of cellular debris and other contaminants. Flow
directors 1334 direct a filtered sample to the outlet, wherein
outlet via 1333 enables the filtered sample to exit the filter
assembly and be directed to amplification module.
[0418] In cartridges described herein, the purification module is
configured to purify a filtered sample to capture nucleic acids
associated with a suspected target pathogen. As exemplified, the
purification module includes a rotary valve 1400 comprising a
porous solid support 1445. In such a configuration, the porous
solid support 1445 allows a filtered lysate to flow through the
porous solid support 1445 to capture nucleic acids while passing
proteins, lipids and other cell debris. The purification module
includes a waste collection element 1470 to which liquid waste from
the filtering module and purification module is conveyed. In this
embodiment, the waste collection element 1470 comprises an output
filter plug 1478 configured to capture aerosolized liquid
particles, thus avoiding contamination of the instrument or
laboratory environment. Furthermore, the waste collection element
1470 is configured to be isolated so as not cause any other areas
of the cartridge or the inside of instrument 2000 to be
contaminated by previously used substances, e.g., liquids, such as
the sample or wash buffer. Another feature of the purification
module, reagent reservoirs, are formed within the fluidics card
1001 to for on-board storage of liquid substances, including a wash
buffer and an elution buffer. Prior to operation of the cartridge,
the reagent reservoirs are sealed by frangible seals to form closed
systems to prevent the cartridge from being fluidically activated
until actuated at the time of the diagnostic test.
[0419] In the exemplified cartridge, the amplification module
provides a plurality of assay chambers 1621 such that the
amplification module can perform an isothermal nucleic acid
amplification on the sample deposited into the loading module. In
this embodiment, each of the assay chambers is a double tapered
chamber which comprises tapered inlet 1641, a tapered outlet 1642,
two curved boundaries, and a reagent plug 1770. In some
embodiments, the plug cap comprises an internal cavity 1774
configured to hold one or more dried reagents for use in an assay
for a diagnostic test to take place in the assay chamber. In such
embodiments, the one or more dried reagents are configured to
produce a visual signal, e.g., fluorescent signal, to indicate the
presence of nucleic acids from a target pathogen within the sample.
The reagent plugs are configured to be transparent such that the
assay within the assay chamber 1621 is optically detectable from
outside of the assay chamber.
[0420] As exemplified, the cartridge includes a rehydration chamber
1520 and a portion of the cartridge is configured to be heat
staked, as described above. The rehydration chamber comprises
tapered inlet 1521, a tapered outlet 1522, two curved boundaries,
and a reagent plug 1770. The reagent plug of the rehydration
chamber comprises an internal cavity 1774 configured to hold one or
more dried reagents. A portion of the cartridge includes a raised
platform feature 1605 to heat stake a cartridge to maintain the
sample level in the plurality of assay chambers therein without
active pressurization. As described herein, heat staking seals the
assay chambers 1621 and the waste collection element 1470 from the
remainder of the features of the cartridge and from the outside
environment. Specifically, a portion of the main channel 1671,
loading channels 1672, channel leading to the waste collection
element 1362, and the vent channel 1472 exiting the waste
collection element are configured to include a raised platform
feature 1605 to melt the two films attached to the fluidics side of
a fluidics card to retain liquids therein.
b) 3 Module Cartridge--Sample Prep
[0421] An alternate configuration of the cartridge is depicted in
FIG. 92 In this alternative configuration, the device comprises a
loading module, a lysing module, and a purification module
configured to receive a sample, lyse cells in the sample, and
subsequently purify nucleic acids from the sample. This cartridge
configuration is intended to be used as a sample preparation device
and is not configured to perform a nucleic acid amplification test,
not to report an assay result. This sample preparation-inly
configuration can be processed using an assay instrument as
described herein, or on an abbreviated sample-preparation
instrument that lacks the chemistry heater assembly and the
reaction imaging assembly.
[0422] In such sample preparation embodiments, the loading module
is configured to accept and seal a sample. As described herein, the
loading module is configured to define a metered sample and
comprises an entry port 1140, a fill chamber 1101, a metering
chamber 1110 and an overflow chamber 1120. Such a configuration
defines the volume of the sample and can accommodate for excess
sample loaded into the fill chamber by directing the excess sample
to the overflow chamber 1120.
[0423] The lysis module, in some embodiments, is configured to lyse
a metered sample generated by the loading module. The lysing module
produces a lysed sample upon mixing a sample with one or more lysis
reagents in the lysis chamber 1371 with a stir bar 1390 as
described above. In the sample preparation cartridge, the lysing
module may further include a filter assembly 1330 to produce a
filtered lysate after flowing the lysed sample through a filter
assembly.
[0424] The purification module of the sample preparation cartridge,
similar to a standard assay cartridge, is configured to purify a
filtered lysate to enrich nucleic acids. For example, the
purification module includes a rotary valve 1400 comprising a
porous solid support 1445. The porous solid support 1445 allows a
filtered lysate to flow through the porous solid support 1445 to
capture nucleic acids while passing proteins, lipids and other cell
debris therethrough. The purification module includes a waste
collection element 1470 to which liquid waste from the purification
module is conveyed. Another aspect of the purification module,
reagent reservoirs, are formed within the fluidics card to for
on-board storage of liquid substances, such as a wash buffer and an
elution buffer. The sample preparation cartridge will include one
or more frangible seals to seal reagent reservoirs, allowing the
cartridge to be fluidically inactive, until actuated by a system,
such as instrument 2000 described herein.
[0425] This embodiment of the device, as described herein, further
comprises a retrieval port for retrieving a purified sample from
the device. In some implementations, the retrieval port comprises a
cap, similar to the cap 1181 configured to cover entry port 1140 of
the loading module. Preferably the cap of the retrieval port is
configured to be opened to permit retrieval of a purified sample
and then resealed prior to disposal of the device. Alternatively,
the sample can be retrieved via a puncturable septa or large
one-way valve. In some implementations, the retrieval port is
enclosed with a film that is cut, punctured or otherwise ruptured
to permit access to the purified nucleic acid. The sample
preparation system can include a sample loader, such as a bulb or
syringe, useful for retrieving a purified sample from the
device.
[0426] Since the sample preparation cartridge does not require any
structures related to the amplification module, a sample
preparation cartridge having the same dimensions as a test
cartridge and designed to be run on an assay instrument can process
larger volumes than the corresponding test cartridge. As
illustrated in FIG. 92, the waste collection element can be
expanded to accept larger volumes of sample, lysis reagent, and/or
wash buffer. As described above, the capacity of the sample
preparation cartridge can be further augmented by increasing the
thickness of the cartridge.
H. Methods of Use--Cartridge
[0427] The cartridge, and any of the cartridges described herein,
can be configured for use in a decentralized testing facility. In a
further embodiment, the device can be a CLIA-waived device and/or
operate in accordance with methods that are CLIA-waived. FIGS. 93
to 102 depict one exemplary method that can be used to prepare a
biological sample to amplify nucleic acid and detect the presence
of a suspected pathogen in a diagnostic test using one embodiment
of a cartridge 1000, as described herein. The features of the
cartridge used to perform the method for a diagnostic test is
depicted in FIG. 93. The relative size of the features and the
routing between features is for illustration of the method and are
not to scale. Each step is summarized in the Table 1 below. Various
processing steps and alternative embodiments are discussed in
greater detail below.
TABLE-US-00001 TABLE 1 Cartridge Test Method Steps Step FIG. Method
0 93 Load Sample 1 94 Cartridge Preparation 2 95 Lyse and Mix
Sample 3 96 Filter and Bind Lysed Sample 4 97 Wash Bound Sample 5
98 Air Dry 6 99 Elute and Meter Purified Sample 7 100 Load Reaction
Chambers 8 101 Isolation (e.g., Heat Stake) 9 102 Assay
[0428] FIG. 93 illustrates the state of a cartridge after a
biological sample is loaded into the sample port assembly 1100,
prior to insertion into an instrument and/or prior to actuation of
any cartridge features by the instrument. Frangible seal 1201 is
configured to maintain the sample within the sample port assembly.
Frangible seals 1202 and 1205 are configured to maintain a lysis
agent solution within the lysis chamber 1371. Frangible seals 1203
and 1204 are configured to maintain wash buffer within the wash
buffer reservoir 1475. Frangible seals 1206 and 1207 are configured
to maintain elution buffer within the elution buffer reservoir
1500. Prior to insertion into and actuation by the instrument, all
of the frangible seals 1201-1207 remain intact. The rotary valve
1400 is positioned such that the rotor and stator are not in
contact (indicated by dashed outline of the rotary valve feature in
FIG. 93). In FIGS. 93-102, channels that conduct only air
(pneumatic pressure) are indicated by dashed lines. Channels that
conduct fluids are indicated by solid lines. When the fluid
channels are active, i.e. subject to a motive force, such as
pneumatic pressure, the solid lines `bolded` (indicated with
thicker solid lines as compared to inactive channels). Liquid
within features of the cartridge is indicated by waved patterning
within the relevant feature. Dried reagents are depicted with
speckled patterning.
[0429] The cartridge is inserted into instrument where cartridge
verification tests are performed to ensure the cartridge is
suitable for use and certain cartridge preparation steps are
performed. The rotary valve 1400 is moved into operational
configuration and the cartridge is clamped by the clamping
subsystem. The frangible seals 1201-1207 are ruptured with pins in
the instrument. After rupture, the fluidic channels are no longer
physically blocked and fluids within the cartridge are free to flow
when exposed to a motive force. Rotary valve 1400 is rotated 360
degrees and indexed to a zero valving position to begin the series
of sample processing steps. FIG. 94 illustrates the status of the
cartridge features after these cartridge preparation steps are
completed. All of the fluids remain in their original positions, as
no motive force has yet been applied to the cartridge features.
[0430] Next, sample is transferred from the sample port assembly to
the lysis chamber 1371 to effect lysis of any cells, including any
suspected pathogen, contained in the sample. Pneumatic pressure is
applied to the main pneumatic via 1193, permitting air to flow
through the liquid trap 1145 positioned in the main pneumatic line
1171 and to the frangible seals. Rotary valve 1400 remains in the
zero valving position to dead-end fill the lysis chamber 1371 under
pressure. The instrument pressurizes the cartridge to transport
sample through exit port 1180, frangible seal 1202, sample transfer
channel 1386 and into the lysis chamber 1371. Pressure is applied
to the cartridge while magnetic mixing assembly 2300 mixes the
sample with lysis buffer by effectuating a magnetic coupling
between the driving magnet system 2310, driven magnet system 2350,
and stir bar 1390 contained in the lysis chamber 1371 to produce a
lysed sample. The pressure applied to the cartridge is turned off
after the sample is mixed for a set period. Due to the vertical
orientation of the cartridge, the liquid lysate settles to the
bottom of the lysis chamber and does not back flow toward the
sample loading assembly when the lysis chamber is no longer under
pressure. FIG. 95 illustrates the status of the cartridge features
after the lysis steps are performed.
[0431] After the lysis step, the rotary valve 1400 is indexed to a
first valving position, thereby fluidically connecting the empty
sample loading assembly 1100, lysis chamber 1371, filter assembly
1330, via 1370 in fluidic communication with the solid support
chamber of the rotary valve, and the waste collection element 1470.
