U.S. patent application number 16/541167 was filed with the patent office on 2019-12-05 for systems and methods for molecular diagnostics.
The applicant listed for this patent is QuanDx Inc.. Invention is credited to Paul Fleming, Ronan Hayes, Xiaojun Lei, Brian Lewis, Bruce Richardson, Qian Xu, Yuan Yuan.
Application Number | 20190366346 16/541167 |
Document ID | / |
Family ID | 62556629 |
Filed Date | 2019-12-05 |
![](/patent/app/20190366346/US20190366346A1-20191205-D00000.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00001.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00002.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00003.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00004.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00005.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00006.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00007.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00008.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00009.png)
![](/patent/app/20190366346/US20190366346A1-20191205-D00010.png)
View All Diagrams
United States Patent
Application |
20190366346 |
Kind Code |
A1 |
Lei; Xiaojun ; et
al. |
December 5, 2019 |
SYSTEMS AND METHODS FOR MOLECULAR DIAGNOSTICS
Abstract
The present disclosure provides systems, devices and methods
associates with processing and analyzing samples for molecular
diagnostics. The system may process samples using assay cartridges
including sample preparation modules and PCR modules. The system
may include thermal cycler modules and optics modules to detect the
specific nucleic acid sequences in the samples.
Inventors: |
Lei; Xiaojun; (San Jose,
CA) ; Yuan; Yuan; (San Jose, CA) ; Xu;
Qian; (Rancho Cucamonga, CA) ; Fleming; Paul;
(San Ramon, CA) ; Hayes; Ronan; (Monte Sereno,
CA) ; Lewis; Brian; (Los Gatos, CA) ;
Richardson; Bruce; (Los Gatos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
QuanDx Inc. |
San Jose |
CA |
US |
|
|
Family ID: |
62556629 |
Appl. No.: |
16/541167 |
Filed: |
August 15, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15385873 |
Dec 21, 2016 |
10427162 |
|
|
16541167 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 7/52 20130101; B01L
2200/0631 20130101; B01L 2300/044 20130101; B01L 3/5085 20130101;
B01L 2300/021 20130101; B01L 3/0275 20130101; B01L 2200/10
20130101; B01L 2300/022 20130101; B01L 2200/16 20130101 |
International
Class: |
B01L 7/00 20060101
B01L007/00 |
Claims
1. A PCR-based molecular diagnostic device for assaying a plurality
of PCR wells, said PCR-based molecular diagnostic device comprising
an optic module for exciting fluorescent dyes in the PCR wells and
detecting fluorescence emitted from the PCR wells, said optic
module comprising: an excitation light source; a plurality of
excitation optic fibers for directing excitation light to the PCR
wells; a fluorescence light detector; a plurality of emission optic
fibers for directing fluorescence within the PCR wells to the
fluorescence light detector, wherein termini of the emission optic
fibers are arranged on a circle on an optic fiber plate; a rotary
plate sandwiched between the optic fiber plate and the fluorescence
light detector, said rotary plate comprising multiple filters each
for a different wavelength, wherein the filters are arranged on a
circle matching the circle of the emission optic fibers on the
optic fiber plate such that when the rotary plate is rotated the
filters are capable of aligning with the termini of the emission
optic fibers.
2. The PCR-based molecular diagnostic device of claim 1, wherein
the excitation light source is a laser or an LED.
3. The PCR-based molecular diagnostic device of claim 2, wherein
the laser is a fixed-wavelength laser or a tunable laser.
4. The PCR-based molecular diagnostic device of claim 2, wherein
the LED is a single wavelength LED, a multi-wavelength LED or a
white LED.
5. The PCR-based molecular diagnostic device of claim 1, wherein
the excitation light is passed through a filter before being
directed to the PCR wells.
6. The PCR-based molecular diagnostic device of claim 1, wherein
the fluorescence light detector is a spectrometer, a single
photo-diode, or a photomultiplier.
7. The PCR-based molecular diagnostic device of claim 1, the rotary
plate comprises five filters.
8. The PCR-based molecular diagnostic device of claim 1, wherein
the optic module comprises a motor driving rotation of the rotary
plate.
9. The PCR-based molecular diagnostic device of claim 8, wherein
the motor is coupled to a drive pulley connected to the rotary
plate.
10. The PCR-based molecular diagnostic device of claim 1, wherein
each of the PCR wells contains multiple fluorescent dyes each
emitting fluorescence of a different wavelength.
11. The PCR-based molecular diagnostic device of claim 1, further
comprising an assay cartridge loading area.
12. The PCR-based molecular diagnostic device of claim 1, further
comprising a control panel.
13. The PCR-based molecular diagnostic device of claim 1, further
comprising a dispense system including a XYZ gantry with a
pipettor.
14. The PCR-based molecular diagnostic device of claim 1, further
comprising a thermal cycler module.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Utility
application Ser. No. 15/385,873 filed on Dec. 21, 2016, the entire
disclosure of which is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention generally relates to systems and
methods for molecular diagnostics.
BACKGROUND OF THE INVENTION
[0003] Many nucleic acid sequences have been used to diagnose and
monitor disease, detect risk and decide which therapies will work
best for individual patient. For example, the presence of nucleic
acid sequences associated with infectious organisms may indicate an
infection by the organism. The presence of an altered nucleic acid
sequence in a patient sample may indicate activation or
inactivation of a pathway related to a disease or disorders.
[0004] Detection of clinically related nucleic acid sequences in a
sample generally involves isolating nucleic acid from the sample
and amplification of specific nucleic acid sequences followed by
detection of the amplified products. However, complexities of the
multi-step process of isolating nucleic acid limit the processing
flexibility and reduce the repeatability. For example, DNA and RNA
have different chemical properties and stability, whose preparation
requires different processing conditions. Further, samples from
different source organism may require different steps to isolate
nucleic acids. For example, isolating DNA from bacteria may use
harsher conditions (e.g., higher temperature, higher concentration
of detergent, etc.) than releasing DNA from relatively labile
mammalian cells. Therefore, there is a need for an analytical
system providing flexible and adjustable operating capabilities to
meet the diverse demands of clinical diagnostics. Moreover,
although amplification increases the sensitivity of the detection
assay by providing sufficient copies of the specific nucleic acid
sequences, it may risk erroneous results born of contamination.
Therefore, there is also a need for an analytical system requiring
minimal user participation to reduce contamination.
SUMMARY OF THE INVENTION
[0005] Embodiments of the present invention are directed to
systems, devices and methods associated with processing and
analyzing samples for molecular diagnostics. Embodiments of the
invention include an automated, random access system for
determining specific nucleic acid sequences in the sample.
[0006] In an aspect, the present invention provides an assay
cartridge for a molecular diagnostic device. In one embodiment, the
cartridge comprising a sample preparation module and a PCR module.
In certain embodiments, the sample preparation module and the PCR
module is detachably coupled.
[0007] In one embodiment, the sample preparation module and the PCR
module is detachably coupled through a snap.
[0008] In one embodiment, the sample preparation module comprises a
sample loading well comprising an inlet opening covered by a
removable cap and an outlet covered by an outlet septum.
[0009] In one embodiment, the assay cartridge further comprises a
marking element. In one embodiment, the marking element is selected
from the groups consisting of a barcode, a dot code, a radio
frequency identification tag (RFID) or a direct reading electronic
memory.
[0010] In another aspect, the present disclosure provides a sample
preparation module for an assay cartridge used in a molecular
diagnostics device, said sample preparation module comprising an
elongated body formed to comprise a sample loading well, wherein
the sample loading well comprises an inlet opening covered by a
removable cap, and an outlet covered by an outlet septum.
[0011] In one embodiment, the sample preparation module further
comprises a formalin-fixed paraffin-embedded (FFPE) capture insert,
wherein the removable cap comprises a plunger.
[0012] In one embodiment, the sample loading well includes a sample
collecting channel having the outlet at the top end and a fluid
collecting area at the bottom end.
[0013] In one embodiment, the sample loading well has a deepest
portion at the fluid collecting area.
[0014] In one embodiment, the elongated body further comprises a
purification well. In one embodiment, the purification well
contains magnetic microparticles capable of binding to nucleic
acid.
[0015] In one embodiment, the elongated body further comprises one
or more reagent compartments.
[0016] In one embodiment, the elongated body further comprises a
pipette tip holder.
[0017] In one embodiment, the pipette tip holder is preloaded with
a pipette tip.
