U.S. patent application number 15/304033 was filed with the patent office on 2017-03-02 for portable nucleic acid analysis system and high-performance microfluidic electroactive polymer actuators.
The applicant listed for this patent is SRI International. Invention is credited to Robert Balog, Nina Sechler.
Application Number | 20170058324 15/304033 |
Document ID | / |
Family ID | 54324515 |
Filed Date | 2017-03-02 |
United States Patent
Application |
20170058324 |
Kind Code |
A1 |
Balog; Robert ; et
al. |
March 2, 2017 |
PORTABLE NUCLEIC ACID ANALYSIS SYSTEM AND HIGH-PERFORMANCE
MICROFLUIDIC ELECTROACTIVE POLYMER ACTUATORS
Abstract
Devices, systems and methods for the parallel detection of a set
of distinct nucleic acid sequences use multiple sequence
amplification and simultaneous hybridization readout. An automated
nucleic acid analysis system comprises in microfluidic connection
sample lysis, purification, PCR and detection modules configured to
detect in parallel distinct nucleic acid sequences via multiple
sequence amplification and simultaneous microarray hybridization
readout. High performance microfluidic electroactive polymer
(.mu.EAP) actuators comprising a dead-end fluid chamber in which
the floor of the chamber is an electrode covered with an EAP layer
of dielectric elastomer are configured for particle sorting.
Inventors: |
Balog; Robert; (Menlo Park,
CA) ; Sechler; Nina; (Menlo Park, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SRI International |
Menlo Park |
CA |
US |
|
|
Family ID: |
54324515 |
Appl. No.: |
15/304033 |
Filed: |
April 14, 2015 |
PCT Filed: |
April 14, 2015 |
PCT NO: |
PCT/US15/25835 |
371 Date: |
October 13, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61979377 |
Apr 14, 2014 |
|
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|
62041430 |
Aug 25, 2014 |
|
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62081525 |
Nov 18, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/087 20130101;
C40B 60/12 20130101; G01N 27/4473 20130101; B01L 2300/0887
20130101; B01L 2200/028 20130101; B01L 2300/0816 20130101; B01L
3/502738 20130101; B01L 2300/0654 20130101; B01L 3/502723 20130101;
B01L 3/502746 20130101; B01L 2200/0684 20130101; B01L 3/502715
20130101; G01N 27/44791 20130101; B01L 2300/168 20130101; B01L
2200/10 20130101; B01L 2300/0819 20130101; B01L 2400/0487 20130101;
B01L 7/52 20130101; B01L 2400/082 20130101; B01L 2300/1822
20130101; C12Q 1/686 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B01L 3/00 20060101 B01L003/00 |
Goverment Interests
[0002] This invention was made with government support under
Defense Advanced Research Projects Agency under contract no.
HR0011-14-C-0082. The government has certain rights in the
invention.
Claims
1-11. (canceled)
12. A microfluidic chip apparatus comprising: a board assembly with
fluidic chambers and channels; at least three portable-modules in
controlled fluidic communication with one another and each
configured and arranged for nucleic acid analysis, the at least
three portable-modules selected from the group consisting of: a
reagent module configured and arranged to contain and deliver
reagents to the other two or more portable-modules, a lysis module
configured and arranged to breakdown a sample input to the board
assembly, a purification module configured and arranged purify
targets in the sample from other material in the sample, a
polymerase chain reaction (PCR) module configured and arranged to
amplify and label the targets, and a detection module configured
and arranged to bind the labeled targets to a surface for detection
of the labeled targets; an input port configured and arranged to
receive the sample and introduce the sample to the fluidic chambers
and channels; a plurality of additional ports and valves configured
and arranged to control a flow of fluid within the fluidic chambers
and channels, the fluid including the sample and the reagents; and
at least one vent configured and arranged to remove bubbles from at
least one of fluidic chambers and channels during the controlled
flow of the fluid.
13. The microfluidic chip apparatus of claim 12, wherein the at
least three or more portable-modules include the lysis module, the
purification module, and the PCR module.
14. The microfluidic chip apparatus of claim 12, wherein the at
least three or more portable-modules include the reagent module,
the lysis module, the purification module, the PCR module, and the
detection module.
15. The microfluidic chip apparatus of claim 12, wherein the fluid
flows through the channels and between the at least three or more
portable-modules via the plurality of additional ports configured
and arranged to introduce pressure on one or more of the plurality
of valves, and the plurality of valves being configured and
arranged to open or shut based on the pressure introduced
thereto.
16. The microfluidic chip apparatus of claim 12, wherein the at
least three or more portable-modules include the PCR module, the
PCR module having an aluminum wall surface.
17. The microfluidic chip apparatus of claim 12, wherein the at
least three or more portable-modules include the detection module
including: a mix chamber configured and arranged to mix a solution,
including the sample, with a hybridization buffer; and a detection
chamber including a detection well having a microarray configured
and arranged to bind hybridized targets having primers, wherein the
solution with the hybridized buffer includes amplified targets with
primers bound thereto.
18. The microfluidic chip apparatus of claim 17, wherein the
detection chamber further includes a transparent substrate located
within the detection well, wherein the microarray is arranged on
the transparent substrate and includes a plurality of probes
arranged in a grid pattern.
19. The microfluidic chip apparatus of claim 17, further comprising
detection optics, including a light source, configured and arranged
to couple light to the microarray, wherein the detection chamber
further includes a transparent substrate located within the
detection well, wherein the microarray is arranged on the
transparent substrate and includes a plurality of probes arranged
in a grid pattern, wherein the light source is configured and
arranged to transmit light through the transparent substrate toward
the microarray.
20. The microfluidic chip apparatus of claim 19, wherein the
transparent substrate and grating are configured and arranged to
steer the light toward the microarray through the transparent
substrate and to excite light sensitive molecules on the microarray
by providing evanescent fields.
21. The microfluidic chip apparatus of claim 12, wherein the at
least three or more portable-modules includes the reagent module
comprising a lysis buffer solution, an elution buffer solution, and
a hybridization buffer solution.
22. The microfluidic chip apparatus of claim 12, wherein the at
least three or more portable-modules includes the purification
module having a frit within a channel, and configured and arranged
to bind with the targets and not bind with the other material in
the sample.
23. The microfluidic chip apparatus of claim 12, wherein the at
least three or more portable-modules includes PCR module including:
a PCR master mix chamber comprising a well and enzymes and
configured and arranged to mix a solution, which includes purified
targets, with the enzymes; a PCR primer chamber comprising a well
and primers and configured and arranged to mix the solution with
the primers; and a PCR chamber comprising a well formed of
passivated metal and configured and arranged to receive the
solution mixed with the enzymes and the primers and to amplify and
label the targets.
24. The microfluidic chip apparatus of claim 12, wherein the at
least three or more portable-modules include: the reagent module
configured and arranged to contain and selectively deliver reagents
to the other two or more portable-modules, the lysis module
configured and arranged breakdown the sample, the sample including
DNA targets, that is input to the board assembly, the purification
module configured and arranged purify DNA targets in the sample
from other material in the sample using a frit, the PCR module
configured and arranged to amplify and label the DNA targets, and
the detection module, including a microarray, configured and
arranged to bind the labeled DNA targets to the microarray for
detection of the labeled DNA targets.
25. The microfluidic chip apparatus of claim 12, further including
a breathable membrane configured and arranged with the at least one
vent to remove bubbles from at least one of fluidic chambers and
channels during the controlled flow of the fluid within at least a
portion of the PCR module.
26. The microfluidic chip apparatus of claim 25, wherein the board
assembly includes a plurality of layers with a first layer
including the channels and a second layer including the breathable
membrane, and wherein the breathable membrane is configured and
arranged to expose the channels to atmospheric pressure.
27. A microfluidic chip apparatus comprising: a board assembly with
fluidic chambers and channels; an input port configured and
arranged to receive a sample have a target and introduce the sample
to the fluidic chambers and channels; a plurality of additional
ports and valves configured and arranged to control a flow of fluid
including the sample within the fluidic chambers and channels of
the board assembly and through a plurality of portable-modules; at
least one vent configured and arranged to remove bubbles from at
least one of fluidic chambers and channels during the controlled
flow of the fluid; and a plurality of portable-modules in
controlled communication with one another and configured and
arranged for nucleic acid analysis, the plurality of
portable-modules including: a lysis module configured and arranged
to breakdown a sample input to the board assembly, a purification
module configured and arranged purify targets in the sample from
other material in the sample, and a polymerase chain reaction (PCR)
module configured and arranged to amplify and label the targets,
wherein the controlled flow of the fluid by the plurality of
additional ports and valves includes flow of the sample from the
input port to the lysis module, from the lysis module to the
purification module, and from the purification module to the PCR
module.
28. The apparatus of claim 27, wherein the controlled flow of the
fluid by the plurality of additional ports and valves includes flow
of the sample from the input port to the lysis module, flow of the
broken down sample from the lysis module to the purification
module, flow of the purified targets from the purification module
to the PCR module.
29. The apparatus of claim 27, further including a detection module
configured and arranged to bind the labeled targets to a surface
for detection of the labeled targets, wherein the controlled flow
of the sample further includes flow of the sample from the PCR
module to the detection module.
30. The apparatus of claim 27, further including a reagent module
configured and arranged to contain and deliver reagents to the
other one or more portable-modules.
31. A method comprising: introducing a sample received at an input
port of a microfluidic chip apparatus to fluidic chambers and
channels of the microfluidic chip apparatus, wherein the
microfluidic chip apparatus includes: a board assembly with the
fluidic chambers and channels, a plurality of additional ports and
valves, at least one vent, and a plurality of portable-module in
controlled communication with one another and configured and
arranged for nucleic acid analysis; and controlling a flow of fluid
including the sample within the fluidic chambers and channels of
the board assembly and through the plurality of portable-modules
using the plurality of additional ports and valves, wherein the
controlled flow includes performing the nucleic acid analysis by:
flowing the sample from the input port to a lysis module and
breaking down the sample using the lysis module; flowing the broken
down sample from the lysis module to a purification module and
purifying targets in the sample from other material in the sample
using the purification module, flowing the purified targets from
the purification module to a polymerase chain reaction (PCR)
module, and amplifying and labeling the targets using the PCR
module, and removing bubbles from at least one of fluidic chambers
and channels during the controlled flow of the fluid using the at
least one vent.
Description
[0001] This application claims priority to Ser. No. 62/081,525;
filed Nov. 18, 2014, to 62/041,430; filed Aug. 25, 2014, and to
Ser. No. 61/979,377; filed Apr. 14, 2014.
INTRODUCTION
[0003] The integration of sample preparation with amplification and
detection in an easy-to-use system remains a significant challenge
for nucleic acid diagnostics. [1] A number of systems have been
approved by the FDA as "moderate complexity" devices for use in
near-POC applications [2], including Cepheid's GeneXpert platform,
Nanosphere's Verigene platform, the BD Max System from Becton,
Dickinson and Company, and Liat's "lab-in-a-tube" disposable
device.
[0004] The research literature is rife with examples of pathogen
detection techniques and devices; however, no one has been able to
develop a multiplex, automated, integrated "sample to result"
system, configured to accept raw biological samples. [3] An early
example used electrophoretic separation and laser-induced
fluorescence to detect the presence of pathogenic DNA extracted and
amplified from whole blood [4]. Xu et al. [5] used real-time
fluorescence readout during amplification to detect as few as 100
copies/.mu.l. Ferguson et al. [6] electrochemically detected viral
RNA hybridized to PNA probes on nanostructured electrodes in the
presence of an electrocatalytic buffer, while Lam et al. [7]
detected hybridization of amplified ssDNA to redox labeled
molecular probes deposited on a gold electrode. Ferguson [6]
demonstrated an LOD equivalent to 10 TCID50 (tissue culture
infective dose), which was 4 orders of magnitude less than clinical
titer values. Lam [7] demonstrated an LOD of 1 bacteria/.mu.l,
although a concentration of 100 CFU/.mu.l was used when performing
analysis on a spiked urine sample. Two groups used a lateral-flow
sandwich assay as the basis for their nucleic acid detection [8,
9], and a Fraunhofer Institutes ivD-Platform [10] used a modular
platform.
