U.S. patent application number 14/653726 was filed with the patent office on 2015-12-03 for portable fluorescence detection system and microassay cartridge.
This patent application is currently assigned to Micronics, Inc.. The applicant listed for this patent is MICRONICS, INC.. Invention is credited to C. Frederick Battrell, Troy D. Daiber, William Samuel Hunter.
Application Number | 20150346097 14/653726 |
Document ID | / |
Family ID | 50979285 |
Filed Date | 2015-12-03 |
United States Patent
Application |
20150346097 |
Kind Code |
A1 |
Battrell; C. Frederick ; et
al. |
December 3, 2015 |
PORTABLE FLUORESCENCE DETECTION SYSTEM AND MICROASSAY CARTRIDGE
Abstract
Disclosed is a compact, microprocessor-controlled instrument for
fluorometric assays in liquid samples, the instrument having a
floating stage with docking bay for receiving a microfluidic
cartridge and a scanning detector head with on-board embedded
microprocessor operated under control of a ODAP daemon resident in
the detector head for controlling source LEDs, emission signal
amplification and filtering in an isolated, low noise, high-gain
environment within the detector head. Multiple optical channels may
be incorporated in the scanning head. In a preferred configuration,
the assay is validated using dual channel optics for monitoring a
first fluorophore associated with a target analyte and a second
fluorophore associated with a control. Applications include
molecular biological assays based on PCR amplification of target
nucleic acids and fluorometric assays in general, many of which
require temperature control during detection.
Inventors: |
Battrell; C. Frederick;
(Wenatchee, WA) ; Daiber; Troy D.; (Auburn,
WA) ; Hunter; William Samuel; (Jan Juc, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MICRONICS, INC. |
Redmond |
WA |
US |
|
|
Assignee: |
Micronics, Inc.
Redmond
WA
|
Family ID: |
50979285 |
Appl. No.: |
14/653726 |
Filed: |
December 20, 2013 |
PCT Filed: |
December 20, 2013 |
PCT NO: |
PCT/US2013/077244 |
371 Date: |
June 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61745329 |
Dec 21, 2012 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/287.2; 702/19 |
Current CPC
Class: |
B01L 7/52 20130101; B01L
2300/1827 20130101; Y02A 90/10 20180101; B01L 3/502715 20130101;
B01L 2400/0487 20130101; G01N 21/6428 20130101; B01L 2300/1844
20130101; B01L 2300/168 20130101; B01L 2300/0816 20130101; G01N
2201/062 20130101; G01N 21/645 20130101; Y02A 90/26 20180101; G01N
2201/12 20130101; B01L 2200/0684 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Claims
1. A microassay system for detecting an optical signal in a sample,
said system comprising a mechanical system enabled for scanning a
detector head across the sample, said detector head comprising at
least one detection channel, said detection channel comprising an
objective lens and optoelectronic circuit elements configured to
capture and amplify optical signals received by said objective
lens, wherein said mechanical system is operated by a controller
and circuitry external to said detector head and said
optoelectronic elements are under control of an autonomous daemon
resident in firmware associated with a microprocessor embedded
within said detector head.
2. The microassay system of claim 1, wherein said optoelectronic
circuit elements are configured for operation in electronic
isolation from said external circuitry.
3. The microassay system of any one of claim 1 or 2, wherein said
optoelectronic elements are configured for signal processing or
signal digitization under control of said daemon.
4. The microassay system of any one of claims 1-3, wherein said
detector head comprises at least one digital transmitter enabled to
transmit said signal in digital form to said external
controller.
5. The microassay system of any one of claims 1-4, wherein said
detector head is enabled to perform a scan across said sample under
control of said external controller, and said daemon in said
detector head is configured to autonomously construct a map of any
optical signals captured during said scan, said map having
datapairs expressing signal intensity as a function of location
relative to at least one reference point.
6. The microassay system of claim 5, wherein said detector head is
configured to scan a sample well on a defined path across a
cartridge body, and said at least one reference point is a spatial
coordinate on said defined path associated with a registration
feature of said cartridge body.
7. The microassay system of claim 6, wherein said registration
feature triggers a mechanical, electrical, or an optical switch to
initialize said reference point of said map.
8. The microassay system of claim 5, wherein a location of said
sample well is determined as a distance from said reference point
along said defined path where a first change in optical signal is
detected, said first change in optical signal correlating with a
first edge of an empty sample well, and a second change in optical
signal is detected, said second change correlating with a second
edge of an empty sample well under control of said autonomous
daemon.
9. The microassay system of claim 8, wherein said scan across said
cartridge body is repeated with a sample in said sample well, and a
change in optical signal associated with the map coordinates
between said first edge and said second edge is analyzed for a
change in signal indicative of an assay result under control of
said autonomous daemon.
10. The microassay system of claim 9, wherein said change in signal
is subjected to electronic conditioning and amplification prior to
digitization to produce a digitized signal under control of said
autonomous daemon.
11. The microassay system of claim 9, wherein said change in signal
is subjected to threshold subtraction prior to digitization.
12. The microassay system of claim 9, wherein said change in signal
is subjected to single-bit digitization.
13. The microassay system of claim 10, wherein said digitized
signal is accumulated in a register as a series of datapairs during
said scan across said well and a summation of signals for said scan
is reported to said external controller.
14. The microassay system of claim 10, wherein said digital signal
is reported to said external controller.
15. The microassay system of claim 5, wherein said detector head is
configured to scan a sample well on a defined path across a
cartridge body, and said at least one reference point is an spatial
coordinate on said defined path associated with a registration
feature of said sample well.
16. The microassay system of claim 15, wherein the daemon is
configured to acquire said reference point on said map by detecting
a change in optical signal associated with a first edge of a sample
well, and the location of said sample well is determined as a
distance from said reference point along said defined path to a
point at which a second change in optical signal is detected, said
second change correlating with a second edge of an empty sample
well.
17. The microassay system of claim 8, wherein said scan across said
cartridge body is repeated with a sample in said sample well, and a
change in optical signal associated with the map coordinates
between said first edge and said second edge is analyzed for a
change in signal indicative of an assay result under control of
said autonomous daemon.
18. The microassay system of claim 17, wherein said change in
signal is subjected to electronic conditioning and amplification
prior to digitization to produce a digitized signal under control
of said autonomous daemon.
19. The microassay system of claim 17, wherein said change in
signal is subjected to threshold subtraction prior to
digitization.
20. The microassay system of claim 17, wherein said change in
signal is subjected to single-bit digitization.
21. The microassay system of claim 18, wherein said digitized
signal is accumulated in a register as a series of datapairs during
said scan across said well and a summation of signals for said scan
is reported to said external controller.
22. The microassay system of claim 18, wherein said digital signal
is reported to said external controller.
23. The microassay system of claim 15, wherein said registration
feature is an edge having autofluorescence, said edge bordering
said sample well.
24. The microassay system of any one of claims 1-23, wherein said
optical signal is a fluorescent signal and said detector channel
comprises an LED for irradiating said mixture with an excitation
light, a lightpath having an excitation filter, emission filter,
dichroic mirror tuned to enable the detection of emissions from a
fluorophore in a defined passband, said objective lens for
condensing said excitation light and for collecting said emissions,
a sensor with sensor lens for receiving said passband emissions,
and a shielded preamplifier and multistage amplifier circuit for
amplifying an output from said sensor.
25. The microassay system of claim 24, wherein said amplifier is a
tri-stage amplifier and said amplifier is shielded from electronic
noise by a Faraday cage, a bypass capacitor, a signal conditioning
pre-amplifier, a separate ground plane, a metallized detector head
housing, or a combination thereof, and said amplifier is in
electrical proximity to said sensor.
26. The microassay system of claim 25, wherein said tri-stage
amplifier has a gain of up to 10.sup.14.
27. The microassay system of claim 25, wherein said tri-stage
amplifier has a gain of 10.sup.12 to 10.sup.14.
28. The microassay system of claim 25, wherein said objective lens
is configured for focusing said excitation light on a liquid sample
having a mixture of fluorophores and said excitation light is
focused on a spot smaller than said sample well.
29. The microassay system of claim 28, wherein said detector head
comprises a plurality of detector channels, each said detector
channel comprises an source LED for irradiating said sample well
with an excitation light, a lightpath having an excitation filter,
emission filter, dichroic mirror tuned to enable the detection of
emissions from a fluorophore in a defined passband, said objective
lens for condensing said excitation light and for collecting said
emissions, a sensor with sensor lens for receiving said passband
emissions, a shielded preamplifier and multistage amplifier circuit
for amplifying an output from said sensor; and said daemon is
configured for strobing each said source LED at a frequency so that
no two source LEDs are simultaneously on, and for multiplexedly
analyzing the signal from each sensor of said plurality of detector
channels according to whether the corresponding LED is on or
off.
30. The microassay system of claim 29, wherein each said LED is
turned on and off at a frequency of 130 Hz.
31. The microassay system of claim 30, wherein said objective lens
is configured for focusing an excitation light on a liquid sample
containing a mixture of fluorophores.
32. The microassay system of claim 29, wherein said daemon is
configured to tabulate a digitized amplifier output and a scanning
position in volatile memory within said detector head.
33. The microassay system of claim 29, wherein said daemon
comprises instructions for comparing a baseline scan of said sample
well with a scan of said sample well containing the mixture, and
reporting a difference indicative of a detection event associated
with detection of at least one fluorophore-specific emission.
34. The microassay system of claim 33, wherein said daemon is
enabled to digitally report said detection event to said host
controller.
35. The microassay system of claim 29, wherein said system
comprises a graphical user interface and programmable instructions
in non-volatile memory for displaying said detection event under
control of said external controller.
36. The microassay system of claim 29, wherein said system
comprises an I/O port and programmable instructions in non-volatile
memory for communicating said detection event to a remote device
under control of said external controller.
37. The microassay system of claim 29, wherein said mixture of
fluorophores comprises a first fluorophore for detecting a target
analyte and a second fluorophore for detecting a control analyte,
and further wherein said target analyte and said control analyte
are present together in the mixture.
38. The microassay system of any one of claims 1-37, wherein said
sensor is a photodiode.
39. The microassay system of claim 24, wherein said irradiated
light received by said objective lens is formed as a divergent
beam, a convergent beam, or a collimated beam for transition
therethrough.
40. A method for automating a microassay, the method comprising
operationally dividing the microassay system into fluidic
electromechanical and thermal processes controlled by a host
controller and optoelectronic processes controlled by an autonomous
daemon resident in a scanning detection head, wherein the motion of
the scanning detection head across a sample is controlled by the
host controller and any optical signal acquisition,
preconditioning, amplification and digitization of a signal
generated in the sample is controlled by the autonomous daemon.
41. An apparatus for performing a microassay, the apparatus
comprising an assay system operationally divided into fluidic
electromechanical and thermal processes controlled by a host
controller and optoelectronic processes controlled by an autonomous
daemon resident in a scanning detection head, wherein the motion of
the scanning detection head across a sample is controlled by the
host controller and any optical signal acquisition,
preconditioning, amplification and digitization of one or more
signals collected from the sample during a scan is controlled by
the autonomous daemon.
42. The apparatus of claim 41, wherein said daemon is enabled to
multiplexedly collect said one or more signals through a plurality
of optical channels.
43. The apparatus of claim 42, wherein said daemon is enabled to
condition and digitize said signals during said scan, recording a
single bit datum as a function of position or time.
44. The apparatus of claim 43, wherein said daemon is enabled to
condition and statistically evaluate digitized optical signals from
said plurality of optical channels and to report a detection event
after validation of the scan by detection of a control signal in
the sample.
45. The apparatus of claim 44, wherein said daemon is enabled to
report said detection event to an external device.
46. The apparatus of claim 45, wherein said plurality of optical
channels is configured for detecting a plurality of fluorophores
comprising a first fluorophore for detecting a target analyte and a
second fluorophore for detecting a control analyte, and further
wherein said target analyte and said control analyte are present
together in the sample.
47. A microassay cartridge for performing a sample assay, said
cartridge comprising a) a first circuit card having a hydraulic
circuit enclosed therein, wherein said hydraulic circuit of said
first circuit card is configured to perform a first sample
processing step; b) an second circuit card having a hydraulic
circuit enclosed therein, wherein said hydraulic circuit of said
first circuit card is configured to form a second sample analysis
step; c) at least one fluid junction between said hydraulic circuit
in said first circuit card and said hydraulic circuit in said
second circuit card; d) an array of pneumatic ports defining a
pneumatic interface, each port for receiving a pneumatic pulse
applied thereto, at least one said port having a via fluidly
joining a pneumatic circuit in said second circuit card to a
pneumatic circuit in said first circuit card, said pneumatic
circuit in said first circuit card for operating the hydraulic
circuit therein, said pneumatic circuit in said second card for
operating the hydraulic circuit therein; and, wherein said
cartridge is enabled such that a pneumatic pulse applied to said
pneumatic interface at said first via forces said processed sample
through said fluid junction from said outboard circuit card to said
inboard circuit card.
48. The microassay cartridge of claim 47, wherein said array
comprises a plurality of pneumatic ports fluidly connected by vias
fluidly joining a pneumatic circuit in said first circuit card to a
pneumatic circuit in said second circuit card, such that said
plurality of pneumatic ports are enabled to receive a plurality of
pneumatic pulses applied according to programmable instructions and
to multiplex those pneumatic pulses to fluidic circuit elements of
said first circuit card and said second circuit card, thereby
reducing the number of ports required to operate said first and
said second hydraulic circuits.
49. The microassay cartridge of claim 48, wherein said pneumatic
interface is configured to enable multiplexed distribution of
stronger pneumatic pulses to circuit elements operative with a
stronger pneumatic pressure and weaker pneumatic pulses to circuit
elements operative with a weaker pneumatic pressure in said first
circuit card and said second circuit card.
50. The microassay cartridge of claim 48, wherein said pneumatic
interface is configured to enable multiplexed distribution of
positive pressure pneumatic pulses to circuit elements operative
with a positive pneumatic pressure and vacuum pressure pneumatic
pulses to circuit elements operative with a vacuum pneumatic
pressure.
51. The microassay cartridge of any one of claims 47-50 which
comprises a single-use sealing gasket configured to join said
pneumatic interface to a host instrument.
52. The microfluidic cartridge of any one of claims 47-51, wherein
said cartridge is configured for being controlled by applying
pneumatic pressure states to the pneumatic circuits, wherein said
pneumatic pressure states are selected from a range of positive and
negative pressures.
53. The microfluidic cartridge of claim 52, wherein said pneumatic
pressure states are selected from about -5 psi, +10 psi, +12 psi,
+20 psi, +7 psi, and 0 psi.
54. The microfluidic cartridge of claim 52, wherein at least one
pneumatic circuit of said first card and at least one pneumatic
circuit of said second card are connected to a common port of said
pneumatic interface port array, and said at least one pneumatic
circuit in said first card is operative on said sample when said
first card receives said sample and said at least one pneumatic
circuit of said second card is operative on said sample after said
sample is transferred from said first card to said second card.
55. The microfluidic cartridge of claim 54, wherein said first card
is configured to extract a nucleic acid extract from a sample, and
said second card is configured to analyze said nucleic acid extract
for a target analyte.
56. A microassay apparatus for performing a sample assay, said
apparatus comprising a) a disposable microassay cartridge
configured for docking with a host instrument, said cartridge
having at least one hydraulic circuit disposed therein, wherein
said at least one hydraulic circuit is configured to operate under
control of at least one pneumatic circuit interfaced thereto; b) an
array of one or more pneumatic ports defining a pneumatic interface
port array, wherein each port is enabled to convey a pneumatic
pressure state from said host instrument to a pneumatic circuit; c)
at least one manifold disposed in said host instrument, said
manifold having a plurality of pneumatic pressure sources fluidly
coupled thereto, wherein a first pressure source is operated to
define a first pressure state in said manifold at a first time
interval in an assay sequence and a second pressure source is
operated to define a second pressure state in said manifold at a
second time interval in said assay sequence, and wherein said
manifold is fluidly connected to said pneumatic circuit through a
port of said pneumatic interface port array; and, d) instructions
for pneumatically controlling said at least one hydraulic circuit,
wherein said instructions are executed under control of a
microprocessor having one or more memory elements, and said
instructions comprise steps for actuating said first pressure
source and said second pressure source according to said assay
sequence.
57. The microassay apparatus of claim 56, wherein said cartridge
comprises at least one hydraulic circuit configured to operate
under control of a plurality of pneumatic circuits interfaced
thereto, said pneumatic interface port array having fluidic
connections to said at least one manifold, wherein said at least
one manifold is configured to be operated at a plurality of
pressure states.
58. The microassay apparatus of claim 57, wherein said at least one
hydraulic circuit is configured to be operated under pneumatic
control of pressure states conveyed by a plurality of pneumatic
circuits, at least one of said pneumatic circuits having a
plurality of pressure states.
59. The microassay apparatus of any one of claims 56-58, which
comprises a single-use sealing gasket configured to join said
pneumatic interface port array of said cartridge to said host
instrument.
60. A host instrument for removably receiving a microassay
cartridge, said instrument comprising: a) a supporting baseplate;
b) a docking bay for receiving a microassay cartridge, wherein said
docking bay is mounted on said baseplate; c) a pneumatic control
manifold disposed within said baseplate, said manifold having
disposed thereon a pneumatic interface port array within said
docking bay; d) attached to said baseplate within said docking bay,
a heater assembly comprising at least one heating member with
spring mount and with superior surface dimensioned for interfacing
with an undersurface of said cartridge; and, wherein said docking
bay comprises a clamping mechanism configured to reposition the
cartridge within the docking bay from first position above the
pneumatic interface port array to a second position wherein said
cartridge is operatively engaged with said pneumatic interface port
array, and further wherein said spring mount is configured to press
an undersurface of the microfluidic cartridge in said second
position against said superior surface of said heating member.
61. The apparatus of claim 60, wherein said spring mount exerts a
spring force of about 1 psi over a heat transfer surface reversibly
contacting said cartridge and said heating member.
62. The host instrument of any one of claims 60-61, wherein said
docking bay with clamping mechanism is configured to simultaneously
engage the pneumatic interface port array of the baseplate with
mating ports of the microassay cartridge and to press the cartridge
against the at least one spring-mounted heating member.
63. The host instrument of any one of claims 60-62, wherein said
host instrument is configured for pneumatically controlling the
operations of the microassay cartridge via said pneumatic control
manifold while clamped in said docking bay against the heating
member.
64. The host instrument of any one of claims 60-63, wherein said
clamping mechanism is configured for disengaging said cartridge
from said pneumatic interface port array and said heating mechanism
of said baseplate so that said cartridge may be removed from said
docking bay.
65. The host instrument of claim 64, wherein said cartridge
comprises a single-use gasket configured to sealingly join said
cartridge to said pneumatic interface port array of said docking
bay.
66. The host instrument of any one of claims 60-65, wherein said
heating member is electropolished and chrome plated.
67. The host instrument of any one of claims 60-66, wherein said
heating member is resistively heated.
68. The host instrument of any one of claims 60-66, further
comprising a first fan for convectively dissipating heat from a
cooling member disposed on said heating member.
69. The host instrument of any one of claims 60-67, further
comprising a second fan for convectively cooling a sample well
disposed in said microassay cartridge.