[0018] In yet another aspect, the present disclosure provides a PCR
module for an assay cartridge used in a molecular diagnostics
device. In one embodiment, the PCR module comprising an elongated
body formed to comprise a push well; and at least one reaction well
connected to the push well through a microfluidic channel.
[0019] In one embodiment, the push well is pre-loaded with a
solution mixture including reagents for PCR reaction.
[0020] In one embodiment, the PCR module further comprises a
barrier film covering the upper ends of the reaction well
formed.
[0021] In one embodiment, the elongated body further comprises a
plurality of reagent wells.
[0022] In one embodiment, the elongated body further comprises a
pipette tip holder. In one embodiment, the pipette tip holder is
preloaded with a pipette tip.
[0023] In another aspect, the present disclosure provides a
cartridge carriage that can load the assay cartridge as disclosed
above into a device for determining specific nucleic acid sequences
in samples. In one embodiment, the cartridge carriage comprises a
cavity configured to hold the assay cartridge. In one embodiment,
the cartridge carriage comprises at least one sample vial holder.
In one embodiment, the PCR wells of the assay cartridge are not
loaded into the cavity when the assay cartridge is loaded into the
carriage.
[0024] In one embodiment, the cartridge carriage comprises
structure that secures the assay cartridge into appropriate
position in the cavity. In one embodiment, the cartridge carriage
comprises a groove located at the distal end of the cavity that
fits a groove runner at the bottom of the assay cartridge. In one
embodiment, the cartridge carriage comprises an opening at the
bottom wall that allows the device to interact with the
compartments of the assay cartridge thought its sides and edges. In
one embodiment, the cartridge carrier includes a proximal fix tab
and a distal fix tab that secures the cartridge carrier in
appropriate location in the device.
[0025] In another aspect, the present disclosure provides a
dispense system including a XYZ gantry with a pipettor for
transferring a reagent between compartments in the assay cartridge
as disclosed above. In one embodiment, the pipettor comprises a
pipettor carriage that supports a pipettor head. In one embodiment,
the pipettor contains a lift that can raise and lower the pipettor
head.
[0026] In another aspect, the present disclosure provides a thermal
cycler module configured to amplify a specific nucleic acid
sequence in the PCR well of the assay cartridge disclosed above. In
one embodiment, the thermal cycler comprises a thermal block and a
receptacle for forming contact surface with a PCR well. In one
embodiment, the receptacle comprises an optical aperture configured
to permit optical communication through optical fibers to the
interior of the receptacle. In one embodiment, the thermal cycler
module further comprises a plurality of heat transfer fins.
[0027] In another aspect, the present disclosure provides an optic
module for exciting dyes in and detecting fluorescence from the PCR
wells in the assay cartridge disclosed above. In one embodiment,
the optical module comprises a rotary plate that includes a
plurality of filters each for a different wavelength, wherein the
rotary plate is stacked on an optical fiber plate. In one
embodiment, the filters are arranged on a circle from the center of
the rotary plate and the terminus of the optical fibers are
arranged on the optical fiber plate on a circle matching the one in
the rotary plate so that when the rotary plate is rotated the
filters can align with the optical fiber termini.
[0028] In another aspect, the present disclosure provides a system
for processing a sample, the system comprising: at least one assay
cartridge comprising at least a first compartment and a second
compartment, wherein the first compartment contains liquid; a
pipettor configured to transfer the liquid from the first
compartment to the second compartment; and a controller configured
to direct the pipettor to transfer the liquid from the first
compartment to the second compartment; wherein the assay cartridge
contains all the reagents needed for processing the sample.
[0029] In one embodiment, the assay cartridge comprises a reaction
vessel for containing a nucleic acid purified from the sample.
[0030] In one embodiment, the system further comprises a thermal
cycler module configured to amplify a nucleic acid sequence in the
sample.
[0031] In one embodiment, the system further comprising an optic
module configured to detect the presence of a nucleic acid sequence
in the sample.
[0032] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, appended claims and accompanying
drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1A shows a top perspective view of a device according
to an embodiment of the invention.
[0034] FIG. 1B shows a top perspective view of the layout of the
components of the device.
[0035] FIG. 1C shows a top plan view of the device.
[0036] FIG. 2A shows a top perspective view of an assay cartridge
according to one embodiment of the invention.
[0037] FIG. 2B shows a cross sectional view of a first half
fastener located on the sample preparation module and a second half
fastener located on the PCR module according to one embodiment of
the invention.
[0038] FIG. 3A shows a top perspective view of a sample preparation
module of an assay cartridge according to one embodiment of the
invention.
[0039] FIG. 3B shows a side, cross-sectional view of a sample
preparation module.
[0040] FIG. 4A shows a top view of a sample loading well according
to one embodiment of the invention.
[0041] FIG. 4B shows a top perspective view of a sample loading
well according to one embodiment of the invention.
[0042] FIG. 4C shows a cross-sectional view of a sample loading
well.
[0043] FIG. 5A shows a top perspective view of a removable cap.
[0044] FIG. 5B shows a side, cross-sectional view of a removable
cap.
[0045] FIG. 5C shows a top perspective view of a cap with a
plunger.
[0046] FIG. 5D shows a side, cross-sectional view of a cap with
plunger as it is used with an FFPE capture insert.
[0047] FIG. 6 shows a side, cross-sectional view of a nucleic acid
purification well.
[0048] FIG. 7A shows a top perspective view of a PCR module
according to an embodiment of the invention.
[0049] FIG. 7B shows a side, cross-sectional view of the PCR
module.
[0050] FIG. 8A shows a top perspective view of a cartridge carriage
according to an embodiment of the invention.
[0051] FIG. 8B shows a side, cross-sectional view of a cartridge
carriage according to an embodiment of the invention.
[0052] FIG. 8C shows a top perspective view of a cartridge carriage
with an assay cartridge loaded in processing lane.
[0053] FIG. 8D shows a side, cross-sectional view of a cartridge
carriage with an assay cartridge loaded in processing lane.
[0054] FIG. 9A shows a top plan view of a dispense head according
to an embodiment of the invention.
[0055] FIG. 9B shows a top perspective view of a dispense head
according to an embodiment of the invention.
[0056] FIG. 10A shows a top perspective view of a thermal cycler
module according to an embodiment of the invention.
[0057] FIG. 10B shows side, cross-sectional view of the thermal
cycler module.
[0058] FIG. 11 shows a top perspective view of an optics module
according to an embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0059] In the Summary of the Invention above and in the Detailed
Description of the Invention, and the claims below, and in the
accompanying drawings, reference is made to particular features
(including method steps) of the invention. It is to be understood
that the disclosure of the invention in this specification includes
all possible combinations of such particular features. For example,
where a particular feature is disclosed in the context of a
particular aspect or embodiment of the invention, or particular
claim, that feature can also be used, to the extent possible, in
combination with and/or in the context of other particular aspects
and embodiments of the invention, and in the invention
generally.
[0060] The term "comprises" and grammatical equivalents thereof are
used herein to mean that other components, ingredients, steps, etc.
are optionally present. For example, an article "comprising" (or
"which comprises") components A, B, and C can consist of (i.e.,
contain only) components A, B, and C, or can contain not only
components A, B, and C but also one or more other components.
[0061] Where reference is made herein to a method comprising two or
more defined steps, the defined steps can be carried out in any
order or simultaneously (except where the context excludes that
possibility), and the method can include one or more other steps
which are carried out before any of the defined steps, between two
of the defined steps, or after all the defined steps (except where
the context excludes that possibility).
[0062] Where a range of value is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictate otherwise, between the upper and
lower limit of that range and any other stated or intervening value
in that stated range, is encompassed within the disclosure, subject
to any specifically excluded limit in the stated range. Where the
stated range includes one or both of the limits, ranges excluding
either or both of those included limits are also included in the
disclosure.
[0063] It will be appreciated that for simplicity and clarity of
illustration, where appropriate, reference numerals have been
repeated among the different figures to indicate corresponding or
analogous elements. In addition, numerous specific details are set
forth in order to provide a thorough understanding of the
embodiments described herein. However, the embodiments described
herein can be practiced without there specific details. In other
instances, methods, procedures and components have not been
described in detail so as not to obscure the related relevant
function being described. Also, the description is not to be
considered as limiting the scope of the implementations described
herein. It will be understood that descriptions and
characterizations of the embodiments set forth in this disclosure
are not to be considered as mutually exclusive, unless otherwise
noted.