[0005] For decades the Department of Defense (DoD) has recognized
the need for field-portable biological analysis. Initially, the
need was for identifying biological threats; however, with the
advent of personalized medicine, the DoD has also recognized the
value of routine health-status monitoring and the availability of
point-of-care (POC) diagnostics in addition to environmental
monitoring. In fact, DARPA has numerous programs aimed at
monitoring biological systems to allow for rapid intervention.
However, despite decades of investment, no commercial instruments
are available that perform nucleic acid processing at the point of
need. To fill this gap, SRI has developed and we disclose here a
portable, integrated, rapidly reconfigurable and automated
biodetection system that performs "sample-in to answer-out"
analysis.
SUMMARY OF THE INVENTION
[0006] The invention provides devices, systems and methods for the
parallel detection of a set of distinct nucleic acid sequences via
multiple sequence amplification and simultaneous hybridization
readout.
[0007] In one aspect, the invention provides an automated nucleic
acid analysis system comprising in microfluidic connection sample
lysis, purification, PCR and detection modules configured to detect
in parallel distinct nucleic acid sequences via multiple sequence
amplification and simultaneous microarray hybridization
readout.
[0008] In embodiments the invention provides the system
wherein:
[0009] the detection module comprises microarray detection optics
comprising a microarray scanner employing evanescent wave
excitation;
[0010] the detection module comprise an automated hybridization
processor configured to provide multiple stringencies via
temperature; and/or
[0011] the PCR module is configured to perform reverse
transcription and PCR in a single reaction.
[0012] In embodiments the invention provides the system wherein
comprising an integrated microfluidics card comprising the modules
and an analyzer comprising a receptacle configured to receive the
card, operators configured to operate the card, and a controller
configured to electronically control the operators, the operators
comprising fluidic actuators, PCR thermal cycler, and automated
hybridization processor and microarray detection optics.
[0013] In embodiments the invention provides the system further
comprising a reagent module configured to contain and deliver
reagents to the lysis, purification, PCR and detection modules.
[0014] In embodiments the invention provides the system wherein
that is:
[0015] portable: less than 1000 in.sup.3 and less than 10 lbs;
[0016] rapid: analysis in less than 120 minutes;
[0017] multiplex: simultaneous analysis of more than 50 target
sequences; and/or
[0018] automated: requiring no user intervention between sample
introduction and result display.
[0019] In embodiments the invention provides the system
wherein:
[0020] the sample comprises protein analytes and the system is
further configured to tag the protein analytes with tags comprising
the nucleic acid sequences;
[0021] anchored probes define the sequences by their spatial
locations;
[0022] the amplification is effected by a number of primers pairs
less than the number of sequences being analyzed;
[0023] the distinct nucleic acid sequences are of multiple
species/organisms;
[0024] the PCR module comprises a metallic (e.g. aluminum) PCR
reaction chamber;
[0025] the microfluidic connection comprises a breathable membrane
configured for bubble removal, wherein the breathable membrane is
underneath the channel layer, so the entire channel can be exposed
to atmospheric pressure (in a particular embodiment, this membrane
spans the card because it is easier to manufacture it as a layer
than individual pieces, though it is only functional under the
channel layers);
[0026] amplification is fully contained in the consumable (no open
tubes, etc.); and/or
[0027] detection is based on probe sets rather than primer sets
(easier to build new tests).
[0028] In embodiments the invention provides the system configured
to:
[0029] amplify in a single vessel (no sample splitting);
[0030] receive and process analyte samples of blood, saliva, GI
samples, urine, wound swabs, spinal tap, nasal swabs, veterinary
and agricultural sources;
[0031] receive samples via a specimen collection tool or transport
media;
[0032] process sample volumes between 1-100 ul;
[0033] be modular (modules can be interchanged to support different
applications);
[0034] be capable of metering (done by channel dimensions and
bubble removal); and/or
[0035] be one directional and self-sealing (prevents sample cross
contamination).
[0036] In embodiments the invention provides the system comprising
an integrated microfluidics card comprising the modules and an
analyzer comprising a housing (box) and within the housing
receptacle configured to receive the card, wherein the
analyzer:
[0037] engages the card to perform the lysis, purification, PCT
(amplification and labeling), and detection;
[0038] interacts with the sample via pressure (e.g. sample
transport), magnetic fields (e.g. sample mixing), temperature (e.g.
amplification, stringency, hybridization) and/or light (e.g.
hybridization detection); and/or
[0039] performs the detection by coupling an evanescant wave with
the sample to observe hybridizations in real time and/or
determining kinetics and possible base-pair mismatch which result
in sequence information.
[0040] In embodiments the invention provides the system comprising
an integrated microfluidics card (cartridge) comprising the
modules, wherein the card is configured:
[0041] to be specific to disease type (ex. respiratory
illnesses);
[0042] to be specific to patient type (ex. pediatric);
[0043] to be specific to pathogen type (ex. biowarfare agents);
[0044] to be specific to individual (ex. pharmacogenomics);
[0045] to contain unique identifiers for patient-specific
information;
[0046] for one-time use to maintain sterility and minimize
cross-contamination;
[0047] to be produced using roll-to-roll manufacturing steps;
and/or
[0048] from a polycarbonate chassis, metallic foil PCR chambers,
acrylic components, breathable membrane materials, and/or
polyurethane seals.
[0049] In embodiments the invention provides the system
functionally integrated with a microfluidic particles sorter, such
as a fluorescence-activated cell sorter (FACS), configured to
provide hydrodynamic and/or inertial focusing for particle or cell
alignment and comprising microscale electroactive polymer (EAP)
actuators configured for sorting.
[0050] The EAP .mu.-sorter may be functionally integrated with or
incorporated as particle-concentration/sorting module of the iMFC
system, and configured to allow the system to increase the
operation envelope by either concentrating a dilute particle
concentration in a large volume (e.g., bacteria present in
environmental samples at a few cells per ml) or sorting out select
cells from a background of many cells (e.g., activated T cells from
a population of peripheral blood mononuclear cells. In addition,
the microscale electroactive polymer actuators are suitable for
alternative applications beyond sorting, including cell trapping,
fluid mixing and pumping, and hence may be provided, configured
and/or operated independent of the subject automated nucleic acid
analysis systems.
[0051] The invention also provides methods of using the disclosed
systems to detect in parallel distinct analyte nucleic acid
sequences via multiple sequence amplification and simultaneous
microarray hybridization readout.
[0052] In another aspect the invention provides a high performance
microfluidic electroactive polymer (.mu.EAP) actuator configured
about a flow channel wherein a voltage pulse applied to the
actuator induces the actuator to create across the flow channel a
transient cross flow that deflects targeted particles within the
flow channel onto a new pathline, wherein the actuator comprises a
dead-end fluid chamber in which one or more surfaces (e.g. wall,
floor, ceiling) of the chamber comprises an electrode covered with
an EAP layer of dielectric elastomer.
[0053] A single uEAP actuator may be paired with a compliant
chamber (i.e., "bellows") that accepts the fluid jet driven by the
actuator. This configuration only requires one active actuator, but
it still allows the generation of a cross flow. The compliant
chamber could just be one of the actuators without an electrical
connection, or it could be a chamber with a different geometry, as
we use for the multi-channel/stage sorters.
[0054] While exemplified primarily with solid electrodes (e.g
indium tin oxide (ITO) electrode on a glass slide), the electrode
could also or alternatively comprise a fluid, such as a conductive
fluid in an adjacent channel.
[0055] In another aspect the invention provides a plurality of such
actuators configured about the flow channel and out of phase with
each other, wherein a voltage pulse applied to the actuators
induces the actuators to create across the flow channel a transient
cross flow that deflects targeted particles within the flow channel
onto a new pathline, wherein each actuator comprises a dead-end
fluid chamber in which a surface of the chamber is an electrode
covered with an EAP layer of dielectric elastomer.
[0056] In another aspect the plurality is a pair of such actuators
configured 180.degree. out of phase with each other.
[0057] In embodiments: [0058] a plurality of surfaces of the
chamber(s) comprise an electrode covered with an EAP layer of
dielectric elastomer; [0059] the flow channel is configured to
provide a combination of hydrodynamic focusing for horizontal
alignment and inertial focusing for vertical alignment of the
particles;
[0060] the new pathline leads to a sort outlet;
[0061] the flow channel comprises a sample input channel and sorted
and unsorted output channels and the new pathline leads to the
sorted output channel;
[0062] the flow channel is configured for fluorescence detection,
wherein upon detection of a targeted particle, the voltage pulse is
applied to the .mu.EAP actuators; [0063] the EAP layer is 1-50 (or
2-25, or 5-15 .mu.m thick);
[0064] the elastomer is silicone; [0065] the actuator(s) are
configured to provide parallel sorting in a multi-channel device;
[0066] the actuator(s) are configured to provide multi-stage serial
sorting into multiple outlets; and/or [0067] the actuator(s) are
functionally integrated in a label-activated particle sorter.
[0068] The invention also provides methods of making and using the
actuators, such as comprising the step of applying a voltage pulse
to induce the actuator(s) to create across the flow channel a
transient cross flow that deflects targeted particles within the
flow channel onto a new pathline.
[0069] The invention specifically provides all combinations of the
recited embodiments, as if each had been laboriously individually
set forth.
BRIEF DESCRIPTION OF THE FIGURES
[0070] FIG. 1: A physical configuration of a molecular diagnostic
system.
[0071] FIGS. 2A and B: A iMGC card.
[0072] FIG. 2C: An input module for a nasal swab.
[0073] FIG. 2D: A TECs are arrangment.
[0074] FIG. 2E: A section of the iMFC card around the PCR and the
detection processing blocks.
[0075] FIG. 2F: A glass substrate with the detection well, the
gratings and the chrome.
[0076] FIG. 2G: A side view of the glass substrate illustrating how
light is coupled into the glass substrate.
[0077] FIG. 2H: A side view of the glass substrate illustrating how
light travels through total internal reflection within the glass
substrate.
[0078] FIG. 2I: An arrangement of the TEC and the camera system in
relation to the microarray.
[0079] FIG. 3: Mechanics to bind nucleic acid to a silica frit.
[0080] FIG. 4: Volume control with a breathable membrane.
[0081] FIGS. 5A and 5B: Valve control mechanics to control
flow.
[0082] FIG. 6: A projection view of a iMFC card
[0083] FIG. 7: A pneumatic system.
[0084] FIG. 8: A functional block diagram of the physical hardware
of device 10.
[0085] FIG. 9: EAP microsorter schematic showing the channel layout
of the .mu.EAP FACS.
[0086] FIG. 10: Streak images of 7-.mu.m green fluorescent
particles.
[0087] FIG. 11: Sorting of a phycoerythrin-labeled B-cell.
[0088] FIG. 12: Integration of multiple sorters into the
dual-channel device.
[0089] FIG. 13: Integration of multiple sorters into a staged
sorting device.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS AND EXAMPLES
THEREOF
[0090] Our invention provides a portable, inexpensive molecular
diagnostic system capable of producing results with no human
intervention--human intervention is only needed to input the sample
and to read the results. The results are also produced very quickly
due to the various techniques described above. The system includes
a method to interface with a disposable member, called the iMFC.
The methods and the systems allow the iMFC to be configured easily
to run different types of tests without the need to modify the
underlying hardware. Finally, the card itself is modular, readily
modifiable for running different types of tests.
[0091] In an aspect the invention provides a self-contained system
that can take a sample as the input, perform molecular diagnostic
steps and display the result of the diagnostic tests. Typically,
the steps include (i) extraction and purification, e.g. where the
DNA sample is extracted from the input sample, which may be blood,
tissue, urine, saliva or other bodily fluid; (ii) amplification and
labeling, e.g. where a specific sequence of the sample is
amplified; (iii) hybridization; (iv) stringency wash to remove
unnecessary molecules that are bound to the target sequence and (v)
readout where the identification step is completed and the specific
sequences are identified. The system generally comprises a
disposable member called the integrated microfluidic card (iMFC)
and a non-disposable base unit. The integrated microfluidic card
may be coupled to the base unit through an interface scheme. Fluid
flow in the iMFC may be achieved by controlling valves that form
the part of the structure of the card. Each card may be designed to
detect and identify a certain set of biological markers.
[0092] In embodiments, the steps between sample input and readout
are carried out without needing any human intervention, the system
does not split the sample, the system is able to identify at least
1,000 targets, the system can identify over 50 targets per run
and/or implements all the testing steps in a single system, and/or
using real-time hybridization the system is able to predict results
before hybridization is complete and can provide information about
target concentration.