This alignment of features permits filtering of the lysed sample
and binding of target analyte, e.g. nucleic acid, to a porous solid
support located in the rotary valve. Pressure applied at the main
pneumatic via provides a motive force. The lysed sample exits the
lysis chamber 1371 through exit channel 1388 passing through
frangible seal 1205. Lysed sample advances to filter inlet via 1332
and flows through filter 1330 to produce a filtered sample. The
filtered sample exits filter outlet via 1333 and into channel 1361
before entering the solid support chamber of the rotor using via
1370. The filter assembly captures and removes undesired cellular
material and debris that may clog the porous solid support to
generate a filtered sample. As the filtered sample passes through
the porous solid support contained within the solid support
chamber, target analyte, e.g. nucleic acid, is bound to the porous
solid support. The remainder of the filtered sample, e.g.,
proteins, lipids, or carbohydrates, exits via 1372 and flows
through channel 1362 to waste collection element 1470. Optionally,
pressure applied to the cartridge is turned off when the pneumatic
subsystem detects pushing the filtered sample over the porous solid
support is complete. FIG. 96 illustrates the status of the
cartridge features after the filtration and binding steps--the
lysis chamber 1371 is empty, and fluid has passed to the waste
collection element 1470.
[0432] In order to remove unbound or loosely bound contaminants
from a porous solid support while continuing to bind the target
analyte, e.g. nucleic acids, a wash buffer is passed through the
porous solid support to remove the contaminants. In an exemplary
embodiment, the porous solid support is a silica resin and the wash
buffer is an aqueous alcohol solution. The rotary valve 1400 is
indexed to a second valving position to flow wash buffer from the
wash buffer reservoir over the matrix. Pneumatic pressure is
applied to the main pneumatic via 1193. Wash buffer contained in
wash buffer reservoir 1475 is pressurized to pass through frangible
seal 1204, wash inlet via 1460, and porous solid support thereby
removing undesired contaminants while target analyte remains bound.
Wash buffer carrying contaminants travels through the wash outlet
via 1461 and channel 1362 where it is directed to waste collection
element 1470. FIG. 97 illustrates the status of cartridge features
after completion of the wash step.
[0433] In implementations using a wash buffer containing volatile
components, such as alcohol, the excess wash buffer occupying the
dead volume of the column advantageously is removed prior to
releasing bound analyte by air drying the porous solid support. To
execute such a step, the rotary valve 1400 is indexed to a third
valving position permitting pressurized air to flow over the porous
solid support through main pneumatic line 1171. Pneumatic pressure
is applied to the main pneumatic via 1193, passes through the
pneumatic line 1177 to air inlet via 1462 on the rotor thereby
drying the porous solid support removing residual volatile
components of the wash buffer from the porous solid support. Air
exits the solid support chamber through air outlet via 1463 and
channel 1362 where it is directed to waste collection element 1470
and ultimately to a vent 1473. Due to the vertical orientation of
the waste collection element, having the inlet and outlet along the
upper boundaries of the element, passing air through the waste
collection element does not disturb fluid waste already stored in
the waste collection element. Further to avoid accidental release
of fluidic contaminants from the cartridge, the vent 1473,
optionally includes an outlet filter plug which is configured to
capture aerosolized liquid particles that may travel through the
waste collection element. FIG. 98 illustrates the status of
cartridge features after completion of the air dry step.
[0434] The bound analyte, e.g. nucleic acid, is then released from
the porous solid support. To effect release, the rotary valve 1400
is indexed to a fourth valving position thereby fluidically
connecting the elution buffer reservoir 1500 to the porous solid
support and then to the rehydration chamber 1520. Elution buffer
exits the elution buffer reservoir 1500 through channel 1551,
frangible seal 1206, channel 1552, elution inlet via 1503 and then
over the porous solid support to release target analyte, thereby
generating a purified analyte solution, e.g. an enriched nucleic
acid solution. The purified analyte solution exits eluate outlet
via 1504 and is directed to the rehydration chamber 1520 using
channel 1553. The fourth valving position permits the filling of
the rehydration chamber with the purified analyte solution. As
previously described herein, in a preferred embodiment, the
rehydration chamber contains one or more dried reagents for
performing an assay. Rehydration of the one or more dried reagents
with the purified analyte solution thus results an analyte/reagent
solution. The exemplary cartridge embodiment further allows the
metering the purified sample to a produce a desired volume.
Pneumatic pressure advances the purified sample to fill the
rehydration chamber and into channel 1554. The purified sample
passes two vias, 1580 and 1581, before flowing through metering via
1582 to fill metering channel 1557 up against a metering vent 1560.
Metering, while optional, is advantageous to avoid generating an
overly dilute solution when the dried reagents are rehydrated. This
implementation is particularly advantageous in assays that require
exact volumes for amplification and detection conducted in the
assay chambers, e.g. when detecting a target pathogen at low
concentrations. In use, while pressure remains on and the purified
sample is pressurized against metering vent 1560, a magnetic
element, e.g. a rehydration motor, within the instrument rotates to
cause a magnetic ball within the rehydration chamber to gyrate,
thereby assisting dissolution and homogenization of dried down
reagents with the purified analyte solution. Completion of this
step generates an analyte/reagent solution. The pressure applied to
the cartridge can be after the metering step is competed. FIG. 99
illustrates the status of cartridge features after the elution and
metering step.
[0435] The analyte/reagent solution is now ready to be passed to
the assay chambers. The rotary valve 1400 is indexed to a fifth
valving position to load the assay chambers within the cartridge
reaction area 1600. Note that in the exemplary cartridge described
herein, indexing to the fifth valving position results a dead
volume of analyte/reagent solution lost. The volume of
analyte/reagent solution present in the metering channel 1577
remains in said channel after indexing to the fifth valving
position, thus resulting in an exact metered volume corresponding
to the sum of the plurality of assay chamber volumes.
[0436] Unlike the aforementioned steps where the rotary valve is
indexed to positions that permit pressurization through main
pneumatic line 1171, the fifth valving position blocks via 1193
(shown in FIG. 75A) to prevent pressurization of the main pneumatic
line. Instead, the fifth valving position allows pressure to be
applied to pneumatic via 1194 (shown in FIG. 75A), thereby
pressurizing pneumatic line 1178. Air is directed first through via
1192 and 1580. The pressurized air subsequently travels through
channel 1554 to push the analyte/reagent solution out of the
rehydration chamber 1520, through channel 1553 to main channel 1671
and then loading channels 1672 (not shown). The analyte/reagent
solution is split and distributed to the plurality of assay
chambers in the reaction area 1600. As previously described herein,
the plurality of assay chambers may be configured to provide one or
more dried reagents, e.g. primers and probes, directed to a
specific target pathogen or process control. In the exemplary
implementation, the plurality of assay chambers contains one or
more dried reagents therein, such that upon distribution of the
analyte/reagent solution into said plurality of assay chambers, an
amplification solution is generated within each assay chamber.
Different cartridge configurations may be designed to provide
different combinations and a variety of dried reagents to the
plurality of assay chambers. For example, an amplification solution
may be generated such that the instrument performs a plurality of
identical assays in multiple assay chambers, i.e. detecting the
same pathogen using multiple chambers. Alternatively, the
amplification solution generated may comprise different dried
reagents, such that the instrument performs two or more distinct
assays in multiple assay chambers, i.e. detecting two or more
target pathogens using multiple chambers. In any case, the
cartridge remains pressurized after all assay chambers are
successful loaded. FIG. 100 illustrates the status of cartridge
features after loading the reaction chambers.
[0437] While it is possible to perform an assay while maintaining
the amplification solution in the assay chambers with a temporary
isolation technique such as pneumatic pressure, it is preferable to
use a form of permanent isolation to physically isolate each of the
assay chambers from the others to avoid cross-contamination as well
as to isolate the reaction from the outside environment. As such
permanent isolation is particularly advantageous when performing
nucleic acid amplification reactions, as amplicon contamination is
a well understood risk of such methods. To isolate the assay
chambers after distributing enriched nucleic acid to two or more
assay chambers, rotary valve 1400 remains in the fifth valving
position during the permanent isolation process and pneumatic
pressure continues to be applied to the pneumatic via 1194. When
using a heat stake for isolation, the pneumatic pressure continues
while the instrument heat stakes the cartridge under pressure by
melting a selected area of the cartridge across the assay chambers,
loading channels, as well as, channel 1362 leading to the waste
collection element 1470, and venting channel 1472 exiting from the
waste collection element to produce heat stake 1603. The heat stake
is illustrated in FIG. 101 with a very heavy straight line across
the channels. Heat stake 1603 seals each of the effected channels
and functions to contain amplified nucleic acids and minimize the
threat of contamination when performing a diagnostic test. FIG. 101
illustrates the status of cartridge features after heat
staking.
[0438] Finally, the cartridge is ready to perform a diagnostic
test. Since the amplification solution is safely contained within
the isolated assay chambers, pneumatic pressure applied to the
cartridge is no longer required. Pneumatic pressure is released
from pneumatic via 1194. The rotary valve remains indexed to the
fifth valving position. FIG. 102 illustrates the status of
cartridge features after release of pressure and during the assay
step. The cartridge 1000 can be configured to produce the visibly
detectable signal within about 30 minutes, more preferably within
about 25 minutes, and most preferably within 20 minutes or less,
from when the sample is received by the loading module. Since the
reacted analytes and waste are contained by the heat stake or other
permanent solution technique, the cartridge can be disposed without
any further processing by the instrument or user.
1. Methods of Use--Instrument
[0439] FIGS. 106A-106E illustrate a detailed process flow chart of
a method 100 running a diagnostic test executed the instruments, as
described herein. The method begins at 110 after insertion of a
cartridge into the instrument to conduct a diagnostic test. Latch
and pin assembly 2210 drops latch 2212 into notch 1021 at the top
of the cartridge to prevent the cartridge from being ejected by the
loading assembly 2230. The instrument verifies that a cartridge is
inserted at 110 using an load position sensor 2236 located within
the loading assembly 2230. The cartridge is verified to be in a
loaded position within the instrument when flag 2237 is detected by
the load position sensor. At step 112 instrument 2000 scans a
cartridge ID code indicating the type of test to be run on the
cartridge. The label imaging system 2770 illuminates and captures
an image of the patient label area during this step. At 114 the
instrument displays the image of the patient label and the type of
diagnostic test about to be run on a graphical user interface
(GUI). At step 120, the user is given the option to abort the test
run, e.g. if the wrong cartridge was loaded. The instrument aborts
a diagnostic test when the user elects to abort the test at 122. In
one implementation, the GUI requires user input to proceed in
running the diagnostic test diagnostic test. In an alternative
implementation, the method proceeds in the absence of user input
within a set period of time, e.g. 10 seconds.
[0440] When the method proceeds, the instrument begins the clamping
sequence to perform a series of verification checks to confirm the
inserted cartridge is unused and suitable to run a diagnostic test.