70. The host instrument of claim 69, wherein said second fan is
configured for performing a thermal annealing function by cooling
said sample well while the sample is fluorescently monitored.
71. A method for co-assaying a sample for a target fluorescent
signal associated with a pathogenic condition and a control
fluorescent signal associated with an endogenous non-pathogenic
component of said sample, said method comprising: a) scanning a
sample well with a detector head of claim 1, wherein said target
fluorescent signal, if present, is detected in a first optical
channel of said detector head and said control fluorescent signal,
if present, is detected in a second optical channel of said
detector head; and b) reporting a valid result of said assay if and
only if said second fluorescent signal is detected.
72. A method for performing a FRET melt curve determination,
comprising a) heating a sample chamber containing a FRET probe and
a target nucleic acid sequence to a temperature above the melt
temperature of the duplex probe; b) turning off said heating; and,
c) using a detector head of claim 1, monitoring a change in
fluorescence of said FRET probe in response to a change in
temperature of said sample chamber caused by turning on a fan and
directing a stream of ambient air onto the sample chamber, thereby
rapidly cooling said sample chamber while monitoring
fluorescence.
73. The method of claim 72, wherein said melting curve is completed
in less than a minute.
Description
BACKGROUND
[0001] 1. Technical Field
[0002] The present invention generally relates to a compact
fluorescence detection instrument with optical, thermal, mechanical
and pneumohydraulic systems for use in diagnostic assays performed
in a microassay cartridge.
[0003] 2. Description of the Related Art
[0004] Although the benefits of the use of fluorophores as probes
for in-vitro diagnostic assays are well known, the most commonly
available forms of equipment for such assays are large, complex to
use, relatively slow and rely on expensive confocal optics. These
attributes make much equipment unsuitable for fully integrated
"sample-to-answer" testing in remote locales and on-site at the
point of care, where such equipment is required to be rugged, fast,
compact, inexpensive, and easy to use. Although automated nucleic
acid amplification in a microfluidic cartridge was first proposed
some years ago (see Wilding: U.S. Pat. Nos. 5,304,487 and
5,635,358), detection of fluorescent assay targets outside
controlled laboratory conditions is still hampered by the lack of
portable and robust equipment. Two decades since their inception,
molecular diagnostics are still relatively uncommon in the absence
of advanced laboratory facilities because of these and other
unsolved problems.
[0005] Needed to promote broader access to molecular diagnostics
are self-contained assay systems designed to operate outside
specialized laboratory facilities. Nucleic acid assays are rapidly
becoming the "gold standard" for the detection of many different
disease types, including infectious diseases, because they offer
both higher sensitivity and specificity. Such assays have proven
highly specific to a broad range of pathogenic conditions and are
useful for tracking genetic strains of a particular disease as is
fundamental to epidemiology, for example in discriminating H5N1
avian influenza from other types of influenza A or B, in
determining whether a particular pathogen target is of a
drug-resistant strain or not, and in detecting toxigenic strains of
an enteric isolate such as E. coli O157:H7. Fluorescence-based
assays have also been shown to be useful for monitoring conditions
such as diabetes, cardiopathies, coagulopathies, immunoassays in
general, and for detection of endotoxin in foods or drug products
for example. Improved equipment is particularly needed for the
large numbers of remote health clinics in the developing world
where access to health care is limited and many infectious diseases
are endemic, and health and life expectancy are poor.
[0006] In a typical fluorescence assay system, a fluorescent probe
or fluorophore absorbs light having a wavelength or range of
wavelengths and becomes excited; and the fluorophore then emits a
fluorescent signal. The activity or inactivity of the fluorophore
is indicative of the assay result. The emission signal has a
wavelength or range of wavelengths that is generally longer than
the exciting light (but may be shorter as in "up-converting
fluorophores"). A dichroic beam splitter or band-pass filter, or
combination thereof, is then used to separate the fluorescent
signal from other light, and the signal is passed to a sensor. The
sensor is often a photodiode, and generates an electrical signal
that can be used to score the assay. Qualitative and quantitative
assays using real time or endpoint fluorometry are feasible.
[0007] In such systems, a liquid sample is conveyed via a
microfluidic channel into a detection chamber or channel of a
microfluidic cartridge where a fluorescent probe admixed with or
native to the sample is excited by an excitation source. Controls
may be run in parallel or multiplexed in the assay channel. Emitted
light is measured to determine the presence or absence of a target.
A plurality of detection channels may be arranged in the detection
region of the microfluidic cartridge. Assays involve making one or
more measurements of fluorescence; fluorophores may be used as
markers for nucleic acid amplicons formed in an amplification step,
or more generally for the presence or absence of a fluorescent
assay target. Real time fluorometry, FRET, qPCR, thermal melt
curves, kinetic and rate endpoints for assay scoring and validation
are also known in the art.
[0008] Known fluorescence detectors typically employ relatively
expensive optical components (such as confocal optics, lasers and
aspheric lenses) in order to pick up and localize fluorescent
emissions present within a microfluidic cartridge or microarray. WO
98/049543 to Juncosa, for example, teaches three dichroic beam
splitters in a single optical train, one for controlling excitation
source power and another for controlling reflectance signal; the
third dichroic beam splitter is used for discriminating
probe-specific fluorescent emission. One or more lenses serve to
focus the excitation beam on the sample. Juncosa further teaches
use of an aperture at the inlet of a photomultiplier and optical
objective lens components of a confocal microscope for controlling
an imaging beam with a resolution of "microlocations" at about
fifty microns. "By restricting the scope of the illumination to the
area of a given microlocation, or a fraction thereof, coupled with
restricting the field of view of the detector to the region of
illumination, preferably through use of an aperture, significant
improvements in signal-to-noise ratio may be achieved." [p 7, lines
10-15]. These teachings are presaged by U.S. Pat. No. 3,013,467 to
Minsky, U.S. Pat. No. 5,296,703 to Tsien, U.S. Pat. No. 5,192,980
to Dixon, U.S. Pat. No. 5,631,734 to Stern, U.S. Pat. No. 5,730,850
to Kambara, and are reiterated in U.S. Pat. No. 6,614,030 to Maher
and U.S. Pat. No. 6,731,781 to Shams, among others. Maher uses
lasers, fiber optics, a quartz plate and aspherical lenses with
mini-confocal optical system in order to optimize focusing and
emission at a ten micron-sized spot at the center of the
microfluidic chamber.
[0009] Similarly, in U.S. Pat. No. 6,635,487, Lee affirms that
focusing the cone of the excitation beam on the plane of the sample
"provides the greatest intensity to enhance analytical detection
measurements on the assay chips.". This teaching thus encapsulates
the prior art.
[0010] In a more recent filing, US Patent Application 2008/0297792
to Kim teaches that an image of an LED serving as a light source
for fluorescence detection in a microfluidic chip is projected onto
a sample as an "optical spot" by an objective lens. The optical
spot is focused at the middle of the depth of a fluid in a chamber
in the microfluidic chip. Fluorescence emitted by the sample is
collimated as nearly as possible to parallel rays by the objective
lens and focused on an avalanche photodiode. The requirement for
high precision in alignment relates to the dichroic mirror because
the stopband will be shifted for light rays that do not enter the
mirror at a 45.degree. angle, as is well known. Thus the teachings
of Kim reflect the generally recognized state of the art.
[0011] In PCT Publication WO2008/101732 to Gruler, where is
described a fluorescence detector head for multiplexing multiple
excitation and detection wavelengths in a single light path, it is
stated that, "A confocal measurement means that the focus of the
illumination optics or the source, respectively intrinsically is
the same as the focus of the detection optics or sensor,
respectively.". Gruler goes on to state, "The confocal optics [of
the invention] . . . secures highest signal and lowest background
intrinsic features of confocal design", i.e., according to Gruler
the highest possible signal and lowest noise are obtained with
confocal optics.
[0012] While the consensus teaching of the prior art arose out of
the specialized use of confocal optics for epifluorescence
microscopy, the teaching has been widely and uncritically applied
to microfluidic, lateral flow, capillary electrophoresis and
microarray applications. However, we have found that this approach
is not well suited to liquid phase microfluidic diagnostic assays
where detection of one or more molecular probes in a fluid-filled
channel is required. Due to effects such as photoquenching,
thermo-convection, and the occasional presence of bubbles or
gradients in a fluid-filled channel, colocalizing the focal point
of the excitation beam and emission cone in the plane of the sample
chamber can lead to unacceptable instability, loss of signal,
quenching, noise, irreproducibility and overall loss of sensitivity
in the results. Because of the higher temperatures of PCR, for
example, outgassing of reagents and sample is not an uncommon
problem, and interference from bubbles entrained in the liquid
sample is a frequent problem. The conventional approach also
requires more expensive optical components and thus is
disadvantageous for widespread application outside advanced
clinical laboratories.
[0013] A second problem is assay validation. Current standards for
validation of infectious disease assays by PCR, for example, have
come to rely on use of spiked nucleic acid templates or more
preferably, co-detection of endogenous normal flora, for example
ubiquitous non-pathogenic Escherichia coli in stools where
pathogens such as Salmonella typhi or E. coli O157 are suspected.
Another ubiquitous endogenous template is human 18S rRNA, which is
associated with higher quality respiratory and blood samples.
Co-amplification and detection of an endogenous template ensures
confidence in the assay results but is difficult to achieve in
practice because of possible crosstalk between the fluorophores
used as markers. When using high gain amplification, some level of
crossover in the spectra of the excitation and emission of
fluorophores commonly selected for multiplex PCR is typical and
expected. Thus a solution that would isolate fluorescent signals
with spectrally overlapping shoulders by using separate optical
channels within a scanning detector head having shared low-noise
electronics for downstream processing would be a technological
advance of benefit in the art.
[0014] A third problem is portability. Use of disposable cartridges
has proved beneficial because cross-contamination due to shared
reagent reservoirs and shared fluid-contacting surfaces is avoided.
However, configuring a precision optical instrument platform for
accepting disposable cartridges is problematic. Problems include
inaccuracies and stackup in mechanical tolerances that affect
cartridge alignment and detector head positioning, the need for
forming a highly conductive thermal interface between the plastic
disposable cartridges and heating sources in the instrument, the
need for sealing the pneumatic interface between control servos on
the apparatus and microvalves on the cartridge, and the necessarily
shorter light path available in a microfluidic cartridge (typically
about or less than 1 millimeter), which without optimization can
lead to loss in sensitivity. A simultaneous solution of these
interlocking problems is only achieved by extensive experimentation
and development, most often guided by trial and error in this
highly unpredictable art. Thus there is a need in the art for
numerous improvements, elements of which are the subject of the
disclosure herein.
BRIEF SUMMARY
[0015] The present invention addresses the problem of reliable and
sensitive detection of fluorescent probes, tags, fluorophores and
analytes in a microfluidic cartridge in the presence of bubbles and
other interfering inhomogeneities in a liquid sample, in a first
aspect of the invention, by providing a reflective mirror face
formed on a heating block that contactingly interfaces with a
thermo-optical window on the underside of the detection channel or
chamber containing the liquid sample. The mirror face is formed on
the top surface of a heating block and contacts the lower optical
window of the detection chamber during use, avoiding the complexity
and expense of manufacturing an integral mirror on the bottom of
each disposable cartridge, and allowing us to use thinner, more
compliant films with lower resistance to heat transfer and
transparent optical characteristics for the thermo-optical window
of the cartridge. The mirror face is optically flat and polished to
improve both heat transfer and fluorescence emission capture. A
scanning objective lens is positioned above an upper optical window
on the top of the microfluidic cartridge. Excitation light is
transmitted through both the upper optical window and the lower
thermo-optical window before striking the mirror and reflecting
back. Direct and reflected emissions are collected by the objective
lens and focused on a detection sensor such as a photodiode,
photocell, photovoltaic device, CMOS or CCD chip.
[0016] Also, and starkly in contrast to the teachings of the prior
art, the problem of fluorescence detection is shown to be solved by
configuring the optics so that the excitation optics are decoupled
from the emission optics on a common optical path. In a preferred
embodiment, by trial and error, when using the back mirror, we have
found that it is advantageous to place the focal point of the
excitation cone near or behind the plane of the reflective mirror
and to independently position the emission cone so that emissions
are preferentially focused on the detection sensor. Optionally, the
excitation cone can be refocused in the near field by directing a
convergent source beam on the objective lens. Surprisingly,
decoupling increases sensitivity, improves limits of detection, and
reduces noise or interference of bubbles and other inhomogeneities
in the sample.
[0017] Contrary to the teachings of the prior art, we find that the
conventional confocal localization of the excitation and emission
signal is less effective in generating a robust signal over a wide
range of sample and operating conditions. Therefore, in one aspect
of the invention, it was found that optimization of signal
detection may be improved by displacement of the focal point of the
excitation light from the plane of the sample to a point behind the
sample, a technological advance in the field of low cost optics for
use with microfluidic fluorescence assays. Decoupling of excitation
and emission optics (i.e., different focal points for excitation
light and emission signals) flies against decades of prior art
dedicated to the principles (first espoused by Minsky in U.S. Pat.
No. 3,013,467) that form the foundation of conventional practice in
confocal microscopy, epifluorescence detection, and microfluidic
fluorescence assays. The prior art teachings have lead to the use
of aspherical lenses, laser diodes, and precise parfocal alignment
of the detection optics with the excitation optics. In contrast,
the optics required for delocalized focus of the excitation cone as
described here are fortuitously of very low cost and do not require
precision assembly or maintenance, as is desirable for
manufacturing a low cost, portable instrument.
[0018] The mirror face on the top surface of the heating block
under the detection chamber is used to increase sensitivity by
improving the light-gathering capacity of the objective lens. As
the objective lens is placed closer to the detection chamber, a
lens of defined angular aperture and numerical aperture becomes
more efficient in collecting emissions. Without the back mirror,
collection efficiency of a typical system of the prior art is less
than 2.5% (assuming for example a 5 mm planoconvex lens). Adding a
back mirror can improve this by as much as 200%, and theoretically
as much as 400%. And focusing the excitation beam behind the sample
chamber can add synergically to any gain in sensitivity by
increasing the excitation pathlength by using a mirror. We have
found that this is especially advantageous in low aspect ratio
microfluidic cartridges, where the optical path length on a z-axis
of a cartridge is typically sub-millimeter in length, a significant
reduction relative to a standard optical cuvette. Happily, this
combination was also found to reduce interferences due to
irregularities in the sample chamber such as the presence of small
bubbles.
[0019] In one embodiment, the mirror is a chromed or polished metal
surface on an aluminum or copper heating block, and is also used to
transmit heat or cooling for temperature controlled assays, thus
achieving another synergy of design. In a preferred embodiment, the
mirror is an electropolished chrome surface on an optically flat
aluminum block, the aluminum selected for its superior heat
transfer characteristics and scaleable thermal inertia. The block
is heated by a resistive heating element in contact with the base
of the block. The smoothness and flatness of the mirror face favors
optimal heat transfer. In this aspect of the invention, the
mirrored face is the upper surface of the heating element used for
example for FRET detection or thermal melting analysis of
fluorescent probes for PCR amplicons. In one embodiment, a combined
application of the optical and thermal properties of the
mirror-faced heating block is illustrated by construction of FRET
melt curves taken by monitoring fluorescence while ramping the
temperature of the assay fluid. In another embodiment, the mirror
faced heating block is used to adjust or control a reaction
temperature in the detection chamber of a microfluidic card while
the cartridge is scanned for fluorescence emission. A mirror-faced
heating block for use with microfluidic cartridges in real-time and
temperature modulated fluorescence assays demonstrates a technical
advance in the art.
[0020] According to another aspect of the present invention, we
have employed a high gain multi-stage amplifier with noise
elimination augmented through the use of downstream signal
processing firmware compactly mounted in the scanning head. Very
high gain amplification and out-of-plane delocalization of the
excitation light cone were found to be synergic in optimizing assay
discrimination and sensitivity, even in the presence of bubbles
which disrupt specular reflection from the mirror face behind the
liquid sample, and happily were implemented with no increase in
cost.
[0021] The complete optical path uses three lenses, the first for
collimating excitation from a light source, the second for
projecting the excitation source onto the mirror and for collecting
a fluorescent emission from any fluorophore in the sample as
collimated emissions, and the third for focusing the emissions on a
detector. Each fluorophore is optically isolated by a separate
optical channel for measurement. A combination of
spectrally-specific LEDs, dichroic mirrors, and barrier filters are
used to achieve near monochromatic excitation light in each optical
channel. The lenses and related optical components, including the
dichroic mirror intersecting the light path for separating
excitation and emissions wavelengths and filters, are provided in a
guiderail-mounted scanning head that moves laterally across the
detection chambers. To minimize noise, the head also includes all
electronic components for amplifying the signal and an on-board
embedded microprocessor for analog and digital signal processing.
Even in the presence of bubble foam interferences that defeat
signal averaging and baseline subtraction methods of data
acquisition, assay scanning data from each optical channel may be
accurately evaluated and reported by conversion to a single bit
output (i.e., a 1 or a 0). This has been found to be a simple and
remarkably effective means for qualitative scoring for the presence
or absence of a signal from a particular fluorophore in a liquid
sample mixture while the detector head is scanned over the
detection chamber or chambers and across the mirror face.
[0022] The scanning head and rails are configured with a drive
chain coupled to a stepper motor for accurate spatial resolution
during scanning. The entire microfluidic cartridge docking bay and
optical bench is mounted in an instrument housing at a pitched
angle, which we have found advantageous in decreasing bubble
entrainment and improving venting during loading, wetout, and
mixing operations on the microfluidic cartridge. In a preferred
embodiment, the entire optical bench is mounted at an angle of
about 15 degrees so that bubbles are displaced from the
microfluidic circuitry, necessitating a complete suspension mount
for the floating optical bench and the docking bay, and a
spring-biased clamping mechanism to ensure active formation of a
thermoconductive interface between on-board heating elements
mounted in the docking bay on the bottom of the optical bench
assembly and the insertable cartridge when the cartridge is loaded
into the instrument. A sealed interface between the angled
cartridge and a gasketed pneumatic interface port must also be
established during docking.
[0023] The detector head may be scanned across the detection
chamber, or conversely, the microfluidic cartridge may be scanned
across the detector. As implemented here, the detector is mounted
with a worm gear- or rack and pinion gear-driven stage under
control of a stepper motor on twin, paired guiderails. Samples may
be scanned on demand by synchronizing data acquisition with motor
control; this may be performed using an on-board or external host
controller in communication with the embedded microprocessor in the
detector head. Surprisingly, such a detector head with integral
co-processor, mounted on a linear motion stage for sampling,
filtering and averaging measurements on the fly, improved the
capability of the system to validate and report assay results
despite many potential interferences.
[0024] It was expected that there will be variations in fluorescent
intensity across the sample well, detection chamber, detection
channel or field, which can be a millimeter to several millimeters
in width. These variations arise, for example, as a result of
inhomogeneities in mixing, from differences in well thickness, from
imperfections in the optical windows, from small temperature
variations (which can cause accompanying variations in
hybridization-dependent fluorescence of amplicons being detected,
for example) and from bubbles or foam which may arise from
degassing and mixing. It was found that these variations can be
minimized by signal digitization over a sampling transect across
the detection chamber. A threshold may be used to discriminate
positive and negative test results, as will be described further
below.