[0064] The following definitions are used in the disclosure:
[0065] The term "at least" followed by a number is used herein to
denote the start of a range beginning with that number (which may
be a range having an upper limit or no upper limit, depending on
the variable being defined). For example, "at least 1" means 1 or
more than 1. The term "at most" followed by a number is used herein
to denote the end of a range ending with that number (which may be
a range having 1 or 0 as its lower limit, or a range having no
lower limit, depending upon the variable being defined). For
example, "at most 4" means 4 or less than 4, and "at most 40%"
means 40% or less than 40%. When, in this specification, a range is
given as "(a first number) to (a second number)" or "(a first
number)--(a second number)," this means a range whose lower limit
is the first number and whose upper limit is the second number. For
example, 25 to 100 mm means a range whose lower limit is 25 mm, and
whose upper limit is 100 mm.
[0066] PCR or "Polymerase Chain Reaction" refers to a method used
to amplify DNA through repeated cycles of enzymatic replication
followed by denaturation of the DNA duplex and formation of new DNA
duplexes. Denaturation and renaturation of the DNA duplex may be
performed by altering the temperature of the DNA amplification
reaction mixture. Reverse-transcriptase PCR (RT-PCR) refers to a
PCR process including a step to transcribing RNA (e.g., mRNA) into
cDNA which is then amplified. Real time PCR refers to a PCR process
in which a signal that is related to the amount of amplified DNA in
the reaction is monitored during the amplification process. This
signal is often fluorescence. However, other detection methods are
possible. In an exemplary embodiment, a PCR subsystem takes a
prepared and sealed reaction vessel and performs a complete
realtime polymerase chain reaction analysis, thermal cycling the
sample multiple times and reporting the intensity of emitted
fluorescent light at each cycle.
Overall System Layout
[0067] In one aspect, the present disclosure provides a fully
automated, random access system for determining specific nucleic
acid sequences in samples. The system can combine two general
functions: sample preparation in the form of isolating nucleic
acids from a sample, and detection of specific sequences within the
isolated nucleic acids. Toward this end, the system includes an
assay cartridge that has at least two distinct functional modules:
one for process samples to isolate nucleic acids and a second for
nucleic acid amplification and detection. The system includes
instrumentation that works on the assay cartridge to carry out the
functions. In some embodiments, the instrumentation is contained in
a single, enclosed device. The system also includes consumables
incorporating necessary reagents for performance of a variety of
assays and transfer devices (e.g., pipette tips). In certain
embodiments, all consumables are contained in an assay cartridge so
that there is no need to store any consumables in the device. The
system may also include holders for samples, connections for power
and information. These are integrated in a single unit to provide a
system that performs major functions of sample handling, nucleic
acid isolation, amplification and detection, and supporting
functions such as supply and consumable management, information
management and maintenance. In some embodiments, the system
includes multiple assay cartridges, each of which can be processed
independently and simultaneously, i.e., in a random access
fashion.
[0068] Combining these functions into a single, highly automated,
self-contained system provides seamless integration of molecular
diagnostics into the workflow of the clinical laboratory. A further
benefit is to perform all steps of nucleic acid determination to
produce clinically acceptable results without the need for user
intervention. The system allows users to load samples as they are
available, and to perform determination on these samples based on
the needs of the patients and physicians, without constraints on
sample or analyte order being imposed by the system.
[0069] FIG. 1A shows a system for molecular diagnostics according
to one embodiment of the invention. Referring to FIG. 1A, the
system includes a device 100 having a generally rectangular housing
101 with sides defining the front, back, left and right sides, top
and bottom as illustrated. The device also has an assay cartridge
loading area 102 and a control panel 103. The housing can be made
of any suitable material known in the art, such as metal, alloy or
plastic. The control panel can include a touch screen through which
user can enter a variety of functions, such as selecting nucleic
acid purification protocols and amplification programs. The touch
screen can also display the status and results of the assays.
[0070] FIG. 1B shows a top perspective view of the embodiment of
FIG. 1A from above, with some components removed to clarify the
basic structural and functional modules. Referring to FIG. 1B, the
system includes a device 100 containing a cartridge loading unit
500 for receiving at least one assay cartridge comprising at least
a first compartment and a second compartment (assay cartridge is
not loaded as shown in FIG. 1B). In use, the assay cartridge is
loaded into the device 100 through a cartridge carriage. The device
100 includes a dispense system 600 having at least one pipettor
620, which may transfer a reagent from the first compartment to the
second compartment. The device 100 also includes a thermal cycler
module for amplification, and an optical module for detecting
products from the amplification.
[0071] FIG. 1C shows a top plan view of the layout of the
embodiment of FIG. 1A from above. Referring to FIG. 1C, the system
includes a device 100 having a cartridge loading unit 500 where a
plurality of assay cartridges 200 are loaded. Each assay cartridge
200 comprises at least a first compartment and a second
compartment. In use, the assay cartridge 200 is loaded with a
sample to be assayed. The assay cartridge 200 contains all
consumables that are needed for the assay so that there is no need
to store any consumables in the device 100. The system also
includes a dispense system 500 having at least one pipettor, which
may perform a variety of functions, such as transferring a reagent
from the first compartment to the second compartment. The system
further includes a thermal cycler module 600 that may assist the
amplification of nucleic acid sequences in the sample loaded in the
assay cartridge 200. The system also includes an optic module 700
responsible for exciting the dyes in the assay and detecting the
fluorescence emitted at each PCR cycle.
[0072] In this embodiment, a method for using the system may
comprise loading a plurality of assay cartridges into the cartridge
loading unit, each assay cartridge loaded with a sample to be
assayed, isolating nucleic acid from the sample by transferring and
mixing the reagents stored in the assay cartridge using a dispense
system having a pipettor, amplifying a specific nucleic acid
sequence in the sample using a thermal cycler module, and detecting
the presence of the nucleic acid sequence using an optic
module.
[0073] This embodiment can provide flexibility in processing a
plurality of samples. The system, in executing a first protocol,
can process a first sample loaded in a first assay cartridge.
Meanwhile, the system, in executing a second protocol, can also
processing a second sample loaded in a second assay cartridge. The
first and second protocols and their sequences of operations may
differ in any suitable manner. For example, the first protocol can
be directed to isolate DNA and the second protocol can be directed
to isolate RNA. Likewise, the first and second protocols may
include common processing steps, but may differ according to
duration processing or the parameters used for processing. For
instance, in some embodiments, two different protocols may have
similar processing steps, but the processing steps may differ
because they are performed at different temperatures and/or for
different periods of time. In another example, two protocols may
have similar steps, but they may be performed in different orders.
For example, a first protocol may include steps A, B, and C
performed in that order. A second protocol may include steps B, A,
and C performed in that order. In yet another example, different
protocols may include different sets of steps. For example, a first
protocol may comprise steps A, B, C, and D, while a second protocol
may comprise steps B, D, E, F, and G.
[0074] Further, the plurality of samples can be processed in any
order. In some embodiments, a plurality of assay cartridges can be
loaded into the device to start processing at about the same time.
Alternatively, the system can execute a first protocol to process a
first sample. During the processing of the first sample and without
stopping the first protocol, the system can receive a second assay
cartridge loaded with a second sample and start to execute a second
protocol to process the second sample.
Assay Cartridge
[0075] In anther aspect, the present disclosure provides an assay
cartridge used in a molecular diagnostic device. The assay
cartridge can be one-time use consumables, or may be reusable. In
certain embodiments, the assay cartridge comprises a sample
preparation module and a PCR module. The sample preparation module
is for purifying nucleic acids (e.g., genomic DNA, total RNA, etc.)
from a sample (e.g., FFPE specimen, blood or saliva, etc.). The PCR
module is for amplifying a target region in the purified nucleic
acids. In certain embodiments, the sample preparation module and
the PCR module are formed in one body. In some embodiments, the
sample preparation module and the PCR module are separated pieces
that can be assembled upon use in the device. This design allows
users to assemble the assay cartridge in their own desired
configuration to combine a sample preparation module with different
PCR modules to perform different assays (e.g., genomic DNA
amplification or reverse transcriptase PCR), or vice versa, and to
detect different target genes. Alternatively, the assay cartridge
can be made as one piece that is functionally divided into a sample
preparation module and a PCR module.
[0076] FIGS. 2A-2B show one embodiment of an assay cartridge 200.