Instrument 2000 first establishes a zero clamping position to set
the reference point from which all other clamping positions are
measured from. The moving bracket assembly 2040 moves in a negative
direction until tab 2047 triggers sensor 2017 fixed to the bottom
of the fixed support bracket 2011. When sensor 2017 is triggered,
the moving bracket assembly subsequently moves a calibrated
distance in the positive or negative direction to define the zero
clamping position at 124. The instrument turns the linear actuator
2014 on to rotate the lead screw 2016 in a first rotational
direction, such that rotating the lead screw pulls the moving
bracket assembly 2040 toward the fixed bracket assembly 2010 in a
positive linear direction to a first clamping position, shown by
126. In the first clamping position, the valve drive assembly 2400
contacts the rotary valve on a cartridge and light frame 2686 of
the thermal clamp assembly 2680 contacts the reaction area 1600. A
first rotary valve verification test on the rotary valve 1400 is
performed in the first clamping position. At step 130, the first
verification test checks that the rotary valve is in the shipping
configuration. As described herein, rotary valves with prematurely
dropped rotors pose the risk of leaking due to gasket deformation
from compressing a gasket for long periods of time prior to
immediate use. In this embodiment, the rotary valve 1400 is
configured for a shipping configuration to prevent the gasket from
sealing against the stator until the time of operation. The valve
drive assembly 2400 checks the shipping configuration of the rotary
valve 1400 by using an interference sensor 2404 and the end of the
valve drive shaft 2405. The valve drive shaft will not trigger the
interference sensor, indicating the rotary valve is in shipping
configuration, when valve drive pins 2402 mate correctly with
engagement openings 1417 on the rotary valve. In this instance
where the valve is confirmed to be in shipping configuration, the
instrument proceeds with the next step of the method 134 to drop
the rotor. An error is detected when the end of the valve drive
shaft 2405 triggers the interference sensor at 132. Triggering the
interference sensor is indicative of the rotary valve not in a
shipping configuration. Conditions that trigger an error include
valve drive pins not fully inserted into engagement openings or
failure to be inserted at all. This condition renders the cartridge
unusable. The instrument aborts the test at step 132, displaying a
shipping configuration error to the GUI and unclamping the
cartridge for ejection.
[0441] After the rotary valve is confirmed to be in the shipping
configuration the valve drive assembly rotates to drop the rotor at
134 to transition the valve from a shipping configuration to an
operational configuration. Rotating the rotary valve drops the
rotor, as described herein, off the threaded retention ring to seal
the gasket between the rotor and stator. At step 140 the instrument
executes a second rotary valve verification test to determine the
valve drop state. The valve drive assembly 2400 checks the state of
the rotary valve using the interference sensor to confirm a
successful rotor drop. The valve drive shaft will not trigger the
interference sensor, indicating a successful rotary valve drop and
proceeds onto step 144 to move the moving bracket assembly 2040 to
a second clamping position. When the interference sensor is
triggered, indicating an unsuccessful valve drop, the instrument
aborts the test at step 142, displaying a failed valve drop error
at the GUI and unclamping the cartridge for ejection.
[0442] The moving bracket assembly 2040 moves in the positive
direction to the second clamping position 142 at which hard stops
2211 contact the first surface of the fixed bracket assembly 2010.
At step 150, the instrument confirms sensor 2019 is triggered by
the hard stops 2211. The second clamping position is the largest
displacement in the positive direction the clamp block 2041 moves
in this exemplified method. In the second clamping position, the
door support assembly 2280 presses against cap 1181, pneumatic
interface 2100 forms a pneumatic seal with the pneumatic interface
cover adaptor 1172, thermal clamp assembly 2680 is engaged and
light frame 2686 forms a seal around the reaction area 1600, and
the valve drive 2401 remains engaged with rotary valve 1400. An
error is detected when sensor 2019 is not triggered by hard stops
2211 contacting the first surface of the fixed support bracket at
152. Failing to trigger the hard stop sensor indicates the moving
bracket assembly 2040 unsuccessfully clamped the cartridge. The
instrument aborts the test at step 152, displaying a failed hard
stop error at the GUI and unclamping the cartridge for ejection.
Completion of all rotary valve verification tests and successful
clamping of the cartridge, as indicated by sensor 2019, signals
instrument may begin the fluidic sequence portion of the
method.
[0443] At steps 154 and 156, the valve drive assembly 2400 of the
instrument rotates the valve 360 degrees (step 154) and then
indexes the valve to a zero valving position (step 156) using
homing sensor 2409. The rotary valve is configured to seal off all
inlet vias and outlet vias in the fluidics card in zero valving
position, such that no fluidic communication is permitted. This
configuration allows the instrument to perform a pneumatic leak
test, step 160. The instrument pressurizes the cartridge and
ensures no pneumatic flow is detected. An error is detected if the
pneumatic subsystem detects any pneumatic flow, thus indicating a
pneumatic leak is present within the cartridge. Such a pneumatic
leak renders the cartridge unreliable and/or unusable. The
instrument aborts the test at step 162, displaying a pneumatic leak
error at the GUI and unclamping the cartridge for ejection. If no
pneumatic leaks are detected, the instrument has completed the
cartridge verification tests, indicating that the cartridge is
competent to perform the diagnostic assay.
[0444] At step 164 of the method (see FIG. 106B), the frangible
seal block 2260 moves in a positive direction to a third clamping
position to break all frangible seals on the cartridge with
frangible seal pins. The frangible seal block is permitted to move
in the positive direction until hard stop 2263 contacts upper rail
2231 of the loading assembly 2230. In the third clamping position,
the clamp block 2041 and all assemblies (i.e. door support assembly
2280, pneumatic interface 2100, valve drive 2400, and thermal clamp
2680) remain stationary in the second clamping position due to hard
stops 2211 contacting the first surface of the fixed support
bracket 2012. As described herein, the frangible seal block 2260
and the clamp block 2041 are separate components mechanically
coupled by linear slide 2264. This configuration separates the
clamping action from the actuation of frangible seals enabling the
instrument to perform rotary valve verification tests prior to
rendering the cartridge fluidically active. When the frangible seal
block 2260 is moved in the positive direction to a third clamping
position at 164, all frangible seals are punctured. In an
alternative embodiment, frangible seal pins can be varying in
length to allow frangible seals to be punctured in a sequence when
the frangible seal block is moved to different positions in the
positive direction. At step 200 the instrument confirms frangible
seals are broken using sensor 2266. An error is detected if sensor
2266 on the frangible seal block is not triggered. This condition
renders the cartridge unusable due to frangible sealing failing to
be actuated, indicating that one or more flow paths within the
cartridge are obstructed by an unruptured seal. The instrument
aborts the test at step 202, displaying a frangible seal actuation
error at the GUI and unclamping the cartridge for ejection. Upon
successful completion of steps 164 and 202, frangible seals are
fluidically active and the cartridge is ready to begin sample
preparation of a sample suspected of containing a target pathogen
at step 204.
[0445] To begin sample preparation, the pneumatic subsystem
pressurizes the cartridge using alternating periods of applied
pressure and zero pressure to draw the sample from the fill chamber
1101 and into the metering chamber 1110 at 204. Camera 2271 of the
label imaging assembly 2770 illuminates the sample window 1050 and
confirms adequate sample volume is loaded into the cartridge at
step 210. The instrument detects the presence of a ball 1114
present in the metering chamber 1110 to determine whether adequate
sample volume is present. An error is detected when the instrument
fails to identify the presence of the ball at a location indicating
a sufficient sample volume is present in the metering chamber. The
instrument aborts the test at step 212, displaying an insufficient
sample volume error at the GUI and unclamping the cartridge for
ejection.
[0446] If sufficient sample volume is present, the instrument
proceeds with pressurizing the cartridge to empty the loading
module 214, forcing the sample into the lysis chamber 1371, which
already contains a lysis buffer comprising at least one chemical
lysis agent. The instrument pressurizes the cartridge for a set
period to transfer the sample from the metering chamber into the
lysis chamber at 214. The lysis chamber is filled by pneumatic
pressure against a dead-end provided by the zero valving position
of the rotary valve, which remains stationary when performing the
lysing step. This configuration enables the instrument to perform a
pressure maintenance check for a constant pressure profile reading
seen at step 300. An error is detected when the cartridge fails to
maintain a constant pressure profile. Inability to maintain a
constant pressure profile indicates a pneumatic leak may be present
within the cartridge, rending the cartridge unreliable or
inoperable. The instrument aborts the test at step 302, displaying
a pneumatic leak error to the GUI and unclamping the cartridge for
ejection. Confirmation of maintenance of a constant pressure
profile for the set period signals the instrument may move onto the
mixing step. The instrument continues to maintain pressure in the
cartridge while drive motor 2330 of the magnetic mixing assembly
2300 turns on for a set period at 304. Magnetic coupling is
effectuated between the driving magnet system 2310, driven magnet
system 2350, and stir bar 1390 of the lysis chamber, such that when
drive motor 2330 rotates, the driven magnet system 2350 and stir
bar 1390 also rotate. Rotation of the stir bar mixes the contents
of the lysis chamber and lyses the sample to produce a lysed
sample.
[0447] Simultaneously during the set period of mixing, a microphone
2380 monitors the audible feedback of the lysis chamber and drive
motor at step 310. An error can be detected when the audible signal
is not within a preset range. Conditions that cause the audible
feedback to not be within the preset range include decoupling of
the stir bar from the magnetic mixing assembly or stalling of the
drive motor. If the audible signal falls outside the preset range,
the instrument aborts the test at step 312, displaying an audible
feedback error to the GUI and unclamping the cartridge for
ejection. The drive motor is subsequently turned off at 314, given
the audible feedback of the magnetic mixing assembly remains in
range for the entire duration of the set period.
[0448] After completion of a successful lysis operation, the valve
drive assembly 2400 indexes the rotary valve to a first valving
position at 316 and the instrument pressurizes the cartridge to
empty the lysis chamber at 318. The lysed sample containing nucleic
acids and other cell debris flow over a porous solid support
contained within the solid support chamber of the rotary valve
according to an embodiment described herein. The porous solid
support captures nucleic acids while permitting cell debris and
lysis buffer to be directed to a waste collection element 1470. The
instrument performs a pressure verification check at 400 to confirm
the pressure profile is achieved in the allotted amount of time. An
error is detected when the pressure profile is not achieved in the
allotted time, indicating a pneumatic leak may be present within
the cartridge. The instrument aborts the test at step 302,
displaying a pneumatic leak error to the GUI and unclamping the
cartridge for ejection.
[0449] The instrument continues to monitor the pressure profile
while the lysed sample is transferred from the lysis chamber 1371
to the porous solid support chamber 1446 of the rotary valve 1400.
In one aspect of the present invention, pneumatic subsystem 2130
lacks a flow sensor and instead uses a feedback control loop based
on an actuation signal sent to the proportional valve to determine
when the lysed sample transfer to the porous solid support chamber
is complete. At step 410 (FIG. 106C), the instrument detects that
lysis buffer transfer is complete using the feedback control look
of the pneumatic subsystem. A transfer time out error is identified
when the instrument fails to detect a successful transfer of the
lysed sample within a preset time period. The instrument aborts the
test at step 412, displaying a timeout error to the GUI and
unclamping the cartridge for ejection. Upon successful detection of
the lysed sample transfer, the applied pressure is turned off at
step 414 and the instrument is ready to perform a washing step.