[0093] FIG. 1 shows the overall system 10. The system comprises a
unit base 20, a unit lid 30, and a disposable integrated
microfluidic card (iMFC) 40. The design of the card is modular such
that it can be customized for accepting sample inputs in various
forms including but not limited to blood drops, nasal swabs, sputum
etc., or customized for conducting different tests. In general, the
testing process begins by first choosing an appropriate card,
placing the sample in the input chamber of the card, placing the
card in the unit base, shutting the lid and choosing the
appropriate software to run. Once the testing process commences, no
human intervention is required. The results may be displayed on a
screen that may be integrated as part of the system or they may be
transmitted to an external device such as a computer or a smart
phone. After the tests are concluded, the card may be disposed of
and a different one may be chosen for the next test. The order of
these steps may be changed as needed.
[0094] Disposable Integrated Microfluidic Card.
[0095] The card generally combines multiple functions, such as
lysis, purification, amplification, labeling and detection--in one
card. In typical on-market systems, these functions are
accomplished with multiple instruments. We achieved consolidation
of the functions by integrating numerous techniques and features
for bubble removal and accurate metering, integration of the
evanescent field imaging system, choice of reactants and
chemistries at various steps and rapid amplification and real time
hybridization, etc. Consolidation of the multiple functions into
one card may allow for CLIA waiver. FIG. 2A illustrates a
perspective view of the card and FIG. 2B illustrates a plan view.
FIG. 2B also illustrates the areas (shown in dashed lines) where
various functions may occur within the card. For example, block 100
may be the lysis block, block 110 may be the purification block,
block 120 may be where the polymerase chain reaction (PCR) process
occurs and block 130 may be where the detection occurs. Fluid
containing the test sample flows from one block to another block in
the order described above through channels that are incorporated
within the card. The flow of liquids through these channels from
one block to another is controlled by micro-valves that may be open
or shut depending on the pressure imposed at the valves.
[0096] Lysis Block.
[0097] Sample input may also occur within the lysis block, FIGS. 2A
and 2B, block 100. Various methods may be used to input the samples
such as using blood drops and nasal swabs. In one embodiment the
card directly accepts the tool used to collect the sample.
Typically, in on-market systems, the tool used to collect the
sample does not directly interface with the testing instrument. By
allowing direct interface of the tool with the test instrument, a
source of contamination and user error may be eliminated. Since the
card is modular, the lysis block may be modified to accept various
methods of input. Referring to FIGS. 2A and 2B, member 102 may be
an input chamber that accepts a sample such as a drop of blood.
FIG. 2C illustrates an advantage of modularity, wherein the lysis
block is configured to accept a nasal swab 250 as an input. For
both these cases of using a blood drop or a nasal swab, the
underlying design of the card 40 may be the same; only the input
module may be different.
[0098] In addition to accommodating various methods of input, the
lysis modules may be configured to carry out different types of
lysis, such as mechanical, chemical or other types. In reference to
FIGS. 2A and 2B, the lysis block is illustrated as having a bead
beater 104, which comprises a bead chamber filled with beads and a
small motor mounted atop the bead chamber which forces the beads to
collide into one another. Cells located between the colliding beads
lyse, freeing up its contents into the lysis buffer solution--an
example of mechanical lysis. Other modules may be configured to
perform other types of lysis, and these modules may have different
or fewer chambers or sub-modules than shown in FIGS. 2A and 2B. For
example a module capable of performing chemical lysis may have,
instead of the bead beater, another chamber where a chemical
(perhaps stored in the reagent block and piped into the chamber at
an appropriate time) may be mixed with the lysis buffer solution
containing the sample. Thus different types of lysis may be
accommodated with the same basic design of the card.
[0099] To start the process of lysis, a lysis buffer solution
stored in the lysis buffer well 142 in the reagent block 140 is
piped into the chamber 108. The method to control the fluid flow
within the card may be achieved through controlling micro-valves.
Chamber 108 may have a gas permeable vent at the top such that as a
liquid touches the vent, it is air-locked due to the pressure
difference between the chamber and atmosphere, letting the air in
the chamber out while it is being filled. This vent may be made of
various materials such as Teflon. When chamber 108 is filled, a
valve between it and input chamber 102 may be opened, letting the
buffer solution mix with the input sample. Once the lysis buffer
and the input sample are mixed, another valve between the input
chamber 102 and the bead beater 104 may be opened to let the
mixture into the bead beater. The motor atop the bead beater is
then turned on for a specific amount of time after which, the valve
between the bead beater 104 and chamber 106 is opened and the
solution now flows into chamber 106. Chamber 106 may be pre-filled
with a reagent such as guanidine-hydrochloride where it mixes with
the lysed solution. After mixing with the reagent is complete, the
valve between the chamber 106 and purification block 110 may be
opened to direct this mixture into the purification block. The
guanidine-hydrochloride enables the DNA in the sample to bind with
a silica based structure in the purification block thus helping in
the process of purification.
[0100] The configurations to provide capability to do different
types of lysis, to accept various methods of input and to accept
the actual tool for the input method are advantageous
characteristics, which may be synergistically combined in device
10.
[0101] Purification Block.
[0102] When the valve between chamber 106 and the purification
block is opened, the solution comes in contact with a silica frit
that is placed within the purification block. The frit and the
structure that holds the frit, are enumerated by 112 and 114
respectively in FIGS. 2A and 2B. The flow of the solution across
the frit is illustrated in FIG. 3. This figure shows a section of
the card around the silica frit 112. The silica frit may be in the
form of a mesh sitting in a channel 410 that may be sandwiched
between a base layer 400 and a top-cap layer 430. The base layer,
the top-cap layer form part of the structure 114 that holds the
frit. The arrows indicate the flow of fluids within the channel
410. The solution containing the nucleic acid in the solution from
chamber 106 containing the guanidine-hydrochloride will bind to the
frit and flow in the direction of the arrows. The flow of the
solution after it flows past the frit may be directed by
micro-valves to either flow into a residue chamber or may be
directed for further processing. Hence the DNA in the solution
binds to the frit but other components of the solution such as
proteins and lipids that do not bind to the frit flow past the frit
and may be directed through channels into a residue collection
chamber 144. After the DNA binding step, a washing step is
performed. Ethanol contained in the ethanol reservoir 146 is
allowed to flow over the frit to perform the washing step to wash
away unwanted components of the lysate that may still be bound to
DNA. The ethanol is then allowed to dry. The next step in the
purification process is to let the elution buffer stored in the
elution buffer well 148, wash over the frit. This step allows the
nucleic acid to uncouple from the frit. The solution is now allowed
to enter the PCR process block 120. At this step, the valve to the
further processing step (PCR process) is opened but the valve to
the residue chamber is shut.
[0103] PCR Block.
[0104] After the purification block polymerase chain reaction (PCR)
may be implemented, preferably with system adaptations such as
described below. PCR is carried out in the PCR block enumerated by
120, and may be carried out in multiple steps as shown in FIGS. 2A
and 2B. After the elution wash from the purification step is
completed, the purified solution containing the nucleic acid is
allowed to flow into the PCR master mix chamber 122. Here, the
purified solution is mixed with lyophilized (freeze-dried) enzymes
that are carried within the structure of the master mix chamber.
These enzymes are needed for the amplification step that occurs
further down in the processing chain. The master mix chamber may
comprise a well where the volume of the solution filling the well
may be controlled, allowing a precise volume of the solution and
avoiding or eliminating air bubbles. The precise volume in the
various stages of processing results in accuracy of the
results.
[0105] After mixing with the enzymes is completed, the solution is
allowed to flow into PCR primer chamber 124, where the solution
mixes with the lyophilized primers. The primer chamber also may be
a well where the volume is controlled precisely in addition to
avoiding or eliminating air bubbles entrapped in the solution. The
separation of the steps involving the mixing with the master mix
and the primers may be advantageous in some situations as this
allows the use of commonly available master mix modules and reduce
the overall cost of the device. In addition, the separation into
the two steps also allows rapid deployment of kits to test for
emerging threats for example as it may be possible to make multiple
cards with different primers which may be used to check for the
presence of different target molecules. The process of
lyophylization of the primer (oligonucleotides) is quick and simple
and may be carried out in a laboratory external to the device 10.
Thus by separating out the two mixing steps, the card may be
modified to test for different substances, though the mixing
process may also be performed in one step if preferred.
[0106] After mixing with the primers is complete, the solution then
flows to the PCR chamber 126. In a departure from traditional
on-market approaches where a PCR chamber may be made of
non-metallic materials, the PCR chamber in the iMFC card may be
made of a passivated metal such as but not limited to aluminum. The
use of metal and particularly of aluminum is advantageous as it
allows rapid control of temperature of the solution within the
aluminum chamber. This rapid control is achieved by having the top
and the bottom of the aluminum chamber to be in close contact with
thermoelectric coolers (TECs). Close contact between the PCR
chamber and the TECs is obtained by locating two TECs above and
below the PCR chamber. The TEC above the PCR chamber is placed on
lid 30 of the device 10 (FIG. 1) within a TEC housing unit 200. The
shape of the face of the TEC 210 and the shape of the PCR chamber
126 are made to match such that when the lid is shut, the TEC face
may be located directly over the top surface of the PCR chamber.
The TEC below the PCR chamber is simply placed within the unit base
20. FIG. 2D illustrates how the PCR chamber may be sandwiched
between two TECs 220 (with TEC face 210) and 230 (with TEC face 240
which is not visible in the figure). Thus this arrangement of the
TECs and the use of the aluminum PCR chamber enable rapid
temperature cycling of the PCR mixture. It has been found through
measurements that this combination allows a temperature ramp of
>15.degree. C./s with accuracy of .+-.1.degree. C. This
configuration then contributes directly to the shortening of the
time between the sample input and result output. In addition to the
advantage obtained in the rapid temperature control of the solution
due to the use of the passivated aluminum, yet another advantage
may be realized in that lower amount of energy is needed to heat
and cool the solution compared to what would have been required if
non-metallic materials were to be used for the PCR chamber. The
lower amount of energy translates directly to needing lower overall
power to run the device, enabling optional battery operation and
field portability.
[0107] Another aspect of the PCR chamber 126 is now explained with
referenced to FIG. 2E, which shows sections of the card around the
PCR chamber and the detection block 130. To preserve the
concentrations of the solution, trapped air should be avoided or
minimized within the PCR chamber while the PCR process occurs. The
top and the bottom sections PCR chamber, as described may be made
of passivated aluminum; hence a breathable membrane may not used
here. Hence an outlet channel 250 from the PCR chamber is provided
that leads to reservoir 255 that in turn opens to the atmosphere
through another channel 257. Hence as the chamber 126 is filling
with solution, air is pushed out and bled to atmosphere. A valve in
channel 260 may be shut while the chamber 126 is filling. During
the filling process, channel 250 and reservoir 255 may also fill
with solution, which prevents air from coming back into the chamber
126. Also, as the solution is driven by a constant pressure of 6
psi, no backflow from the channel 250 or the reservoir 255 occurs.
After the chamber is filled, the PCR process is commenced. At the
end of the PCR process, the required target sequences are amplified
and labeled for detection within the detection block.
[0108] Detection Block--General Description.
[0109] After the PCR process is complete, referring to FIG. 2E,
valve 262 in channel 260 may be opened and the solution with the
amplified components (amplicons) in the PCR chamber 126 may be
flowed into a mix chamber 275. Here the solution is mixed with a
hybridization buffer that may be stored in the hybridization buffer
well 150 in the reagent block. The solution from the hybridization
buffer is metered before it is allowed to mix with the PCR product.
After the mixing process in the mix chamber 275 is complete, the
solution is allowed to flow into the detection chamber 325 via
channel 330. The detection chamber 325, the mix chamber 275, the
channels which transport solutions in and out of the chambers all
form part of the detection block 130 illustrated in FIG. 2B. Next,
within the detection chamber, the PCR product mixed with the
hybridization buffer may be allowed to flow over a DNA microarray.