[0025] In order to further provide noise elimination, extraneous
noise is removed by strobing the excitation beam at a frequency
known to prevent interference by AC line fluctuations and other
ambient electric or RF interference; resulting in cleaner signal
modulation, a modulated signal that also can be filtered to remove
the effects of any ambient light leaking into the instrument
housing. We have achieved this result by configuring the optics
printed circuit board with a dedicated co-processor, and using
independent clock frequency and firmware, which efficiently
minimizes traffic on a databus connected to the host instrument.
The on-board optical signal processor has dedicated instructions
stored in EEPROM resident on the optics card, and synchronizes
pulses sent to the source LED with interrogation of the sensor
photodiode at a frequency selected to limit electromagnetic
interference. The firmware is designed to evaluate the difference
in signal between strobe illumination bursts of excitation light
and background between strobe bursts, and any background due to
ambient or extraneous light is readily subtracted by this
method.
[0026] The resulting optics modules are packaged in a sealed
detector housing and can be expanded as a series of optics modules
with multiple optical channels for simultaneously scanning multiple
samples or fluorophores in series or in parallel. Signal processing
is routed to an autonomous co-processor embedded in the detector
head, and from there to the host instrument controller. Thus the
invention comprises single head/single channel embodiments, dual
head/dual channel embodiments, and multi-head/multichannel
embodiments and may perform single and multiplex assays on single
samples or on multiple samples in parallel.
[0027] In general, the assay system comprises a mechanical system
enabled for scanning the detector head across the sample, the
detector head housing at least one detection channel, and more
preferentially a plurality of detection channels, each detection
channel comprising an objective lens and optoelectronic circuit
elements configured to capture and amplify optical signals received
by the objective lens, wherein the mechanical system is operated by
a controller and circuitry external to the detector head and the
optoelectronic elements are under control of an autonomous daemon
resident in firmware associated with a microprocessor embedded
within said detector head. Advantageously, segregation of the
optical signal acquisition and processing within a shielded
detector head where all the components are in close proximity
realizes a system for multi-stage amplification with negligible
signal noise that would otherwise be associated with the analog
functions occurring in the host instrument outside the detector
head. The optoelectronic circuit elements are operated in
electronic isolation from any external circuitry in the host
instrument; including optoelectronic elements configured for signal
processing and/or signal digitization under control of the
autonomous daemon. We believe that this is an advance in the
art.
[0028] The detectors were found to function well when housed as
dual head and multi-head detectors, where two or more channels in a
single housing were configured with fully independent optics,
fluorophore-specific filters, dichroic mirrors and source LEDs,
reducing crosstalk between multiple fluorophores. The head thus
will optionally contain a plurality of light sources mounted on a
first circuit board and a plurality of objective lens and
associated detectors mounted on a second circuit board, and will
collect signals for each of a plurality of fluorophores
independently using shared signal processing capability and
firmware embedded in the detector head. To reduce noise, no analog
signals are transmitted from the detector head to the host
instrument.
[0029] In this way, light sources for excitation in each channel
can be matched to the individual fluorophores. It is no longer
necessary to provide white light and an excitation filter to ensure
a narrow pass beam of excitation light striking the sample. This
simplification proved useful in assay protocols calling for paired
collection of "biplex" or multiplex target and control signals.
Where, as for FDA CLIA waiver requirements, both target and control
templates are amplified, a positive control signal must be present
before an assay result on a test sample can be reported or billed.
In the absence of a detectable control signal, any target signal
detected is not a valid result. In order to meet CLIA waiver
requirements, it is necessary that the fluorescence detector be
able to detect not only the presence, for example, of a target
infectious organism amplified by PCR but also of an endogenous
human control organism co-existing with the target and amplified by
the same PCR reaction or a PCR reaction conducted in parallel in
the instrument, for example.
[0030] Such an approach preferably requires that the fluorescent
detector have the capacity to determine the presence of both the
target and the control fluorophores as a "biplex" ("duplex")
amplification reaction mixture in a common detection chamber. A
positive amplification control fluorophore is typically used which
has fluorescent excitation and emission spectra which is shifted
(in wavelength) to be well resolved by selective band pass filters
from the fluorescent excitation and emission spectra for the target
fluorophore (see FIG. 25 for example). However, according to the
present invention, it proved possible to achieve superior
resolution by using a dual head detector and by scanning each
detection chamber twice in series, once with each optical channel,
detecting first the control fluorescence signature, then the test
sample fluorescence signature--each scanning pass utilizing
separate excitation and emission optics as described above.
[0031] A benefit was found by configuring these detectors with
fully separated and independent light paths. In this aspect of the
invention, the use of a duplex head design ensures that the
presence of an amplification control fluorophore in a sample does
not inadvertently create a signal in the target channel due to
"crosstalk". Such a situation would result in the sample being
classed as a "false positive". Conversely, it is also important
that there is no crosstalk from the target channel to the control
channel, which is likely when multiple signals share a common
optical path. Such a situation could result in the positive
amplification control being inadvertently deemed present, when this
may not be the case, and would lead to reporting of an invalid
assay, an unacceptable outcome.
[0032] These principles are exemplified by the use of fluorescein
or equivalent fluorophore as a molecular probe for the target, and
Texas Red or equivalent fluorophores as a molecular probe for the
control. A dual head detector, with one detection channel optimized
for detection of fluorescein and the other detection channel
optimized for Texas Red, each with separate excitation and
detection optics, was found to be surprisingly sensitive, accurate
and robust. The detector head was moved to position each optical
channel in turn over the sample and separate fluorescence readings
were made. Surprisingly, this improved resolution and minimized
cross talk but did not contribute to higher noise or loss of
sensitivity due to the mechanics of moving the head. Because
excitation is not performed with white light, but is instead
performed at a wavelength specific for an individual fluorophore,
quenching of the second fluorophore is not an issue.
[0033] In another aspect of the invention, we have found that
multiple microfluidic channels can be scanned by sequential
traverse of a multihead detector across multiple sample wells, each
head being configured with independent optics for excitation and
emission of a single fluorophore. Although the excitation and
detection optics are separated for each optical channel, signal
processing is performed in circuitry shared within the detector
head. The detector head perform scans under control of an external
controller, while the daemon in the detector head autonomously
constructs a map of the sample wells encountered during the scan,
the map having datapairs expressing signal intensity as a function
of location relative to at least one reference point. In one
instance the reference point is a spatial coordinate on a defined
scanning path taken by the detector head, where the reference point
is associated with a registration feature of said cartridge body,
and may be associated with a feature that triggers a mechanical,
electrical or optical switch, for example. Sample wells are
detected by a change in optical signal strength associated with a
wall or edge of the well, or other fiducial that identifies the
well edge. In some instances, the reference point is taken as a
first edge of a first well, and the map includes information about
the width from a first edge to a second edge of each of the wells,
so that when liquid is introduced, a background scan for each map
point can be subtracted from any optical signals associated with
the sample.
[0034] Thus in yet another embodiment the invention provides a
robust multi-head independent channel fluorescent detection system
for a point of care molecular diagnostic assay which has a high
degree of specificity directed towards the presence of both a
target and an nucleic acid amplification control co-existing in a
common or parallel detection chamber. The cleaner signals permit
higher gain electronic amplification without a corresponding
decrease in signal-to-noise ratio. A first embodiment in this
invention realizes a fluorescence detection system having more than
two channels, for example to detect target and control mixed in a
single detection chamber, and having multiple assay channels for
detection of multiple targets. Optionally, the heads may be
positioned side-by-side, in an array, or radially, as in a
cylinder.
[0035] Use of the independent optical pathways fortuitously
resulted in reduced need for precision in assembly of lenses,
dichroic beam splitters, and associated filter elements, improving
the manufacturability of the apparatus. Embodiments of the present
invention incorporate inexpensive non-precision optics, plastic
lenses, a mirror on the heating block behind the sample window,
moveable stage elements, strobed excitation and emission, noise
suppression, on-board continuous signal processing over a movable
detection field, and more than one optical channel for biplex assay
validation by use of paired target and control fluorophores.
Despite its high amplification gain, the instrument has proven
advantageously resistant to interferences such as electrical noise
and bubbles in the detection chamber.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0036] FIG. 1 is a perspective view of an instrument of the
invention and a microfluidic cartridge in the docking bay.
[0037] FIG. 2 is an animated view showing the insertion of the
microfluidic cartridge in the docking bay.
[0038] FIG. 3 is a block diagram providing an overview of the
functional units, software and firmware of the apparatus.
[0039] FIG. 4A is a simplified representation of a floating stage
with docking bay, optical bench, and clamping mechanism for
thermally contacting the microfluidic cartridge with the heater
module and scanning the microfluidic cartridge.
[0040] FIG. 4B demonstrates conceptually that the floating stage,
docking bay, optical bench and microfluidic cartridge are mounted
in the instrument chassis at a defined angle "theta" relative to
the ground plane, where the ground plane is horizontal.
[0041] FIGS. 5A and 5B are front and rear interior perspective
views from above and below, showing the suspension-mounted stage
with docking bay, optical bench, and scanning detector head.
[0042] FIG. 6A is a front interior perspective view from below the
docking bay, showing the underside heater module and cooling
fan.
[0043] FIG. 6B is a detail of the heater module with heating block
elements and mirror face.
[0044] FIGS. 7A and 7B are perspective views of the floating stage
with insertable cartridge in place in the docking bay. For clarity,
the docking saddle and accessory mounting elements have been
removed.
[0045] FIG. 8 is a detailed view of the docking bay and floating
stage suspended from the underside of the docking saddle. The
floating stage is fitted with a four point spring suspension.
[0046] FIGS. 9A and 9B are anterior subassembly views of the
clamping mechanism. FIG. 9B illustrates the worm drive operation on
the clamping gear rack.
[0047] FIGS. 10A and 10B are posterior subassembly views of the
clamping mechanism. FIG. 10B illustrates the worm drive operation
of the clamping gear rack and the platen arm.
[0048] FIGS. 11A and 11B are perspective views of an insertable
microassay cartridge for use with the detection system of the
invention.
[0049] FIG. 12 is an exploded view showing the internal components
of a microassay cartridge.
[0050] FIGS. 13A and 13B are plan and elevation views of an
insertable microassay cartridge for use with the detection system
of the invention.
[0051] FIG. 14A isolates the mounting plate with internal pneumatic
manifold, pneumatic interface multiport, and heater module; and
FIG. 14B depicts the position occupied by a microassay cartridge
when in use.
[0052] FIG. 15 is an exploded view of the microassay cartridge nose
with central pneumatic inlet port array.
[0053] FIG. 16 is a plan view of the mounting plate with microassay
cartridge, showing the position of a sectional view through the
pneumatic interface multiport.
[0054] FIG. 17 is a cross-sectional view through the pneumatic
interface multiport.
[0055] FIG. 18 is a plan view of the mounting plate with microassay
cartridge, showing the position of a sectional view through the
cartridge and heater module.
[0056] FIG. 19 is a cross-sectional view through the heating
manifold and microassay cartridge.
[0057] FIG. 20 shows the heater module assembly in exploded
view.
[0058] FIG. 21 is a schematic of spring forces exerted on the
cartridge in the docking bay.
[0059] FIG. 22 is a partial assembly of the apparatus showing a
case-mounted blower with fan outlet directed at the nose of the
cartridge and heater module.
[0060] FIG. 23 is a perspective view of a detector head with dual
optical channels and electronically isolated circuit boards for
excitation and emissions detection. One half of the housing is
removed in order to view the internal components.
[0061] FIGS. 24A and 24B are schematic views of the internal
optical components of a fluorescence detector with dual optical
channels, heating block-mounted mirror and microfluidic cartridge.
Excitation optics are mounted on one circuit board and detection
optics on another to reduce noise interference.
[0062] FIGS. 25A and 25B show emission and excitation wavelengths
for two fluorophores in a liquid sample, and illustrate dual head
optical isolation for removal of crosstalk.
[0063] FIGS. 26A through 26D are views of electronic traces from
the amplifier of the detector head under assay conditions with
control and target signals.
[0064] FIG. 27 is a block diagram of the detector head electronics
used for controlling the fluorescent excitation, and receiving,
processing, and digitally communicating fluorescence emission
signals to the host instrument.
[0065] FIGS. 28A and 28B are representations of raw input and
digitized output showing digital removal of bubble
interference.
[0066] FIG. 29A is a representation of an excitation cone and
planoconvex objective lens relative to the cartridge detection
chamber and mirror-faced heating block.
[0067] FIG. 29B is a representation of emission collection with a
planoconvex objective lens at short working distance relative to
the cartridge detection chamber and mirror-faced heating block.
Shown are primary and reflected fluorescent emissions.
[0068] FIG. 30 is a schematic representation of an optical pathway
with decoupled excitation and emission optics where L1'<L1 and
the source beam is divergent.
[0069] FIG. 31 is a schematic representation of an optical pathway
with decoupled excitation and emission optics where L1'>L1 and
the source beam is convergent.
[0070] FIG. 32A plots experimental results demonstrating
enhancement of signal output by varying the height of the objective
lens above the mirror. FIG. 32B graphs the integrated output signal
strength with and without the back mirror. Output signal was found
to be optimized by focusing the excitation beam at a focal point
behind the mirror as shown in FIG. 29A and collecting emissions at
a shorter working distance as shown in FIG. 29B.
[0071] FIG. 33A demonstrates nested thermal melt curve data for a
positive assay result and control, showing fluorescence resulting
from a molecular beacon hybridized to an amplicons as a function of
temperature. FIGS. 33B and 33C analyze the thermal melt profile,
calculating a first derivative indicative of the T.sub.m.
DETAILED DESCRIPTION
[0072] Although the following detailed description contains
specific details for the purposes of illustration, one of skill in
the art will appreciate that many variations and alterations to the
following details are within the scope of the claimed invention.
The following definitions are set forth as an aid in explaining the
invention as claimed.
DEFINITIONS
[0073] "Daemon" refers herein to a programmable instruction set for
optical data acquisition and processing (ODAP) that is stored in
firmware which is executed by an embedded microprocessor as an
autonomous background process rather than under the direct control
of an interactive user or by the host controller. The daemon can be
viewed as a virtual machine which operates and controls electronic
circuitry that is electronically isolated from noise and is
localized in the detector head. Conditioned outputs from multiple
amplifiers associated with the detection optics are conditioned and
amplified in a tri-stage amplifier, and one of the three amplifier
outputs is selected for digitization followed by signal processing
and tabulation with positional data. The daemon then compares
optical data with background scans and makes a complex
determination as to whether the fluorescence is a positive assay
result before reporting data to the host system for display to the
user. During operation, the host system multitasks to perform
various assay functions such as temperature and pneumatics control,
detector head scanning, user interface operability, and fault
monitoring, and the tasks related to emission signal acquisition
and processing are delegated to the daemon, herein termed an "ODAP
daemon".
[0074] "Angular aperture"--is the angle between the most divergent
rays from a single point that can enter the objective lens and
participate in image formation.
[0075] "Back focal length"--is defined for a lens with an incident
beam of collimated light entering the lens as the distance L from
the back surface of the lens to the focal point of a cone of
focused light. "Back focal positions" indicates that non-collimated
rays may be focused at alternate distances from the back of the
lens by decoupling the optics from the native focal length of a
lens.
[0076] Target analyte: or "analyte of interest", or "target
molecule", may include a nucleic acid, a protein, an antigen, an
antibody, a carbohydrate, a cell component, a lipid, a receptor
ligand, a small molecule such as a drug, and so forth. Target
nucleic acids include genes, portions of genes, regulatory
sequences of genes, mRNAs, rRNAs, tRNAs, siRNAs, cDNA and may be
single stranded, double stranded or triple stranded. Some nucleic
acid targets have polymorphisms, deletions and alternate splice
sequences. Multiple target domains may exist in a single molecule,
for example an immunogen may include multiple antigenic
determinants. An antibody includes variable regions, constant
regions, and the Fc region, which is of value in immobilizing
antibodies. Target analytes are not generally provided with the
cartridge as manufactured, but are contained in the liquid sample
to be assayed; in contrast, "control analytes" are typically
provided with the cartridge and are assayed in order to ensure
proper performance of the assay. Spiked samples containing target
assay may be used in certain quality control testing and for
calibration, as is well known in the art.
[0077] Means for Amplifying: The grandfather technique was the
polymerase chain reaction (referred to as PCR) which is described
in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159,
Ausubel et al. Current Protocols in Molecular Biology, John Wiley
and Sons, Baltimore, Md. (1989), and in Innis et al., ("PCR
Protocols", Academic Press, Inc., San Diego Calif., 1990).
Polymerase chain reaction methodologies require thermocycling and
are well known in the art. Briefly, in PCR, two primer sequences
are prepared that are complementary to regions on opposite
complementary strands of a target sequence. An excess of
deoxynucleoside triphosphates are added to a reaction mixture along
with a DNA polymerase, e.g., Taq polymerase. If the target sequence
is present in a sample, the primers will bind to the target and the
polymerase will cause the primers to be extended along the marker
sequence by adding on nucleotides. By raising and lowering the
temperature of the reaction mixture, the extended primers will
dissociate from the template to form reaction products, excess
primers will bind to the template and to the reaction products and
the process is repeated. By adding fluorescent intercalating
agents, PCR products can be detected in real time.
[0078] Other amplification protocols include LAMP (loop-mediated
isothermal amplification of DNA) reverse transcription polymerase
chain reaction (RT-PCR), ligase chain reaction ("LCR"),
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA), "Rolling Circle", "RACE"
and "one-sided PCR", also termed "asymmetrical PCR" may also be
used, having the advantage that the strand complementary to a
detectable probe is synthesized in excess.
[0079] These various non-PCR amplification protocols have various
advantages in diagnostic assays, but PCR remains the workhorse in
the molecular biology laboratory and in clinical diagnostics.
Embodiments disclosed here for microfluidic PCR should be
considered representative and exemplary of a general class of
microfluidic devices capable of executing one or various
amplification protocols.
[0080] Typically, nucleic acid amplification or extension involves
mixing one or more target nucleic acids which can have different
sequences with a "master mix" containing the reaction components
for performing the amplification reaction and subjecting this
reaction mixture to temperature conditions that allow for the
amplification of the target nucleic acid. The reaction components
in the master mix can include a buffer which regulates the pH of
the reaction mixture, one or more of the natural nucleotides
(corresponding to A, C, G, and T or U--often present in equal
concentrations), that provide the energy and nucleotides necessary
for the synthesis of nucleic acids, primers or primer pairs that
bind to the template in order to facilitate the initiation of
nucleic acid synthesis and a polymerase that adds the nucleotides
to the complementary nucleic acid strand being synthesized.
However, means for amplification also include the use of modified
or "non-standard" or "non-natural" bases such as described in U.S.
Pat. No. 7,514,212 to Prudent and U.S. Pat. Nos. 7,517,651 and
7,541,147 to Marshall as an aid to detecting a nucleic acid
target.