The assay cartridge 200 comprises a sample preparation module 300
and a PCR module 400. The sample preparation module 300 and the PCR
module 400 can be engaged through a snap structure 201. The snap
structure 201 comprises a first half fastener 202 located on the
sample preparation module 300 and a second half fastener 203
located on the PCR module 400. The sample preparation module 300
and the PCR module 400 can be engaged by pressing the first half
fastener 202 and the second half fastener 203 together.
[0077] A. Sample Preparation Module
[0078] In one embodiment, the sample preparation module comprises
an elongated body comprising a proximal end and a distal end, and a
plurality of compartments arranged between the proximal end and the
distal end, wherein at least one of the compartments is a sample
loading well and at least one of the compartments is a purification
well. The sample loading well is where a sample is loaded for
procession before nucleic acids are extracted from the sample. The
processed sample is transferred to the purification well to extract
nucleic acids.
[0079] At least one of the compartments is a reagent storage well
for storing reagents for nucleic acid (e.g., DNA or RNA) extraction
from a sample. In one embodiment, the various compartments in the
sample preparation module include all reagents needed for
extracting nucleic acid from a sample. The reagents can include
cell lysis solution, wash buffer and elution buffer.
[0080] The sample preparation module can include a pipette tip
holder preloaded with a pipette tip (e.g., a microtip or a
millitip) for transferring the fluids between the various
compartments in the sample preparation module and/or between the
sample preparation module and the PCR module.
[0081] FIG. 3A shows one embodiment of a sample preparation module
300. The sample preparation module 300 comprises an elongated body
301 formed to include multiple compartments, which may hold fluids
(e.g., reagents) and devices (e.g., pipette tips) needed to process
various samples. Examples of compartments may include one or more
sample loading wells 310, one or more purification wells 320, one
or more reagent storage wells 330, one or more pipette tip holders
340, and one or more waste disposal wells 350. In certain
embodiments, the sample preparation module 300 can be in the form a
monolithic body, and may be formed of plastic (or any other
suitable material). In certain embodiments, the sample preparation
module 300 is made by a plastic injection molding process.
Alternatively, the sample preparation module 300 is made by
assembling individual components into a rigid framework. In one
embodiment, several pieces of the sample preparation module 300,
including a base formed to have the compartments and wells, and a
cover plate having holes corresponding to each compartments and
wells are made by a plastic injection molding process. To make the
sample preparation module, the base and the cover plate are
assembled to sandwich a barrier film (as described in detail
infra).
[0082] The sample preparation module 300 can have a proximal end
302 and a distal end 303 at opposite ends of the elongated body
301. The orientation of the compartments defines the top and bottom
portion of the sample preparation module 300. In certain
embodiments, compartments can be open at the top and closed on the
bottom and sides.
[0083] The sample preparation module 300 may also include a cap 360
that covers the opening of the sample loading well 310, optionally
an FFPE insert for holding FFPE samples (see FIGS. 3B and 4B), a
cover (e.g., a barrier film) that is disposed around various
compartments, features to facilitate handling (e.g., a half
fastener 202), selected reagents and labeling.
[0084] As shown in FIG. 3A, compartments within an sample
preparation module 300 can be arranged in a generally linear
layout, with the sample loading well 310 located near the proximal
end 302, followed by the purification well 320, reagent storage
wells 330, pipette tip holders 340, and waste disposal well 350 at
the distal end 303. This layout allows simple motion of the
dispense system (described in detail infra) to transfer the fluids
among various compartments. Alternatively, the sample preparation
module 300 can take different shape and arrangement of the
compartments (e.g., an arc, a single-row linear, or a circle),
depending on the overall system design, such as on the number and
sequence of operative locations that need access to the individual
compartments within a sample preparation module.
[0085] In some embodiments, the top ends of various compartments of
a sample preparation module form openings that align at a common
height. In some embodiments, compartment bottom ends generally do
not align because various compartments differ in depth and
shapes.
[0086] Compartments of the sample preparation module can perform a
variety of functions. For example, the purification well 320 can
provide a site for nucleic acid extraction. In addition, some
compartments may perform more than one function. For example,
reagent storage wells 330 initially contain reagents used in
extracting nucleic acids may later hold wastes produced during
purification process. And pipette tip holders 340 may later hold
discarded pipette tips.
[0087] In some embodiments, various compartments lack common walls
to prevent the creeping of liquids between compartments. This has
the benefit of reducing the possibility of contamination between
compartments. In some embodiments, the external profile of each
compartment closely tracks the cavity internal profile, i.e., the
walls of the compartment can be of relatively constant thickness
and can be thin compared to the size of the compartment. One of the
benefits of such design is to reduce the amount of material used
and hence reduces the manufacturing cost of the module.
[0088] FIG. 3B shows a side cross-section view of a sample
preparation module 300. Referring to FIG. 3B, the sample
preparation module 300 contains at least one sample loading well
310 where a sample for diagnostic analysis is loaded and processed.
The sample loading well 310 is covered by a removable cap 360. The
sample loading well 310 has a faceted shape designed to contain a
relatively large reaction volume, to permit effective mixing of its
contents, to permit aspiration with minimal dead volume. The sample
loading well 310 can have a capacity of about 1000 microliters. In
certain embodiments, the sample preparation module 300 includes a
formalin-fixed paraffin-embedded (FFPE) sample insert 370 disposed
in the sample loading well 310. The FFPE insert 370 can be used to
hold FFPE sample when the sample is processed in the sample loading
well 310. In such embodiment, the removable cap 360 includes a
plunger 364 to push FFPE samples to the bottom of the FFPE insert
370.
[0089] FIG. 4A shows a top view and a perspective view of a sample
loading well according to an embodiment of the invention. As shown
in FIG. 4A, the sample loading well 310 can have a generally
rhombus cross-section in the horizontal plane with one diagonal
axis of the rhombus aligned with the long axis of the sample
preparation module. The sample loading well 310 can have an
essentially vertical collecting channel 311 configured to allow a
pipette tip to be inserted to the bottom of the sample loading well
310. The collecting channel 311 is arranged off-center and
partially formed by the wall of the sample loading well 310. The
structure of the collecting channel 311 is also illustrated in FIG.
4C, which is a cross-sectional view of the sample loading well
through the plane (a).
[0090] FIG. 4B shows a perspective view of the sample loading well
of FIG. 4A as shown above. Referring to FIG. 4B, the sample loading
well 310 has an inlet opening 313 and an outlet 314. The inlet
opening 313 can be covered by the removable cap 360. The bottom of
the sample loading well 310 is configured to form a fluid
collecting area 312 at the bottom end of the collecting channel
311. The collecting channel 311 has an outlet opening 314 at the
top end, which optionally is covered by an outlet septum 315. The
outlet septum 315 is thin enough and contains a slit 316 and has a
cracking pressure, which in certain embodiments plays two
functions. When fluid is pipetted into the sample loading well 310
through the inlet 313, the outlet septum allows air to leak through
the outlet septum. On the other hand, the outlet septum 315 is used
to insert a pipette tip to remove fluid after processing. The
outlet septum 315 seals when there is no pipetting-action taking
place.
[0091] FIG. 4C shows a cross-sectional view of the sample loading
well of FIG. 4A as shown above along the section plane (a).
Referring to FIG. 4C, the bottom of the sample loading well 310 is
configured to form a fluid collecting area 312 at the bottom end of
the collecting channel 311, with an outlet opening 314 at the top
end. As shown in FIG. 4C, in the cross-section along the section
plane (a), the sample loading well 310 can be asymmetric, with a
deepest portion at the fluid collection area 312. The deepest
portion fits a pipette tip so that the pipette tip can reach the
deepest portion without touching the sidewalls when the tip is in
an aspirate position.
[0092] In certain embodiments, the sample loading well 310 is
covered by a removable cap to protect contents in the well and
prevent cross-contamination. The cap may be made of plastic or
other suitable material known in the art.
[0093] FIGS. 5A and 5B show a top perspective view and a side
cross-section view of the cap, respectively, according to one
embodiment. Referring to FIG. 5A, the cap includes an inlet 361 for
samples to be pipetted into the sample loading well. The inlet 361
is covered by an inlet septum 362. When a pipette tip is inserted
into the sample loading well through the inlet 361, the inlet
septum 362 seals around the tip, allowing fluid to be pushed and
pulled into the well. The inlet septum 362 is thin enough and
contains a slit 363 and has a cracking pressure that allows fluid
to be pipetted in through the inlet septum, but seals when there is
no pipetting-action taking place.