This occurs in the detection well 317 as seen in FIG. 2F, where the
well holds the solution and the microarray 315 is placed at the
bottom of the well. Thus with this configuration, the solutions is
located on top of the microarray. FIG. 2F illustrates the
arrangement of the DNA microarray 315 and explains how the light is
coupled into the microarray as this light forms part of the optics
system that is used to read the microarray. The DNA microarray may
contain several probes arranged in a grid pattern over a glass
substrate 310. Each probe may contain a strand of a DNA (or RNA)
and is used to detect the presence of nucleotide sequences in the
sample solution that are complementary to the sequence in the
probe. As stated, these probes are located in a grid pattern on top
of a glass substrate 310. Light is coupled into the glass substrate
via gratings 305 that are etched within the glass.
[0110] Referring to FIG. 2G, the gratings are illustrated along
with an incident light 306. The incident light 306 may be at a
specific wavelength. Due to the presence of the gratings, the
incident light may travel in multiple directions; some light may be
transmitted right through as shown by 307, some light may be
transmitted at specific angles as shown by 308 and 309. Light may
be transmitted at additional and steeper angles with respect to the
light 307, but they are ignored in this disclosure as the intensity
of the light at the steeper angles tends to be lower. The angles at
which light is transmitted (for light not transmitted in a straight
line) are determined by the well-known grating equation. The angle
of light 308 and 309 may be adjusted by adjusting the wavelength of
the incident light 306 and by the patterning of the gratings. Next,
to couple light into the glass substrate, the process of total
internal reflection is used. This concept is illustrated in FIG.
2H. In this figure, the light 307 that is transmitted straight
through and light 309 transmitted at an angle is ignored. Light 308
is illustrated by a beam constrained by the size of the gratings
305. Thus the beam of light 308 is illustrated by a solid line 308L
emanating from the left edge of the grating and by a dashed line
308R emanating from the right edge of the grating. The solid line
and the dashed line are used to distinguish the two edges of the
beam; no other difference is indicated. Depending on the angle of
beam 308 and the indices of refraction of the glass substrate and
of the surrounding environment (essentially air), total internal
reflection may be set up at the top surface of the glass substrate
region 1 marked as R1. This reflected light may reach the bottom
surface of the glass substrate and may again be totally internally
reflected at region 2 (marked as R2). Thus though total internal
reflection, the light may be steered behind the detection well 317,
below the microarray 315. As stated earlier, the microarray 315 is
located at the bottom of the detection well 317. Now that the light
is steered to the location of the microarray, another phenomenon
called evanescent fields is made use of to excite the light
sensitive molecules in the DNA microarray. It is well known in
optics that at boundaries where total internal reflection occurs,
an evanescent field is set up on the other side of the boundary.
These evanescent fields are a near-field phenomena and the
intensity drops exponentially further away from the boundary.
However, very close to the boundary, the evanescent fields are able
to excite the light sensitive molecules and since the solution in
the detection well 317 is located at and near the boundary, the
detection method of using evanescent fields becomes possible.
Returning back to FIG. 2H, the detection well is seen to be placed
within an enclosing layer 320; this enclosing layer is made of
chrome. The chrome layer is also shown in FIG. 2F, surrounding the
detection well on all four sides. The chrome is included as part of
the design so that stray light from the immediate vicinity of the
detection well is reduced or eliminated. The possibility of
erroneous light is further removed by coupling a black plastic
sheet 327 over the detection well. Materials other than plastic may
be used as well.
[0111] The grating design and the subsequent angle of light 308,
the thickness of the glass substrate, the index of refraction of
the glass substrate, the distance between the grating and the
detection well are parameters we have optimized for this device and
provide synergistic functionality. In addition, the angle of light
308 may be selected so that it not only totally internally reflects
from regions such as R1 and R2 (essentially from a glass-air
boundary), but also from the glass-solution boundary in the
detection well. The index of refraction of the solution in the
detection well is approximately 1.33 while that of air is
approximately 1. The grating pitch, grating material, and substrate
index can be altered to use different wavelength laser light. In
one example, a 633 nm wavelength light was used with a 150 nm
silicon nitride grating at a 195 nm pitch on a 750 micrometer fused
silica substrate. The light is coupled into the substrate with a
10.degree. divergence. The distance between the grating and the
microarray is chosen to such that the microarray is positioned at a
distance corresponding to an integral number of total internal
reflectance bounces. All parameters specified above may be adjusted
as required. For example other wavelengths of light may be used
which may then require a different grating spacing than specified
above.
[0112] A TEC 365 may be placed over the detection well 317 to
control the temperature of the solution. Finally a CCD camera 360
may be placed beneath the microarray so that a one to one image of
the microarray may be formed on the camera detectors. The camera
system takes photographs of the solution over the microarray. The
photographs reveal the areas within the microarray that may be
fluorescing. This information is used in the identification and
detection process.
[0113] The invention exploits synergistic combinations of the
described improvements and adaptations to implement the detection
function.
[0114] Detection Block--Quality Control.
[0115] In addition to identifying the target sequences within the
sample, the microarray may be used to also ensure that the steps
(lysis, purification, mixing with the enzymes and the primers etc)
occurred as desired. Markers may be added at each step and presence
or absence of the markers may be tested optically within the
microarray. Thus by analyzing the presence or absence of markers,
quality control may be achieved to identify if and where the tests
may not have run appropriately.
[0116] Detection Block--Pan-Amplification.
[0117] The device 10 may be used to avoid sample splitting to test
for various nucleic acid sequences. Sample splitting is commonly
used in on-market systems; it requires that the sample be divided
into multiple samples where each divided sample may be tested for a
certain sequence. Since the original sample is split into multiple
samples, this method reduces the detection limit by a factor equal
to the number of sample splits. Instead of using sample splitting,
we implement a process called pan-amplification, by which the
variations within a species of bacteria or virus may be identified
without splitting the sample. This type of testing becomes possible
because some sections of the DNA of the variants within a species
may be same or similar. Knowing that certain sequences are present,
the primers may then be designed to identify the variants.
[0118] Typically PCR is limited to about 20 different primer sets,
which limits the number of targets that the on market systems can
detect. Our system overcomes the limitation by pan-amplification,
wherein the PCR primer set targets DNA sequences that are common
across many organisms, while the region in between the primers
contains DNA sequences that are highly variable between organisms.
The pan amplification approach allows for differentiation of the
sample type at the microarray where high multiplexing is possible.
For example conserved regions of coronavirus polymerase gene allow
for amplification of 6 different coronavirus serotypes with a
single primer set, while each serotype is distinguishable on the
microarray by analyzing the variable region amplified between the
primer sets.
[0119] One advantage of the pan-amplification is that fewer primers
are needed than a typical on-market device doing PCR. Use of more
than 20 primers can lead to a phenomenon called "primer dimer"
where primer molecules hybridize to each other due to the strings
of the complementary bases in the primers, whereas our
pan-amplification process allows the use of fewer primers hence
resulting in an advantageous configuration.
[0120] Detection Block--Real Time Hybridization.
[0121] In another advantageous configuration, a real time
hybridization approach is implemented within device 10 that results
in shortening the time between sample input and results output. In
typical on-market approaches, hybridization is allowed to continue
until stable concentrations of the sample or samples being tested
are achieved. In contrast, in device 10, a real time approach is
implemented where the concentration of the sample or samples is
estimated repeatedly during the time the concentration of the
sample or samples may be rising soon after the start of the
hybridization. This technique is based on the observation that the
strength of the signal from the CCD camera may be related to the
concentration according to the kinetic curve equation, supra. Using
the kinetic model, the fluorescence signal increase with respect to
time may be monitored. This signal may be modeled or fitted as an
exponential from which a time constant may be estimated. With an
estimate of the time constant, the analyte concentration may be
estimated.
[0122] Detection Block--Use of Multistage Temperature Control.
[0123] In typical on-market hybridization procedures, after the
hybridization is complete, a stringency wash is performed so that
unbound probes may be washed off. Typically, stringency washes are
done by changing the salt concentration, however in device 10, a
similar effect may be achieved by changing the temperature of the
sample using the TECs while maintaining a constant salt
concentration. Hence after hybridization is complete and an initial
set of pictures are takes, the temperature of the TECs may be
increased so that some of the unbound molecules may be removed.
This allows for an advantageous way to perform a check on the
results as changing the temperature of the solution is easier than
changing the salt concentrations of the hybridization buffer.
[0124] Volume Control.
[0125] Testing procedure require accurate volume, which may be
disrupted by air bubbles present in a fill chamber. In typical
on-market systems, various devices such as peristaltic pumps are
used, but the addition of these devices increases the cost and
complexity of the device. Our device provides volume control that
achieves high accuracy and precision (preferably 10% or less) and
is inexpensive to implement.
[0126] FIG. 4 illustrates a fill chamber 450 in cross-section,
along with the structures around the fill chamber. The fill chamber
may be located on top of a base layer 490. The sides of the chamber
are indicated by 460. Fluid may enter the chamber along arrow 480.
The outlet of the fill chamber is marked by a dashed arrow 495.
Member 470 may be a valve; if it is shut, no flow occurs along
dashed arrow 495. A breathable membrane 440 may be mounted on top
of the fill chamber as illustrated. With the valve closed, fluid
may flow in the direction of 480, into the fill chamber 450. The
breathable membrane may let the trapped air out and as the fill
chamber fills, all the air is forced out. The breathable membrane
does not let any air back in due to the pressure differential
between the chamber and the atmosphere; thus with the valve 470
closed, a precise quantity of fluid may be collected within the
fill chamber. The accuracy of the volume within the fill chamber
may be determined by the accuracy of the dimensions of the chamber,
and the dimensions of the chamber may be controlled using
well-known techniques such as but not limited to machining and
etching. The breathable membrane may be made of materials such as
but not limited to microporous polypropylene. Thus with a
combination of the precisely made chambers and the breathable
membranes, accurate and precise volume control may be achieved.
[0127] The fill chambers along with the membranes are located in
various locations within the iMFC card. Thus for example, fill
chambers with breathable membrane may be used for the PCR master
mix chamber 122 and the PCR primer chamber 124. In these two
locations in particular, the lyophilized compounds may be located
as a pellet within the chamber; thus the rehydration process within
these chambers may occur in a volume controlled environment.
[0128] Valve Control.
[0129] FIGS. 5A and B illustrate how the valves and flow of fluid
may be controlled within the iMFC card, including a fluid flow
channel 520 and an air flow channel 540, and a flexible membrane
530. FIG. 5A shows two additional arrows 550 to indicate fluids may
flow through the fluid flow channel 520. The fluid may be pumped
through the system at a certain pressure such as 6 psi. If the
membrane 530 is not deformed as illustrated in FIG. 5A, then
channel 520 may allow free flow of fluids. However if a higher
pressure such as 18 psi is applied to the air flow channel, the
membrane may deform as illustrated in FIG. 5B, which effectively
stops the flow through the channel 520. Thus by controlling the
pressure on either side of a membrane flow through flow channels
may be controlled. The pneumatic system that applied the
differential pressure is described below.
[0130] iMFC Layers.
[0131] The iMFC card, as stated above, may be disposable and may
constructed of inexpensive materials such as polycarbonate,
acrylic, and polyethylene terephthalate. The card may be thought of
as a "mother board" where different modules may be accommodated,
such as where the card may be designed to have various different
types of input modules to accommodate different methods of input.
Not only can the card accommodate various modules, but the channel
configuration may also be amended to accommodate different
diagnostic tests or to add or subtract steps from a diagnostic
test. Thus each card may be designed to accommodate a specific test
or a set of tests, without needing to make changes to the
underlying hardware. Each card may be made of one or multiple
layers. One of the main functions of the card is to route fluids
from one place to another at the appropriate time, through a system
of fluid flow channels and air flow channels built into the card.
Other functions include but are not limited to metering the flow
and proving a temperature controlled environment.
[0132] FIG. 6 illustrates a composite "projection" image of all the
layers of a iMFC card. In general, the card has one or multiple
channels such as 600, such that fluids can flow from one location
to another in these channels. The channels may be formed by cutting
grooves into the material of a layer within the card. In addition,
the card may also have one or multiple valves such as 630 and one
or multiple channels or reservoirs such as 620 where volume may be
metered. Some layers or section of the layers may be composed of
the breathable membrane. The card may contain a number of ports
such as 610. The ports may form an interface between the hardware
and the card. These ports are used to apply drive pressure (to
drive the fluids) or valve pressure (to control the valves). The
location of these ports may remain the same for the cards, which
prevents any need to modify the underlying hardware. However the
card may be designed in any convenient manner and the functions on
the card may be placed in any convenient manner. This aspect makes
the card versatile as it may be designed for doing various tests
without the need to change the underlying hardware.