[0081] "Means for detection": as used herein, refers to an
apparatus for displaying an endpoint, i.e., the result of an assay,
and may include an instrument equipped with a spectrophotometer,
fluorometer, luminometer, photomultiplier tube, photodiode,
nephlometer, photon counter, voltmeter, ammeter, pH meter,
capacitive sensor, radio-frequency transmitter,
magnetoresistometer, or Hall-effect device. Magnifying lenses in
the cover plate, optical filters, colored fluids and labeled probes
may be used to improve detection and interpretation of assay
results. "Labels" or "tags" include, but not limited to, dyes such
as chromophores and fluorophores; and chemoluminescence as is known
in the prior art. QDots, such as CdSe coated with ZnS, decorated on
magnetic beads, or amalgamations of QDots and paramagnetic Fe3O4
microparticles, are a convenient method of improving the
sensitivity of an assay of the present invention. Fluorescence
quenching detection endpoints are also anticipated. A variety of
substrate and product chromophores associated with enzyme-linked
immunoassays are also well known in the art and provide a means for
amplifying a detection signal to improve the sensitivity of the
assay, for example "up-converting" fluorophores. Fluorescence and
optical detectors may include photodiodes, photovoltaic devices,
phototransistors, avalanche photodiodes, photoresistors, CMOS, CCD,
CIDs (charge injection devices), photomultipliers, and reverse
biased LEDs. Detection systems are optionally qualitative,
quantitative or semi-quantitative.
[0082] "Molecular beacon"--is a single stranded hairpin-shaped
oligonucleotide probe designed to report the presence of specific
nucleic acids in a solution. A molecular beacon consists of four
components; a stem, hairpin loop, end-labeled fluorophore and
opposite end-labeled quencher. When the hairpin-like beacon is not
bound to a target, the fluorophore and quencher lie close together
and fluorescence is suppressed. In the presence of a complementary
target nucleotide sequence, the stem of the beacon opens to
hybridize to the target. This separates the fluorophore and
quencher, allowing the fluorophore to fluoresce. Alternatively,
molecular beacons also include fluorophores that emit in the
proximity of an end-labeled donor. `Wavelength-shifting Molecular
Beacons` incorporate an additional harvester fluorophore enabling
the fluorophore to emit more strongly. Current reviews of molecular
beacons include Wang K et al, 2009, Molecular engineering of
DNA:molecular beacons. Angew Chem Int Ed Engl, 48(5):856-870;
Cissell K A et al, 2009, Resonance energy transfer methods of RNA
detection, Anal Bioanal Chem 393(1):125-35 and Li Y, et al, 2008,
Molecular Beacons: an optimal multifunctional biological probe,
Biochem Biophys Res Comm 373(4):457-61. Recent advances include
Cady N C, 2009, Quantum dot molecular beacons for DNA detection.
Methods Mol Biol 554:367-79.
[0083] Fluorescence nucleic acid assays include amplification with
tagged primers and probe-based detection chemistries. Fluorescent
products can be assayed at the end of the assay, or by measuring
the amount of amplified product in real time. While not limiting,
TaqMan Probe (Applied Biosystems) which relies on displacement and
polymerase-mediated hydrolysis of a 5' reporter dye with 3'
quencher construct, FRET hybridization probes, dual oligo
FRET-based probes (Roche), minor groove binder-conjugated
hybridization probes (MGB probes, Applied Biosystems), Eclipse
probes, Locked NA Probes (Exiqon/Roche), Amplifluor primer
chemistries, Scorpions primer chemistries, LUX primers, Qzyme
primers, RT-PCR, among others, are all suitable in the present
invention. Intercalation dyes may also be used. Reverse
transcriptase is used to analyze RNA targets and requires a
separate step to form cDNA. Recent advances include Krasnoperov L N
et al. 2010. Luminescent probes for ultrasensitive detection of
nucleic acids. Bioconjug Chem 2010 Jan. 19 epub.
[0084] In addition to chemical dyes, probes include green
fluorescent proteins, quantum dots, and nanodots, all of which are
fluorescent. Molecules such as nucleic acids and antibodies, and
other molecules having affinity for an assay target, may be tagged
with a fluorophore to form a probe useful in fluorescent assays of
the invention.
[0085] "FRET" (Fluorescence Resonance Energy Transfer)--is a
fluorescence technique that enables investigation of molecular
interactions. It depends on the transfer of energy from one
fluorophore to another fluorophore (i.e., a donor and a quencher)
when the two molecules are in close proximity such a when
hybridized. Recent advances include Carmona A K et al [2009, The
use of fluorescence resonance energy transfer (FRET) peptides for
measurement of clinically important proteolytic enzymes, An Acad
Bras Cienc 81(3):381-92].
[0086] Unless the context requires otherwise, throughout the
specification and claims which follow, the word "comprise" and
variations thereof, such as, "comprises" and "comprising" are to be
construed in an open, inclusive sense, that is, as "including, but
not limited to". Reference throughout this specification to "one
embodiment", "an embodiment", "one aspect", or "an aspect" means
that a particular feature, structure or characteristic described in
connection with the embodiment or aspect may be included one
embodiment but not necessarily all embodiments of the invention.
Furthermore, the features, structures, or characteristics of the
invention disclosed here may be combined in any suitable manner in
one or more embodiments. "Conventional" is a term designating that
which is known in the prior art to which this invention relates.
"About" and "generally" are broadening expressions of inexactitude,
describing a condition of being "more or less", "approximately", or
"almost" in the sense of "just about", where variation would be
insignificant, obvious, or of equivalent utility or function, and
further indicating the existence of obvious minor exceptions to a
norm, rule or limit.
[0087] "Crosstalk"--in fluorescence imaging occurs when the
excitation and/or emission spectra of two or more fluorophores
(and/or autofluorescence of a substrate) in a sample well overlap,
making it difficult to isolate the activity of one fluorophore
alone.
[0088] Turning now to the figures, FIG. 1 is a perspective view of
the instrument 100 with a microfluidic cartridge 200 in the docking
bay. Shown are membrane panel 104 and touch screen display surface
108 and compact chassis or housing 106. Because all reagents are
provided in the microfluidic cartridge, the instrument has full
standalone operability. FIG. 2 complements this exterior view by
animating the insertion of the microfluidic cartridge anterior nose
105 into the docking bay 103. The docking bay is suspension-mounted
and may be tilted at an angle relative to the instrument base, as
will be discussed in more detail below.
[0089] FIG. 3 is a block diagram providing an overview of the
functional units, software and firmware of the assay apparatus.
Mechanical, pneumohydraulic, temperature control, optical, and
input/output (I/O) systems are coordinated by two digital
processors, the first is termed the "host controller", which
receives user commands and outputs assay results, and also directs
the mechanical, thermal, and pneumohydraulic process steps of an
assay, and the second is termed the "embedded microprocessor",
which exerts localized control over optical systems, including
signal acquisition and processing within the detector head and
communicates digital data to the host controller. The optical
systems controlled by the embedded processor are shielded within
the detector head from the noisy analog circuits within the
apparatus housing, permitting high gain amplification in a
relatively noiseless environment. Each processor is provided with a
separate clock, volatile and non-volatile memory, and performs
autonomously of the other.
[0090] As an overview, a floating stage (1000, dotted line) within
the instrument supports a docking bay (here 1001) for receiving and
reversibly clamping a microfluidic cartridge and is provided with a
scanning detector head 311. The detector head is scanned across
optical windows of the microfluidic cartridge under control of the
host controller. The detector head contains subassemblies for
providing excitation light and sensors for detecting, amplifying
and processing fluorescent emission signals under control of a
daemon resident in firmware associated with embedded microprocessor
1841. The floating stage rides on a spring suspension mounted to a
stationary baseplate and instrument stand.
[0091] Affixed to the stationary baseplate and positioned for
interfacing with the floating stage are a heater module with
separately controllable heating blocks under control of the host
controller 1003, a pneumatics interface connected to pneumatic
servos mounted on the base plate 330, which also serves as a
pneumatic distribution manifold, a wire harness connecting the
stepper motor and the host controller, and wiring harnesses for the
clamp motor and related sensors, including pressure switches for
measuring the position of the clamp and the microfluidic cartridge,
a barcode reader, and temperature monitors. Power is distributed to
all systems from a rechargeable battery mounted in the instrument
stand, or by direct connection to an AC converter or to a DC source
such as an automobile.
[0092] The host controller 1003 is mounted on a motherboard which
also contains a touch pad panel for operation of the instrument and
an LCD display panel. The instrument may transmit data to an
outside network or device via a variety of digital serial I/O
links, including a wireless networking card or other remote
communication device. A special digital junction is provided for
service access to the RAM registers and programming, which is
software encoded in solid state ROM.
[0093] General instructions for operation of the instrument, such
as the sequence of pneumatic pulses and valve logic required to
operate a particular microassay card having the capability to
diagnose a particular disease or pathology from a liquid sample,
are provided by programmable software associated with the host
controller, which is supplied with volatile and non-volatile memory
for executing assays. If for example, the barcode reader detects a
particular microassay cartridge, the device is programmed to
perform a particular assay recognized from with that barcode and to
interpret and display the results in a designated format.
[0094] However, the operation of the signal acquisition optics,
including modulation of source intensity, signal amplification and
filtering, is under control of a daemon resident in the embedded
microprocessor 1841 on the sensor PCB within the detector head 311.
Thus electrical signals that are highly sensitive to noise are
shielded in the detector head from the more noisy environment of
the host instrument, and transmission of analog signals from
detector head to host A/D converters is completely avoided. This
unconventional separation of functions has happily proved highly
advantageous in reducing noise susceptibility of the instrument, as
is needed for full portability and field operation, and
unexpectedly permits use of a high gain tri-stage amplifier in what
would be expected to be a noisy electronic environment.
Mechanical Systems
[0095] The floating stage with on-board optical bench and docking
bay is a distinctive feature of the instrument. This feature is
introduced conceptually in FIG. 4. FIG. 4A is a conceptual
representation of the primary optothermomechanical subsystems of
the instrument. The floating stage 300 consists of a tray-like
chassis 301 (dashed box) that is suspended on an inclined plane by
a four-point spring-mounted suspension (indicated by 302,303) and
supports a docking bay 103 for receiving a microfluidic cartridge
200. Also supported on the floating stage 300 is a scanning
detector head 311 mounted on paired guiderails (308,309). The
cartridge is not part of the instrument 100, but interfaces with
the instrument after insertion into the floating docking bay
103.
[0096] During operation, the floating stage is clamped (indicated
by 320) against a mounting plate (330) and engages contacting
surfaces of zone heating blocks (341,342,343,344) of a heater
module 340 and associated resistive heating elements and circuits.
A fan 345 is provided to dissipate excess heat during cooling. The
inclined mounting plate is also provided with a pneumatic interface
port 350 for sealedly docking to the base of the microfluidic
cartridge. Pneumatic pressure is delivered to the cartridge through
the pneumatic interface port from an integral pneumatic
distribution "manifold" or system embedded in the inclined mounting
plate 330. The pneumatic manifold supplies negative and positive
pressure from sources mounted on the inclined mounting plate. A
motherboard-mounted, programmable host controller directs pneumatic
driving pressure, vacuum, and control pulses to pumps and valves on
the cartridge via the internal manifold in the base plate 330 and
pneumatic interface port 350.
[0097] The detector head 311 is motorized and scanning of the
cartridge is performed under the control of the host controller. To
scan the detector head along paired guiderails (308,309) the host
controller engages a worm-gear driven by stepper motor 307. The
detector head is fitted with an external window with objective lens
315 which scans optical windows in the anterior nose of the
microfluidic cartridge and collects raw optical signals. The
detector head 311 has its own embedded microprocessor 1841 with
resident daemon which functions independently of the host
controller for optical signal acquisition. The host controller also
regulates temperature in heating elements (341,342,343,344) of
heater module 340 and controls a set of solenoid valves and
positive pressure and vacuum pump reservoirs linked to the
pneumatic interface. The instrument is supplied with a display
panel and touch panel for user interactions. Power input is
flexible, and is optionally supplied by an AC adaptor, car adaptor,
or from a rechargeable battery mounted under the instrument. Also
included are optional wireless IO and digital IO ports.
[0098] FIG. 4B demonstrates conceptually that the floating stage
301, docking bay 103, detector head 311, and microfluidic cartridge
200 may be mounted in the instrument chassis 106 at a defined angle
relative to the ground plane. Tilting the cartridge at an angle
from the ground plane improves venting during fluid loading and
minimizes air entrainment during wetting and mixing operations.
Bubble accumulation, which interferes with heat transfer and
optical interrogation of assay results, is avoided by this and
other innovations disclosed here. The inclined mounting plate 330
establishes the angle of the floating stage 301, cartridge 200, and
mechanical components of the clamping 800 and optical scanning 310
subassemblies. We found that bubble accumulation interfered with
nucleic acid amplification, and was limited by the angular mount of
the stage. This angle "theta" has been found to be advantageous in
the range of 10-45 degrees from the ground plane, more preferably
10-20 degrees, and is most preferentially about 15 degrees.
[0099] As shown in FIG. 4B, the detector head 311 includes a
clamshell housing with mating half shells (312,313). The detector
head slides on lateral guide rails 308 and 309 and is under host
control of a stepper motor 307 with worm drive. The floating stage
chassis 301 is springedly mounted in a four-point suspension and
has no direct connection to the inclined mounting plate 330 until
clamped. The clamping mechanism is indicated here figuratively by
an arrow 320 and will be discussed in more detail below.
[0100] One of the two scanning guiderails 308 is readily visible in
this view, and is supported at either end by the floating stage
chassis or tray 301. The docking bay is indicated by a dashed box
(103) and marks the opening for insertion of the nose of the
microfluidic cartridge under the detector head 311, which is
enabled to scan from side to side as shown (double arrow). The
pneumatic interface port 350, shown here as a raised platform under
the docking bay, blocks the view of the heater module 340 and
heating blocks 341-344 located immediately inferior and in line
with the docking bay. Power conditioning, AC adaptors and battery
storage functions are mounted beneath the inclined mounting plate
330 above the underside of the instrument stand 329, which is
designed to rest on a flat surface.
[0101] FIGS. 5A and 5B are front and rear interior CAD views from
above, showing the suspension-mounted stage with floating chassis
301 with optical bench and detector head 311, and docking bay
occupied by a microfluidic cartridge 200. The floating stage is
suspended from a saddle shaped support, docking saddle 400, which
is rigidly bolted to the inclined mounting plate 330. Also shown is
a gear rack 401 that provides clamping pressure, as will be
described in more detail below. In FIG. 5B, the guiderails
(308,309) and stepper motor 307 of the optical bench are readily
recognized. The inclined mounting plate 330 is populated with
pumps, vacuum and pressure storage tanks, solenoids and pneumatic
control circuitry, which are not shown for clarity of presenting
cartridge docking mechanism and scanning detector head.
[0102] FIG. 6A is a front interior perspective view from below the
docking bay, showing the underside heater module 340 and cooling
fan 345. It can be seen that on insertion of the microfluidic
cartridge 200 into the docking bay, the heater module 340 is
brought into alignment with the underside of the cartridge on
alignment pins 510 and resting on step 511 as shown in FIG. 6B.
Heating System
[0103] FIG. 6B is a detail of the heater assembly 340 with heating
block elements ("thermal elements" 341,342, 343,344) and mirror
face (500). The superior aspect of the heater module consists of
one or more heating blocks, each of which forms a thermal interface
with a defined zone on the underside of the microfluidic cartridge
for proper operation of the biochemical or molecular biological
reactions that occur in the enclosed channels and chambers of the
cartridge during the assay. These reactions can be as simple as
immunobinding or hybridization, or as complex as nucleic acid
amplification or enzymatic dehydrogenation coupled to the formation
or consumption of nicotinamide adenine dinucleotide and adenosine
triphosphate, or cascading clotting factors, and generally require
relatively stringent temperature control for optimal reactivity and
specificity. The heating blocks (341,342,343,344, although the
invention is not limited to this configuration) may be
spring-mounted and are urged upward in opposition to the downward
pressure of the clamping mechanism to establish a high thermal
diffusivity contact zone for heat transfer. Each heating block is
in thermal contact with a resistive (Coulombic) heating element,
generally by means of a compliant thermal pad for good thermal
conductivity. Each heating block contacts a thermal window in the
microfluidic cartridge. Each window is generally a thin layer of a
flexible plastic film, may be less than 3 mils in thickness, and
most commonly of a compliant transparent material such as
polyethylene terephthalate (Mylar.RTM.), although optionally of a
cyclic polyolefin or polyimide with good optical transparency,
while not limited thereto (see U.S. Pat. No. 7,416,892, which is
co-assigned), and also having good thermal conductivity. Thus in
one embodiment the invention is a thermo-optical interface for
reflective transillumination of a detection chamber in a
microfluidic card while controlling or modulating the temperature
of a liquid sample in the detection chamber. This feature is of
benefit for performing a reaction or reactions associated with an
analyte while monitoring an optical signal associated with the
analyte. In one exemplary application, thermal melt curves are used
to verify FRET hybridization results.
[0104] Also shown is the fan housing 345, which is used to
dissipate heat from heat sinks below the heating blocks and as an
adjunct for PID control of temperature in the blocks in combination
with resistance heating circuits (FIG. 20). A second fan (FIG. 22)
is used to cool the reaction chambers of the assay cartridge, as
will be described below.
[0105] Heating block 341 in this case is modified by fabrication
with a polished chromium mirror face 500 on the upper aspect which
contacts and aligns with thermo-optical windows in the microfluidic
card 200 during the assay. The thermo-optical window in this case
corresponds to a detection chamber enclosed in the cartridge body.
The mirror face reflects light from the detector head 311, which
scanningly transilluminates the cartridge, back into the objective
lens 315, and also reflects any fluorescent emission from the
cartridge detection chambers back into the detector head and from
there to the detection sensor, which is typically a photodiode, as
will be discussed in more detail below. Heating block 341 in this
example differs from the other heating blocks, and is generally
machined from aluminum, then polished and coated with an underlayer
of copper under nickel before application of the chromium mirror
face. Electropolishing and/or buffing may be used to form a highly
reflective optical finish on the chrome surface. The optically flat
superior surface of the block aids in heat transfer and improves
sensitivity of fluorescence assays. Happily, the use of the mirror
permits simultaneous heating and optical interrogation of the fluid
contents of the detection chamber, as is useful for example in
optically assaying melting curves.
[0106] Other heating zones may be modified similarly to permit
optical monitoring with simultaneous temperature control or
modulation. The configuration of heating zones and mirrors may be
modified or adapted for particular assay/cartridge requirements,
and is not limited to the configuration shown here.
[0107] FIG. 6B also shows pneumatic interface port 350, here with
ten outlets, each independently ported to a source of positive or
negative pressure from the pneumatic distribution manifold of the
host instrument and independently under the control of a
programmable host controller. These outlets interface and seal to
mated inlets in the underside of the microfluidic card, and a timed
pattern of intercommunicating pneumatic pressure, vacuum and
pressure pulses are routed through the pneumatic interface to drive
and control the assay in the cartridge.
[0108] FIGS. 7A and 7B are perspective views of the floating stage
subassembly 300 with insertable cartridge in place in the docking
bay 103. For clarity, the docking saddle 400 and accessory mounting
elements have been removed. It can be seen in FIG. 7A how the
inferior surface of the microfluidic cartridge 200 is shaped to be
contacted with the mated superior surfaces of the heater module 340
of FIG. 6B. The cartridge is secured and supported under the
floating stage 301 by two attached lateral flanges 609 and 610,
which are bolted in place and guide insertion.