[0450] To align the wash buffer reservoir with the porous solid
support chamber, the valve drive assembly 2400 indexes the rotary
valve to a second valving position at 416 and the instrument
pressurizes the cartridge to empty the wash buffer reservoir 1475
at 418. Wash buffer flows out of the wash buffer reservoir and
passes through the porous solid support 1445 contained within the
solid support chamber 1446 of the rotary valve 1400 to remove
unbound cell debris or other contaminants remaining in the porous
solid support. The wash buffer is directed to the waste collection
element 1470 leaving predominantly nucleic acids bound to the
porous solid support 1445. In a manner similar to step 400, the
instrument performs a pressure verification check at 420 to confirm
the pressure profile is achieved in the allotted amount of time
like the test performed during the lysed sample transfer. An error
is detected when the expected pressure profile is not achieved in
the preset allotted time, potentially indicating a pneumatic leak
within the cartridge. The instrument aborts the test at step 302,
displaying a pneumatic leak error to the GUI and unclamping the
cartridge for ejection. The instrument continues to monitor the
pressure profile while the wash buffer is transferred from the wash
buffer reservoir 1475 to the porous solid support chamber 1446 of
the rotary valve 1400. The feedback control loop of the pneumatic
subsystem described above monitors the actuation signal sent to the
proportional valve to determine when wash buffer transfer to the
porous solid support chamber is complete at 422. An error is
detected when the instrument fails to detect a successful transfer
of wash buffer in the allotted time. The instrument aborts the test
at step 412, displaying a timeout error to the GUI and unclamping
the cartridge for ejection. Upon successful detection of the wash
buffer transfer, the applied pressure is turned off at 424 and the
instrument is ready to perform an air drying step.
[0451] To air dry the porous solid support, the valve drive
assembly 2400 indexes the rotary valve to a third valving position
at 426 and the instrument pressurizes the cartridge at 428 to
perform an air drying step. Pressurized air is blown through the
porous solid support contained within the solid support chamber
1446 of the rotary valve and directed to the waste collection
element 1470 for a set period. Performing the air drying pushes
residual fluids and evaporates lingering volatile compounds that
may be present in the solid support chamber after the washing step.
One of ordinary skill in the art would recognize the advantages of
minimizing the residual presence of lysis buffer and/or wash buffer
in a final assay. During the air drying step, the instrument again
performs a pressure verification check at 430 to confirm the
pressure profile is achieved in the allotted amount of time. An
error is detected when the pressure profile is not achieved in the
allotted time, indicating a pneumatic leak may be present within
the cartridge. The instrument aborts the test at step 302,
displaying a pneumatic leak error to the GUI and unclamping the
cartridge for ejection. Upon successful completion of the air
drying process, the pressure is turned off at step 432 and the
instrument is ready to perform an elution step.
[0452] To align the elution buffer reservoir with the porous solid
support chamber, the valve drive assembly 2400 indexes the rotary
valve to a fourth valving position at 434 and the instrument
pressurizes the cartridge to empty the elution buffer reservoir
1475 as seen by step 436. Eluent flows out of the elution buffer
reservoir 1475, passing through the porous solid support 1445
contained within the solid support chamber of the rotary valve to
release nucleic acids bound to the porous solid support thereby
generating an enriched nucleic acid solution. The enriched nucleic
acid is directed to the rehydration chamber 1520 to rehydrate dried
reagents deposited within the chamber. The cartridge remains
pressurized while the instrument performs another pressure
verification check at 500 to confirm the pressure profile is
maintained for the preset period. An error is detected when the
cartridge fails to maintain a constant pressure profile. Inability
to maintain a constant pressure profile indicates a pneumatic leak
may be present within the cartridge. The instrument aborts the test
at step 302, displaying a pneumatic leak error to the GUI and
unclamping the cartridge for ejection.
[0453] The instrument continues to pressurize the cartridge to fill
the rehydration chamber 1520 with purified sample and proceeds to
push the purified sample to the metering channel 1557 producing a
metered purified sample volume. While pressure remains applied,
rehydration motor 2510 is turned on at step 502 for a set period to
gyrate the magnetic mixing element (i.e., a ball 1524) within the
rehydration chamber to dissolve and mix dried reagents with the
metered purified sample (see FIG. 106D). At step 504 of the method,
rehydration motor is turned off. Pressure is turned off at 506 and
the chemistry heater 2601 of the chemistry heater assembly 2600 is
simultaneously turned on at 508 to preheat the chemistry heater to
a loading temperature for filling the assay chambers 1621.
[0454] While preheating the chemistry heater 2601, the reaction
imaging assembly 2700 captures an image of the dry assay chambers
at step 510. Subsequently, the valve drive assembly 2400 indexes
the rotary valve to a fifth valving position at 512 to align the
rehydration chamber 1520 with the dry assay chambers. The
instrument pressurizes the cartridge at 514 to pass the solution
from the rehydration chamber into the assay chambers, thereby
loading the assay chambers. In one implementation, the pneumatic
subsystem 2130 pressurizes the cartridge using a stepwise ramping
function to load the assay chambers. In an alternative
implementation of the method, the assay chambers are loaded using a
constant pressure profile. Pressure remains applied while the
reaction imaging assembly captures an image of the loaded assay
chambers at step 516. The instrument uses the image to verify the
assay chambers successfully loaded 600. An error is detected when
the instrument identifies incomplete loading of the assay chambers.
The instrument aborts the test at step 602, displaying an
incomplete loading error to the GUI and unclamping the cartridge
for ejection.
[0455] After confirmation of loaded assay chambers, the heater 2661
of the heat staker assembly 2640 is activated to bring the staker
bar assembly 2641 up to a preset staking temperature at step 604.
The instrument uses motor 2642 to move the staker bar assembly 2641
toward the cartridge until the staker blade 2660 contacts the
cartridge. The motor releases a spring 2643, which applies the
force required to press the staker bar assembly into the film side
of the cartridge to heat stake the cartridge at 606, as described
herein. The hot staker blade 2660 melts selected areas of the
cartridge, e.g. across u-bend 1607, loading channels 1672, channel
1362 leading to the waste collection element, and venting channel
1472 exiting from the waste collection element 1470. Heat staking
these specific channels prevent liquids from escaping the cartridge
thereby mitigating the risk for release of amplicon or potentially
contaminated biological waste into the outside environment. The
heat staker assembly 2640 heat stakes the cartridge for a set
period 606 and then turns off the heater 2661 of the heat staker
assembly. Fan 2644 turns on at 608 and the heat staker blade 2660
is actively cooled by the fan until the instrument detects the
staker bar is cooled to the desired temperature. At step 610, motor
2642 retracts the heat staker assembly 2640 from the cartridge and
the pneumatic pressure applied to the cartridge is turned off at
612.
[0456] At step 614, the reaction imaging assembly captures an image
of the assay chambers 1621 after heat staking and release of
pneumatic pressure. The image verifying assay chambers remain
loaded with the sample mixture at 700 is used to confirm a
successful sealing by the heat stake 1603. Failure of the assay
chambers to remain loaded indicate a cartridge leak due to an
unsuccessful heat stake. The instrument aborts the test at step
702, displaying a failed heat stake error to the GUI and unclamping
the cartridge for ejection. Confirmation of a successful heat stake
allows the instrument to proceed onto an amplification step.
[0457] At this step in the method, the chemistry heater 2601 of the
chemistry heater assembly 2600 has come up to the loading
temperature and is ready to facilitate an amplification of nucleic
acids within the assay chambers. In an alternative embodiment, the
chemistry heater 2601 may be fluctuated between a high and low
temperature one or more times prior to being set to a reaction
temperature shown by step 704. The chemistry heater is warmed until
the chemistry heater reaches the high temperature. Thereafter, the
chemistry heater is turned off and actively cooled by fan 2603
until the assay chambers 1621 cool to the low temperature to
complete one cycle. The assay chambers optionally may be fluctuated
one or more times before being set to the reaction temperature.
[0458] The chemistry heater 2601 is then set to the reaction
temperature for the duration of the test at 706. At a predetermined
frequency, the reaction imaging assembly 2700 captures images of
the assay chambers 1621 during amplification allowing the
instrument to process the images of the assay chambers. In one
implementation, the instrument turns on the excitation LED 2731 of
the excitation lens cell 2730 and reaction camera 2701 captures an
image of the assay chambers every 20 seconds of the amplification.
In an alternative embodiment, the instrument turns on the
excitation LED 2731 and reaction camera 2701 captures an image of
the assay chambers every 15 seconds of the amplification. The
instrument processes the sequence of images captured by the
reaction imaging assembly 2700 to determine the presence of a
signal, such as a fluorescent signal, indicative of the presence of
target nucleic acids in each of the plurality of assay chambers, as
shown by step 800. In embodiments where the device is configured to
perform a multiplex assay, the instrument may detect a positive or
negative signal for each of the plurality of assay chambers. In
certain embodiments, e.g. cartridges containing a process control
expected to generate a positive signal in at least one assay
chamber, the instrument may produce a timeout error, as shown by
802, when the expected signal is not determined within the allotted
time. The instrument aborts the test at step 802, displaying an
error to the GUI and unclamping the cartridge for ejection at
900.
[0459] Upon completion of the amplification steps, either by
detecting a positive signal in each well or after an allotted time
for amplification elapses, the unclamping and ejection sequence
begins at with linear actuator 2014 on the fixed bracket assembly
2010 rotating the lead screw 2016 in a second rotational direction
to first push the frangible seal block 2260 away from the
cartridge. The lead screw continues to rotate in the second
rotational direction as the frangible seal block 2260 contacts
ledge 2046 of the clamp block 2041 and pushes entire moving bracket
assembly 2040 in a negative direction away from the cartridge to a
fourth clamping position at 900. As the moving bracket assembly
moves in the negative direction, latch release arm 2214 contacts
the end of pin 2216 to lift latch 2212 out of notch 1021 on top of
the cartridge at 902. The loading assembly 2100 ejects the
cartridge at 904 using spring 2235 to pull the pusher carriage 2234
and the cartridge to a forward most loading position towards the
slot 2072 of the instrument to eject the cartridge. Steps 900
through 904 are executed each time an error is detected that leads
to aborting the method and ejecting a cartridge. When the method is
successfully completed, the last step of the method displays the
result of the diagnostic result to the GUI, shown by step 906.
METHODS OF USE--BIOLOGY
[0460] One aspect of the invention provides methods of testing a
sample suspected of containing a target pathogen, comprising (a)
accepting a cartridge having a loading chamber containing the
sample suspected of containing the target pathogen, (b) advancing
the sample to a lysis chamber having at least one lysis reagent
therein, (c) mixing the sample with the at least one lysis agent to
generate a lysed sample; (d) passing the lysed sample through a
porous solid support to capture a nucleic acid on the porous solid
support, (e) releasing the captured nucleic acid from the porous
solid support to generate an enriched nucleic acid, (f)
distributing the enriched nucleic acid to two or more assay
chambers and combining the enriched nucleic acid with one or more
amplification reagents, (g) isolating each one of the two or more
assay chambers from each one of all the other two or more assay
chambers, and (h) performing an isothermal amplification reaction
within each one of the two or more assay chambers while
simultaneously detecting amplification product, wherein presence of
an amplification product is an indication of a presence, an absence
or a quantity of the target pathogen in the sample suspected of
containing the target pathogen. The method is implemented on a
modular assay system comprising a loading module, lysis module,
purification module and amplification module.
A. Loading Module
[0461] In some cases, the cartridge comprises a sample entry port,
a sample input well, or a fill chamber. Given the pressurization
inherent to the devices described herein, the entry port preferably
is air tight when sealed by a cap. In certain implementations, the
cap is configured to be opened to permit addition of a sample and
then resealed prior to the sample being loaded to the device.
Alternatively, the sample can be loaded via a puncturable septa or
large one-way valve. The diagnostic system can include a sample
loader, such as a bulb or syringe, useful for loading a sample into
the device. The cartridge can be packaged with a sample collection
device, such as a syringe, bulb, swab, scraper, biopsy punch, or
other tool for a user to collect a sample.