[0133] Pneumatic System.
[0134] The pneumatic system (FIG. 7) is responsible for regulating
the flow within the card. The pneumatic system may have a pump that
may be located within the unit base. This pump compresses air in an
accumulator to a desired pressure, such as 16 psi. The figure also
indicates a feedback loop from the accumulator to the pump so that
the pressure within the accumulator is kept constant. From the
accumulator, one path leads to a regulator where the relatively
high pressure of the accumulator is regulated down to a lower
pressure, such as 6 psi. This lower pressure is used to drive the
fluids within the entire system. A solenoid valve may be included
at the output of the regulator, to turn on the fluid drive. Another
path from the accumulator may go through one or multiple solenoid
valves to control the opening or closing of the valves. Thus the
relatively high pressure (16 psi) of the accumulator may be used to
control the valves. As indicated in the figure, each solenoid valve
may control one or multiple valves. FIGS. 5A and 5B depict how the
flow of fluid is controlled through the design of the fluid
channels and the air flow channels. In the context of FIG. 7, the
fluid channels may be connected to the "Drive" output and the air
flow channels may be connected to one of the "Valve" outputs.
[0135] The pneumatic system may be part of the hardware; hence some
of the pathways for supplying the pressure for the fluid flow or
for valve control may not be modified easily. However to achieve
flexibility of design of the card, only the ports are required to
be at the same location; these ports are how pressure is supplied
to the air flow channels to control the valve or to the fluid drive
channels. These ports are shown by 610 on FIG. 6. Thus by requiring
the cards to have the ports at the same location, the same hardware
unit may be used, but the cards themselves may be designed for
different purposes.
[0136] Hardware.
[0137] As illustrated in FIG. 1, the testing system described may
be built into a portable box, including a unit base 20 and a unit
lid 30. Various functions and capabilities may be physically
configured within the base and the lid. FIG. 8 illustrates a
functional block diagram of the hardware. The hardware may include
a processor and a power source such as a battery. The processor may
interface with the other members such as the TECs, the motors, the
optics system, the pneumatic system, the display and the
communications system. With regards the display system, the device
10 may have a screen that may relay messages or the results. With
regards the communication system, the unit 10 may be interfaced to
an external computer via any suitable communication method such as
ethernet and bluetooth. The display and communication subsystems
these may be implemented using well-known techniques. FIG. 8 also
illustrates that the card may be mechanically interfaced with some
of the subsystems such as the TEC, the motors etc. These interfaces
are illustrated by dashed lines.
[0138] Embodiments of our claimed invention include:
[0139] 1. A molecular diagnostic device, system or method that
detects a large set (>6, >10, >20 or >50) nucleic acid
sequences without any human intervention to carry out the testing
process, except for the purposes of injecting or inserting a sample
and reading the results.
[0140] 2. A device, system or method herein where the steps of
lysis, purification, PCR and hybridization are integrated into one
system and where these steps are carried out in a disposable
card.
[0141] 3. A device, system or method herein where the disposable
card may contain modules so that by changing the modules, the card
can be configured for different tests.
[0142] 4. A device, system or method herein where the analytes may
be detected in samples from a variety of sources including, for
example: blood, saliva, GI samples, urine, wound swabs, spinal tap,
nasal swabs, veterinary and agricultural sources.
[0143] 5. A device, system or method herein where the tool used to
collect the sample may be directly input into the card.
[0144] 6. A device, system or method herein where the amplification
step of the PCR process is done via a process called
pan-amplification rather than sample splitting.
[0145] 7. A device, system or method herein where the detection
step uses a microarray.
[0146] 8. A device, system or method herein where the microarray
includes controls that verifies if each step of the testing process
occurred appropriately.
[0147] 9. A device, system or method herein where the detection
step uses a real time hybridization method which predicts the
concentration of the samples based on a kinetic model, wherein
readout time is reduced.
[0148] 10. A device, system or method herein where the PCR process
is carried out in a passivated metallized chamber such that the
temperature within the chamber can be controlled rapidly by
locating the metallized chambers adjacent to and in contact with
TECs, preferably where the metal used is aluminum.
[0149] 11. A device, system or method herein where the volume of
the solutions is metered through the use of channels or chambers
coupled with a breathable membrane so that as the channel or the
chamber fills up, air is forced out of the breathable membrane;
hence the error from having air in a metered volume may be
minimized or removed.
[0150] 12. A device, system or method herein where a pneumatic
system controls the fluid flow by applying different pressures to
drive the fluids and to control the valves.
DETAILED DESCRIPTION OF ADDITIONAL EMBODIMENTS AND EXAMPLES
THEREOF
[0151] Disclosed is a portable system that can analyze nucleic acid
sequence content in a variety of samples. The system is capable of
taking in a variety of raw samples (diagnostic or environmental)
and performing nucleic acid extraction, purification,
amplification, labeling, and sequence analysis by microarray in a
self-contained unit. The system is automated and rapid to allow for
point of site analysis of samples by users who are not trained
laboratory technicians.
[0152] In embodiments: the system comprises (1) a reusable hardware
platform and (2) a consumable integrated microfluidics card (iMFC)
that determines the assay to be performed;
[0153] the system is small (<150, <250, or 400 in3),
light-weight (<3, 5 or 10 lb), fast, and intuitive to use;
[0154] the reusable hardware controls and provides the pneumatics,
pressure regulation, temperature control, laser control, and
imaging of the final microarray;
[0155] the iMFC consumable comprises a card that performs sample
lysis, purification, PCR, and detection and houses all the required
dry reagents; and/or
[0156] the iMFC further comprises a reagent storage element that
holds all the liquid reagents separately so as not to compromise
the integrity of the dry reagents.
[0157] In embodiments the system provides:
[0158] Ease of Use: easy to operate and the results simple to
interpret, for field use by untrained operators; configured to be
Clinical Laboratory Improvement Amendments (CLIA)-waived; and/or
provides sample-in to answer-out capability without user
intervention.
[0159] Configurability: the iMFC is modular, enabling stocking of
the main iMFC and attaching separate, quickly produced, lower-cost
modules that define the end product assay functionality; since the
main iMFC remains the same, the system provides just-in-time
development of new assays as new threats emerge.
[0160] Assay Flexibility; flexibility in sample type (from hardy
spores to easily ruptured mammalian cells) and/or analyte class
(DNA, RNA, and protein).
[0161] Manufacturability and Cost; the iMFC and the modular
components are manufacturable using low-cost injection molding or
an advanced manufacturing laser converting process; high-speed
laser converting and precision lamination allows manufacture of an
iMFC with features as small as 125 .mu.m and tolerances of less
than 50 .mu.m at production rates approaching 50 feet per minute;
the combination of older injection molding technology and advanced
laser converting technology allows production of complicated
disposable cartridges for less than $10 per card at volume.
[0162] In embodiments our system is fully automated from raw sample
input to answer out, and/or configurable to allow multiple analysis
types.
[0163] In embodiments our system provides a portable bioanalysis
platform to detect nucleic acids, typically DNA or RNA, such as
microbial, typically bacterial, viral or fungal detection, and
health monitoring via mRNA and protein detection, and cell
selection and concentration. In an embodiment the system consists
of two elements: (1) a reusable hardware platform and (2) a
consumable integrated microfluidics card (iMFC) that determines the
assay to be performed.
[0164] In embodiments our system is demonstrably adaptable to
diverse applications:
[0165] Bacterial Agent Detection.
[0166] Our DNA detection iMFC operates in a manner similar to that
of a laboratory work flow: lysis, DNA purification, DNA
amplification and labeling, and hybridization. Our microfluidic
technical approach to each step is highlighted below.
[0167] Lysis is accomplished using silica beads and low-cost
disposable motors for robust lysis of sample types, from hardy
spores to mammalian cells.
[0168] We purify DNA by binding the DNA to a silica frit, washing
away impurities, and eluting in a polymerase chain reaction
(PCR)-ready solution. Our DNA purification module and procedure
allow elution of high DNA concentration in the first 6 .mu.l in
.about.5 min with no user intervention. In contrast, a laboratory
bench approach for spore lysis and DNA purification takes 30 min
over 7 steps and requires laboratory centrifuges.
[0169] We perform DNA amplification in an aluminum-walled chamber
between two custom thermoelectric coolers (TECs). The TECs and
aluminum chambers allow for rapid heat transfer between the TECs
and the PCR mix. We have demonstrated PCR duplex of the two genes
(AGG and STX2) associated with the E. Coli O104:H4 pathogen in 12
min and have detected down to 10 genomic copies.
[0170] We hybridize DNA using a custom DNA microarray and optical
system. Light is coupled into a glass slab using a grating and
remains confined by total internal reflectance. The evanescent wave
on the surface is used to excite the target sequences hybridized to
their complement on the surface of the microarray. Use of the
evanescent wave allows us to visualize hybridization in real time.
A custom optical relay and CCD are used to image the surface of the
microarray.
[0171] We can verify multiplex detection of multiple potential
biological warfare agents and establish the receiver operator
characteristic (ROC) curves for our assay and hardware
platform.
[0172] Viral RNA Detection.
[0173] Our technical approach to detecting viral RNA is the same as
our approach to DNA detection, except we use a single
reverse-transcription/PCR mix. We have demonstrated a single
master-mix reverse transcription and PCR in less than 30 min for
the influenza H3N2 and H1N1 viruses. The field-portable capability
can directly leverage the information obtained from DARPA's
Prophecy program and allow early detection of potentially pandemic
causing mutations in viral populations in domesticated animal herds
or flocks.
[0174] mRNA Detection.
[0175] We can address mRNA analysis using the same hardware,
updating the iMFC to allow for mRNA capture using poly-T beads and
imaging of the microarray in real time to capture kinetic data. The
use of kinetic measurements allows us to determine the
concentration of each analyte well before equilibrium is reached,
thereby reducing the hybridization time that typically drives gene
expression analysis. Our capability to analyze a blood sample
rapidly and at the point of need for mRNA leverages the investment
DARPA made in the Predicting Health and Disease program.
[0176] Protein Detection.
[0177] We can transduce a protein-binding event into a nucleic acid
readout, thereby giving our same platform the ability to assay for
both nucleic acids and proteins. Plasma protein concentrations are
indicative of health status or environmental exposure. We have
developed a four-host-response protein panel indicative of ionizing
radiation exposure; similar panels for other environmental exposure
diagnostics can also be integrated into the platform. We used the
same bead-capture microfluidic module as that for mRNA detection to
capture protein analytes on a bead. The beads are functionalized
with antibodies for particular protein analytes instead of poly-T
oligonucleotides. A second antibody labeled with an oligonucleotide
is used as the reporter molecule as in a traditional immunoassay.
Once the sandwich assay has undergone a stringent wash, the
reporter oligonucleotide is amplified, labeled, and hybridized
using the same modules as for DNA detection. We call this approach
"microsphere-immune-PCR" (MSiPCR).
[0178] Cell Selection and Concentration.
[0179] Our front-end module allows specific cell selection and
concentration using a microfluidic electroactive (EAP) polymer cell
sorter, analogous to laboratory-scale fluorescence-activated cell
sorters (FACS). The module increases the operational envelope of
the system by either concentrating a dilute cell concentration in a
large volume (a few bacterial cells per ml) or sorting out select
cells from a background of many cells (only activated T cells from
all peripheral blood mononuclear cells). In this module we align
cells using hydrodynamic and/or inertial focusing, and then sort
based on a fluorescent trigger using EAP actuators. We have
demonstrated the technology's potential to sort at speeds in excess
of 25,000 cells/second.
[0180] Baseline Handheld Analyzer
[0181] The hardware platform provides all necessary hardware
actuation required to support iMFC processing of a microbial DNA
sample. The user interacts with the hardware platform via a
computer USB port. Once the iMFC is inserted into the hardware and
the lid is closed, the user selects a specific processing script
for the desired assay. After the script is initiated, the handheld
runs without user interaction, until completion. During the run,
images are transferred from the handheld to the host PC, where they
are analyzed. At the completion of the run, analysis and results
will display on the screen for user review.