[0094] In certain embodiments, the removable cap 360 comprises a
plunger 364 that is inserted into the FFPE sample insert. FIGS. 5C
and 5D show a top perspective view and side cross-section view of
the removable cap 360 with a plunger 364 according to one
embodiment. Referring to FIGS. 5C and 5D, the removable cap 360 has
a plunger 364 attached to the cap. In one embodiment, the plunger
364 has a well structure of a cylindrical shape and has a diameter
small than the FFPE sample insert 370. Referring to FIG. 5D, in
use, a solid FFPE sample is placed in the FFPE sample insert 370
before the removable cap 360 with a plunger 364 is mounted to push
the FFPE sample to the bottom of the FFPE sample insert 370. The
FFPE sample insert 370 has a mesh filter 371 at the bottom end to
prevent the solid FFPE sample from passing the FFPE insert 360 to
the sample preparation well 310. FFPE lysis buffer is then loaded
into the plunger 364 through the inlet 361, which is covered by the
inlet septum 362. The FFPE lysis buffer passes through the plunger
364 into the FFPE sample insert 370 via at least one hole 365 (see
FIG. 5C) at the bottom of the plunger 364, and then passes into the
sample loading well 310 via the mesh filter 371. In some
embodiments, the FFPE sample has a density lower than the FFPE
lysis buffer, causing the FFPE sample to float on the top of the
lysis buffer. As a result, the FFPE sample may stick to the side of
the holder and cannot be effectively lysed. The plunger 364 pushes
the FFPE sample down to the lysis buffer so that it can be
effectively lysed.
[0095] FIG. 6 shows a cross-sectional view of a purification well
according to an embodiment of the invention. As shown in FIG. 6,
purification well 320 is cylindrical with conically tapered
bottoms. This shape minimizes dead volume and allows a pipettor to
collect all, or nearly all, of the contained reagent. In some
embodiments, purification well within sample preparation module may
hold the solid phase microparticles (e.g., magnetic nanoparticles).
In some embodiments, the system stores solid phase microparticles
in suspension, but dry storage may extend shelf life. In either
case, solid phase microparticles may require mixing before use
either to resuspend microparticles that settle in storage or to
disperse a rehydrated suspension.
[0096] In some embodiments, the device mixes contents in the
purification well using tip mixing. Tip mixing can include one or
more cycles of aspiration and redispense of the contents. For
example, the tip could be a microtip and aspiration and redispense
of the contents may be performed using the microtip. Tip mixing
agitates the contents so that different elements of the fluid
interact on a small scale. The conical bottoms of the purification
wells support agitation and limited rotation of the redispensed
contents with a minimum of uninvolved volume. The redispense
process uses the kinetic energy of the redispensed fluid to impel
fluid agitation. The purification well has a diameter that reduces
the effects of capillary forces on mixing. The purification well
has a depth greater than its diameter to better contain any
splashing. In some embodiments, the depth of the purification well
is at least twice its diameter.
[0097] While the device operates on other compartments in the
sample preparation module primarily from the top, the purification
well can also interact with a magnet through its sides and edges
(e.g., the bottom). In certain embodiments, when the assay
cartridge is loaded into the device and the solid phase
microparticles need to be collected, a magnet is pushed up to
contact closely to the purification well. The magnet can be
controlled to set up a magnetic field that collects and pellets
magnetically responsive microparticles on the wall of the
purification well. The magnet can be turned off (i.e., to remove
the magnetic field) when needed so that the magnetically responsive
microparticles can be mixed with other contents in the purification
well or be collected by a pipettor. In certain embodiments, when
needed, the magnet stays at a home position that is low on the
bottom to avoid affecting the solid phase microparticle in the
purification well.
[0098] In one embodiment, to isolate DNA or RNA from a sample that
has been lysed in the sample loading well, proper binding buffer is
added to allow DNA or RNA to bind to magnetically responsive
microparticles. A magnet is then pushed up to contact closely to
the purification well to apply the magnet field and collect the
microparticles on one side of the purification well. The liquid is
removed using the pipettor system. The magnet field is then removed
and the wash buffer is added into the purification well and fully
mixed with the microparticles. The magnet field is again applied to
collect the microparticles and the wash buffer is removed. Elution
buffer is added to the purification well to mix with the
microparticles. Purified DNA or RNA is then eluted from the
microparticles for downstream application.
[0099] Reagent storage wells within sample preparation modules may
hold discrete components used in the extraction and purification
process, including cell lysis buffer, wash buffer and elute
buffer.
[0100] Reagent storage wells with sample preparation modules may be
of various sizes and shapes. In some embodiments, the reagent
storage wells have a filled volume of 100 uL-1000 u. In certain
embodiments, the reagent storage wells may be cylindrical with
conically tapered bottoms. This shape minimizes dead volume and
allows a pipettor to collect all, or nearly all, of the contained
reagent. In some embodiments, the bottoms of the reagent storage
wells may have a central deepest point, and may be rounded,
conical, or pyramidal.
[0101] A barrier film may seal the reagent storage wells
individually to preserve the reagents and to prevent reagent
cross-contamination. In some embodiments, a single barrier film may
cover all reagent storage wells. In another embodiment, the reagent
storage wells of the sample preparation module may have individual
seals. The barrier film may be a multilayer composite of polymer
(e.g., rubber) or sticky foil. In some embodiments, the barrier
film includes cross cut at the center of each compartment that has
both sufficient stiffness and flexibility to cover the opening of
the compartments when piercing device (e.g., a microtip) is
removed. The barrier film can be a continuous piece spanning all of
the reagent wells. In operation, a pipette tip pierces the barrier
film from the cross cut to access contents in the reagent storage
well. In some embodiments, the manufacturing process may fix the
barrier film to the reagent storage well with methods known in the
art, e.g., laser welds, heat sealing, ultrasonic welding, induction
welding, and adhesive bonding.
[0102] In some embodiments, the device uses materials from reagent
storage wells in a sequence that is roughly based on the position
of the reagent storage wells in the sample preparation module. The
device may limit transfers to a single aspiration from each reagent
storage well in order to avoid use of material possibly
contaminated by an earlier aspiration. The device may first use
materials from reagent storage wells nearest the purification well.
When removing wastes, the device first deposits its waste materials
in empty wells closest to the purification well. The sequencing of
well usage may reduce the possibility of contamination. Any drips
falling from the pipettor can only fall in wells that the device
has already used.
[0103] B. PCR Module
[0104] In one embodiment, the PCR module comprises an elongated
body comprising a proximal end and a distal end, and a plurality of
compartments arranged between the proximal end and the distal end,
wherein at least one of the compartments is a push well and at
least one of the compartments is a PCR well. The push well is where
nucleic acid extracted and purified in the sample preparation
module is loaded. In certain embodiments, the push well is
pre-loaded with a solution mixture including reagents for PCR
reaction, e.g., primers, PCR reaction buffer, polymerase and
fluorescence dye. The nucleic acid loaded in the push well mixes
with the solution mixture, which then flows through a microfluidic
channel into the PCR well where PCR reaction is carried out.
[0105] FIGS. 7A and 7B show the top perspective view and a side
cross-section view, respectively, of a PCR module according to one
embodiment of the invention. Referring to FIGS. 7A and 7B, the PCR
module 400 comprises an elongated body 401 formed to include
multiple compartments, which may hold fluids (e.g., reagents) and
devices (e.g., pipette tips) needed to perform various PCR
reactions. Examples of compartments may include one or more push
wells 410, one or more PCR wells 420, and one or more pipette tip
holders. In certain embodiments, the PCR module 400 can be in the
form a monolithic body, and may be formed of plastic (or any other
suitable material). In certain embodiments, the PCR module 400 is
made by a plastic injection molding process. Alternatively, the PCR
module 400 is made by assembling individual components into a rigid
framework.
[0106] The PCR module 400 can have a proximal end 402 and a distal
end 403 at opposite ends of the elongated body 401. The orientation
of the compartments defines the top and bottom portion of the PCR
module 400. In certain embodiments, compartments can be open at the
top and closed on the bottom and sides.
[0107] The push well 410 can be of various shape. In one
embodiment, the push well 410 is cylindrical with conically tapered
bottom. In another embodiment, the push well 410 is generally
rectangular.