Clamping Mechanism
[0109] In FIG. 7B, four vertical posts (601,602,603,604) forming
the male elements of the four-point suspension 600 are apparent on
the anterior section of the floating stage 300. These posts are
fitted with coil springs (603a in FIG. 8) and inserted into
cylindrical suspension housings (605, 606,607,608) formed as part
of the docking saddle member 400. Suspension 600 serves to suspend
the floating stage 300 and optical scanning assembly as will be
described in more detail in the next figure, FIG. 8. The entire
optics bench and docking bay subassembly (shown in FIGS. 7A and 7B)
floats on this suspension and is rigidly brought into contact with
the rest of the instrument only on downward action of the clamping
mechanism as will be described in FIGS. 9 and 10.
[0110] FIG. 8 is an exploded view of the floating stage 300 with
detector head 311 and docking bay 103 suspended from the underside
of the docking saddle 400. The floating stage is fitted with a four
point spring suspension with coil springs (replicates of 603a) on
each of the four supporting posts (601,602,603,604). Each post is
received in a mated suspension housing (605, 606,607,608) of the
docking saddle. The microfluidic cartridge 200 is not shown, but it
can be seen in this view that the docking bay is configured for
receiving the nose of the cartridge at the projecting lip 515 of
the docking bay, so that the cartridge rests on lower lateral
flanges (609,610). Alignment pins (616,617) ensure that the docking
bay seats true when pressed down against the heater module 340. The
posterior section of the floating stage, which contains the
guiderails (308,309) and detector head 311 for fluorescence
scanning, is free of any support other then at the docking saddle
and is cantilevered from the docking saddle during operation. The
role of guiderails in supporting motion of the scanning detector
head 311 is apparent in this view.
[0111] The docking saddle is provided with brackets 712 and 713 for
attaching the clamping mechanism 800 as will be discussed below,
and a bar code reader as is useful in automated operation. Linker
arm 710 with slot 711 is engaged by the clamping gear mechanism as
discussed below and the floating stage 300 raised or lowered as a
single assembly. Linker arm 710 operatively presses the cartridge
against the heating blocks.
[0112] FIG. 9A is a frontal view of the clamping mechanism. The
bridging shape of the docking saddle 400 is seen to rest above the
anterior nose 515 of the floating docking bay 103. Immediately
under the docking bay mouth is the pneumatic interface port 350,
visible between lateral flanges 609 and 610 forming the channel for
receiving the microfluidic cartridge 200. During docking of the
loaded cartridge, the function of the clamping mechanism is to urge
the floating stage and spring-mounted chassis 301 with inserted
cartridge downward onto the pneumatic interface port and heater
module as described above.
[0113] Docking saddle 400 is bolted on mounting plate 330, and
floating stage chassis 301 is suspended on the four-point
suspension 600. The suspension springs apply a downward pressure on
the floating stage, which is opposed by the suspending action of
clamping assembly 800 when in the raised position. Then, when
clamping the cartridge, clamp gear piece 804 is driven by worm gear
801 and worm gear motor 802, driving travelling axle 803 in a
downward arc. The axle pin 803a is attached to a cam block (901,
visible in FIG. 9B). Linker arm (710, visible in FIGS. 8 and 10A)
follows the cam-action of the clamping gear 401 and slider block
901 up or down on the four-point suspension. When disengaging the
cartridge, the action is reversed. Worm gear motor 802 is run
clockwise, raising pin 803a and cam block 901, thus lifting the
linker arm 710, which is part of the stage chassis assembly 301,
and then reversed (double arrow). When the stage chassis is in the
uppermost resting position, the microfluidic cartridge can be
removed from the instrument. Mechanical fiducials or alignment pins
are used to register the cartridge in the instrument docking bay
during the assay.
[0114] FIG. 9B is a detailed view of the front side of the clamp
gear piece 804 with gear comb 804a and worm drive gear 801, showing
also cam follower surfaces 805 and 806 on the anterior edge of the
gear member that are used by pressure switches to monitor the
position of the gear and actuate coordinated mechanical functions
by the host controller. For simplicity, the pressure switches are
not shown. Axle 810 is the center of rotation for the piece and
rotates on pin 810a. Axle 803 travels during rotation of the gear
piece, driving a slider block which engages with the stage chassis
as shown in the following figure.
[0115] FIGS. 10A and 10B are mechanical drawings showing the action
of the clamping mechanism assembly 800. The purpose of the clamp
gear-driven cam is to raise and lower the floating stage 301. The
clamping gear piece 804 pivots on stationary axle 810, so that
travelling axle 803 scribes an arc upward or downward, propelling
the linker arm 710 up or down vertically (double arrow). Slider
block 901 captive on pin 803a slides left to right in a slot 711 in
the linker arm (710, see FIG. 8) to accommodate the lateral vector
of the motion of the clamp gear while raising or lowering the
floating stage 300. The upward movement of the floating stage is
opposed by springs 603a as shown in the preceding figures. FIG. 10B
is a detailed view of the rear side of the clamp gear piece 804 and
worm drive gear 801 showing the central axle 810a and eccentric cam
block axle 803a and cam slider block (901).
[0116] The mechanism illustrated here is not limiting, insofar as
the invention can be realized in alternate ways, for example by
clamping up from the bottom rather than down from the top, or by
magnetically clamping rather than mechanically clamping. Other
spring means may be selected from coil spring, leaf spring, torsion
spring, helical spring, and alternatives such as pneumatic
canisters (e.g. gas springs) and elastomeric materials or other
equivalent means known in the art.
[0117] FIGS. 11A and 11B are perspective views of an insertable
microassay cartridge 1100 for use with the apparatus of the
invention. The cartridge shown here consists of a housing 1102 and
coverplate 1103 with internal workings. Port 1104 is for receiving
a liquid sample and anterior nose 1105 is for inserting into the
docking bay of the host instrument. Window 1101 in the cartridge
housing is an optical window formed over the sample wells 1201, as
shown in FIG. 13. Gasket 1106 is for sealedly interfacing with the
pneumatic interface multiport 350 of the host instrument. Shown
from underneath is the inboard circuit card 1200 and the outboard
circuit card 1204.
Pneumohydraulic Systems
[0118] FIG. 12 is an exploded view showing the internal components
of a microassay cartridge 1100. This particular cartridge 1100 is
designed for PCR with FRET or molecular beacon detection. It is
preferably left to the skill of the artisan to determine the
details of the cartridge design and analytical reactions to be
employed according to the diagnostic application and specific
requirements to be met, and the following detailed description is
supplied only for illustration.
[0119] Sample is added through inlet port 1104. An optical window
1101 on the nose of the housing nose 1105 inserts into the host
instrument and is aligned so that the windows of the FRET or
molecular beacon detection chambers 1201 of the microassay inboard
circuit card 1200 are scanned by the detector head, which follows a
linear path that transects the optical window longwise. Added
processing related to sample preparation is supplied on outboard
card 1204. All liquid reagents are enclosed in sealed rupturable
pouches 1206 and are dispensed when needed under pneumatic control.
Other reagents are provided on-card in dry form. Fluid waste is
sequestered in an adsorbent batting 1207 that is sealed in place
under the plastic coverplate 1103. The details of any particular
biochemical or molecular microassay are beyond the present scope,
but microassay circuit 1200 with internal microassay channels and
wells for thermocycling and amplifying a nucleic acid target
includes detection chambers 1201 for FRET detection of any
resultant amplicon.
[0120] The pneumatic supply directed through 10 ports in gasket
1106 is fluidly connected to circuitry in both card 1200 and 1204.
During initial sample processing in card 1204, pneumatic elements
in card 1200 are actuated, but to no effect because the card is
empty. Then when processed sample is transferred to card 1200 for
analysis, pneumatic elements in card 1204 are actuated, but to no
effect because the card is no longer needed. Serendipitously, this
achieves multiplex valve logic without the need for extra pneumatic
ports to control the two cards 1200, 1204 separately. Ten ports are
found to be sufficient for most assays and are associated
individually with separate pressures for pumps, valves, and vents,
including both positive pressure and suction pressure, without the
need for additional complexity and redundancy in the external
pneumatics supply manifold contained in baseplate 330.
[0121] FIGS. 13A and 13B are plan and elevation views of an
insertable microassay cartridge 1100 for use with the apparatus of
the invention. Shown are optical windows 1101 on anterior nose 1105
of the cartridge (the nose inserts into the hose instrument and the
optical windows are scanned by the detector optics). Also shown is
platen surface 1107 for compressing the cartridge against heater
assembly 340 and gasketed pneumatic control interface port 350.
[0122] The cartridge as shown is a disposable cartridge 1100 that
inserts into a docking bay 103 in the host instrument when in use.
Lateral ribs 1110 of the cartridge housing are for structural
reinforcement and aid in the docking process. FIG. 14A is a
perspective view of the inclined mounting plate 330 with enclosed,
internal pneumatic manifold, heater module 340, and pneumatic
interface multiport 350; FIG. 14B depicts the microassay cartridge
when docked in the host instrument. In this view, the cartridge is
fluidly engaged onto the pneumatic control interface multiport and
is thermally contacted with the four zone heaters of heater module
340. Pressure on platen surface 1107 urges the cartridge against
the thermal and pneumatic interfaces. The mounting plate includes
an internal pneumatic control manifold that supplies regulated
pneumatic pressure to the cartridge through the pneumatic interface
multiport 350. In a complete instrument, mounting plate 330 is
populated with pressure regulators, accumulators, and solenoids for
controlling card pneumatics.
[0123] FIG. 15 is an exploded view of the nose 1105 of a microassay
cartridge 1100, the nose containing the optical window 1101 and the
inboard card member 1200. Nested inboard 1200 and outboard circuit
cards 1204 share a common pneumatic control interface (1111a,
1111b), aligning here with sealing gasket 1106, and share ports by
fluidly connecting inputs through vias to both the inboard and
outboard fluidic circuits. The outboard fluidic circuit in this
case is responsible for sample preprocessing; the inboard circuit
receives the preprocessed sample and analyzes it for a target
analyte. Reduced interface complexity is achieved by multiplexing
the pneumatic supply ports in the pneumatic interface between the
inboard and outboard cards. The pneumatics may be used first for a
fluidic operation in the outboard circuit, and then for a fluidic
operation in the inboard circuit, a surprising solution to the
problem of managing complex circuitry economically.
[0124] More generally, the pneumatic interface array comprises a
plurality of pneumatic ports fluidly connected by vias fluidly
joining a pneumatic circuit in a first circuit card to a pneumatic
circuit in a second circuit card, such that the plurality of
pneumatic ports are enabled to receive a plurality of pneumatic
pulses applied according to programmable instructions and to
multiplex those pneumatic pulses to fluidic circuit elements of the
first circuit card and the second circuit card, thereby reducing
the number of ports required to operate said first and said second
hydraulic circuits. For example, a pneumatic pulse applied to the
pneumatic interface at a first via forces processed liquid sample
through the fluid junction from the outboard circuit card to the
inboard circuit card, such as is useful when the first card is
designed for extracting nucleic acid from a sample and the second
card is designed for amplifying and detecting a molecular target,
as will be described in more detail below.
[0125] Bosses 1108 have a narrow cross-section to limit unwanted
heat transfer in the inboard card, and also serve to transfer
compressive loading to a defined circumference around the larger
pneumatic diaphragm members. Pin receiving holes 1109a, 1109b are
used to align the cartridge in the docking bay 103 on pins 510a,
510b when loading a new cartridge into the host instrument.
[0126] The particular cartridge illustrated is designed for PCR
with thermocycling and FRET detection. The outboard card 1204 is
intended for isolating a nucleic acid fraction from a sample.
Fluidic circuit card 1200 with internal channels and chambers
(1115, 1116) for thermocycling, and amplifying a nucleic acid
target includes sample wells 1201 for FRET detection of any
resultant amplicon. Also depicted are chambers 1114 for cDNA
synthesis. Three parallel assay channels are shown. Other circuit
configurations may be used with the microassay detection system of
the invention and the assays are not limited to three channels or
to nucleic acid detection by PCR.
[0127] FIG. 16 is a plan view of the mounting plate 330 without
microassay cartridge, showing the position of a sectional view
through the pneumatic interface multiport 350. Also shown are
alignment pins 510 for receiving the cartridge and the heater
assembly 340. Other holes are ports intended for use in mounting
pneumatic system components in fluidic communication with a
pneumatic manifold extending through the mounting plate, which may
be made of layered acrylic for example that has been fused by
solvent welding or made by a process of three-dimensional
printing.
[0128] FIG. 17 is a sectional view through the pneumatic control
interface 350 at the section cut in FIG. 16, showing ports for
engaging vias (cf 1120a, 1120b) leading to pneumatic channels in
the inboard card 1200. The ten ports 351 connect through the vias
to the pneumatic manifold enclosed in mounting plate 330, of which
pneumatic channel 1121 is representative. Also shown are cartridge
registration pins 510a, 510b for seating the pneumatic interface
port of the card against the card pneumatic control interface
350.
[0129] The card 1200 shown here in block outline has internal
pneumatic workings which operate in response to pressurized gas
pulses applied from the pneumatic distribution manifold 330 via
pneumatic interface multiport 350. Internal pneumatic workings in
the card include a valve tree operable under control of valve logic
supplied by the host controller, diaphragm pumps for moving fluids
through the circuitry, and fluid reservoirs for dispensing fluids
under pneumatic control. The need for multiple ports relates to the
way in which valve logic is laid out on the card and the need for
multiple pressures. Pneumatic supply at both a positive and a
negative pressure is generally needed, and several intermediate
pressure levels have been found to be advantageous. Pressures that
have been found to be useful include +10 psig for operating pumps,
-5 psig for opening valves (such as the diaphragm valves described
in WO Publ. No. 2002/081934), and zero pressure vents, while not
limited thereto. As shown here, as many as ten pneumatic
interconnects with the cartridge may be engaged. Depending on the
complexity of the cartridge, fewer or more ports may be used by
resizing or reconfiguring the pneumatic interface if desired.
[0130] Separating the pneumatic channels of the microassay
cartridge 1100 and the corresponding ports of the pneumatic
manifold 350 is a silicon rubber gasket 1106, or a generally
compliant elastomeric gasket, which seals the pneumatic
interconnections under compression when the cartridge is loaded in
the instrument and spring pressure is applied. Gasket 1106 serves
as a single-use sealing gasket and is advantageously supplied with
the disposable cartridge, not as part of the instrument as per
conventional wisdom. By including the gasket with the disposable
cartridge 1100, a better and cleaner seal at the pneumatic
interface is obtained and the need to periodically replace the
gasket is eliminated. Gaskets are generally formed of silicone
rubber or other compliant material.
[0131] Using the pneumatic circuits of the invention, the circuitry
may also be multiplexed by supplying multiple pneumatic sources in
the host instrument, where pneumatic supply circuits in the
connecting manifold 350 may be switched from one pneumatic pressure
to another, such as from a positive pressure to a negative
pressure, without the need for additional pneumatic circuits. Thus
each manifold subcircuit in the baseplate 350 may be operated at a
plurality of pressure states. In this way it is possible to
multiplex distribution of stronger pneumatic "states" to circuit
elements operative with a stronger pneumatic pressure and weaker
pneumatic states to circuit elements operative with a weaker
pneumatic pressure via a common pneumatic interface port array.
Analogously, it is possible to multiplex distribution of stronger
pneumatic "states" to circuit elements operative with a stronger
pneumatic pressure and vacuum pneumatic states to circuit elements
operative with a vacuum pressure card via a common pneumatic
interface port array. By joining a first circuit card and second
circuit card with a common pneumatic interface, dual functionality
of the pneumatic circuitry is achieved.
[0132] Pressure states are generally selected from a range of
positive and negative pressures, including while not limiting to -5
psi, +10 psi, +12 psi, +15 psi, +20 psi, +7 psi, and 0 psi, where
higher pressures are used for rupturing reagent packs, for example,
and negative pressure are used for opening valves, and so forth.
These pressures may be supplied from buffered pressure reservoirs.
Dial up variable pressure supplies may also be used. The pneumatic
states are supplied to the cartridge through manifold subcircuits
under microprocessor control. Each assay consists of a series of
steps to be executed according to pneumatic valve and pump logic
needed to move the fluid through the hydraulic circuitry. Vents are
also a necessary part of the circuit and pressurizing, reversing
pressure, and venting are all within the capacity of the instrument
and associated microassay circuitry according to the requirements
of each individual assay type.
[0133] FIG. 18 is a plan view of the mounting plate 330 with
superimposed cartridge 1100 in position for use (heater module 340
and pneumatic interface 350 contacting the underside of microassay
cartridge 1100 are under the cartridge); showing the position of a
sectional view cut through the cartridge and heater module.
Temperature Control Systems
[0134] FIG. 19 is a view of the heater module and the nose 1105 of
cartridge 1100 in section, referencing the cross-section position
marked on FIG. 18. The cartridge is raised above heating blocks
341-344 for purposes of labeling. The pneumatic interconnect module
350 (two rows of ports are indicated) engages disposable gasket
1106 of the cartridge, which was described functionally with
reference to FIGS. 17 and 15. Also shown are four resistive zone
heating blocks (341, 342, 343, 344) configured to contact
individual chambers of the nucleic acid amplification circuit.
Paired registration pins 510 align the cartridge housing so that
the thermal surfaces of the blocks properly engage the fluidics.
Step 511 supports the nose 1105 of the cartridge.
[0135] Zone heating block 341 is a thermo-optical interface and
comprises a mirror face 500 contacting the fluidic circuit member.
An optical window 1101 in the cartridge housing aligns with the
mirror face and the detector head slides along rail 308, 309 (FIG.
4) so that the objective lens assembly 315 of the detector head 311
runs down the middle of the long axis of heating block 341.
Detection wells in the circuit are positioned to line up on the
thermal block and mirror face under the optical window.
[0136] Block 341 is temperature controlled for FRET determinations.
Resistive heating block 342 is adjusted to a double stranded
nucleic acid denaturing temperature before the assay is started,
and block 343 is set to an annealing temperature. The annealing
block 343 is provided with a cooling fin 343a and a blower 345 so
that hot fluid conveyed from the denaturing chambers can be rapidly
cooled, thus speeding the thermocycling time. Block 344 is used for
cDNA synthesis if needed.
[0137] The fluidic circuit may include a pair of fluid chambers
overlying heating blocks 342 and 343. The paired chambers include
pneumatically actuated diaphragms operated cooperatively as
described in U.S. Pat. Nos. 7,955,836 and 7,763,453 (which are
co-assigned) for reciprocating fluid flow between the denaturing
and annealing temperatures as is useful for amplification of
nucleic acids by thermocycling. Each heating block is spring loaded
and presses against the underside of the fluidic circuit. The
springs shown here are wire springs chosen for their
compactness.
[0138] FIG. 20 is an exploded view of the heater module. Four
heating blocks are shown, each block associating with a printed
circuit strip 347 having resistive heating elements (347a) and a
centrally positioned thermistor (347b), the resistive heaters being
under PID-type feedback loop control. Supporting circuitry on the
printed circuit is rendered schematically and is controlled from
the host controller.