[0462] Samples can be obtained from a subject (e.g., human
subject), a food sample (e.g., including an organism), or an
environmental sample (e.g., including one or more organisms).
(e.g., microbiological cultures). A sample may include a specimen
of synthetic origin (e.g., microbiological cultures). Samples may
be obtained from a patient or person and includes blood, feces,
urine, saliva or other bodily fluid. Exemplary, non-limiting
samples include blood, plasma, serum, sputum, urine, fecal matter
(e.g., stool sample), swab (e.g. of skin, wound, mucosal membrane,
cervix, vagina, urethra, throat or nasal cavity), sweat,
cerebrospinal fluid, amniotic fluid, interstitial fluid, tear
fluid, bone marrow, tissue sample (e.g., a skin sample or a biopsy
sample), a buccal mouthwash sample, an aerosol (e.g., produced by
coughing), a water sample, a plant sample, or a food sample. The
sample can include any useful target or analyte to be detected,
filtered, concentrated, and/or processed.
[0463] Analysis can indicate the presence, absence, or quantity of
an analyte of interest. For example, nucleic acid amplification can
provide qualitative or quantitative information about a sample,
such as the presence, absence, or abundance of a cell, cell type,
pathogen (e.g., bacteria, virus), toxin, pollutant, infectious
agent, gene, gene expression product, methylation product, genetic
mutation, or biomarker (e.g., nucleic acid, protein, or small
molecule).
[0464] Analysis can indicate the presence, absence, or quantity of
an analyte of interest. For example, nucleic acid amplification can
provide qualitative or quantitative information about a sample,
such as the presence, absence, or abundance of a cell, cell type,
pathogen (e.g., bacteria, virus), toxin, pollutant, infectious
agent, gene, gene expression product, methylation product, genetic
mutation, or biomarker (e.g., nucleic acid, protein, or small
molecule). Analytical targets of interest can include indicators of
diseases or illnesses such as genetic diseases, respiratory
diseases, cardiovascular diseases, cancers, neurological diseases,
autoimmune diseases, pulmonary diseases, reproductive diseases,
fetal diseases, Alzheimer's disease, bovine spongiform
encephalopathy (Mad Cow disease), chlamydia, cholera,
cryptosporidiosis, dengue, giardia, gonorrhea, human
immunodeficiency virus (HIV), hepatitis (e.g., A, B, or C), herpes
(e.g., oral or genital), human papilloma virus (HPV), influenza,
Japanese encephalitis, malaria, measles, meningitis,
methicillin-resistant Staphylococcus aureus (MRSA), Middle East
Respiratory Syndrome (MERS), onchocerciasis, pneumonia, rotavirus,
schistosomiasis, shigella, strep throat, syphilis, tuberculosis,
trichomonas, typhoid, and yellow fever. Analytical targets can
include biomarkers indicative of traumatic brain injury, kidney
disease, cardiovascular disease, cardiovascular events (e.g., heart
attack, stroke), or susceptibility of certain infectious agents
(such as bacteria or viruses) to certain therapeutic agents.
Analytical targets can include genetic markers, such as
polymorphisms (e.g., single nucleotide polymorphisms (SNPs), copy
number variations), gene expression products, specific proteins or
modifications (e.g. glycosylation or other post-translational
processing) of proteins. In preferred implementations, the analyte
of interest is a nucleic acid useful in the identification of
microbes including viruses, bacteria, unicellular fungi and
parasites.
[0465] In many implementations, it is desirable to subject the
sample to one or more treatments before attempting to lyse the
target pathogen. In some implementations, the treatment occurs
prior to passing the sample to the lysis chamber. In other
implementations, the treatment occurs after passing the sample to
the lysis chamber, but prior to mixing the sample with at least one
lysis reagent.
[0466] The diagnostic system comprising a device and instrument
described herein can be used to detect target pathogens from any
biological sample. Solid samples or semi-solid, such as tissue
samples, require chemical, enzymatic, physical and/or mechanical
treatment to release the pathogen into a fluid sample suitable to
flow through the test cartridge. Similarly, other biological sample
types may preferably be subjected to a chemical, enzymatic,
physical or mechanical pre-treatment prior to mixing with one or
more lysis agents. Such pre-treatment can be performed within a
cartridge or prior to loading the sample into the cartridge.
Chemical pretreatments include, e.g. n-acetylcysteine to break up
mucus in sputum samples or lysis of animal cells with saponin to
release intracellular pathogen or debulk the sample. Dithiothreitol
is also commonly used to break up mucus as well as disintegrate
solid tissue samples. In another implementation, the sample can be
enzymatically pre-treated, e.g. with an elastase, collagenase or
proteinase K to preferentially degrade connective tissues in a
solid tissue sample. In yet another implementation, the sample can
be treated with a nuclease, e.g. a DNase or RNase, to remove
extracellular nucleic acid from the sample prior to lysis. Such
nucleases can be deactivated by subsequent addition of a nuclease
inhibitor or by denaturation with a chaotropic lysis agent.
Finally, certain samples can be disrupted with bead beating prior
to addition of a chemical lysis agent.
[0467] In some implementations, an undesired contaminant can be
physically separated from the target pathogen(s), e.g. by
filtration. Filtration enables the separation of one component or
fraction of a sample from another component or fraction. For
example, a filter can enable the solid components, such as, e.g.,
cells, debris or contaminant, to be separated from the liquid
components of the solution. Alternatively, a filter can enable
larger solid components, such as, e.g., proteinaceous aggregates,
aggregated cell debris, or larger cell, to be separated from
smaller components, e.g. virus, bacterial cells or nucleic acid,
from a solution. In aspects of this embodiment, a filter useful for
separating components contained in a solution can be, e.g., a
size-exclusion filter, a plasma filter, an ion-exclusion filter, a
magnetic filter, or an affinity filter. In other aspects of this
embodiment, a filter useful for separating components contained in
a solution can have a pore size of, e.g., 0.1 .mu.m, 0.2 .mu.m, 0.5
.mu.m, 1.0 .mu.m, 2.0 .mu.m, 5.0 .mu.m, 10.0 .mu.m, 20.0 .mu.m, 30
.mu.m, 40 .mu.m, 50 .mu.m, 60 .mu.m, 70 .mu.m, 80 .mu.m, 90 .mu.m,
100 .mu.m, or more. In yet other aspects of this embodiment, a
filter useful for separating components contained in a solution can
have a pore size of, e.g., at least 0.2 .mu.m, at least 0.5 .mu.m,
at least 1.0 .mu.m, at least 2.0 .mu.m, at least 5.0 .mu.m, at
least 10.0 .mu.m, at least 20.0 .mu.m, at least 30.0 .mu.m, at
least 40.0 .mu.m, at least 50.0 .mu.m, at least 60.0 .mu.m, at
least 70.0 .mu.m, at least 80.0 .mu.m, at least 90.0 .mu.m, or at
least 100.0 .mu.m. In still other aspects of this embodiment, a
filter useful for separating components contained in a solution can
have a pore size of, e.g., at most 0.1 .mu.m, at most 0.2 .mu.m, at
most 0.5 .mu.m, at most 1.0 .mu.m, at most 2.0 .mu.m, at most 5.0
.mu.m, at most 10.0 .mu.m, at most 20.0 .mu.m, at most 30.0 .mu.m,
at most 40.0 .mu.m, at most 50.0 .mu.m, at most 60.0 .mu.m, at most
70.0 .mu.m, at most 80.0 .mu.m, at most 90.0 .mu.m, or at most
100.0 .mu.m. In other aspects of this embodiment, a filter useful
for separating components contained in a solution can have a pore
size between, e.g., about 0.2 .mu.m to about 0.5 .mu.m, about 0.2
.mu.m to about 1.0 .mu.m, about 0.2 .mu.m to about 2.0 .mu.m, about
0.2 .mu.m to about 5.0 .mu.m, about 0.2 .mu.m to about 10.0 .mu.m,
about 0.2 .mu.m to about 20.0 .mu.m, about 0.2 .mu.m to about 30.0
.mu.m, about 0.2 .mu.m to about 40.0 .mu.m, about 0.2 .mu.m to
about 50.0 .mu.m, about 0.5 .mu.m to about 1.0 .mu.m, about 0.5
.mu.m to about 2.0 .mu.m, about 0.5 .mu.m to about 5.0 .mu.m, about
0.5 .mu.m to about 10.0 .mu.m, about 0.5 .mu.m to about 20.0 .mu.m,
about 0.5 .mu.m to about 30.0 .mu.m, about 0.5 .mu.m to about 40.0
.mu.m, about 0.5 .mu.m to about 50.0 .mu.m, about 1.0 .mu.m to
about 2.0 .mu.m, about 1.0 .mu.m to about 5.0 .mu.m, about 1.0
.mu.m to about 10.0 .mu.m, about 1.0 .mu.m to about 20.0 .mu.m,
about 1.0 .mu.m to about 30.0 .mu.m, about 1.0 .mu.m to about 40.0
.mu.m, about 1.0 .mu.m to about 50.0 .mu.m, about 2.0 .mu.m to
about 5.0 .mu.m, about 2.0 .mu.m to about 10.0 .mu.m, about 2.0
.mu.m to about 20.0 .mu.m, about 2.0 .mu.m to about 30.0 .mu.m,
about 2.0 .mu.m to about 40.0 .mu.m, about 2.0 .mu.m to about 50.0
.mu.m, about 5.0 .mu.m to about 10.0 .mu.m, about 5.0 .mu.m to
about 20.0 .mu.m, about 5.0 .mu.m to about 30.0 .mu.m, about 5.0
.mu.m to about 40.0 .mu.m, about 5.0 .mu.m to about 50.0 .mu.m,
about 10.0 .mu.m to about 20.0 .mu.m, about 10.0 .mu.m to about
30.0 .mu.m, about 10.0 .mu.m to about 40.0 .mu.m, about 10.0 .mu.m
to about 50.0 .mu.m, about 10.0 .mu.m to about 60.0 .mu.m, about
10.0 .mu.m to about 70.0 .mu.m, about 20.0 .mu.m to about 30.0
.mu.m, about 20.0 .mu.m to about 40.0 .mu.m, about 20.0 .mu.m to
about 50.0 .mu.m, about 20.0 .mu.m to about 60.0 .mu.m, about 20.0
.mu.m to about 70.0 .mu.m, about 20.0 .mu.m to about 80.0 .mu.m,
about 20.0 .mu.m to about 90.0 .mu.m, about 20.0 .mu.m to about
100.0 .mu.m, about 30.0 .mu.m to about 40.0 .mu.m, about 30.0 .mu.m
to about 50.0 .mu.m, about 30.0 .mu.m to about 60.0 .mu.m, about
30.0 .mu.m to about 70.0 .mu.m, about 30.0 .mu.m to about 80.0
.mu.m, about 30.0 .mu.m to about 90.0 .mu.m, about 30.0 .mu.m to
about 100.0 .mu.m, about 40.0 .mu.m to about 50.0 .mu.m, about 40.0
.mu.m to about 60.0 .mu.m, about 40.0 .mu.m to about 70.0 .mu.m,
about 40.0 .mu.m to about 80.0 .mu.m, about 40.0 .mu.m to about
90.0 .mu.m, about 40.0 .mu.m to about 100.0 .mu.m, about 50.0 .mu.m
to about 60.0 .mu.m, about 50.0 .mu.m to about 70.0 .mu.m, about
50.0 .mu.m to about 80.0 .mu.m, about 50.0 .mu.m to about 90.0
.mu.m, or about 50.0 .mu.m to about 100.0 .mu.m.