[0108] The PCR well 420 is cylindrical with a conically tapered
bottom.
[0109] The PCR module 400 has a microfluidic channel that connects
the push well 410 and the PCR well 420. In one embodiment, the
microfluidic channel connects to the push well 410 through an
opening located at the bottom of the push well 410. In one
embodiment, the microfluidic channel connects to the PCR well 420
through an opening located at the top of the PCR well 420.
[0110] The PCR module 400 may also include a cover (e.g., a barrier
film) that is disposed around various compartments and the
microfluidic channel, features to facilitate handling (e.g., a half
fastener 203), selected reagents and labeling.
[0111] As shown in FIGS. 7A and 7B, compartments within a PCR
module 400 can be arranged in a generally linear layout, with the
pipette tip holder 430 located near the proximal end 402, followed
by the push well 410, and the PCR well 420 at the distal end 403.
This layout allows simple motion of the dispense system to transfer
the fluids among various compartments. Alternatively, the PCR
module 400 can take different shape and arrangement of the
compartments (e.g., an arc, a single-row linear, or a circle),
depending on the overall system design, such as on the number and
sequence of operative locations that need access to the individual
compartments within a PCR module.
[0112] In some embodiments, the top ends of various compartments of
a PCR module form openings that align at a common height. In some
embodiments, the bottom ends of multiple PCR ends align at a common
depth and fit to the receptacles in the thermal cycle module.
[0113] In some embodiments, various compartments lack common walls
to prevent the creeping of liquids between compartments. This has
the benefit of reducing the possibility of contamination between
compartments. In some embodiments, the external profile of each
compartment closely tracks the cavity internal profile, i.e., the
walls of the compartment can be of relatively constant thickness
and can be thin compared to the size of the compartment. Such
design has the benefits of reducing the amount of material used and
hence reducing the manufacturing cost of the module, and improving
thermal contact/temperature control of the compartments.
[0114] A barrier film may seal the push wells and PCR wells
individually to preserve the reagents and to prevent reagent
cross-contamination. In some embodiments, a single barrier film may
cover all compartments within the PCR module. In another
embodiment, the compartments of the PCR module may have individual
seals. The barrier film may be a multilayer composite of polymer
and foils, and can include metallic foils. In some embodiments, the
barrier film includes at least one foil component that has both a
low piercing force and sufficient stiffness to maintain an opening
in the barrier film once the piercing device (e.g., a pipette tip)
is removed. Additionally, the barrier film may be constructed such
that no fragments of the foil component are released from the
barrier film upon piercing. A suitable material for the barrier
film may be stick foil. The barrier film can be a continuous piece
spanning all of the push wells and PCR wells. In operation, a
pipette tip pierces the barrier film to load purified nucleic acid
in the push well. In some embodiments, the manufacturing process
may fix the barrier film to the push well and PCR well with methods
known in the art, e.g., laser welds, heat sealing, ultrasonic
welding, induction welding, and adhesive bonding.
[0115] In order to keep the PCR well sealed during thermal cycling,
the sample fluid is pushed into the PCR well through a microfluidic
channel from an adjacent push well. This prevents cross
contamination and evaporation. The sample volume is added to the
push well and pressure applied using the pipette tip causes the
fluid to flow into the PCR well. In some applications, oil may be
pushed after the sample or provide an oil overlay for condensation
prevention.
[0116] In some embodiments, different types of PCR module may be
combined with the sample preparation module depending on the
application. Some PCR modules may have multiple PCR wells for
thermal cycling. Some PCR wells can be used to perform the reverse
transcription reaction or any other thermal process prior to the
polymerase chain reaction. Extra reagent storage wells can be added
to modules requiring additional thermal cycling wells.
[0117] C. Marking and Packaging
[0118] Assay cartridges may include marking elements to transfer
information. Marking may include human readable information such as
text or illustrations. Marking may also include machine readable
information in any of a variety of forms such as barcodes, dot
codes, radio frequency identification tags (RFID) or direct reading
electronic memory. In some embodiments, each module of an assay
cartridge includes a barcode (e.g., on the side of the sample
preparation module and the side of the PCR module). The marking may
include information about module type, manufacturing information,
serial numbers, expiration dates, use directions, etc.
[0119] Prior to loading on the device, assay cartridges may be
stored in transport boxes. Sample preparation modules and PCR
modules may be stored in one package or in separate packages.
Typically, a transport box retains several modules in common
orientation, grouped for easy grasping of several at a time to
load. In some embodiments, transport boxes include a supporting
base, labeling, and a clamshell lid to protect the modules during
handling. Manufacturing processes useful for producing transport
boxes include at least plastics thermoforming and plastics
injection molding.
Cartridge Loading Unit
[0120] In some embodiments, the assay cartridges can be loaded into
the device through a cartridge loading unit. The cartridge loading
unit serves as an area for loading and temporary storage of assay
cartridges in the system. In use, assay cartridges can be loaded
into the system at the cartridge loading unit without interrupting
normal device operation, such as the processing of the assay
cartridges loaded earlier. After loading, the cartridge loading
unit may read marking elements, such as a barcode, that are
attached to the loaded assay cartridges. In certain embodiments, a
barcode reader attached to the dispense system is used to read the
barcode. In certain embodiments, a barcode reader installed in the
loading channel is used to read the barcode. A proper protocol may
then be launched to direct the processing of the sample.
[0121] In some embodiments, the cartridge loading unit comprises a
plurality of cartridge loading lanes accommodating cartridge
carriages, each of which receives an assay cartridge. FIG. 8A shows
a top perspective view of a cartridge carriage according to an
embodiment of the invention. FIG. 8B shows a side cross-sectional
view of the cartridge carriage of FIG. 8A. Referring to FIGS. 8A
and 8B, the cartridge carriage 501 has an elongated body having a
proximal end 502 and a distal end 503. The cartridge carriage 501
can include a storage location near the distal end 503 comprising a
cavity 504 configured to hold assay cartridge. In some embodiments,
the cartridge carriage 501 includes at least one sample vial holder
505. In use, the sample vial holder 505 may receive a vial of
sample, which can be added to the assay cartridge loaded in the
cartridge carriage 501, either by a user or by the device.
[0122] FIGS. 8C and 8D shows a top perspective view and a side
cross-section view, respectively of a cartridge carriage according
to an embodiment of the invention, with an assay cartridge loaded
in the cartridge carriage. Referring to FIGS. 8C and 8D, the assay
cartridge 200 can be loaded into the cavity of the cartridge
carriage 501. In one embodiment, the PCR wells 420 of the assay
cartridge 200 are not loaded into the cavity. This design allows
the PCR wells 420 to be received in the receptacles of the thermal
cycler module. In one embodiment, the cartridge carriage 501 has a
structure that secures the assay cartridge into the appropriate
position in the cavity 504. In one embodiment, the structure
includes a groove located at the distal end of the cavity that fits
a groove runner at the bottom of the assay cartridge. In one
embodiment, the cartridge carriage 501 has an opening 505 at the
bottom wall. The opening 505 allows the device to interact with the
sample loading well 310 and the purification well 320 of the assay
cartridge 200 through its sides and edges. For example, when the
assay cartridge 200 is loaded into the device, a magnet is
positioned to contact closely to the side of the purification well
320, which assists to pellet the magnetically responsive
microparticles in the purification well 320. For another example, a
heater can be positioned close to sample loading well 310 to assist
the lysis of a sample, e.g., a FFPE sample.
[0123] In some embodiments, the cartridge carrier 501 includes a
proximal fix tab 506 and a distal fix tab 507 that secures the
cartridge carrier 501 in appropriate location in the device when
cartridge-loaded carrier is loaded into the device. In one
embodiment, the proximal fix tab 506 and the distal fix tab 507 are
designed such that the cartridge carrier 501 can be removed from
the device when a user pulls the cartridge carrier out of the
device.
Dispense System
[0124] In some embodiments, the systems disclosed herein use a
dispense system including a XYZ gantry with a pipettor to perform a
variety of functions, such as transferring a reagent between
compartments in assay cartridges.