[0139] Each block floats on a spring support 348 and is provided
with compliant electrical connections to move freely in a limited
vertical range. The blocks are thermally insulated for each other
by an insulator jacket 346 with cut-out pockets. The heating blocks
are clipped into slots formed in a supporting mounting plate 349.
The wire springs 348 are configured to urge the block against the
compliant plastic undersurface of the card 1200 (FIG. 15) with a
mild force to decrease resistance to heat transfer.
[0140] FIG. 21 is a schematic depicting the balance of clamping and
spring forces responsible for thermal contact between the cartridge
and the heating blocks. As shown with reference to FIGS. 5 through
10, the docking bay 103 of floating stage 301 is mounted with a
4-point suspension 600 such that springs mounted on each of four
corner posts (601, 602, 603, 604) are biased to press down on the
cartridge once the cartridge has been seated. The clamping
mechanism 800 is worm gear driven and squeezes the cartridge
against registration pins 510 and step 511 in the heater module.
Each heating block is a floating member which is urged upward
against the undersurface of the inboard circuit card by independent
spring members under each block. Wire springs mounted in the heater
module press the blocks up against the underside of the inboard
fluidic member 1200 and gasket 1106 to seal the card pneumatic
interface against multiport 350 mounted on the pneumatic
distribution manifold 330. In this way, the cartridge is locked in
place by the stronger suspension springs of the clamping assembly
and the heating blocks are thermally contacted with the inboard
card by weaker springs of the heater module assembly, requiring
less precision in manufacturing. The cartridge is sandwiched in the
docking bay by the two opposing spring forces. Lateral reinforcing
ribs 1110 strengthen the cartridge; ensuring that the more pliant
the inboard circuit cards are in thermal contact with the heating
members. The downward spring suspension force is about 5 psi and
upward spring force is about 1 psi across the surface of the
heating member, the springs acting cooperatively to clamp the
cartridge and to press the heating blocks against the undersurface
of the inboard card where heating is intended. Also shown for
illustration are card 1200 features including cDNA well 1114,
annealing well 1115, denaturing well 1116, and detection well 1201
aligned under optical window 1101.
[0141] Also shown in FIG. 21, bosses 1108 are formed on the inside
surface of the cartridge housing abutting the fluidic chambers. The
bosses concentrate the spring loading forces around fluidic
diaphragm elements where compression is needed to ensure low
thermal resistance between the fluid contents of the circuit and
the underlying heating blocks. These bosses 1108 are configured for
low thermal cross-section and are formed by removing mass from the
housing that would otherwise increase parasitic heat losses and
thermal capacitance around the cDNA synthesis and thermocycling
chambers. Low thermal cross-sections are also presented laterally
because the inboard fluidic member is suspended under the cartridge
housing with an air gap above the amplification circuits, thus
reducing conductive heat losses and thermal crosstalk between the
zone heaters. An insulative jacket 346 inserted between the heating
blocks is also used to isolate and maintain each thermal zone at
the correct temperature and avoid convective thermal crosstalk
between the resistive heating elements.
[0142] The nose 1105 of the microassay cartridge containing the
sample wells is also formed to be thin to avoid excess thermal
mass. A blower 360 shown in FIG. 22 is directed via baffled duct
361 onto the nose of the cartridge and is useful for FRET
determinations. In FRET, a molecular beacon is mixed with single
stranded amplicons in a detection chamber at a temperature above
the melting temperature of the target analyte species. The
temperature is then reduced using forced cool air while fluorescent
emissions are collected. The blower is mounted on an external wall
359 of the instrument chassis.
[0143] In one illustrative embodiment, as the mixture cools,
molecular beacon anneals to the target sequences, quenching
fluorescence in the probe. Real time monitoring of the fluorescence
results in a melt curve that can be used to validate the identity
of the target amplicons. To perform the test, zone heater 341 is
brought to a temperature above the melting point of the target
species, the heater is turned off and blower 360 is turned on and
is directed at nose 1105 of the cartridge around and underneath the
optical window 1101. Temperature in the reaction mixture and
fluorescence are monitored. Temperature falls very rapidly in
response to the circulating air while changes in fluorescence are
optically monitored, allowing replicate FRET determinations to be
rapidly performed on each sample well.
[0144] Thus in one embodiment, the invention is a method for
performing a FRET melt curve determination, comprising a) heating a
sample chamber containing a FRET probe and a target nucleic acid
sequence to a temperature above the melt temperature of the duplex
probe; b) turning off said heating; and, c) using a shielded
detector head having an autonomous ODAP daemon, scanning the
detector head across a sample chamber while monitoring a change in
fluorescence of the FRET probe in response to a change in
temperature of the sample chamber caused by turning on a fan and
directing a stream of ambient air onto the sample chamber, thereby
rapidly cooling said sample chamber while monitoring fluorescence.
Analysis of the derivative of fluorescence intensity as a function
of temperature yields a melting point characteristic of the
amplicon of interest. It has proved possible to make these
determinations in less than a minute using the low thermal mass of
the cartridges of the invention. The heating block 341 may be
provided with a cooling fin and independently air cooled to
accelerate the measurement, if desired, but generally this has not
been needed.
Optical Systems
[0145] FIG. 23 is a perspective view of a dual channel detector
head 1300 with two optical channels and electronically isolated
circuit boards (1301,1302) for excitation and for emissions
detection respectively. In this view, the upper half of the housing
1303 is removed in order to show the internal components of the
detector head. The dual channels are marked by objective lenses
(1310, 1330) and optic pathways A and B (open arrows marked A and
B). The SMD LED excitation light sources (1311, 1331) are mounted
on a source LED printed circuit board (1301), which is connected at
right angles to sensor PCB (1302) via an edge-type resistive
pin-connector (1304). The photodetection components are mounted on
the sensor PCB (1302). A Faraday cage element (1306) is used to
shield the photodiodes (1317) and (1337) and surrounding high gain
amplification circuitry.
[0146] Fluorescent excitation is provided in the target channel
(Arrow A) by a surface mounted LED (1331) which is chosen to match
the excitation spectrum of the target fluorophore. Source LED
(1331) emits a divergent light beam, and the radiated light beam is
then collimated by source excitation lens (1332). Source lens
(1332) is a planoconvex lens having its flat surface facing the
LED. The collimated light beam may then be passed through an
excitation bandpass filter (1333), the purpose of which is further
explained in the description associated with FIG. 20. The
collimated, filtered excitation light beam is then reflected from a
dichroic mirror element or beamsplitter (1334), which is installed
at a forty-five degree angle to the incident beam, and is passed
through a planoconvex objective lens (1330) and through an external
window in the detector housing (Arrow A). After passing lens 1330,
the excitation light is focused through a detection chamber (not
shown, see FIGS. 14-17) embedded in a microassay cartridge, which
contains a sample liquid with any target fluorophore. The path
length of the excitation light through the sample liquid is doubled
by use of a back mirror behind the microassay cartridge. The target
fluorophore is excited by the incident light beam. The emission of
the fluorophore is generally at a longer wavelength than the
excitation wavelength and is shifted by an amount equal to the
Stokes shift of the target fluorophore.
[0147] A portion of the returning emission from the target
fluorophore in the detection chamber is collected by planoconvex
sampling lens 1330 and is collimated before striking dichroic
mirror 1334. Optionally, a Fresnel lens may be use to further
reduce the working distance between the lens and the sample to
optimize collection of emitted light, which is further enhanced by
back mirror mounted on a heating block behind the detection
chamber. Because dichroic beamsplitter 1334 has a wavelength cutoff
between the excitation and emission wavelength, the dichroic mirror
1334 now acts as a pass-band beam splitter for the emitted
fluorescent light beam and a stopband filter for the excitation
light. It transmits the emitted fluorescent light while reflecting
reflected excitation light and any ambient light entering the light
path through the objective lens window. Emitted light passing
through the dichroic beamsplitter 1334 then passes through an
emission filter 1335, the purpose of which is further explained in
the description associated with FIG. 20. Light exiting emission
filter 1335 then passes through planoconvex sensor lens 1336, where
it is focused onto the surface of a photo-sensor 1337 which is
surface mounted to PCB 1302 and is protected from electrical noise
by Faraday cage 1306.
[0148] The above described optical pathways are repeated in a
second (control) channel having control excitation LED 1311,
planoconvex excitation lens 1312, excitation filter 1313, dichroic
beamsplitter 1314, objective lens 1310, control emission filter
1315, planoconvex sensor lens 1316, and control photodiode 1317.
Outputs from both photodiodes are amplified by three-stage
trans-impedance amplifiers built into the board next to the
photodiodes and grounded to an embedded microprocessor on the
sensor PCB via carefully shielded pins from the amplifiers.
[0149] In one embodiment, as exemplified by the use of fluorescein
and Texas Red as fluorophores, excitation LED 1331 is a 470 nm LED
with band-pass excitation filter 1333 for delivering essentially
monochromatic light of 485.+-.12 nm used for the target channel and
a 590 nm LED 1311 with band pass filter 1313 was used for the
control channel. The excitation LEDs are modulated or "strobed" on
and off using a strobe rate of 130 Hz to filter AC power-related
noise at 50 or 60 Hz and at harmonic frequencies associated with
fluorescent overhead illumination, also filtering phantom signal
related to stray ambient light and electrical noise that may be
present at 30 or 60 Hz. Local feedback sensors are used to monitor
and stabilize source LED output intensity. Detection monitoring of
fluorophore emission is coordinated with movement on rails of the
detector head under power of a stepper motor controlled by a host
controller. An embedded microprocessor and associated circuitry in
the detector head is provided with RAM memory, ROM memory, an A-D
converter, a three-stage trans-impedance amplifier, and signal
processing and command sequence firmware to handle these
functions.
[0150] Each of the photo-sensors 1317 and 1337 are mounted on a
common PCB 1302. The output signal legs from each of these
photo-sensors are connected directly to a pre-amp or first stage of
respective tri-stage trans-impedance amplifiers (not shown). PCB
1302 makes extensive use of hardware noise-reduction components, in
particular an embedded ground plane and a Faraday Cage 1306 to
minimize the unwanted effects of any RF or electromagnetic
interference on the input signals. The amplifier is shielded from
electronic noise by a Faraday cage, a bypass capacitor, a signal
conditioning pre-amplifier, a separate ground plane, a metallized
detector head housing, or a combination thereof, and the amplifier
is in close electrical proximity to the sensor. Optical signal
acquisition, preconditioning, amplification and digitization is
controlled by an autonomous daemon operating in a shielded
environment. The combination of these hardware noise-reduction
elements with optical data acquisition, digitization and processing
method under local control in the detection head, leads to a
detector design which is essentially immune from the effects of
unwanted noise. The tri-stage amplifier may be configured for a
gain of up to 10.sup.14, and is selectably configured for gain of
10.sup.2, 10.sup.3, 10.sup.6, 10.sup.10, or 10.sup.12, as
desired.
[0151] We have surprisingly found that packaging of signal
processing in the scanning head achieves an isolated, low noise
environment with improved signal-to-noise ratio and sensitivity by
minimizing signal pathlengths and permitting effective use of
Faraday shielding where necessary, such as around the sensor diode
leads and at the junction between the excitation and sensor circuit
boards. Optionally the detector housing may be fabricated from
aluminum or coated with a conductive polymer and grounded to
further shield the internal electronics from unwanted interference.
Advantageously, higher signal amplification is realized in this
environment,
[0152] FIGS. 24A and 24B are schematic views of the internal
optical components of a fluorescence detector head 1300, showing
the external optical interface with optical windows in a microassay
cartridge 1402 and back mirror 1400 mounted behind the cartridge on
the surface of a heating block 1410 that is used to control or ramp
the temperature in detection chambers enclosed in the cartridge.
Unconventionally, multiple independent optic pathways or "channels"
are formed in a single detection head and share electronic PCBs and
downstream signal processing circuitry, but excitation optics are
mounted on one circuit board and detection optics on another to
reduce noise interference. The two boards are electrically coupled
by a corner mounted pin junction 1304 and are electronically
isolated using bypass capacitors mounted on separate ground
planes.
[0153] Shown in FIG. 24A is the optical transition for the
excitation of a fluorophore in a detection or sample well (1403a or
1403b) embedded within a microassay cartridge 1402. The head is a
scanning head and moves across microassay cartridge 1402 (double
arrow). Light from excitation LED 1331 on PCB 1301 is collimated by
lens 1332 and made essentially monochromatic by band-pass filter
1333. Any fluorophore or fluorophores in detection well 1403a
(whether the control or the target fluorophore) are excited by
incident light 1420 focused on the sample by objective lens 1330.
In FIG. 24B, the emission of the fluorophore(s) is collected by
objective lens 1330 and transmitted to sensor 1337 after passing
through dichroic beamsplitter 1334, emission filter 1335 and sensor
lens 1336. Sensor 1337 is in direct electrical contact with the
base of a high gain transistor that amplifies the output signal and
is shielded in a Faraday cage 1306. The emitted fluorescent light
is generally at a longer wavelength according to the Stokes shift
of the fluorophore, enabling the emitted light to pass through
dichroic bandpass mirror 1334 and emission band-pass filter 1335
without losses. Mirror face 1400 is used to increase the amount of
excitation light on the target, doubling the excitation path
length, and to improve emission collection efficiency. The light
returned from sample chamber 1403a to objective lens 1330 is thus a
mixture of emitted and reflected fluorescence 1421 and reflected
excitation light 1420. Light traps (not shown) are provided to
capture stray reflections. Reflected light 1420 does not pass
dichroic mirror 1334 and is returned to the source, and does not
interfere with the measurement of emission intensity at sensor
1337. The optic elements of a single channel, including excitation
source, source collimating lens, excitation filter, dichroic
mirror, objective lens, excitation filter, sensor lens, and
detector with amplifier make up an optics module having an
essentially monochromatic source wavelength and a highly specific
sensor for detecting fluorescence at a particular wavelength
characteristic of the target (or control) fluorophore. One optics
module or channel may be used for an assay target, the other module
for a control channel. Tandem mounted optics channels may be used
to collect data on a plurality of fluorophores, where electrical
processing is multiplexed through an embedded microprocessor under
control of a resident daemon before transmission to the host
instrument. As shown, each of the two channels shares circuitry on
each of the two circuit boards, but has separate optics.
Optionally, additional channels may be incorporated into the
detector head by a process of duplication of the optical elements
shown.
[0154] The microassay cartridge 1402 is movable (double arrow)
relative to detector head 1300 and motorization of the detector
head or cartridge tray or mounting chassis permits scanning: a
transect across cartridge 1402 permits measurements to be made on
sample chambers 1403a and 1403b, for example. By using multiple
detection optics modules mounted side-by-side in a detector head,
the sample chambers can be scanned for multiple fluorophores in
series.
[0155] According to one embodiment, the excitation electronics are
mounted on a printed circuit board (1301) and the detection
electronics are mounted on a second PCB (1302). An edge-connector
1304 electrically joins the boards. Faraday cage 1306 protects the
sensor and associated high gain amplifier from stray
electromagnetic noise. Mirror 1400 is fabricated on the upper
surface of heating block 1410, which also functions in heat
transfer and controls the temperature of the sample fluid during
the assay. The temperature of heating block 1410 can be ramped
during scanning, for example as in a FRET melt determination with
temperature and motor functions under control of the host
controller while optical data is acquired by the embedded processor
in the detection head in an autonomous process.
[0156] FIGS. 25A and 25B show the excitation and emission spectra
of a typical system of mixed fluorophores for the target and
control, here illustrated by fluorescein and Texas Red. Shown in
FIG. 25A, curve 2001 is the excitation spectrum for fluorescein
(dashed line); curve 2002 is the emission spectrum (solid). Shown
in FIG. 25B, curve 2003 is the excitation spectrum for Texas Red
(dashed line); curve 2004 is the corresponding emission spectrum
(solid). Here control is Channel B (FIG. 25B) and target is Channel
A (FIG. 25A), but the assignment is arbitrary.
[0157] Boxed area ExA indicates the wavelength band that is allowed
to pass through the target excitation bandpass filter 1333. Box EmA
indicates the emission pass band that is allowed to pass through
the target emission bandpass filter 1335. Box ExB indicates the
wavelength band that is allowed to pass through the control
excitation bandpass filter 1313. Box EmB indicates the emission
pass band that is allowed to pass through the control emission
bandpass filter 1315. The boxes indicate the presence of stopbands
on either side of the maxima. It can be seen from FIGS. 25A and 25B
that, given the spectra for these two fluorophores and optical
filters having the passband characteristics configured as shown,
the following error conditions are corrected: a) Long wavelength
excitation light from the target LED 1331 (greater than the
wavelength of the LED peak excitation) cannot be mistakenly
confused for target fluorescent emission, due to these longer
wavelengths being cut off by the LED excitation filter 1333; b)
Long wavelength excitation light from the control LED 1311 (greater
than the wavelength of the LED peak excitation) cannot be
mistakenly confused for control fluorescent emission, due to these
longer wavelengths being cut off by the LED excitation filter 1313;
c) Target fluorescent emissions cannot be inadvertently triggered
by the (excitation filtered) control LED 1311. This error condition
would otherwise lead to the control photosensor 1317 receiving
unwanted contemporaneous signals from both the target and control
fluorophores; and d) Control fluorescent emissions cannot be
inadvertently triggered by the (excitation filtered) target LED
1331. This error condition would otherwise lead to the target
photosensor 1337 receiving unwanted contemporaneous signals from
both the target and control fluorophores.
Assay Validation
[0158] Serendipitously, the autonomous detector head functions can
be multiplexed in dual head and multi-head detectors containing
separate optical channels for detection of individual fluorophores,
such that each channel comprises an LED for irradiating excitation
light, at least one lightpath having an excitation filter, emission
filter, dichroic mirror tuned to enable the detection of emissions
from a fluorophore in a defined passband, an objective lens for
condensing said excitation light and for collecting said emissions,
a sensor for receiving any passband emissions, and a high gain
amplifier for amplifying the output from the sensor, wherein each
LED is configured to irradiate light in a frequency range so that
the irradiation frequencies of the channels do not overlap, each
optical channel is configured so that the emission passbands of the
channels do not overlap; and the outputs of said amplifiers of said
plurality of channels are multiplexedly digitized and tabulated in
volatile memory under control of an autonomous daemon resident in
firmware executed by a microprocessor embedded within the detector
head.
[0159] For assay validation, two sensor channels (or more) are
provided for monitoring two or more fluorophores, the two sensor
channels are configured so that the emission passbands from each
fluorophore do not overlap. In a preferred embodiment, a first
detection channel is for the purpose of detecting a target signal
and a second detection channel is for the purpose of detecting a
control signal, and an assay result is reported if and only if a
valid control signal is reported.
[0160] This system has proved useful in validation of assay
protocols calling for paired collection of "biplex" or multiplex
target and control signals. Where, as for FDA CLIA waiver
requirements, both target and control templates are amplified in
parallel, a positive control signal must be present before an assay
result on a test sample can be reported or billed. In the absence
of a detectable control signal, any target signal detected is not a
valid result. If this condition is met, under the Clinical
Laboratory Improvement Amendments of 1988 (CLIA), regulations
governing simple, low-risk tests can be waived and the tests
performed without oversight in physicians' offices and various
other locations. In order to meet these CLIA waiver requirements,
it is necessary that the fluorescence detector be able to detect
not only the presence, for example, of a target infectious organism
amplified by PCR but also of an endogenous human control organism
co-existing with the target and amplified by the same PCR reaction
or a PCR reaction conducted in parallel in the instrument.