[0468] In certain implementations, the size-exclusion filter can be
a depth filter. Depth filters consist of a matrix of randomly
oriented, bonded fibers that capture particulates within the depth
of the filter, as opposed to on the surface. The fibers in the
depth filter can be comprised of glass, cotton or any of a variety
of polymers. Exemplified depth filter materials may include, type
GF/F, GF/C and GMF150 (glass fiber, Whatman), Metrigard.RTM. (glass
fiber, Pall-Gelman), APIS (glass fiber, Millipore), as well as a
variety of cellulose, polyester, polypropylene or other fiber or
particulate filters, so long as the filter media can retain a
sufficient contaminant to allow further processing of the
sample.
[0469] In alternate implementations, the size-exclusion filter can
be a membrane filter, or mesh filter. Membrane filters typically
performs separations by retaining particles larger than its pore
size on the upstream surface of the filter. Particles with a
diameter below the rated pore size may either pass through the
membrane or be captured by other mechanisms within the membrane
structure. Membrane filters can support smaller pore sizes,
including small enough to exclude bacterial cells. Membrane filters
can be used to concentrate solutions, e.g. bacterial cell
suspensions, by filtering a first larger volume through the
membrane filter, thereby holding the bacterial cells to the
upstream surface of the membrane filter (or suspended in residual
fluid retained on the upstream side of the filter). The bacterial
cells can then be resuspended in a second small volume of fluid by
either passing the suspension fluid in the reverse direction to
float the bacterial cells off the membrane surface or by washing
the suspension fluid across the upstream surface of the filter to
wash the bacterial cells off the filter. Exemplified membranes may
include, polyethersulfone (PES) membranes (e.g., Supor.RTM. 200,
Supor.RTM. 450, Supor.RTM. MachV (Pall-Gelman, Port Washington,
N.Y.), Millipore Express PLUS.RTM. (Millipore)). Other possible
filter materials may include, HT Tuffryn.RTM. (polysulfone), GN
Metricel.RTM. (mixed cellulose ester), Nylaflo.RTM. (Nylon), FP
Verticel (PVDF), all from Pall-Gelman (Port Washington, N.Y.), and
Nuclepore (polycarbonate) from Whatman (Kent, UK).
[0470] In some embodiments, an undesired contaminant can be removed
from a sample by exposing the sample to a capture agent, such as a
capture antibody, is immobilized on a solid substrate. The solid
substrate can be contacted with the sample such that contaminant in
the sample can bind to the immobilized antibody. In some
embodiment, a capture antibody can be used that has binding
affinity for red blood cells. The antibody may be a monoclonal
antibody or a polyclonal antibody. Suitable solid substrates to
which a capture antibody can be bound include, without limitation,
membranes such as nylon or nitrocellulose membranes, and beads or
particles (e.g., agarose, cellulose, glass, polystyrene,
polyacrylamide, magnetic, or magnetizable beads or particles). In
an alternate implementation, the capture agent can be any protein
having specific and high affinity for binding to an undesired
contaminant.
B. Lysis Module
[0471] Cell lysis refers to a process in which the outer boundary
or cell membrane is broken down or destroyed in order to release
inter-cellular materials such as nucleic acids (DNA, RNA), protein
or organelles from a cell. Lysis resulting in release of nucleic
acids can be achieved by chemical, enzymatic, physical and/or
mechanical interventions.
[0472] In one implementation, the lysis agent is a chemical lysis
agent. Chemical lysis methods disrupt the cell membrane, e.g., by
changing pH or by addition of detergents and/or chaotropic agents
to solubilize membrane proteins and thereby rupture the cell
membrane to release its contents. These chemical lysis solutions
can include one or more chemical lysis agents such as anionic
detergents, cationic detergents, non-ionic detergents or chaotropic
agents. Non limiting examples of non-ionic detergents include
3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS),
Triton X, NP-40, Tween, and
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
(CHAPSO), a zwitterionic detergent. Suitable chaotropic agents
include, but are not limited to, urea, guanidine (e.g. guanidinium
isothiocyanate or guanidinium hydrochloride),
ethylenediaminetetraacetic acid (EDTA) and lithium perchlorate. In
a preferred implementation, the suspected pathogen is a virus or a
gram-negative bacteria and the chemical lysis reagent is a
chaotropic agent.
[0473] The lysis agent can be an enzyme, or enzymatic lysis agent.
Enzymatic cell lysis advantageous can allow for selective lysis of
certain types of cells. For example, the enzymatic lysis agent can
selectively cleave peptidoglycans found only in bacterial cell
walls. Exemplified enzymatic lysis agents include achromopeptidase,
lysostaphin, lysozyme, mutanolysin. Alternatively, a lyticase or
chitinase, specific to yeast cells, can be used as the enzymatic
lysis agent. In some cases enzymes with broad specificity and no
specific target cell type, such as proteinase K, can be used as the
lysis agent. Any of the enzymatic enzymes can be used in
combination with mechanical or chemical lysis agents to promote
faster and/or more complete lysis.
[0474] For use in the cartridges described here, bead beating is a
preferred mechanical lysis mechanism, in which cells are disrupted
by agitating, e.g. mixing, tiny beads made of glass, steel or
ceramic with the cell suspension at high speeds. Bead beating is
capably of lysing a variety of cells, including yeast and
gran-positive bacteria. A preferred lysis method combines both
mechanical and non-mechanical (e.g. chemical) methods, for example
bead beating in a solution containing guanidine and/or Triton
X-100. In some implementations, the mechanical lysis agent is
ceramic beads, glass beads or steel beads, and mixing comprises
rotating a stir bar at at least 500 rpm, at least 1000 rpm, at
least 2000 rpm or at least 3000 rpm for at least 30 seconds, at
least one minute, or at least two minutes. Bead beating is a
preferred method for use in disrupting cells with significantly
structured cell walls. Accordingly, in some implementations
utilizing a mechanical lysis agent comprised of ceramic, glass or
steel beads, the suspected pathogen is a gram-negative bacterium, a
fungus such as a yeast, or a plant cell.
[0475] In samples having high organic loads, such as stool samples,
blood samples, sputum samples or swab samples collected from mucus
membranes, significant debris may be present in the lysed sample.
In such cases, it is advantageous to filter the lysed sample prior
to passing it through the porous solid support. In a preferred
implementation, the lysed sample is passed through a size-exclusion
filter, wherein nucleic acid passes through the filter. In a more
preferred implementation, the lysed sample is passed through a
depth filter. Preferably such post-lysis filters have a pore size
of 20 .mu.m or less, more preferably 10 .mu.m or less.
C. Purification Module
[0476] After lysis, the lysed sample is passed through a first
porous solid support thereby capturing nucleic acid. In some
implementations, the porous solid support may preferentially bind
DNA more than RNA or RNA more than DNA or certain lengths of
nucleic acid (e.g. fragmented genomic DNA more than complete
genomic DNA). However, a porous solid support for capturing nucleic
acid in the devices described herein, preferably, binds nucleic
acid regardless of the sequences present in the nucleic acids. When
the lysed sample is passed through porous solid support having
affinity for nucleic acids, the nucleic acids are captured by the
porous solid support while proteins, lipids, polysaccharides, and
other cell debris that can inhibit nucleic acid amplification pass
through the column and to the waste chamber. In some
implementations, after capturing the nucleic acid, a wash solution
is passed through the porous solid support to further remove
contaminants. Captured nucleic acid is then released from the
porous solid support with an elution buffer to generate an enriched
nucleic acid.
[0477] In a preferred implementation, the porous solid support is a
silica resin, e.g. silica fibers. Salt is important to binding
nucleic acid to silica resins. In implementations where a chemical
lysis agent, such as guanidinium isothiocyanate, is used, the lysis
agent can provide the necessary salt. In other implementations, it
is advantageous to supplement the lysed sample with a chaotropic
salt. The addition of alcohol, such as ethanol or isopropanol, can
further enhance and influence the binding of nucleic acids to the
silica resin. In a preferred implementation, the silica resin
column is washed with a dilute salt and/or alcohol solution. If a
dilute salt solution is used, preferably a send wash buffer
containing alcohol but no salt is also passed over the silica
resin. Prior to elution, preferably excess alcohol is removed,
e.g., by drying with forced air. Finally, enriched nucleic acid is
released from the silica resin with water or buffered (e.g. 10 mM
Tris) water. Higher molecular weight DNA can be preferably released
from the resin with 10 mM Tris at pH 8-9. RNA can be preferentially
released from the silica resin with water.
[0478] In some implementations, it may be desirable to remove
additional contaminants from the enriched nucleic acid by passing
is through a second solid support. In such implementations, prior
to distributing the enriched nucleic acid to the assay chambers,
the method further comprises passing the enriched nucleic acid
through a second porous solid support. The second porous solid
support can be the same as the first solid support. In cases in
which the first and second solid support are comprised of the same
material, the enriched nucleic acid is mixed with a matrix binding
agent prior to passing through the second solid support. For
example, when the first and second solid support are a silica
resin, the matrix binding agent can be a salt and/or alcohol
solution as described above. In an alternate implementation, the
second solid support is different than the first solid support. In
some such implementations, the second solid support has an affinity
for nucleic acid and the method further comprises releasing the
captured nucleic acid from the second solid support to generate a
twice enriched nucleic acid. In other implementations, the second
solid support does not have an affinity for the nucleic acid, but
rather captures one or more contaminants, thereby removing the
contaminant from the enriched nucleic acid.
[0479] In an alternate embodiment, it may be desirable to remove
contaminant from the lysed sample prior to passing it through the
first porous solid support. In such cases, the method further
comprises passing the lysed sample through a second solid support,
wherein the second solid support does not bind nucleic acid, but
rather has affinity for one or more contaminants, thereby removing
the one or more contaminants from the lysed sample.
[0480] In order to implement a method using more than one solid
support on the devices described herein, the rotor can comprise a
plurality of flow channels, each flow channel comprising an inlet
1441, an outlet 1442, and a porous solid support 1445. In certain
implementation, the rotor comprises a main body and a cap 1430
operably connected to the main body, and wherein one wall of the
flow channel is defined by the cap. The rotor comprises an outer
face 1413 opposite the rotor valving face, wherein the outer face
can comprise an opening for engaging a spline. The multiple flow
channels can have the same or different dimensions. Similarly, the
multiple flow channels can contain the same or different porous
solid supports. Accordingly, nucleic acid may be purified from a
particularly contaminated same by binding the nucleic acid to a
first column, washing the bound nucleic acid and eluting a
partially purified nucleic acid solution. The partially purified
nucleic acid can be mixed with a binding buffer and passed through
a second solid support, binding the nucleic acid to the second
support, while allowing contaminants to pass. The bound nucleic
acid is washed and then eluted to generate a double-purified
nucleic acid solution. Alternatively the second solid support can
be specific for a contaminant, allowing nucleic acid to pass, but
retaining the undesired contaminant, thereby generating a more
purified nucleic acid solution.