[0125] FIG. 9A shows a perspective view of a dispense system
according to an embodiment of the invention. Referring to FIG. 9A,
the dispense system 600 includes a XYZ gantry 610 and a pipette
pump assembly (pipettor) 620. The XYZ gantry 610 has an "L" shape
structure on the horizontal plane and is configured to control the
three-dimensional movement of the pipettor 620. In one embodiment,
the XYZ gantry 610 has an X-axis track 611 that is perpendicular to
the axes of the cartridge-loading lane. The XYZ gantry 610 also has
a Y-axis track 612 that is perpendicular to the X-axis track (i.e.,
parallel to the axes of the cartridge-loading lane). In one
embodiment, the X-axis track 611 has a fixed location in the device
while the Y-axis track 612 is attached to the X-axis track 611 and
is freely movable along the X-axis track 611. The pipettor 620 is
attached to and freely movable on the Y-axis track 612. In one
embodiment, the dispense system 600 uses at least one motor coupled
to a pulley system 613 to control the location of the pipettor. In
one embodiment, the motor is attached to the gantry near one
terminus of a track. The pulley system 613 contains a drive pulley
that coupled to the motor and an idler pulley attached to the
gantry near the opposite terminus of the track. A timing belt
substantially parallel to the track may connect the drive pulley to
the idler pulley. Rotation of the motor drives the timing belt and
adjusts the separation between the drive pulley and the idler
pulley, thus moves the pipettor along the track. The combination
movement of the Y-axis track 612 and the pipettor 620 allows the
pipettor 620 to be positioned appropriately on a horizontal plane.
Alternatively, the XYZ gantry 610 may have any suitable structure
capable of directing the movement of the pipettor 620 such as a
rotary transport or an articulated arm.
[0126] In one embodiment, the pipettor 620 contains a pipettor
carriage 621 that supports a pipettor head 622. In one embodiment,
the XYZ gantry 610 also includes an elevator 614 that can raise and
lower the pipettor 620 as required for pipetting, mixing,
resuspension, and transfer. In one embodiment, the pipettor 620
also contains a lift 623 that can raise and lower the pipettor head
622. This allows the fine tuning of location of the pipettor head
as required for pipetting, mixing, resuspension and transfer
without using the XYZ gantry 610 to move the pipettor 620.
[0127] The pipettor 620 can be used to transfer liquids from one
location to another throughout the system. The pipettor 620 may
transfer liquids that include patient samples stored in sample
vials, which may include serum, plasma, whole blood, urine, feces,
cerebrospinal fluid, saliva, tissue suspensions, and wound
secretions. The pipettor 620 may also transfer liquids, such as
reagents, between compartments in the assay cartridge 200.
[0128] In order to reduce contamination, the pipettor 620 typically
uses disposable pipette tips to contact liquids. A pipettor mandrel
may act as the point for the attachment of disposable pipette tips
to the pipettor. Attachment can be held in place actively by a
gripper or held in place passively by friction between the inner
surface of the pipette tip and the outer surface of the pipettor
mandrel.
[0129] In one embodiment, the pipettor 620 has a pipette pump that
is specifically constructed to accurately aspirate and dispense
fluids within a defined range of volumes, e.g., 1-20 uL, 10-200 uL
200-1000 uL.
Thermal Cycler Module
[0130] In some embodiments of the invention, the system disclosed
herein comprises a thermal cycler module used to amply a specific
nucleic acid sequence through PCR.
[0131] As disclosed above, PCR or "Polymerase Chain Reaction" is a
process used to amplify DNA through repeated cycles of enzymatic
replication followed by denaturing the DNA duplex and formation of
new DNA duplexes, i.e., thermal cycles. Denaturing and annealing of
the DNA duplex may be performed by altering the temperature of the
DNA amplification reaction mixture. Reverse transcription PCR
refers to a process that converts mRNA into cDNA before DNA
amplification. Real time PCR refers to a process in which a signal
(e.g., fluorescence) that is related to the amount of amplified DNA
in the reaction is monitored during the amplification process.
[0132] In certain embodiments, a thermal cycle can refer to one
complete amplification cycle, in which a sample moves through a
time versus temperature profile, also known as a temperature
profile, that includes: heating the sample to a DNA duplex
denaturing temperature, cooling the sample to a DNA annealing
temperature, and exciting the sample with an excitation source
while monitoring the emitted fluorescence. A typical DNA denaturing
temperature can be about 90.degree. C. to 95.degree. C. A typical
DNA annealing temperature can be about 50.degree. C. to 70.degree.
C. A typical DNA polymerization temperature can be about 68.degree.
C. to about 72.degree. C. The time required to transition between
these temperatures is referred to as a temperature ramping time.
Ideally, each thermal cycle will amplify a target sequence of
nucleic acid by a factor of two. In practice, however,
amplification efficiency is often less than 100%.
[0133] In some embodiments of the invention, the system disclosed
herein includes a PCR subsystem that takes a prepared PCR well and
performs a complete real-time PCR analysis, thermal cycling the
sample multiple times, and reporting the intensity of emitted
fluorescent light at each cycle. In certain embodiments, the PCR
subsystem comprises a thermal cycler module, one or more PCR wells
and an optic module.
[0134] As noted supra, a prepared PCR well may contain RNA or DNA
isolated from a sample, target sequence specific primers and
probes, a "master" mix that includes nucleotide monomers and
enzymes necessary for synthesis of new DNA strands. Total fluid
volume contained in the PCR well is small (typically 40 .mu.L to 50
.mu.L) to facilitate rapid heat transfer.
[0135] FIG. 10A shows a top perspective view of a thermal cycler
module according to an embodiment of the invention. FIG. 10B shows
a side cross-sectional view of the thermal cycler module of FIG.
10A. Referring to FIGS. 10A and 10B, the thermal cycler module 700
comprises a thermal block 701 with a substantially planar thermal
mass for transferring thermal energy, and a receptacle 702 for
forming a thermal contact surface with a PCR well. The thermal
block 701 may be composed of a highly thermally conductive material
such as copper, copper alloy, aluminum, aluminum alloy, magnesium,
gold, silver, or beryllium. The thermal block 701 may have a
thermal conductivity of about 100 W/mK or greater and a specific
heat of about 0.30 kJ/(kgK) or less. In some embodiments, the
thermal block 701 has a thickness between about XX inches and about
XX inches. The thermal block 701 can also comprise a heating
element that provides the heat that is transferred to the PCR well.
The heating element can be a thin film heater affixed to the back
surface of the planar thermal mass, although other heat sources
such as resistance heaters, thermoelectric devices, infrared
emitters, streams of heated fluid, or heated fluid contained within
channels that are in thermal contact with the thermal block may
also be used. The thermal block may also include one or more
temperature sensors that are used in conjunction with a controller
to control the temperature of the thermal block by, for instance, a
proportional--integral--derivative (PID) loop. These temperature
sensors may be imbedded in the thermal block. The receptacle may
comprise an optical aperture, where the optical aperture is
positioned to permit optical communication through optical fibers
to the interior of the receptacle.
[0136] In certain embodiments, the thermal cycler module 700 may
have a plurality of heat transfer fins 703, which facilitates the
release of heat from the thermal block 701. The receptacle 702 may
have any suitable characteristics necessary to secure the PCR well
and ensure good thermal contact with it. For example, in some
embodiments, the walls of the conical receptacle 702 have an angle
of about 1 degree to about 10 degrees, an angle of about 4 degrees
to about 8 degrees, or an angle of about 6 degrees. The decreasing
internal radius of the receptacle ensures that as the PCR well that
is pressed into the receptacle 702 the exterior of the PCR well is
brought into intimate contact with the interior of the receptacle
702. The receptacle 702 can comprise a frustum of a conical shape
and having an upper opening and a lower opening. The receptacle 702
is affixed to the front surface of the thermal block 701. The upper
opening allows for insertion of the PCR well. The lower opening
acts as an optical window for the optics assembly (as disclosed
infra).
Optic Module
[0137] The systems of the present disclosure can also include an
optic module responsible for exciting the dyes in the assay and
detecting the fluorescence emitted at each PCR cycle. Both
excitation and emission can occur over a range of wavelengths.
Light used to excite the fluorescent dyes can, for example, range
from 400 nm to 800 nm. The detector used to measure light emitted
form the dyes can, for example, be sensitive to light ranging from
400 nm to 800 nm. In some embodiments, the optical module can
detect a plurality of emitted wavelengths from the PCR well and to
perform the detection asynchronously across multiple PCR wells. In
certain embodiments, up to 5 different dyes can be detected
asynchronously among up to 30 different PCR wells.
[0138] The optical module includes hardware and software components
from the light sources through to the detection on the CCD camera.