[0161] Such an approach requires that the fluorescent detector used
for such a molecular diagnostic assay may have the capacity to
determine the presence of both the target and the control
fluorophores as a "biplex" amplification reaction mixture in a
common detection chamber. In one aspect, a positive amplification
control fluorophore is used which has fluorescent excitation and
emission spectra which is positioned to be well resolved (in
wavelength) by selective band pass filters from the fluorescent
excitation and emission spectra for the target fluorophore (see
FIGS. 25A-25B for example). However, according to the present
invention, it proved possible to achieve superior resolution by
using a dual head detector and by scanning each detection chamber
twice, once with each head, detecting first the control
fluorescence signature, then the target sample fluorescence
signature--each scanning pass requiring separate excitation and
emission optics. A benefit was found by configuring these detectors
with fully separated and independent light paths. In this aspect of
the invention, the use of the duplex head design ensures that the
presence of an amplification control fluorophore in a sample does
not inadvertently create a signal in the target channel due to
"crosstalk". Such a situation would result in the sample being
classed as a "false positive". Conversely, it is also important
that there is no crosstalk from the target channel to the control
channel, which is likely when multiple signals share a common
optical path. Such a situation could result in the positive
amplification control being inadvertently deemed present, when this
may not be.
[0162] These principles are exemplified by the use of fluorescein
or equivalent fluorophore as a molecular probe for the target, and
Texas Red or equivalent fluorophores as a molecular probe for the
control. A dual head detector, with one detection channel optimized
for detection of fluorescein and the other detection channel
optimized for Texas Red, each with separate excitation and
detection optics, was found to be surprisingly sensitive, accurate
and robust for assaying biplex amplification mixtures. Separate
fluorescence readings are made by the resident daemon for each
optical channel. Surprisingly, this procedure both improved
resolution and minimized cross talk but did not contribute to
higher noise or loss of sensitivity due to the mechanics of moving
the detector head.
[0163] FIG. 26A is a plot of the signal level received by the
target channel of the detector in the case where a fluorescent
target (in this instance indicating the presence of malarial
nucleic acids from a molecular diagnostic assay) is present in the
detection chamber embedded within a microfluidic cartridge. In this
case it can be seen from the trace that the target channel receives
a fluorescent signal from the detection chamber in the range 900
mV. The "malaria positive" signal in this case from the target
channel is equal to approximately 14 times the "background" signal
level of an empty detection chamber. The traces are smooth and
indicate the low level of electronic noise in the amplification
circuit within the shielded detector head.
[0164] FIG. 26B is a plot of the signal level received by the
control channel of the detector in the case where a fluorescent
target (in this instance indicating the presence of malaria from a
molecular diagnostic assay) is present in the detection chamber. In
this case it can be seen from the trace that the control channel
has an output in the range 40-50 mV, essentially equal to pre-scan
baseline, as is indicative of a negligible level of crosstalk
between the target optical channel and the control optical channel
when scanning a detection well having only a target signal and no
control fluorophore.
[0165] FIG. 26C is a plot of the signal level received by the
target optical channel of the detector in the case where a
fluorescent target (malaria, Texas Red fluorophore) and a
fluorescent control (endogenous human control, fluorescein
fluorophore) are amplified together as a mixture in a single
detection chamber embedded within a microfluidic cartridge. In this
case, the upper trace shows that a target signal level of
approximately 680 mV was received in a first optical channel, where
the target signal is associated with Texas Red-tagged malaria
amplicon. This is equivalent to a level of approximately 10.5 times
the signal from a blank detection chamber (lower trace). This
indicates the detector functions correctly in identifying the
presence of the "malaria positive" component of the biplex
sample.
[0166] FIG. 26D is a plot of the signal level received by a second
optical channel of the detector in the case where a fluorescent
target (malaria) and a fluorescent control (endogenous human
control) are present together in the detection chamber embedded
within a microfluidic cartridge. The biplex sample mixture is the
same as for FIG. 26C, and the signal is associated with a
fluorescein-tagged endogenous control amplicon, demonstrating no
crosstalk from the target amplicon and good signal stability and
freedom from electrical noise.
[0167] In this case, the trace shows that a signal level of
approximately 160 mV was received by the control optical channel.
This is equivalent to a level of approximately 3.6 times the signal
from a blank detection chamber. This indicates the detector
functions correctly in terms of identifying the presence of the
"control positive" component of the biplex sample in a separate
optical channel. Thus target amplicons and control amplicons are
detected in a single well, as is indicative of a successful
"biplex" assay per the CLIA waiver requirements.
[0168] Because excitation is not performed with white light, but is
instead performed at a wavelength specific for an individual
fluorophore, quenching of the second fluorophore is not an issue.
The systems described here use a combination of physical methods
and signal processing methods to achieve robust assay performance
such as is needed for reliable operation outside the controlled
environment of a clinical laboratory.
[0169] FIG. 27 is a block diagram of the detector head 1300
electronics and optics used for controlling the fluorescent
excitation and for receiving, processing and digitally
communicating fluorescence emission signals to the host instrument.
Optical channels are again identified by open arrows A and B and
terminate in sensors 1817 and 1837. Channel A is taken as a control
channel and channel B as a target analyte channel, but the roles
are interchangeable. The electronic functional blocks in each
channel, source (1811,1831 and associated circuit block) and sensor
(1817,1837 and associated circuit block) are identical (with the
exception that channels A and B are configured for different
wavelengths).
[0170] The detection system can be configured for wavelengths in
the UV, visible region, and near infrared spectrum. Available light
sources having near monochromatic output, filters, chromophores and
fluorophores allow for tuning excitation and emission passbands in
the range of 300 to 900 nm. For applications in fluorescence
detection mode, which is one of the preferred operating modes of
the invention, the apparatus can be configured for specific
fluorescence dyes with excitation spectrum in the UV and visible
spectrum and for emission in the UV, visible and near infrared
spectrum. While a red shift is more typical, up-converting
fluorophores may also be used.
[0171] Within the detector head 1300, each of the LEDs associated
with source excitation circuits (1811, 1831) are modulated by a
square wave at a frequency of 130 Hz. The reason for this
modulation is related to noise reduction measures from the
following potential noise sources: 1) 50/60 Hz AC mains; 2) 100/120
Hz second mains harmonic from the fluorescent lights; 3) Third and
higher harmonics of 50/60 Hz AC; 4) Differential frequencies
(rumble) of 130 Hz and higher; and 5) Wide band white noise from
the photodiode sensor, first stage feedback resistor, and
amplifier. In order to retrieve a useful signal from the noisy
source, the following mitigation methods are employed: 1) Fast
sampling and averaging of taken data in order to avoid aliasing and
at the same time limit noise bandwidth; 2) Modulation of the LED
light at 130 Hz and correlation with the detected fluorescence
signal at 130 Hz in order to reject all uncorrelated components (a
130 Hz modulating frequency was selected to provide at least 10 Hz
difference from the 50/60 Hz mains and harmonics at 100, 120, 150
and 180 Hz and higher); and 3) Further filtering and processing of
the correlated data to eliminate electromagnetic noise from mains
power supplies of either 50 Hz or 60 Hz or harmonics thereof. The
excitation LEDs are turned on and off using a 130 Hz square wave
driven by FAN5612 LED-drivers. Each driver is capable of sinking
current up to 120 mA (40 mA on each of three outputs) at the
required frequency. The clock frequency of the detector head
microprocessor is used to strobe the excitation LEDs and to
synchronize pulse collection in the sensor diodes. These features
are found to produce a quiet output from the sensors in the absence
of specific fluorescence signal and enable higher
amplification.
[0172] The circuit board 1801 for the source LEDs can support
multiple LEDs, and the circuit board 1802 for the sensor circuit
can support multiple photodiodes. The excitation and detection
circuit are electronically isolated using bypass capacitors on
separate PCB boards (1801, 1802) on separate ground planes to
further reduce any possible crosstalk through pin junction 1804.
Each photodiode is shielded and intimately associated with a
multiple stage high gain amplifier (1818, 1838) and the two circuit
boards are electronically isolated with separate grounds. In the
detector head, electrical output from the sensor optics is
conditioned in a preamplifier and fed into a three-stage amplifier.
Each of three outputs from the amplifier has a multiple log scale
gain, with the highest amplification factor being up to 10.sup.14.
The three outputs are fed into A/D converters integrated into the
embedded microprocessor, and one digital output (the output
selected by firmware) is bussed as a digitized score to memory.
[0173] Advantageously, the isolation of the analog and digital
signal processing circuitry within a shielded detection head
realizes low noise conditions that permit very surprisingly high
gain factors, up to 10.sup.14, and in selected embodiments between
10.sup.12 and 10.sup.14 in three stages. By localizing the
digitization electronics within the detection head next to the
sensors, amplified signals having high gain are achieved with a
surprising lack of noise interference. Buffered and filtered
reference voltages are also supplied to the op amps to ensure
maximum stability in amplified signal output, which is digitized in
an A/D converter associated with the embedded microprocessor 1841;
thus digitization occurs within the scanning detector head.
[0174] While the above is a description of the hardware, the actual
operation of amplifier electrooptics and the signal processing
algorithms in the detector head is conducted by an "ODAP daemon"
coded in the firmware, which functions as a "virtual machine". The
daemon is essentially a set of instructions, but is resident in the
detector head in non-volatile memory digitally associated with the
embedded microprocessor and operates independently, once called
out, of the host controller. The firmware, a dedicated on-board
instruction set for control of signal acquisition and the
optoelectronic functions of the scanning detector head, is
typically resident in a socketed EEPROM chip 1842. The daemon
controls the embedded microprocessor 1841 in the detector head.
After digitization of the amplified signal, the daemon is capable
of further signal processing, for example comparing optical data
with background scans and/or making a complex determination as to
whether the fluorescence is a positive assay result before
reporting data to the host system for display to the user.
[0175] Surprisingly we have found that the two processors, the host
controller and the embedded microprocessor in the detector head,
can be coordinated so that the ODAP daemon is able to construct a
spatial map of the sample area while the detector head is moved
under external control of the host controller. ODAP functions
proceed autonomously in collecting signal data, for example, while
the host controller performs a melt/anneal subroutine on the
temperature in the detection chamber, so that assay reprogramming
in the host controller routines does not affect or impact the
operation of data acquisition and reporting under control of the
daemon, a distinct advantage where, depending on a barcode read
from the assay cartridge, any of several assay protocols may be
performed by the host controller.
[0176] One advantage of using an embedded microprocessor 1841
inside the detector head is that a proprietary method of "optical
data acquisition and processing" (ODAP) may be programmed into the
firmware in the detector head to eliminate noise before
transferring a clean, digitized signal to the host controller. The
ODAP daemon is thus an independent subroutine or subroutines that
is invoked by the host processor when needed--but that once
initiated the daemon and associated optoelectronics run
autonomously under independent control of the embedded firmware. In
practice, it has proved possible to complete the entire analysis
within the detector head and to simply communicate a summary score
or assay result to the host instrument. Unlike analog signals,
digital transmissions from the detector head to the host instrument
are not susceptible to interference caused by the noisy analog
machinations occurring within the instrument housing.
[0177] As currently realized, the ODAP daemon runs continuously and
autonomously as the detection head is scanned linearly across the
cartridge. "Daemon" refers here to the operation of a programmable
instruction set for optical data acquisition and processing that is
stored in firmware which is executed by an embedded microprocessor
as an autonomous background process rather than under the direct
control of an interactive user or by the host controller. The
daemon is in part a virtual machine which operates and controls
electronic circuitry that is localized in the detector head so that
it can be protected from external electronic noise associated with
host operations.
[0178] Thus the invention includes a method for automating a
microassay, which comprises operationally dividing the assay into
fluidic, electromechanical, and thermal processes controlled by a
host controller and optoelectronic processes controlled by an
autonomous daemon resident in a scanning detection head, where the
motion of the scanning detection head is controlled by the host
controller and any optical signal acquisition and processing is
controlled by the autonomous daemon.
[0179] By way of illustration and example, three detection
chambers, each for analyzing a liquid sample, are disposed in an
optical window that is scanned by the detection head. Scan time is
about 30 sec and the scan length is divided into about 500 steps.
The detector is operating at 130 Hz, but the digitization rate is
higher, so that 12000 to 24000 measurements of fluorescence may be
taken during each scan. Sensor diode output is stored and averaged
so that the dark half-cycle can be subtracted from the light
half-cycle as the LED is strobed at 130 Hz to eliminate
non-specific photodiode output. The dataset is further processed
under control of the on-board firmware in 100 ms increments. Thus
the digitized score tabulated in local memory in the detection head
consists of a processed signal acquired over 100 ms and corresponds
to one or only a few steps in the linear scan, depending on the
characteristics of the stepper motor and the length of the scan.
The "spot size" corresponding to the signal is thus fairly small
but not so fine as a high resolution digital pixel. Each digital
score has a value that corresponds to all the amplified current
accumulated during the LED ON time intervals less any ambient
signal during the LED OFF time intervals.
[0180] The daemon is synchronized to track stepper motor actuations
and accumulates a table of fluorescence output scores versus scan
distance (position) over the width of the optical window. By
performing a baseline scan prior to assay, any "new" signal (i.e.,
any change over baseline) that results from dyes, chromogens or
fluorophores associated with the assay reactions is then
quantitated. After background subtraction is complete, a threshold
value may also be used to further filter signal changes over
baseline if desired.
[0181] Data sampling, filtering, digital smoothing and
conditioning, may be achieved by a combination of hardware and
firmware means, the firmware referring to the daemon instruction
set. Programmable statistical analysis of the digital scores
acquired during a scan across a sample well may be parametric or
non-parametric. From each dataset specific to each sample well, the
distribution of the scores over the scan transect is a better
predictor of the assay truth value ("positive" or "negative" for
target analyte or control) than an average, a median or a mode
value taken over the well. A result based on a distribution
function having many points is also superior to an optically
averaged signal taken by interrogating the well at low resolution.
Averages can lead to false negatives, whereas we have found that a
pattern of high signal spikes, even if localized to a particular
part of the well, is highly likely to correspond to a positive
assay. The firmware is configured to assess and correlate the
intensity of the digital scores and the frequency of signal spikes
with expected ranges for positive versus negative assay results.
Datasets may be considered as population statistics, where a
population of 30 measurements, ranked according to intensity, for
example, is compared to a distribution expected according to a null
hypothesis according to statistical analysis of variation.
Statistical tools for assessing the data non-parametrically are
also applicable and may be encoded in firmware. Thus in a preferred
embodiment, the on-board capabilities of the detection head
encompass outputting an assay result as a digital "one" or "zero"
corresponding to whether an analyte has been detected, true or
false, and also the status of the corresponding controls.
Surprisingly, we have found that aberrations in the datasets due to
bubbles and other irregularities can be managed by a conversion to
a 1-bit digital score, generally under control of the daemon with
embedded firmware.
[0182] Inputs into the embedded microprocessor are multiplexed so
that several target analytes and controls may be assayed
simultaneously. The detector head is provided with at least two or
more optical channels, each having independent excitation optics
and emission optics which are operative at discrete wavelengths.
White excitation light is not used to eliminate possible crosstalk
between different fluorophores in a multiplexed assay, such as when
a target fluorophore and internal control fluorophore are mixed in
a common liquid sample. The use of "one pot" conditions for target
and control results from the need to prove that conditions for a
positive control signal are present in each sample well (avoiding
false negatives). But by separating target and control channel
optics, crosstalk that could lead to false positive tests or test
rejection due to invalid control results was eliminated.
[0183] For comparison, the host controller 1800 is responsible for
the operator interface, including display of results, and for
operation of mechanical and pneumatic functions required to perform
an on-cartridge assay and detector head scan. A separate clock in
the host controller is used to drive the stepper motor during
scanning. To correlate detector head position with fluorescence
signals acquired during the scan, stepper motor activity is
monitored by the firmware in the detector head.
[0184] Well position recognition is achieved by an "edge detection"
process operated under control of the embedded daemon in the
scanning detector head. In a first instance, a pair of mechanical
switches is mounted on the underside of the detector head with
leads digitally communicating to the embedded controller. In a
first scan of an empty cartridge, the detector head glides along
its rail under host instrument control of a stepper motor, and when
a first mechanical switch is tripped by a detent formed in the
instrument chassis alongside the rail, then an electronic signal is
sent to the daemon. The daemon is programmed to initiate its
activities and will start capturing stepper motor data from the
host instrument and using this data to map an x-axis transect
across the optical windows of the cartridge, where each step of the
motor corresponds to a position and a distance increment n defined
by the stepper motor such that (x.sub.i=x.sub.0+(ni)) for any
distance x.sub.i from a reference point x.sub.0. The daemon also
stores optical output during the first scan. Thus the daemon
tabulates a position x.sub.0 and an intensity I.sub.0 datapair and
continues to increment x.sub.i as the stepper motor advances the
detector head. By virtue of discontinuities in the cartridge optics
that are associated with sample wells, an optical scan of the
cartridge along the x-transect results in signals that are useful
for well edge detection. These signals are characterized by a peak
where a positive slope is separated from a negative slope by a peak
condition dx/dt=0, or by a sudden step change (drop or increase) in
the baseline optical output as the detection head crosses over a
nearside edge of the well. If a threshold value is crossed, then
the edge effect associated with the entry into the well is scored
by the daemon as a well start position having an x-coordinate
(motor step number) on the linear map or x-transect. Because the
detector head is mounted on a rigid guideway, the x-transect path
can be reversed, and the scanning head can precisely retrace its
route in a subsequent scan across the well from the same start
point x.sub.0. In this way, a definite physical distance is
established by the mapping process from the position on the rail
where the mechanical detent was tripped to the well edge.
Similarly, a farside edge of a well is identified. The background
optical readings of the empty well between the nearside edge and
the farside edge positions are then processed and stored as a
representative background signal. This process is repeated for all
wells, and is repeated again for each optical channel in the
detector head. With appropriate offsets, the mechanical switches
may be used for more than one channel, so that an x-transect is
collected for each optical channel during a single scan. Similarly,
multiple wells may be scanned one at a time as the scanning head
scans along the optical window.
[0185] The detector head then returns to its rest position, and on
command, starts a scan that will detect any changes in optical
output associated with a sample filling the wells. The daemon
collects data from the detection wells according to the predefined
well edge positions in memory and performs calculations to evaluate
changes in fluorescence (or other optical signal), associated with
the contents of the wells. These data are further processed by
signal processing algorithms operated by the daemon. Changes or
lack of changes in fluorescence in each of the wells after
introduction of sample may be indicative of the presence of a
control signal and/or the presence or absence of a target analyte
signal depending on the assay design.