[0182] The hardware is designed to allow for cell lysis,
purification, amplification and detection, in a small portable
device, preferably with a total volume of less than 1 ft.sup.3.,
preferably less than 200 in.sup.3. In one embodiment the hardware
dimensions are 4.75 in. deep.times.6.25 in. wide.times.5 in. high
for a total volume of 148 in..sup.3.
[0183] On the iMFC, membrane valves and liquid fluid flow are
controlled by positive pressure. The pneumatic subsystem is
centered on a custom acrylic manifold that interconnects all
pneumatic components in the handheld analyzer (HHA) and routes
those component outputs to the input ports on the iMFC. This
manifold contains an accumulator to hold a volume of air at a
regulated pressure (18 psi) appropriate for microfluidic valve
membrane actuation on the iMFC. A single solenoid is used to
trigger drive pressure to the iMFC at any time during assay
processing. This drive pressure port is routed through a pressure
regulator (mounted to the integrated manifold), so that drive
pressure can be adjusted between 0-10 psi for any given HHA.
[0184] Table 1 shows functional components and their purposes and
implementationin the baseline system. The hardware that supports
the amplification (PCR) and detection modules are described in
detail below as part of the baseline iMFC description. The 2nd
column lists the purpose of that functional component and the final
column lists our technical approach implemented on the hardware
platform for achieving the desired capability.
TABLE-US-00001 TABLE 1 Functional Component Purpose Components
Pneumatics Solenoids control air flow to actuate Air accumulator,
22 pneumatic membrane valves on iMFC to stop and solenoids,
pressure sensor, and start fluid flow. integrated manifold to route
air to iMFC pneumatic inputs. Drive Regulate drive pressure used to
drive Pressure regulator, 1 pneumatic pressure fluid flow on iMFC.
solenoid. regulator Amplification Rapid thermocycling of small
volume Two thermoelectric coolers (TEC) (PCR) reaction. Support for
near continuous and heat sinks. thermocycling. Hybridization
Temperature-controlled stringency to One TEC and heat sink. station
improve hybridization specificity. Detection Normal incidence 635
nm laser 10-mW, 635 nm laser diode, station illumination of optical
grating on iMFC focusing optics, line-generating microarray. 647 nm
filtering and CCD optics, turning minor, 647 nm image capture of
hybridized sample interference filters, relay lens, CCD label
fluorescence. chip, and supporting electronics. Lid Small form
factor enclosure to hold Hinging lid mechanism and Enclosure iMFC
with sufficient force to ensure translating lid to allow for iMFC
pneumatic sealing. thickness variability. Electronics Power and
electrical signaling required Circuit boards, wiring, Li-ion and
power by all powered components. battery. External I/O Power plug
input port and laptop AC power supply input, USB communication
port. Type-B, battery connection.
[0185] Baseline iMFC and iMFC Modules
[0186] The iMFC consumable comprises (1) a card that performs
sample lysis, purification, PCR, and detection and houses all the
required dry reagents and (2) a reagent storage element that holds
all the liquid reagents separately so as not to compromise the
integrity of the dry reagents. Because the iMFC was designed in a
modular fashion--i.e., application-specific modules are assembled
onto a generic card--the iMFC for new applications can be easily
and quickly developed by simply interchanging the modules. This
versatility feature eliminates the need to redesign, develop, and
manufacture new cards, the most complex component. Once fabricated,
the same cards can be used for a wide range of applications,
thereby reducing cost and development time.
[0187] Microfluidic Card.
[0188] The card utilizes positive-pressure-driven flow and consists
of three functional layers that contain (1) fluid channels, (2) 22
membrane valves to control fluid flow, and (3) vents for bubble
removal from the fluid channels. These functional layers together
are composed of nine laminate layers surrounded by two
injection-molded parts. Seven modules are pre-mounted on the card,
four on the top surface (lysis module, purification filter, PCR
master mix chamber, and primers chamber) and three on the bottom
surface (PCR chamber, stir bar mixer chamber, and detection
chamber). Affixed to the detection chamber is the optical waveguide
chip, which contains a DNA microarray to sense the targets of
interest.
[0189] Reagent Block.
[0190] When the user starts the program, an inflatable bladder in
the handheld hardware presses the reagent block onto the sharps to
pierce the foil seals, which, in turn, releases the liquid
reagents. With positive-pressure-driven flow, air enters each
chamber in the reagent block and drives the liquid reagents through
the outlet vias and into the card. Compressible gaskets on the card
prevent fluid or air leakage at the reagent block-card interface.
Table 2 identifies liquids that can be stored in the reagent block
for DNA analysis. Cards designed for different application (mRNA or
protein analysis) will have different reagents. The reagent block
also includes a waste reservoir that contains absorbent material to
collect the reagents that flow through the card. The reagent block
preferably contains all the liquid reagents in a single prepackaged
format required for biological assay analysis. The table lists the
reagents currently used for DNA analysis. The buffer name is
specified along with the purpose of the buffer and the exact
chemical formulation. The reagent block is general purpose, and the
required reagents will change depending on the biological assay
being performed by the iMFC. Our technical approach is to use the
same package but fill it with different reagents to support the new
assay capabilities for RNA virus detection, mRNA detection, and
protein detection.
TABLE-US-00002 TABLE 2 Reagent Purpose Contents Lysis buffer
Prepare sample for lysis Bead-beating solution from MO BIO
PowerSoil DNA Isolation kit modified with 1% phenol (w/v) to add a
denaturant and 1% n-octanol (w/v) to prevent foaming Wash buffer
Wash sample during purification 80% (v/v) Ethanol Elution buffer
Remove and collect purified 10 mM Tris buffer, pH 9.2 sample from
purification filter Water Dilute residual PCR inhibitors in
Deionized water purified sample (if necessary) Hybridization
Prepare amplicons for Saline-sodium phosphate EDTA (SSPE) buffer
20x buffer hybridization on the microarray concentrate
[0191] Lysis.
[0192] Following the release of liquids from the reagent block, the
next step in the automated program is lysis. The lysis module,
which can handle even spore samples, consists of three chambers:
the sample chamber, the bead beating chamber (for spore lysis), and
the binding agent chamber. After lysis buffer from the reagent
block flows into the sample chamber, a motor turns on to mix the
lysis buffer with the sample. The mixed sample then flows into the
bead-beating chamber, where a second motor runs for 3 min to
agitate glass beads and lyse the spore sample via bead beating. The
lysed sample then enters the binding agent chamber, where a solid
mixture of guanidinium hydrochloride and sodium bisulfate (the
binding agent) dissolves in the sample to facilitate binding of DNA
to the purification filter in the next step.
[0193] As a proof of concept for this approach, we obtained lysis
efficiency data from samples of B. subtilis spores lysed by the
lysis module. Spores represent the most challenging lysis
situation. For non-spore applications, the bead-beating chamber can
be exchanged with a motorless chamber module to simplify the design
and reduce costs.
[0194] Purification.
[0195] We faced several challenges in developing a technical
approach for microfluidic DNA purification. For example, we needed
to develop an approach that replicated a laboratory procedure
requiring 7 steps and 30 min with multiple centrifugation steps to
successfully lyse and purify a spore sample. Additionally, we
needed to elute the DNA in the first 6 .mu.L fraction. In a
laboratory assay the purified DNA is typically eluted in a larger
volume (e.g., 20 .mu.L), and then a smaller volume aliquot (1-3
.mu.L) is used for PCR amplification. In this situation the DNA is
mixed and therefore elution rate is averaged over the entire 20
.mu.L. A microfluidic approach involves little mixing since most of
the flow is laminar; therefore, the highest concentration of eluted
DNA needs to be in the first fraction.
TABLE-US-00003 TABLE 3 We performed three replicates of B. subtilis
spore lysis using our lysis module. The results are shown below in
percentages from the starting stock of 107spores (based on a
viability count). The control was a Claremount bead-beating device.
Our microfluidic results are comparable to the control using
laboratory pipettes and tubes. Recovery after Lysis B. subtilis
Sample Number (%) Based on Viable Count Input of 10.sup.7 1 48.4 2
31.6 3 18.6 Average 32.9 Control 39.6
[0196] On the iMFC after lysis, the sample flows through the
purification module, DNA binds to the filter, and the contents of
the remaining lysed sample flow to the waste reservoir. Wash buffer
from the reagent block then flows through the filter to remove
residual impurities and also collects in the waste reservoir. Air
blows through the purification module to dry the filter, followed
by elution buffer (a key aspect to achieve significant elution in
the first fraction is the elution buffer pH) from the reagent
block, which, as it flows through, removes the purified DNA from
the filter in preparation for PCR. Table 4 presents the data for
three replicates of E. coli samples purified on the microfluidic
card and compares the results with those from a standard
purification filter. In each case it is clear the highest
concentration is coming out in the first fraction. Each fraction
represents .about.6 .mu.l of DNA in elution buffer.
TABLE-US-00004 TABLE 4 We repeated three replicates of E. Coli
sample purification using our iMFC protocol. We developed the
protocol to elute most of the DNA in the first fraction, since we
will not have an opportunity to pool the entire elution and select
just a fraction with a pipette as with typical laboratory bench
approaches. The results clearly indicate the largest concentration
comes in the first 6-.mu.l fraction. Sample 1 Sample 2 Sample 3
Fraction 1 in ng/.mu.l 7.5 1.5 0.7 Fraction 2 in ng/.mu.l 5.5 1.3
0.4 Fraction 3 in ng/.mu.l 3.2 1.1 0.4 Laboratory bench control in
ng/.mu.l 11.0 3.3 2.6 Percent compared to laboratory 67.8% 44.0%
27.8%
[0197] PCR Amplification and Labeling.
[0198] Carrying the purified DNA from the filter, the elution
buffer fills the PCR master mix chamber, which contains lyophilized
master mix. Then the rehydrated master mix flows into the primers
chamber and rehydrates the dried primers. We have separated the
primers and master mix to allow for reuse of generic PCR master mix
modules. The lyophylization of oligonucleotides is quick, and this
approach allows us to support rapid deployment of kits to test for
emerging threats. Following the rehydration steps, the
sample--along with the master mix and primers--enters the PCR
chamber, where the sample DNA is then amplified.
[0199] Two key features enable us to accomplish rapid PCR: (1)
aluminum PCR module surfaces and (2) a novel TEC assembly that can
ramp >15.degree. C./s with accuracy of .+-.1.degree. C.
[0200] The PCR module is sandwiched between two TEC assemblies. The
PCR master mix is held in a 1-mm-tall acrylic chamber enclosed by
two 25-.mu.m aluminum walls. The aluminum surfaces of the PCR
chamber enable fast conductance of heat to and from the TEC
assembly to the liquid PCR mix. As a comparison, a 50-.mu.m-thick
plastic slows the ramp rate down by .about.3.times..
[0201] SRI designed and tested a custom TEC assembly. We have
transitioned the design to RMTltd for fabrication. The custom SRI
assembly combines a heat sink, a TEC, a feedback sensor
(thermistor), and an aluminum nitride (AlN) thermal spreader that
surrounds the sensor. Each thermistor sensor is calibrated to allow
the HHA to compensate for any sensor manufacturing error
tolerance.
[0202] As a proof of concept for our PCR system, we created a
duplex PCR assay for the aggregative adherence fimbriae (AGG) and
Shiga toxin (STX2) genes associated with the E. coli O104:H4
pathogen from the 2011 German outbreak. The primer and probes were
selected for unique regions based on sequence analysis uploaded by
BGI shortly after the outbreak began. A laboratory assay using
benchtop equipment was used to establish probe selection and primer
optimization for AGG and STX2 targets. Once established, the assay
was transitioned to the SRI microfluidic PCR system. Since the SRI
PCR system takes advantage of the aluminum PCR chambers optimized
for heat conduction and temperature uniformity and the novel
thermistor-driven TEC design for rapid temperature cycling, a
custom master-mix formulation with adjuvants used to passivate
aluminum chambers allowed for duplex amplification of AGG and STX2
targets in 16 min.
[0203] Results provided an overlay of 40 PCR cycles (95.degree. C.
denature step, 62.degree. C. anneal and 73.degree. C. extension).