Typically, the optical module includes at least the following
components: an excitation light source, assemblies for directing
excitation light to the PCR wells, assemblies for directing light
emitted by fluorescent dyes within the PCR wells to a detector, and
one or more detectors for measuring the emitted light.
[0139] The excitation light source can be lasers (including
fixed-wavelength lasers and tunable lasers) and LEDs (including
single wavelength LEDs, multi-wavelength LEDs and white LEDs). In
some embodiments, the light from the light source is passed through
filters (e.g., multibandpass filter) to remove light that is
outside of the nominal wavelength range before being directed to
the PCR wells.
[0140] The light from the light source can be directed to
individual excitation optical fibers, which then direct the
excitation light to individual PCR wells. In some embodiments, an
assembly of 30 excitation optical fibers is used to supply
excitation light to each of 30 PCR wells. A variety of optical
fibers can be used to carry the excitation light. In some
embodiments, the optical fibers are about 200 um in diameter.
Excitation optical fibers carrying the excitation light terminate
in the excitation optics assembly of the thermal cycler module,
which is described above.
[0141] Light emitted from the PCR wells as a result of exposure to
the excitation light is collected by the emission optics assembly
of the thermal cycler module, which is described above. In some
embodiments, the emitted light is directed to the input end of an
emission optical fiber, which subsequently directs emitted light to
a detector.
[0142] In some embodiments, the detector can be a spectrometer. The
spectrometer may be a multi-channel or an imaging spectrometer,
which permits simultaneous reading of multiple optical fibers and
reduce the need for switching. The spectrometer can include a
multi-bandpass filter between the output terminus of the emission
optical fibers and the detector to selectively remove emission
excitation wavelengths. In some embodiments, the detector may be a
single photo-diode, photomultiplier, channel photomultiplier, or
similar device equipped with an appropriate optical filter, which
can be a set of optical filters or a tunable filter.
[0143] FIG. 11 shows a top perspective view of an optics module
according to an embodiment of the invention. Referring to FIG. 10A,
the optical module contains a rotary plate that includes multiple
filters each for a different wavelength. The filters are arranged
on a circle from the center of the rotary plate. The rotary plate
is stacked on an optical fiber plate where one terminus of each
optical fiber is attached. The optical module also contains a motor
coupled to a drive pulley connected to the rotary plate through a
belt. Rotation of the motor drives the belt to rotate the rotary
plate. The termini of the optical fibers are arranged on a circle
matching the one in the rotary plate so that when the rotary plate
is rotated the filters can align with the optical fiber termini.
This design allows asynchronous detection of fluorescent signals
from multiple PCR wells. For example, the rotary plate can contain
five filters, each for detection of a different dye. The optical
fiber plate contains termini of 30 optical fibers, each for a
different PCR well. When the rotary plate rotates above the optical
fiber plate, the filters can align with termini of 5 optic fibers.
As a result, excitation light is sent to the 5 PCR wells, the
fluorescent signal from the 5 PCR wells are received. Then the
motor drives the rotation of the rotary plate so that the filters
align with the next 5 termini. When the rotary plate completes one
full circle, the fluorescent signals from all 30 PCR wells can be
detected.
EXAMPLE 1
[0144] The following is an example of detecting a target nucleic
acid using a device disclosed herein.
[0145] A 15 um BRAF Wild Type FFPE DNA reference standard scroll
(Horizon Discovery, cat # HD266) was used as the sample input. The
scroll was inserted into the sample loading well 310 of a sample
preparation module 300 as illustrated in FIG. 3A, which was coupled
to a PCR module 400 (FIG. 7A). The sample loading well 310 was
capped with a removable cap 360 with a plunger 364 (FIG. 5C) and
loaded onto the device 100 (FIG. 1A). The sample loading well 310
was preloaded with an FFPE DNA deparafinization (DP) solution
(MagBio Genomics, HighPrep.TM. FFPE Tissue DNA Kit). To extract the
DNA from the scroll, the sample loading well 310 was incubated at
65.degree. C. for 15 min. The DP solution was then removed from the
sample loading well 310 and replaced with digestion buffer (MagBio
Genomics, HighPrep.TM. FFPE Tissue DNA Kit) and Protease K
solution. The solution was incubated at 55.degree. C. for 45
min.
[0146] The lysate was then transferred into the purification well
320 (see FIGS. 3A and 3B) which was preloaded with the magnetic
beads (Nvigen) in DNA binding buffer (MagBio Genomics, HighPrep.TM.
FFPE Tissue DNA Kit) and incubated at room temperature for 10 min.
Magnet force was applied to collect the beads onto the side of the
purification well 320, and the liquid was removed from the
purification well 320.
[0147] The beads were washed once with wash buffer 1 (MagBio
Genomics, HighPrep.TM. FFPE Tissue DNA Kit) and twice with wash
buffer 2 (MagBio Genomics, HighPrep.TM. FFPE Tissue DNA Kit). The
beads were air dried and eluted with 50 uL elution buffer (MagBio
Genomics, HighPrep.TM. FFPE Tissue DNA Kit).
[0148] The purified DNA was then transferred to a push well 410
(FIG. 7A) that was loaded with the PCR supmermix, including the
hotstart PCR polymerase, dNTP and buffer with PCR primer/probe
designed to target house-keeping GUSB gene, and loaded into the PCR
well. Oil was then loaded on top of the PCR mix to prevent
evaporation. PCR started with denaturation at 95.degree. C. for 3
min, followed by 40 cycles of 95.degree. C. for 20 s and 60.degree.
C. for 45 s. Fluorescence data was collected at the 60.degree. C.
annealing temperature. The collected fluorescence signal was
plotted vs cycle number. The Ct value for the run is around 22,
which is comparable to the result from manual prep.
[0149] The previous description provides exemplary embodiments
only, and is not intended to limit the scope, applicability, or
configuration of the disclosure. Rather, the previous description
of the exemplary embodiments will provide those skilled in the art
with an enabling description for implementing one or more exemplary
embodiments. It is understood that various changes may be made in
the function and arrangement of elements without departing from the
spirit and scope of the invention. Several embodiments were
described herein, and while various features are ascribed to
different embodiments, it should be appreciated that the features
described with respect to one embodiment may be incorporated within
other embodiments as well. By the same token, however, no single
feature or features of any described embodiment should be
considered essential to every embodiment of the invention, as other
embodiments of the invention may omit such features.
[0150] Specific details are given in the previous description to
provide a thorough understanding of the embodiments. However, it
will be understood by one of ordinary skill in the art that the
embodiments may be practiced without these specific details. For
example, circuits, systems, networks, processes, and other elements
in the invention may be shown as components in block diagram form
in order not to obscure the embodiments in unnecessary detail. In
other instances, well-known circuits, processes, algorithms,
structures, and techniques may be shown without unnecessary detail
in order to avoid obscuring the embodiments.
[0151] Also, it is noted that individual embodiments may be
described as a process which is depicted as a flowchart, a flow
diagram, a data flow diagram, a structure diagram, or a block
diagram. Although a flowchart may describe the operations as a
sequential process, many of the operations can be performed in
parallel or concurrently. In addition, the order of the operations
may be re-arranged. A process may be terminated when its operations
are completed, but could have also included additional steps or
operations not discussed or included in a figure.
[0152] Furthermore, not all operations in any particularly
described process may occur in all embodiments. A process may
correspond to a method, a function, a procedure, a subroutine, a
subprogram, etc. When a process corresponds to a function, its
termination corresponds to a return of the function to the calling
function or the main function.
[0153] Furthermore, embodiments may be implemented, at least in
part, either manually or automatically. Manual or automatic
implementations may be executed, or at least assisted, through the
use of machines, hardware, software, firmware, middleware,
microcode, hardware description languages, or any combination
thereof. When implemented in software, firmware, middleware or
microcode, the program code or code segments to perform the
necessary tasks may be stored in a machine readable medium. A
processor(s) may perform the necessary tasks.
[0154] While detailed descriptions of one or more embodiments have
been give above, various alternatives, modifications, and
equivalents will be apparent to those skilled in the art without
varying from the spirit of the invention. Moreover, except where
clearly inappropriate or otherwise expressly noted, it should be
assumed that the features, devices, and/or components of different
embodiments may be substituted and/or combined. Thus, the above
description should not be taken as limiting the scope of the
invention. Lastly, one or more elements of one or more embodiments
may be combined with one or more elements of one or more other
embodiments without departing from the scope of the invention.
* * * * *