[0186] Edge detection may include filters such as a triangular
filter or other signal filters known in the art. Fidicials
associated with the edges of the cartridge wells may include
colored layers deposited or laid down around the wells, physical
edges that result in optical scattering, cassette materials having
autofluorescence, and so forth, as are known in the art. Thus the
mapping routine may alternatively be indexed to a fiducial
associated with the cartridge body or a sample well such that an
initial reference point is established optically, electrically or
mechanically. A set of datapairs for the initial position x.sub.0
and associated intensity I.sub.0 is recorded. Subsequent datapairs
(x.sub.i,I.sub.i) are collected as well mapping proceeds stepwise
(x.sub.i=x.sub.0+(ni)) where n is the number of steps of a stepper
motor and i is a step distance. Algorithms are used to detect
signal changes characteristic of sample well edges and these are
indexed so that one or more sample wells in a scanning transect are
indexed. Thus a pre-scan may be performed and digitized baseline
optical signal data collected; then the sample wells are flooded
with liquid containing the analytes of interest and a second scan
is performed: the difference between the two scans is
representative of the target analyte and/or control analyte of
interest. Signal processing and digitization and subsequent
analysis are performed under the control of the autonomous daemon
within the detector head.
[0187] FIGS. 28A and 28B are representations of raw input and
digitized output and demonstrate a method for digital removal of
bubble interference by the daemon. In FIG. 28A, output (1900) from
the photodiode after amplification is represented. The level of
noise is generally low, but signal deteriorates at 1901 and 1902
due to the presence of two small bubbles in the detection chamber
(1501, see FIG. 29), for illustration. The daemon applies a
threshold to the output signal and scores the signal "high" (i.e. a
one) if the signal is above the threshold and "low" (i.e. a zero)
if the signal is below the threshold. Because no signal output can
be above the threshold except in the presence of a fluorophore
matched to the emission optics and filters, any positive signal
(1903) is a positive assay for the presence of fluorophore. The
threshold comparator can be adjusted based on experience with
clinical samples in the assay. Thus a "1-bit" digital
transformation of the scanning image data removes any interference
from bubbles. We have found surprisingly that when a fluorophore is
present but multiple bubbles fill the chamber, light refracted
around or through the bubbles will result in a positive signal. The
system is thus very error resistant and robust for qualitative
testing, such as is needed in diagnostic assays for infectious
disease. The signal comparator is a digital function of the
microprocessor and firmware embedded in the detector head and is
independent of host controller function.
Host Controller Functions
[0188] Again with reference to FIG. 27, the host controller 1800 is
responsible for actuating the daemon, but does not control its
operations. During operation of the daemon, the host system
multitasks to perform various assay functions such as temperature
and pneumatics control, detector head scanning, user interface
operability, and fault monitoring.
[0189] Among other functions, the host controller actuates system
hardware for controlling pneumatic logic and pulse train routines
needed to perform the assay, including valves and diaphragm pumps
in the microassay cartridge, any resistive or Peltier-type heating
elements associated with thermal cycling of the sample, and
optionally may perform melt curves in the detection chamber by
actuating resistive heating elements during melting and a blower
during cooling (FIG. 22). Optional components interfacing with the
host controller include a bar code reader for sensing information
printed on the insertable microassay cartridges.
[0190] The host controller is provided with non-volatile memory and
programmable instructions for coordinating the steps of an assay
process and for transforming and formatting a signal, result or
other data from the embedded daemon into a machine-readable or
graphically displayable report in a form that will be made
available to the user. A graphical user interface 1820 for
displaying test data can be integrated into the detection system as
shown here or can be connected to it through data lines or wireless
interfaces as part of a network, intranet or internet. Generally, a
serial asynchronous communications interface is provided for
communication with the host controller on the instrument
motherboard or on an external network.
[0191] Similarly, results, data, error codes, status updates, and
so forth can be sent via common electronic interfaces and data
lines such as USB, RS232 or FireWire and via a wireless
transmission system such as IR-transmission, Bluetooth, GSM, GPRS,
and RSID. Programming, reprogramming, calibration, and
recalibration as well as system diagnosis of the device is possible
via common electronic interfaces and data lines such as USB, RS232
or FireWire and via a wireless transmission system such as
IR-transmission, Bluetooth, GSM, GPRS, RSID, and so forth.
Decoupled Optics
[0192] FIG. 29A is a representation of a planoconvex objective lens
1500 in excitation mode. Excitation cone 1501 is shown relative to
the cartridge detection chamber 1403 and heating block 1410 with
mirror face 1400. The convergent excitation cone is formed by
diverging rays illuminating the objective lens from the source,
whereby the focal position distance L2' is greater than the native
focal length L2 of the lens. By convention, the native back focal
length L2 of the lens is determined using collimated light.
Shifting the focal position is termed "decoupling", and was
achieved in this instance by moving the source closer to the source
lens, but more generally decoupling can be achieved by using
non-collimated light.
[0193] Interposed between the lens 1500 and the mirror face 1400 is
a microassay cartridge 200 with detection chamber 1403. The
detection chamber is bounded by an upper optical window 1502 and a
lower thermo-optical window 1503. In operation, the intervening
volume is taken up by a liquid sample, shown here with two
entrained bubbles 1505. The focal cone is seen to reflect from the
mirror face, forming a real image (1510, solid rays) of the source
in the detection chamber and a virtual image (1511, dashed rays) of
the source below the mirror face. The back focal position L2' is
thus generally equal to or greater than the distance between the
lens and the mirror. Excitation light striking the mirror is
reflected as a focused beam in the fluid volume of the detection
chamber, thus doubling the length of the light path of the
excitation light through the sample and increasing the excitation
fluorescence yield. The back focal position L2' is not equal to the
back focal length L2 of lens 1500; the two are decoupled, generally
by illuminating the lens with a divergent beam from the source
1512.
[0194] FIG. 29B is a representation of the objective lens of FIG.
28 in emission collection mode. Shown are primary and reflected
fluorescent emissions (solid and dashed lines from a real image
1510 and a virtual image 1511). Again shown are bubbles in the
chamber 1403. Rays striking the planar back surface of the
objective lens 1500 will be collimated (1522) and transmitted to a
detector. The quantity of fluorescence signal captured depends on
the angular and numerical apertures of the lens. The native back
focal length of lens 1500 is L2. The back focal position of the
lens can be manipulated or "decoupled" relative to the native focal
length of the lens (L2 versus L2') by repositioning the source as
shown in FIGS. 30 and 31 or by changing the shape or refractive
index of the source lens.
[0195] FIG. 30 is a schematic representation of an optical pathway
1700 with decoupled excitation and emission optics. In this figure,
excitation rays are shown as solid lines and emission rays are
shown as dashed lines. Here the light source 1701 may be an LED as
shown, an SMD LED without lens housing and reflector ring, an SLED,
a pumping laser diode, a ridge-waveguide (Fabry Perot) laser diode,
a tunable laser, and so forth, such source of illumination
preferably having a narrow bandwidth. LEDs of various narrow
bandwidths are available, for example with peak emission at 630 nm
(red), 470 nm (blue), 525 nm (green), 601 nm (orange), 588 nm
(yellow) and so forth. White light LEDs may be used if desired, but
generally the LED output is matched with the fluorophore of the
target in the assay and an optional excitation barrier filter (not
shown) may be used to sharpen the bandpass as required. Light
output of light source 1701 is transmitted by source lens 1702,
shown here as a planoconvex lens, although molded aspheres may also
be used, before striking dichroic mirror element 1704, where the
excitation beam is reflected onto objective lens 1705. The
excitation cone striking a sample fluid volume 1720 in microassay
cartridge 1402 is shown with solid lines. Unconventionally, the
focal point of the excitation cone has been projected past the
sample chamber and back mirror 1400 by moving the source 1701
closer to the source lens 1702 (i.e., shortening distance L1' in
order to increase distance L2', where distance L1 would be the
native back focal length of lens 1702). As distance L1' is
shortened, the source rays striking the objective lens are not
collimated by the source lens and are caused to diverge, thus
increasing distance L2'. The native back focal length of objective
lens 1705 is L2, where L2 is less than or equal to the distance
separating the back of the objective lens and the mirror, i.e.,
preferably is within the plane of the sample chamber, so that any
emissions from a fluorophore in the sample chamber and any
reflected emissions forming a virtual image of the fluorophore in
the mirror will enter the objective lens. The emissions cone has a
focal point that is decoupled from the focal point of the
excitation light (which is focused behind the mirror). Fluorescent
emissions within the focal plane L2 and any virtual image of light
originating in the focal plane are effectively collimated by the
objective lens, are effectively transmitted through dichroic mirror
1704, bandpass filter 1706, and are then focused by sensor lens
1707 onto sensor 1708, which is typically mounted on a PCB or other
solid support 1709. Lens 1707 has a back focal length L3 that
generally is equal to back focal length L2 of objective lens 1705.
However, a larger lens 1707 may be used to better utilize the
surface area of the sensor 1707, which is for example a photodiode
or CCD chip. Optimization of signal may require independent
adjustment of each lens according to the principles of decoupled
optical systems outlined here, and may use non-collimated
excitation light, contrary to the teachings of the prior art.
[0196] The cone of excitation light emerging from objective lens
1705 and the cone of emission light entering objective lens 1705 on
a common optical axis are operably decoupled at different focal
points (indicated by focal positions L2' versus L2 respectively). A
distance separates the actual focal plane of the excitation light
and the native focal length of the objective lens. The objective
lens will capture light in a broad plane of origin of the
fluorescent emissions when excited using a mirror and an extended
focal position of the excitation cone 1501. This phenomenon, termed
"decoupling" was found to increase capture of fluorescent emissions
when a back mirror is used, and controverts earlier teachings in
favor of the confocal approach of the prior art.
[0197] While the teachings of the prior art strongly support making
the excitation and emission confocal, there is in fact a previously
unseen advantage in decoupling the focus of the source from the
emission cone and using a back mirror 1400. Emitted light arises
from a greater area and depth throughout the sample cartridge, thus
overcoming any lack of signal from dead spots or inhomogeneities as
would be due to small bubbles, unmixed areas, or quenched probe.
Greater reliability is achieved at the expense of some selectivity
for excitation originating at the point of focus, but spatial
selectivity for excitation is in fact not desirable in an assay
device. This is a technological advance in the art.
[0198] To summarize, in certain embodiments L2' may be greater than
L2 and L3. L1 advantageously may be configured so that the cone of
excitation light falls behind the sample chamber 1403 and most
preferentially close to or behind the back mirror 1400. In a
preferred embodiment, the focal point of the excitation cone falls
on or behind the back mirror. The objective lens is configured,
generally, so that emitted light is efficiently collected and
collimated for projection onto the detection sensor by a
symmetrical cone of emitted light from sensor lens 1707 (i.e.
L2=L3).
[0199] Accordingly, in another embodiment the apparatus of the
invention employs lenses configured so that excitation optics and
the emission optics are decoupled. In a first embodiment of this
apparatus, the light source is positioned at a distance L1' from
the source lens, where L1'<L1, whereby the excitation optics and
emission optics are decoupled by transitioning the excitation cone
to a focal position L2' at or behind the mirror face, such that
L2'>L2. More generally, the source lens is configured to form a
diverging beam of light incident on the objective lens, thereby
positioning the excitation cone at a focal position L2', whereby
L2'>L2.
[0200] Advantageously, L2 is configured to be symmetrical to L3
(i.e., L2=L3), so that the operation of detector 1708 is optimized
and robust. The sensor photodiode is preferentially configured to
be large enough with reference to the cone of focus of lenses 1705
and 1707 to accommodate some degree of misalignment without loss of
assay validity.
[0201] In an alternate embodiment of decoupled optics, the native
focal point of the objective lens and the actual focal point of the
excitation light are separated by a distance such that L2>L2'.
This is illustrated in FIG. 31, where the source 1701 is moved away
from the source lens 1702 such that the native back focal length of
the lens is exceeded (L1'>L1). The effect of such a
repositioning is to cause rays emitted from the source lens to
converge as they approach the objective lens 1705 as shown, and the
ultimate effect is to shorten L2'. By placing the excitation cone
focal position directly in the middle of the sample chamber 1403
between optical windows 1502 and 1503, and by using a mirror 1400,
excitation of fluorophores in the sample liquid is achieved. But in
this instance of the use of decoupled optics, the objective lens is
also moved closer to the sample chamber and the native back focal
length of the lens L2 is now positioned behind the mirror. The
effect of this is to increase the apparent angular aperture of the
lens, i.e., by moving the lens closer to the fluorophores, more
light is collected and redirected to the sensor. As before,
collimated output from the objective lens is focused on sensor
1708, and thus typically L2 can equal L3. Thus the principles of
decoupling are shown in FIGS. 30 and 31 to encompass two
complementary methods for using non-collimated excitation light to
optimize signal collection efficiency when used with the mirrored
thermo-optical interface that is also an aspect of the
invention.
[0202] Decoupling is useful not only in assays where
differentiation of a positive or negative assay result is required,
but also in assays were some level of quantitation is required, as
for example schizont or merozoite copy number in the case of
Plasmodium falciparum. It should be recalled that the original
purpose of confocal optics, as articulated by its inventor, Minsky
in 1957 (U.S. Pat. No. 3,013,467), was to create a three-dimension
image of a thick solid specimen by rastoring a focal point of
excitation across and through the specimen (xyz axis rastoring)
while monitoring emission only from the area of the specimen where
the excitation cone is focused at any given time. In contrast, in a
fluid mixing specimen that is generally homogeneous, an opposite
effect is desired, that of measuring the overall fluorescence of
the specimen with the highest possible fluorescence capture. In
systems where multiple measurements are taken during a traverse of
the specimen, statistical methods can be used to overcome
interferences such as caused by bubbles, and a strong positive
signal, regardless of where it originates in the sample chamber, is
likely to indicate a positive assay result. Thus by reformulating
the problem, we have been able to design a novel optical system
with back mirror, with decoupling of the focal plane of excitation
and emission, with the happy result that fluorescence detection is
more sensitive and more robust in the presence of occasional
interferences.
[0203] FIG. 32A plots experimental results demonstrating
enhancement of fluorescent signal output by varying the height of
the objective lens above the mirror. Briefly, fluorescent beads
(Thermo Fisher Scientific, part number G0300, Pittsburgh Pa.) were
inserted into a microassay detection chamber and the detection
chamber mounted under the objective lens 315 of the detector. Using
a digital micrometer, the height of the detector head above the
microassay cartridge was then varied to construct the plot. In a
second paired experiment, the mirrored surface was removed. The
solid line (2100) shows the effect of varying the lens height in
the presence of a mirror on the heating block; the dashed line
(2101) shows the effect of varying lens height in the absence of a
back mirror. As can be seen, the presence of the mirror seems to
shift the optimal emission maximum behind the mirror plane (i.e. a
composite of the real and virtual fluorescent emissions captured in
the lens).
[0204] FIG. 32B is a bar graph showing the output signal strength
with (2103) and without (2102) the back mirror. Output signal was
found to be optimized by focusing the excitation beam at a focal
point behind the mirror as shown in FIG. 29A and collecting
emissions at a shorter working distance as shown in FIGS. 29B and
30.
[0205] In a second experiment, the detection chamber is filled with
a liquid sample containing a soluble fluorophore and pumped through
the chamber at constant rate to avoid quenching artifact. The
detector head height is then varied as before and the optimal
detector height determined. In related experiments, the working
distance of the light source from the source lens is also varied in
order to optimize sensitivity and limit of detection. We learn that
optimal configuration is not achieved when the objective lens is
centered in the detection chamber and the other lenses are made
confocal. When a mirror is used, decoupled optics achieves
advantageous results, a technological advance in the field.
[0206] FIG. 33A shows scanning data collected for a molecular
beacon hybridized to an amplicon. The scanning axis transects
detection wells (2200, 2201) representing positive and negative
test conditions respectively, and it can be seen that signal is
limited to the detection wells. The sample is scanned repetitively
as the temperature in the detection or sample chamber is
systematically varied. The scans are overlaid in the plot to
illustrate the spatial resolution of the data. Fluorescence scans
for 35.degree. C., 65.degree. C., 70.degree. C., 75.degree. C. and
80.degree. C. test conditions are marked. Test plots at 40, 45, 50,
55, and 60.degree. C., and the 85 and 90.degree. C. plots were not
well differentiated, as expected, and are not individually marked.
It can be seen that fluorescent signal is a function of
temperature. Fluorescence quenching is observed to increase as the
double stranded probe-target is melted, i.e., signal is greatest at
35.degree. C. and is essentially not present at 80.degree. C. In
FIG. 33B, the data is plotted for signal versus temperature for the
positive (2301, solid line) and negative (2302, dashed line) test
conditions. In FIG. 33C, a first derivative is plotted, indicating
a FRET melt temperature of about 70.degree. C.
Example I
[0207] In this example, the apparatus of the invention is shown to
be useful in diagnosis of infectious disease by detection of the
nucleic acids of a pathogen in a human sample such as blood. Using
on-board dry and liquid reagents, a blood sample is processed and
DNA associated with Plasmodium falciparum is detected in about 30
minutes or less. DNA purified from the sample is subjected to PCR
using two chambers with dual temperature zones as described in U.S.
Pat. Nos. 7,544,506, 7,763,453, and 7,955,836, which are
co-assigned. Amplicon is then detected using a FAM
fluorescence-tagged molecular beacon directed at the amplified
target. Optionally, a control consisting of a California Red-tagged
RNAase P leukocyte exon sequence, with multiplex amplification, is
used to validate the assay. A representative thermal melt curve
obtained using the thermo-optical interface of the invention is
shown in FIG. 33B.
Example II
[0208] The apparatus of the invention is useful in the diagnosis of
coagulopathies. Using on-board dry and liquid reagents, a blood
sample is assayed for Coagulation Factor VIIa deficiency by
incubating plasma with a fluorogenic substrate such as
(D-Phe-Pro-Arg-ANSNH-cyclohexyl-2HC1; F.W.=777.81, Haematologic
Technologies, Essex Junction Vt.) where ANSN is fluorophore
6-amino-1-naphthalene-sulfonamide, which lights up when the amide
bond between the dye and the peptide is cleaved. Tissue Factor (TF)
is obtained from Calbiochem (LaJolla Calif.) and incorporated into
phosphatidylcholine or phosphatidylserine vesicles before use. TF
is used in excess. A 100 uL substrate reaction mixture consisting
of 20 mM Hepes buffer, pH 7.4, 0.15 M NaCl, with 5 nM TF and
containing 20 uM EDTA is incubated with a plasma sample for 10 min
to form the active enzyme complex. The ANSH substrate is then
added. The rate of hydrolysis of substrate is linear over the
normal range of Factor VIIa, and can be determined from a standard
curve. Descriptions of assay development may be found in US Pat.
Appl. Publ. No. 2009/0325203 and other experimental literature.
[0209] While the above is a description of certain embodiments of
the present invention, it is possible to use various alternatives,
modifications and equivalents. Therefore, the scope of the present
invention should be determined not with reference to the above
description but should, instead, be determined with reference to
the appended claims, along with their full scope of equivalents.
The appended claims are not to be interpreted as including
means-plus-function limitations, unless such a limitation is
explicitly recited in a given claim using the phrase "means
for."
[0210] All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or cited in an accompanying application data
sheet, including but not limited to U.S. Patent Application No.
61/745,329, are incorporated herein by reference, in their
entirety. Aspects of the embodiments can be modified, if necessary
to employ concepts of the various patents, applications and
publications to provide yet further embodiments.
* * * * *