D. Amplification Module
[0481] The amplification module comprises a plurality of assay
chambers of defined volume, each configured to receive a purified
nucleic acid. The amplification module includes a heater such that
the amplification module can perform an isothermal or thermocycling
amplification reaction on the target nucleic acid. The
amplification module further is configured to detect a signal
indicative of target amplicon generated from the nucleic acid. In
one implementation, the distributing step is performed prior to
combining the enriched nucleic acid with an amplification reagent.
Alternatively, the enriched nucleic acid is combined with one or
more amplification reagents before the distributing step. The
amplification reagent can be any reagent that is necessary or
beneficial for nucleic acid synthesis, including, but not limited
to, a DNA polymerase, a reverse transcriptase, a helicase,
nucleotide triphosphates (NTPs), a magnesium salt, a potassium
salt, an ammonium salt, a buffer, or combinations thereof. In many
implementations the one or more amplification reagents comprise a
primer or primer set. The primer set can be specific to a first
nucleic acid sequence present in one of the one or more target
pathogens. In some implementations, a first reaction well contains
a first primer set specific to a first nucleic acid sequence and a
second reaction well contains a second primer set specific to a
second nucleic acid sequence. The first nucleic acid sequence can
be present in one or more of the target pathogen or present in a
process control.
[0482] In addition to the primary amplification assay, the method
can comprise the step of pre-amplifying the enriched nucleic acid.
Such preamplification is particularly useful when a very limited
amount of target nucleic acid is in the sample, either due to few
pathogen cells and/or to low copy of the target nucleic acid within
the pathogen cells. Using a cartridge with a large number of wells,
i.e. highly multiplexed, also benefits from pre-amplification. For
such implementation, the isothermal amplification is initiated
prior to distributing the enriched nucleic acid to the two or more
assay chambers. Optionally, after the distributing step, but prior
to performing the isothermal amplification reaction, the method
further comprises combining the enriched nucleic acid with a primer
set specific to one of the one or more target pathogens.
E. Alternate Workflows
[0483] The instruments and cartridges described herein can be
adapted to analyze a variety of biological samples, including
cerebrospinal fluid (CSF), urine, throat or nasal swabs, blood,
genital swabs (e.g. vaginal, cervical or urethral swabs), sputum,
stool or solid tissue sample. In each case the method of testing a
sample suspected of containing a target pathogen, comprising the
following basic steps (a) accepting a cartridge having a loading
chamber containing the sample suspected of containing the target
pathogen 180, (b) advancing the sample to a lysis chamber having at
least one lysis reagent therein, (c) mixing the sample with the at
least one lysis agent to generate a lysed sample 380; (d) passing
the lysed sample through a porous solid support to capture a
nucleic acid on the porous solid support 480, (e) releasing the
captured nucleic acid from the porous solid support to generate an
enriched nucleic acid 484, (f) distributing the enriched nucleic
acid to two or more assay chambers 784 and combining the enriched
nucleic acid with one or more amplification reagents 780, (g)
isolating each one of the two or more assay chambers from each one
of all the other two or more assay chambers and (h) performing an
isothermal amplification reaction within each one of the two or
more assay chambers while simultaneously detecting amplification
product 786, wherein presence of an amplification product is an
indication of a presence, an absence or a quantity of the target
pathogen in the sample suspected of containing the target pathogen.
For certain sample types, between steps (a) and (c), the method
further comprises pretreating the sample 182. The pretreatment can
be a chemical, physical, mechanical or enzymatic pretreatment as
described above. For some sample types, subsequent to step (c) but
prior to step (d), the method further comprises filtering the lysed
sample 382, preferably by passing the lysed sample through a
size-exclusion filter. In many cases, after step (d) and prior to
step (e), the method further comprises washing the porous solid
support 482. Some samples will contain a high level of
contaminants, in such case it may be advantageous to repeats steps
(d) and (e) on passing the lysed sample and then enriched nucleic
acid through a first and second porous solid support.
[0484] Some sample types, e.g. blood, is expected to contain the
target pathogen at very low concentrations. Some cartridges will
comprise a large number of assay chambers. In cases where the
concentration of pathogen is very low and the number of assay
chambers is high, can lead to false negative determinations due to
partitioning. For example, when an enriched nucleic acid that
contains five copies of nucleic acid associated with the suspected
pathogen is assayed in a cartridge having eight wells, at least
three assay chambers will not receive a copy of the nucleic acid
associated with the suspected pathogen. If the primer set
associated with the target pathogen is in one of those three wells,
the cartridge will falsely report that no pathogen is present. This
result can be avoided by pre-amplifying certain nucleic acid
targets prior to distributing the enriched nucleic acid to the
plurality of reaction well 782.
[0485] For simple, clear patient samples, such as cerebrospinal
fluid, urine or cell suspensions extracted from throat or nasal
swabs (FIG. 108), the method comprises (a) accepting a cartridge
having a loading chamber containing the sample suspected of
containing the target pathogen 180, (b) advancing the sample to a
lysis chamber having at least one lysis reagent therein, (c) mixing
the sample with the at least one lysis agent to generate a lysed
sample 380; (d) passing the lysed sample through a porous solid
support to capture a nucleic acid on the porous solid support 480,
(e) releasing the captured nucleic acid from the porous solid
support to generate an enriched nucleic acid 484, (f) distributing
the enriched nucleic acid to two or more assay chambers 784 and
combining the enriched nucleic acid with one or more amplification
reagents 780, (g) isolating each one of the two or more assay
chambers from each one of all the other two or more assay chambers
and (h) performing an isothermal amplification reaction within each
one of the two or more assay chambers while simultaneously
detecting amplification product 786, wherein presence of an
amplification product is an indication of a presence, an absence or
a quantity of the target pathogen in the sample suspected of
containing the target pathogen. Typically, the lysis agent is a
chemical agent such as a detergent, a chaotropic agent or a
combination thereof. If the target pathogen is resistant to
chemical lysis, e.g. a yeast or gram positive bacterium, the one or
more lysis agent further comprises a mechanical lysis agent such as
beading beating.
[0486] Exemplified pathogens that may be suspected in CSF include,
but are not limited to, Brucella, Haemophilus influenzae, Bacillus
anthracis, Listeria, Streptococcus pneumoniae, Leptospira, Borrelia
burgdorferi (Lyme's disease), Mycobacterium tuberculosis,
Cryptococcus, and Candida. Exemplified pathogens that may be
suspected in urine include, but are not limited to, Escherichia
coli, Klebsiella, Enterobacter, Serratia, Pseudomonas sp. (e.g.,
Pseudomonas aeruginosa), Enterococcus sp. (e.g., Enterococcus
faecalis or Enterococcus faecium), Leptospira, Chlamydia sp. (e.g.
Chlamydia trachomatis), Mycoplasma sp. (e.g. Mycoplasma
genitalium), and Trichomonas vaginalis. Exemplified pathogens that
may be suspected in a throat or nasal swab include, but are not
limited to, Haemophilus influenzae, Bordetella pertussis,
Corynebacterium diphtheriae, Streptococcus sp. (e.g. Group A or
Group B strep), Mycoplasma sp. (e.g. Mycoplasma pneumoniae),
Candida sp. (e.g. Candida albicans), Influenza, and coronavirus
(e.g. MERS, SARS, or SARS-CoV-2).
[0487] Sputum is the thick mucus or phlegm that is expectorated
from the lower respiratory tract (bronchi and lungs) and is
important for the investigation of certain respiratory diseases,
e.g. tuberculosis. Other exemplified pathogens that may be detected
in sputum include, but are not limited to, Klebsiella sp.,
Enterobacter sp., Serratia sp., Legionella sp., Bordetella
pertussis, Yersinia sp. (e.g. Yersinia pestis), Pseudomonas sp.
(e.g., Pseudomonas aeruginosa), Streptococcus pneumoniae,
Mycoplasma sp. (e.g. Mycoplasma pneumoniae), Blastomyces
dermatitidis, and Mycobacterium sp., (e.g. Mycobacterium
tuberculosis).
[0488] Given the high viscosity of sputa in some patients, e.g.
from patients with advanced cystic fibrosis, sputa must first be
liquefied mechanically by bead beating or chemically with mucolytic
agents such as n-acetylcysteine (Mucomyst; Bristol) or
dithiothreitol (Sputolysin). Referring to FIG. 111, the invention
provides methods of identifying one or more suspected pathogens in
a sputum sample, the method comprising the steps of: (a) accepting
a cartridge having a loading chamber containing the sample
suspected of containing the target pathogen 180, (b) pretreating
the sputum sample with a mucolytic agent 182, (c) advancing the
sample to a lysis chamber having at least one lysis reagent
therein, (d) mixing the sample with the at least one lysis agent to
generate a lysed sample 380; (e) filtering the lysed sample 382,
preferably by passing the lysed sample through a size-exclusion
filter, (f) passing the lysed sample through a porous solid support
to capture a nucleic acid on the porous solid support 480, (g)
passing a wash solution through the porous solid support 482, (h)
releasing the captured nucleic acid from the porous solid support
to generate an enriched nucleic acid 484, (i) distributing the
enriched nucleic acid to two or more assay chambers 784 and
combining the enriched nucleic acid with one or more amplification
reagents 780, (j) isolating each one of the two or more assay
chambers from each one of all the other two or more assay chambers
and (k) performing an isothermal amplification reaction within each
one of the two or more assay chambers while simultaneously
detecting amplification product 786, wherein presence of an
amplification product is an indication of a presence, an absence or
a quantity of the target pathogen in the sputum sample suspected of
containing the target pathogen. Optionally, the enriched nucleic
acid is pre-amplified 782 prior to distributing the nucleic acid to
the two or more assay chambers.
[0489] Referring to FIG. 110, the invention provides methods of
identifying one or more suspected pathogens in a sample extracted
from a genital swab (e.g. vaginal, cervical or urethral swab), the
method comprising the steps of: (a) accepting a cartridge having a
loading chamber containing the sample suspected of containing the
target pathogen 180, (b) advancing the sample to a lysis chamber
having at least one lysis reagent therein, (c) mixing the sample
with the at least one lysis agent to generate a lysed sample 380;
(d) filtering the lysed sample 382, preferably by passing the lysed
sample through a size-exclusion filter, (e) passing the lysed
sample through a porous solid support to capture a nucleic acid on
the porous solid support 480, (f) passing a wash solution through
the porous solid support 482, (g) releasing the captured nucleic
acid from the porous solid support to generate an enriched nucleic
acid 484, (h) distributing the enriched nucleic acid to two or more
assay chambers 784 and combining the enriched nucleic acid with one
or more amplification reagents 780, (i) isolating each one of the
two or more assay chambers from each one of all the other two or
more assay chambers and (j) performing an isothermal amplification
reaction within each one of the two or more assay chambers while
simultaneously detecting amplification product 786, wherein
presence of an amplification product is an indication of a
presence, an absence or a quantity of the target pathogen in the
sample suspected of containing the target pathogen. Optionally, the
enriched nucleic acid is pre-amplified prior to step (h).
Exemplified pathogens that may be suspected in a urogenital swab
include, but are not limited to, Chlamydia sp. (e.g. Chlamydia
trachomatis), Mycoplasma sp. (e.g. Mycoplasma genitalium), Candida
sp. (e.g. Candida albicans), human papilloma virus (HPV),
Trichomonas vaginalis, Gardnerella vaginalis, Lactobacillus sp.,
Bacteroides sp., Prevotella sp, Mobiluncus sp., and
Peptostreptococcus sp., Atopobium vaginae, and Sneathia
(Leptotrichia).