Each cycle lasts 21 s for 40 cycles, totaling 14 min of PCR
thermocycling. The remaining time is for initial denature and
uracil-DNA glycosylase (UNG) treatment. Our custom master mix
contains dUTPs as part of the amplification, and the UNG step
ensures there is no contamination between different uses of the
hardware platform. We tested the 16-min PCR at five different input
copy numbers (10, 50, 100, 500, and 1,000) and quantified the
amplification factor for both the AGG and STX2 genes. Additionally
we tested a 500 copy input using a 12-min PCR protocol and observed
that the cycle time was reduced from 21 s per cycle to 15 s. Again,
2 min of UNG treatment and initial denature were used, for a
cycling time of only 10 min. Table 5 shows the results of nested
PCR against amplicon standards to quantify the amplicons in each
sample and determine amplification factors. Overall we had an
amplification range of 1.6.times.10.sup.9 to 3.4.times.10.sup.11
for modular PCR. In addition, amplicons generated with modular PCR
systems have been detected with the SRI modular hybridization
system.
TABLE-US-00005 TABLE 5 Quantitation results of modular PCR samples.
Results indicate all samples would have >3.4 nM of amplicon in
the hybridization, which is above the 1-nM LOD. PCR AGG AGG STX2
STX2 STX2 Input PCR Total Amplicon Molarity AGG Amp Amplicon
Molarity Amp (copies) Time (min) Conc (ng/.mu.l) (nM) Factor Conc
(ng/.mu.l) (nM) Factor 500 12 5.2 55.8 2.33E+09 3.7 39.59 1.65E+09
10 16 0.1 1.0 2.08E+09 0.6 6.68 1.40E+10 50 16 76.5 816.2 3.41E+11
11.0 116.93 4.89E+10 100 16 110.6 1180.0 2.47E+11 26.6 284.01
5.93E+10 500 16 43.6 464.9 1.94E+10 11.5 122.62 5.12E+09 1000 16
129.6 1382.9 2.89E+10 34.3 365.57 7.64E+09
[0204] Detection.
[0205] To detect the presence of specific targets, the amplified
sample is hybridized to the microarray on the optical waveguide
chip. To facilitate hybridization, SSPE buffer from the reagent
block is first added to the amplified sample. Two metering chambers
are utilized to achieve the optimal ratio of sample to SSPE
buffer.
[0206] The SSPE and PCR product are mixed and then pushed into the
detection chamber, which incubates the sample over the microarray
on the optical waveguide chip. After 5 min of
temperature-controlled hybridization using our custom TEC assembly,
additional SSPE buffer flows into the detection chamber to wash out
the sample and remove unhybridized amplicons, followed by a rise in
temperature to remove cross-hybridization or any amplicons that are
nonspecifically bound to the wrong probes.
[0207] Finally, the DNA microarray is imaged using a custom
illumination, collection, and imaging optical block. The optics
block is designed as a standalone subcomponent that includes
everything necessary to laser illuminate and image the microarray
hybridization. The optics block can be pre-aligned and adjusted
before installation into the HHA. Once this pre-alignment is
performed, there is no need for further adjustment at the time of
installation.
[0208] The laser illumination optics consists of a line-generating
laser diode module and a turning mirror. The target for the laser
illumination is the grating on the microarray chip. The
line-generating laser diode emits a 10-mW beam at 635 nm in a
rectangular pattern to excite the Alexa Fluor 647 nm dye molecules
used to visualize hybridization to the microarray. The
line-generating diode module has focusing and line-generating
optics integrated into an off-the-shelf package (Coherent).
[0209] The microarray imaging optics comprises a folded 1:1 relay
lens and interference filters. The custom-designed relay lens group
is fast (at f/1.5) for maximum light-gathering capacity. It is
small in size (12 mm in diameter and <27 mm long). The relay
lens is designed to sufficiently collimate light into the two
interference filters (made of dielectric stacks) used to reject
laser excitation light and scattered light from reaching our
monochrome CCD imager.
[0210] The CCD chip and electronics are connected directly to the
microarray imaging optics to reduce noise and minimize signal loss.
The CCD is capable of sufficient readout speeds for microarray
analysis (4 frames/s), and sufficient signal-to-noise for
microarray imaging without the need for CCD cooling.
[0211] As a proof of concept for the optical system, we hybridized
the PCR amplicons generated by the SRI aluminum PCR chambers and
the TEC assemblies and read out the results using the described
optical system. The test protocol included spotted probes (as
identified by our rapid DNA synthesis instrument), on-board
incubation at 37.degree. C. for 5 min, multiple stringency washes
at increasing temperatures from 37.degree. C. to 60.degree. C., and
the optics package for automated image capture. We hybridized a
negative control with only control (A3) analyte, the resulting
amplicon from 10 copies into PCR, and the amplicon from 50 copies
into PCR. Assay results have proven selectivity by hybridizing
negative control samples and samples amplified from individual
primers for AGG and STX2. The images of the hybridization
demonstrated that the target probes are clearly visible in both the
10- and 50-input template cases and not present in the A3 control
case.
[0212] Low-Cost High-Volume Manufacturing of the iMFC
Consumable.
[0213] We developed a manufacturing process plan to drive down the
costs of iMFC fabrication for high-volume production. Because
injection molding is a simple, inexpensive method to create parts,
we injection-mold both the top and bottom layers of the card, as
well as the lysis module and reagent block. The remaining laminate
layers of the card are fabricated in an automated roll-to-roll
process, in which rolls of materials are laminated together, laser
cut, and then rewound into another roll, all on the same equipment
system in a rapid pipelined fashion. With high production volumes
on the order of millions of cards, the cost may become as low as
$10 per card.
[0214] DNA Detection Approach.
[0215] Our system has the capability to photolithographically
synthesize oligonucleotide arrays with all possible probes to an
amplicon in less than 10 hours, so we can select appropriate probes
and transition a bench PCR assay to our platform in about 2 weeks.
As shown herein, we have successfully demonstrated this capability
to develop an amplification assay, transition it to iMFC PCR, and
select probes for the E. Coli O104:H4.
[0216] Viral RNA Detection Approach.
[0217] RNA virus detection uses all the same modules as for DNA
detection, with a microfluidic assay for reverse-transcription of
an RNA virus genome and amplification. We have demonstrated proof
of concept of this approach using an assay that can distinguish
seasonal and swine influenza. The approach taken to develop the
influenza assay is generalizable, and can use the same approach for
select agent RNA viruses.
[0218] For identification of influenza, our first step was to
identify primers that would selectively amplify a segment of the
influenza A matrix gene as well as portions of the hemagglutinin
gene that distinguish H1 (swine) and H3 (seasonal) strains. Our
RT-PCR protocol involves a 5-min reverse-transcription (RT) step at
42.degree. C., where the reverse primers anneal to the RNA target
and initiate synthesis of the first DNA strand. A 2-min
reverse-transcriptase deactivation and simultaneous Taq polymerase
"hot start" then allows the forward primers to anneal to the first
DNA strand and synthesize the second DNA strand. At this point, 40
cycles of PCR commence with 10-s denature at 95.degree. C., 20-s
anneal at 62.degree. C., and a 5-s extension at 75.degree. C. Once
good amplification is achieved in a benchtop instrument we move to
our modular amplification system, which simulates amplification in
our iMFC. Because of fast temperature ramping in this system, the
RT-PCR protocol described above takes <33 min RT-PCR products
are evaluated first using gel electrophoresis and then on a
microarray. Gel electrophoresis results for modular amplification
of matrix and hemagglutinin genes for CA 2009 swine flu strain
(H1N1) and HK 68 seasonal strain H3N2 (a triplex amplification)
were obtained. The 244 bp amplicon indicates the influenza A matrix
gene. The hemagglutinin amplicon for the H1 strain is 173 base
pairs, while the H3 amplicon is 177 base pairs. Hybridization to
sequence specific probes on a DNA microarray gives unambiguous
differentiation of the H1 and H3 strains.
[0219] The first step in achieving sensitive and selective
detection on a microarray is preparation of a photolithographically
synthesized probe selection chip containing 20-25 mer probes that
cover the entire amplicon for our targets of interest. Our ability
to synthesize in less than a day all possible probes to an amplicon
of interest means we can empirically test for sensitive and
specific probes. Next we performed hybridization experiments to the
microarray to select the most sensitive and specific probes. A
selection of probes that readily distinguish the H1 and H3 were
identified; there are a few regions of the amplicon that easily
differentiate between the two amplicons. The best probes can be
fitted with an amine modification for spotting on epoxy
functionalized microscope slides or waveguide chips to be used in
our handheld device.
[0220] mRNA Detection Approach.
[0221] Gene expression profiling of peripheral mononuclear cells
(PBMCs) is a useful method of monitoring disease status,
environmental exposure, and pre-symptomatic diagnosis of infection.
Our iMFC capable of mRNA expression analysis uses a slightly
modified version of the hardware platform--an extra TEC and
increasing illumination uniformity on the microarray, and can
perform:
[0222] PBMC selection and lysis: using commercial size exclusion
filters for PBMC purification from whole blood, and the cell
selector module described herein.
[0223] mRNA purification: capture the mRNA from lysed cells using
microspheres coated with poly-T oligonucleotides.
[0224] T7 linear amplification and labeling: use the current PCR
chamber for T7 amplification and labeling and commercially
available kits.
[0225] Rapid Hybridization: use kinetic measurements of
hybridization to determine analyte concentration; reduce the time
required for gene expression hybridizations from many hours to
minutes.
[0226] T7 amplification leverages existing iMFC PCR modules and
commercially available kits.
[0227] mRNA Purification.
[0228] For mRNA purification we use a microfluidic purification
module that can hybridize mRNA to polyT coated beads, wash away
containments, and then release the mRNA from the polyT beads. We
use a mixer that keeps .about.5 .mu.m microsphere beads in solution
and a new TEC to melt the captured mRNA from the beads after a wash
step. We have tested the device to verify that 5-.mu.m beads move
well between the bellow mixing chamber and the filter. We have also
demonstrated that the fluid can be heated to .about.80.degree. C.
to allow for denaturing of the polyT:mRNA duplex.
[0229] Rapid Hybridization.
[0230] To decrease hybridization time and improve repeatability we
infer concentration from the kinetic rate parameter. In situations
where the number of target transcripts is in excess compared to the
number of probes, the signal value vs. time should follow a kinetic
curve:
Y=C(1-e.sup.-rt),r=k.sub.off+[A]k.sub.on
[0231] In the equation, Y is the background-subtracted signal;
k.sub.off and k.sub.on are kinetic parameters dependent on
temperature, gene sequence, and probe shift; [A] is the
concentration of target gene in solution; and C is a scale factor
depending on multiple factors including light intensity, probe
density, target concentration, and kinetic parameters. Our probe
selection technique estimates koff and kon by fitting the equation
to the time series of hybridization signals across multiple
concentrations. These estimates can be stored and used in real time
to estimate [A]. Because variations in light intensity and probe
synthesis density affect the parameter C but not the variables
inside the exponential, this kinetic technique significantly
improves chip-to-chip and same-chip replicate repeatability.
[0232] As a proof of concept, we have experimented with the kinetic
rate approach, starting with synthesized 25-base-pair oligomer
targets. In one probe's kinetic response to the A3 control labeled
oligonucleotide. Each line represents the response to a different
concentration of A3: 50 nM, 100 nM, 300 nM. A nonlinear
least-squares fit is used to estimate the parameters C and r for
each time series. Then, experiments at multiple concentrations [A]
reveal the affine relationship between r and [A].
[0233] We identify conditions that create a sensitive, repeatable
relationship between r and [A]. Our data show that the relationship
can occur in the right direction, with r=(0.0031, 0.0033, and
0.0058) s-1 for [A]=(50, 100, 300) nM. An additional benefit of
kinetic curve fitting is that even if the equilibrium value C
proves to be better than the kinetic rate r, the fitting process
averages out short-term noise. We also note that the ability to
measure kinetic parameters depends on the iMFC's use of an
evanescent wave to excite only fluorescent molecules that are bound
to the chip surface, giving a high signal-to-background ratio even
while the target solution is in place.
[0234] Protein Detection Approach.