[0490] Blood samples can be particularly difficult for nucleic acid
amplification testing, as heme (a component of hemoglobin in red
blood cells) is a well-known inhibitor of nucleic acid
amplification. According, blood samples will require additional
processing prior to the amplification steps. Referring to FIG. 109,
the invention provides methods of identifying one or more suspected
pathogens in a blood sample, the method comprising the steps of:
(a) accepting a cartridge having a loading chamber containing the
sample suspected of containing the target pathogen 180, (b)
subjecting the blood sample to one or more chemical, enzymatic or
physical pretreatments 182, (c) advancing the sample to a lysis
chamber having at least one lysis reagent therein, (d) mixing the
sample with the at least one lysis agent to generate a lysed sample
380; (e) filtering the lysed sample 382, preferably by passing the
lysed sample through a size-exclusion filter, (f) passing the lysed
sample through a porous solid support to capture a nucleic acid on
the porous solid support 480, (g) passing a wash solution through
the porous solid support 482, (h) releasing the captured nucleic
acid from the porous solid support to generate an enriched nucleic
acid 484, (i) pre-amplifying the enriched nucleic acid 782, (j)
distributing the enriched nucleic acid to two or more assay
chambers and combining the enriched nucleic acid with one or more
amplification reagents 784, (k) isolating each one of the two or
more assay chambers from each one of all the other two or more
assay chambers and (l) performing an isothermal amplification
reaction within each one of the two or more assay chambers while
simultaneously detecting amplification product 786, wherein
presence of an amplification product is an indication of a
presence, an absence or a quantity of the target pathogen in the
blood sample suspected of containing the target pathogen.
Optionally steps (f), (g) and (h) are repeated with a first and
second porous solid support. Exemplified pathogens that may be
suspected in a blood sample include, but are not limited to,
Brucella, Campylobacter sp., Escherichia coli, Haemophilus
influenzae, Klebsiella, Enterobacter, Serratia, Yersinia (e.g.
Yersinia pestis), Pseudomonas (e.g., Pseudomonas aeruginosa),
Salmonella sp. (e.g. Salmonella typhimurium or Salmonella typhi),
Francisella tularensis, Bacillus anthracis, Listeria,
Staphylococcus aureus (e.g. MRSA or MSSA), Streptococcus sp. (e.g.
Group A or Group B strep), Treponema pallidum (syphilis),
Leptospira, Borrelia burgdorferi (Lyme's disease), Coccidioides
immitis (Valley fever), coronavirus (e.g. MERS, SARS, or
SARS-CoV-2), hepatitis, and human immunodeficiency virus (HIV).
[0491] As with blood samples, fecal samples (e.g. stool samples)
contain a high concentration of contaminants, such as organic
matter and high commensal bacterial load, and may require
additional processing prior to the amplification steps. Referring
to FIG. 112, the invention provides methods of identifying one or
more suspected pathogens in a fecal sample, the method comprising
the steps of: (a) accepting a cartridge having a loading chamber
containing the sample suspected of containing the target pathogen
180, (b) subjecting the fecal sample to one or more enzymatic or
mechanical pretreatments 182, (c) advancing the sample to a lysis
chamber having at least one lysis reagent therein, (d) mixing the
sample with the at least one lysis agent to generate a lysed sample
380; (e) filtering the lysed sample 382, preferably by passing the
lysed sample through one or more size-exclusion filters, (f)
passing the lysed sample through a porous solid support to capture
a nucleic acid on the porous solid support 480, (g) passing a wash
solution through the porous solid support 482, (h) releasing the
captured nucleic acid from the porous solid support to generate an
enriched nucleic acid 484, (i) distributing the enriched nucleic
acid to two or more assay chambers and combining the enriched
nucleic acid with one or more amplification reagents 780, (j)
isolating each one of the two or more assay chambers from each one
of all the other two or more assay chambers and (l) performing an
isothermal amplification reaction within each one of the two or
more assay chambers while simultaneously detecting amplification
product 786, wherein presence of an amplification product is an
indication of a presence, an absence or a quantity of the target
pathogen in the fecal sample suspected of containing the target
pathogen. Typically, the mechanical pretreatment is required to
homogenize and liquify the fecal sample. Such homogenization can be
achieved within the cartridges described herein by stirring the
fecal sample with ceramic, glass or steel beads in the lysis
chamber prior to exposing the fecal sample to the lysis agent. The
enzymatic pretreatment of step (b) can be incubating the fecal
sample with a protease and/or nuclease. Optionally steps (f), (g)
and (h) are repeated with a first and second porous solid support.
Optionally, prior to step (i), the method further comprises
pre-amplifying the enriched nucleic acid 782. Exemplified pathogens
that may be suspected in a fecal sample include, but are not
limited to, Campylobacter sp. (e.g., Campylobacter jejuni), Vibrio
sp. (e.g. Vibrio cholerae), Salmonella sp. (e.g. Salmonella
typhimurium or Salmonella typhi), Shigella, and Bacillus
anthracis.
[0492] Finally, the cartridges and instruments described herein can
be used to detect suspected pathogen in solid tissue samples. Such
tissue samples require additional processing to separate the cells
of the tissue sample. Referring to FIG. 113, the invention provides
methods of identifying one or more suspected pathogens in a tissue
sample, the method comprising the steps of: (a) accepting a
cartridge having a loading chamber containing the tissue sample
suspected of containing the target pathogen 180, (b) subjecting the
tissue sample to one or more enzymatic, chemical or mechanical
pretreatments 182, (c) advancing the sample to a lysis chamber
having at least one lysis reagent therein, (d) mixing the sample
with the at least one lysis agent to generate a lysed sample 380;
(e) filtering the lysed sample 382, preferably by passing the lysed
sample through one or more size-exclusion filters, (f) passing the
lysed sample through a porous solid support to capture a nucleic
acid on the porous solid support 480, (g) passing a wash solution
through the porous solid support 482, (h) releasing the captured
nucleic acid from the porous solid support to generate an enriched
nucleic acid 484, (i) distributing the enriched nucleic acid to two
or more assay chambers and combining the enriched nucleic acid with
one or more amplification reagents 780, (j) isolating each one of
the two or more assay chambers from each one of all the other two
or more assay chambers and (l) performing an isothermal
amplification reaction within each one of the two or more assay
chambers while simultaneously detecting amplification product 786,
wherein presence of an amplification product is an indication of a
presence, an absence or a quantity of the target pathogen in the
tissue sample suspected of containing the target pathogen.
Typically, the mechanical pretreatment is required to disintegrate
and liquify the tissue sample. Such homogenization can be achieved
within the cartridges described herein by stirring the tissue
sample with ceramic, glass or steel beads in the lysis chamber
prior to exposing the tissue sample to the lysis agent. The
enzymatic pretreatment of step (b) can comprise incubating the
tissue sample with an elastase, collagenase or proteinase K. The
chemical pretreatment of step (b) can comprise incubating the
tissue sample with dithiothreitol (DTT). Optionally steps (f), (g)
and (h) are repeated with a first and second porous solid support.
Optionally, prior to step (i), the method further comprises
pre-amplifying the enriched nucleic acid 782. Exemplified pathogens
that may be suspected in a solid tissue sample include, but are not
limited to, Bacillus anthracis (e.g. from skin scraping),
Corynebacterium diphtheriae, and Aspergillus (lung).
EXAMPLES
[0493] To demonstrate functionality of the diagnostic system of the
invention for the qualitative detection of Chlamydia trachomatis
(CT) and Neisseria gonorrhoeae (NG), the instrument described
herein was paired with an integrated diagnostic cartridge populated
with CT- and NG-specific RTLAMP reagents. The embodiment of the
integrated diagnostic cartridge used is shown in FIGS. 69A, 70A and
89 and is described herein. The CT-specific reagents are described
in detail in U.S. Pat. No. 10,450,616 B1, incorporated herein by
reference. The NG-specific reagents are described in detail in U.S.
patent application Ser. No. 16/523,609, filed 26 Jul. 2019, said
application incorporated herein by reference.
[0494] Fresh urine samples from healthy non-infected donors were
co-spiked with live CT and NG and used as samples loaded into the
cartridge loading assembly. Specifically, frozen, single use
aliquots of titered bacterial stocks, either grown in-house (NG) or
purchased from ATCC (CT), underwent a 30 second thaw at
37.quadrature.C followed by serial dilution at room temperature in
Mueller Hinton cation-adjusted growth medium. Bacteria were diluted
1:10 into pools of negative urine to achieve a final concentration
of 1 IFU/mL of CT and 1 CFU/mL of NG.
[0495] Urine samples were mixed by short vortex, 1 mL of sample
removed and loaded onto the integrated diagnostic cartridge via the
fill chamber using a pipette. To initiate the method (time, T=0),
cartridges were inserted into the instrument. Using pneumatic
pressure, the instrument advanced the urine sample to the lysis
chamber, which held a chemical lysis buffer including, inter alia,
guanidinium isothiocyanate and isopropanol. The urine sample and
lysis buffer were mixed in the lysis chamber at 1300 rpm for 30
seconds to generate a lysed sample.
[0496] The valve drive assembly of the instrument rotated the
rotary valve to fluidically connect the sample exit channel of the
lysis chamber, the porous solid support chamber of the rotor,
contained therein a silica fiber matrix, and the waste collection
element. The instrument then pressurized the lysis chamber to
forcibly pass the lysed sample through the silica fiber matrix
(i.e. the porous solid support), capturing nucleic acid on the
matrix and passing cell debris, urine and other contaminants to the
waste collection element. The matrix was washed to further rid the
matrix of contaminants, and then eluted with buffered water to
release nucleic acid from the matrix to generate an enriched
nucleic acid. The enriched nucleic acid was used without dilution
to rehydrate a dried amplification reagent solution in a
rehydration chamber with agitation from a mixing ball for 20
seconds. The nucleic acid/amplification reagent solution was then
distributed to five assay chambers, such that the assay chambers
are fully loaded at T=6:12 (mm:ss).
[0497] The instrument captured an image of the filled assay
chambers and then isolated the assay chambers, here by forming a
heat stake across the loading channels leading to the assay
chambers while under pressure. Pneumatic pressure to the assay
chambers was subsequently released and the reaction imaging
assembly of the instrument captured another image of the assay
chambers to confirm that the contents of the assay chambers do not
leak out of the reaction area. At T=9:53, the instrument initiated
the amplification reaction and images were collected for an
additional 18 minutes. For these test runs, image acquisition time
was extended to collect additional amplification information. Total
run time, including image processing was approximately 27 minutes.
In this initial test, 12 cartridges were run, presenting 10
contrived (CT+/NG+) samples and 2 clean urines. In addition to CT-
and NG-specific reagents, one assay well in the cartridge contained
primers and probes specific for human beta actin, which is present
in human urine, as a positive control. The CT and NG were detected,
as expected, in each of the contrived samples. The human beta actin
was detected in both contrived and clean urine samples.
Amplification results are summarized in Table 1.
TABLE-US-00002 TABLE 1 Sample-to-Answer Test Results Target Time to
positive (T.sub.p) 1 CFU NG 7.44 .+-. 0.33 1 IFU CT 10.59 .+-. 0.45
b-actin 6.32 .+-. 0.41
[0498] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications can be made thereto without departing
from the spirit or scope of the appended claims. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
[0499] Accordingly, the preceding merely illustrates the principles
of the invention. It will be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
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