[0235] One way to expand the detection capabilities of the iMFC
platform to include protein biomarkers is the microsphere
immunoassay, which uses antibodies labeled with an oligonucleotide
to create a nonlinear amplifiable DNA target when the target
antigen is present, and we have developed an immunoassay module
with the iMFC platform based on SRI's demonstrated microsphere
immune-PCR assay (MSiPCR). Biotinylated target-specific antibodies
are conjugated onto streptavidin-coated 6-.mu.m polystyrene beads
for initial capture of target antigen. Separate target-specific
antibodies are chemically conjugated with one oligonucleotide for a
second capture event of the target protein. Extensive washing
between the two protein capturing events wash away any unbound
target as well as free conjugates. The remaining bead portion is
then amplified using a specific labeled primer set along with a
taqman probe. The amplified nucleic acid signal is subsequently
hybridized onto a microarray for detection and readout. We have
migrated our benchtop assay to the iMFC platform: our
proof-of-concept modular system for the iMFC platform incorporates
a bellows mixer for washing and incubation steps, along with a
filter to catch the beads after nucleic acid amplification
[0236] System Improvements.
[0237] The optics block can be modified to provide a more uniform
pattern into the microarray grating, and to use 532 nm excitation
to improve detection signal-to-noise ratio. The process to switch
from a 635 nm to 532 nm excitation light source is only a matter of
changing the grating pitch.
[0238] This modified optics block consists of both laser target
illumination optics and microarray imaging optics. The laser target
illumination optics uses a small diode-pumped solid-state (DPSS)
laser module, scanning and focusing optics, and a 2D scanning
microelectromechanical systems (MEMS) mirror. The DPSS laser module
can output a 40-mW laser beam at 532 nm for exciting Cy3 dye
molecules. The beam can be focused on the MEMS mirror using the
focusing optic. The 2D MEMS mirror can scan the spot over a
rectangle and aim the beam at the grating. After reflecting off the
scanning mirror, the beam hits the scan lens, which collimates the
beam before it enters the grating on the microarray chip.
[0239] This system scans the same laser spot over the entire
grating, and thus over the entire microarray. This eliminates
illumination nonuniformity from the line-generating optics which
can vary as much as 25% in intensity. The scan lens also allows us
to collimate the beam before it enters the microarray chip grating.
We validated with a proof-of-concept demonstration of the new
scanning technical approach. The microarray imaging optics are
unchanged from the current optics block, with the exception of
filters for the different excitation (532 nm) and emission (570 nm)
wavelengths.
REFERENCES
[0240] 1. Niemz, A., T. M. Ferguson, and D. S. Boyle, Point-of-care
nucleic acid testing for infectious diseases. Trends Biotechnol,
2011. 29(5): p. 240-50. [0241] 2. Bissonnette, L. and M. G.
Bergeron, Infectious Disease Management through Point-of-Care
Personalized Medicine Molecular Diagnostic Technologies. Journal of
Personalized Medicine, 2012. 2(4): p. 50-70. [0242] 3. Foudeh, A.
M., et al., Microfluidic designs and techniques using lab-on-a-chip
devices for pathogen detection for point-of-care diagnostics. Lab
Chip, 2012. 12(18): p. 3249-66. [0243] 4. Easley, C. J., et al., A
fully integrated microfluidic genetic analysis system with
sample-in-answer-out capability. Proc Natl Acad Sci USA, 2006.
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all-in-one cartridge for sample preparation and real-time PCR in
rapid influenza diagnosis. Lab Chip, 2010. 10(22): p. 3103-3111.
[0245] 6. Ferguson, B. S., et al., Genetic analysis of H1N1
influenza virus from throat swab samples in a microfluidic system
for point-of-care diagnostics. J Am Chem Soc, 2011. 133(23): p.
9129-35. [0246] 7. Lam, B., et al., Polymerase chain reaction-free,
sample-to-answer bacterial detection in 30 minutes with integrated
cell lysis. Anal Chem, 2012. 84(1): p. 21-5. [0247] 8. Chen, D., et
al., An integrated, self-contained microfluidic cassette for
isolation, amplification, and detection of nucleic acids. Biomed
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[0250] Embodiments of our claimed invention include:
[0251] 1. An automated nucleic acid analysis system comprising in
microfluidic connection sample lysis, purification, PCR and
detection modules configured to detect in parallel distinct nucleic
acid sequences via multiple sequence amplification and simultaneous
microarray hybridization readout.
[0252] 2. The system of a foregoing or subsequent claim wherein:
the detection module comprises microarray detection optics
comprising a microarray scanner employing evanescent wave
excitation and detection; the detection module comprise an
automated hybridization processor configured to provide multiple
stringencies via temperature; and/or the PCR module is configured
to perform reverse transcription and PCR in a single reaction.
[0253] 3. The system of a foregoing or subsequent claim comprising
an integrated microfluidics card comprising the modules and an
analyzer comprising a receptacle configured to receive the card,
operators configured to operate the card, and a controller
configured to electronically control the operators, the operators
comprising fluidic actuators, PCR thermal cycler, and automated
hybridization processor and microarray detection optics.
[0254] 4. The system of a foregoing or subsequent claim further
comprising a reagent module configured to contain and deliver
reagents to the lysis, purification, PCR and detection modules.
[0255] 5. The system of a foregoing or subsequent claim that
is:
[0256] portable: less than 2000, 1000 or 500 in.sup.3 and less than
50, 25 or 10 lbs;
[0257] rapid: analysis in less than 60, 120, 180 or 240
minutes;
[0258] multiplex: simultaneous analysis of more than 10, 50, 500
target sequences; and/or
[0259] automated: requiring no user intervention between sample
introduction and result display.
[0260] 6. The system of a foregoing or subsequent claim
wherein:
[0261] the sample comprises protein analytes and the system is
further configured to tag the protein analytes with tags comprising
the nucleic acid sequences;
[0262] anchored probes define the sequences by their spatial
locations;
[0263] the amplification is effected/achieved by a number of
primers pairs less than the number of sequences being analyzed;
[0264] the distinct nucleic acid sequences are of multiple
species/organisms;
[0265] the PCR module comprises a metallic (e.g. aluminum) PCR
reaction chamber;
[0266] the microfluidic connection comprises a breathable membrane
configured for bubble removal, wherein the breathable membrane is
underneath the channel layer, so the entire channel can be exposed
to atmospheric pressure (in a particular embodiment, this membrane
spans the card because it is easier to manufacture it as a layer
than individual pieces, though it is only functional under the
channel layers);
[0267] amplification is fully contained in the consumable (no open
tubes, etc.); and/or
[0268] detection is based on probe sets rather than primer sets
(easier to build new tests).
[0269] 7. The system of a foregoing or subsequent claim configured
to:
[0270] amplify in a single vessel (no sample splitting);
[0271] receive and process analyte samples of blood, saliva, GI
samples, urine, wound swabs, spinal tap, nasal swabs, veterinary
and agricultural sources;
[0272] receive samples via a specimen collection tool or transport
media;
[0273] process sample volumes between 1-100 ul;
[0274] be modular (modules can be interchanged to support different
applications);
[0275] be capable of metering (done by channel dimensions and
bubble removal); and/or
[0276] be one directional and self-sealing (prevents sample cross
contamination).
[0277] 8. The system of a foregoing or subsequent claim comprising
an integrated microfluidics card comprising the modules and an
analyzer comprising a housing (box) and within the housing
receptacle configured to receive the card, wherein the
analyzer:
[0278] engages the card to perform the lysis, purification, PCT
(amplification and labeling), and detection;
[0279] interacts with the sample via pressure (e.g. sample
transport), magnetic fields (e.g. sample mixing), temperature (e.g.
amplification, stringency, hybridization) and/or light (e.g.
hybridization detection); and/or
[0280] performs the detection by coupling an evanescant wave with
the sample to observe hybridizations in real time and/or
determining kinetics and possible base-pair mismatch which result
in sequence information.
[0281] 9. The system of a foregoing or subsequent claim comprising
an integrated microfluidics card (cartridge) comprising the
modules, wherein the card is configured:
[0282] to be specific to disease type (ex. respiratory
illnesses);
[0283] to be specific to patient type (ex. pediatric);
[0284] to be specific to pathogen type (ex. biowarfare agents);
[0285] to be specific to individual (ex. pharmacogenomics);
[0286] to contain unique identifiers for patient-specific
information;
[0287] for one-time use to maintain sterility and minimize
cross-contamination;
[0288] to be produced using roll-to-roll manufacturing steps;
and/or from a polycarbonate chassis, metallic foil PCR chambers,
acrylic components, breathable membrane materials, and/or
polyurethane seals.
[0289] 10. The system of a foregoing claim functionally integrated
with a microfluidic fluorescence-activated cell sorter (.mu.FACS)
configured to provide hydrodynamic and/or inertial focusing for
particle or cell alignment and comprising microscale electroactive
polymer (EAP) actuators configured for sorting.
[0290] 11. A method comprising using the system of a foregoing
claim to detect in parallel distinct analyte nucleic acid sequences
via multiple sequence amplification and simultaneous microarray
hybridization readout.
Detailed Description of Additional Embodiments and Examples
Thereof: High-Performance Fluorescence-Activated Sorting (FACS) for
Novel Point-of-Care, Portable, and Highly-Integrated
Applications
[0291] High-end commercial FACS instruments achieve sort rates of
10,000 to 40,000 sorts/s by electrostatically deflecting cells
contained within charged droplets. [1, 2] They can be applied to a
wide range of applications, for example, isolation of
cancer-targeting T cells for immunotherapy, enrichment of stem
cells for tissue engineering, and separation of specific cells for
manipulation or further analysis (e.g., DNA sequencing, RNA
expression, and fluorescence in-situ hybridization). However, they
are large, expensive instruments that require expert operators, and
thus are not suitable for point-of-care or portable applications.
Additionally, their monolithic nature means they cannot be easily
integrated with other instruments or processes.
[0292] In an attempt to address these limitations, researchers have
developed many varieties of microfluidic FACS. Until recently,
these devices were slow (more than an order of magnitude slower
than conventional FACS), and most were difficult to fabricate or
otherwise inappropriate for manufacturing and practical
application. With the development of pulsed laser-activated cell
sorting (PLACS), the microfluidic state of the art increased to
.about.10,000 cells/s with high purity [3]. In PLACS, cells are
sorted using a fluid jet produced by a rapidly expanding and
contracting plasma bubble. While the fluidic device is simple,
inexpensive, and disposable, the system requires a
high-repetition-rate pulsed laser, which is both expensive and
large. Thus, PLACS fails to address the challenges of scale and
expense.
[0293] We disclose a simpler approach to fluid actuation based on
EAPs, which are polymers that change shape in response to
electrical stimulation. They have been used in microfluidic devices
to change the cross-sectional geometry of channels, generating
small injections for electrophoretic separations [4], modifying the
fluidic resistance of a channel and clearing blockages [5]. We have
developed a novel, high-performance microfluidic EAP (.mu.EAP)
actuator and integrated it into a micro-FACS.
[0294] Electroactive Polymer Actuation.
[0295] Our fluidic actuator comprises a dead-end fluid chamber in
which one or more surfaces comprise an EAP. In one implementation
the floor of the chamber is an electrode covered with a thin
(.about.12 .mu.m) EAP layer of dielectric elastomer (silicone).
Conceptually, the silicone acts like a flexible capacitor. It
distorts when a voltage is applied to the electrode, increasing the
chamber volume and drawing fluid into the chamber. When the voltage
is released, the silicone relaxes and pushes fluid from the
chamber.
[0296] EAP actuators are easily fabricated using proven microscale
manufacturing techniques. To create actuators, we pattern
electrodes onto an indium-tin-oxide-coated slide, which becomes the
base substrate. We then spin a layer of uncured silicone onto the
slide and thermally cure it. To create the channel layer, we mold
microfluidic channels using conventional soft lithography. We then
complete the devices by aligning and plasma-bonding the channel
layer to the silicone-coated slide. The actuators' compatibility
with soft lithography means they can be readily integrated into a
large existing library of microfluidic devices. It also enables
rapid prototyping. The devices are inexpensive because they require
only a voltage source for actuation, and the fabrication approach
is amenable to low-cost manufacturing.
[0297] In particular exemplification we fabricated actuators <1
mm.sup.2 that demonstrate response times of 10 .mu.s; however, the
actuator size can also be micron scale (e.g. less than 1, 10 or 100
.mu.m.sup.2) and response times can be less than 10, 1, 0.1 .mu.s.
that shorter response times. Their size options makes them
particularly amenable to both handheld and integrated applications
and for parallel operation to increase.
[0298] The rapid response rate of these exemplifed EAP actuators
(<20 .mu.s) also indicates that they sorting rates of >25,000
cells/s. The devices are inexpensive because they require only a
voltage source for actuation and are built using low-cost
micro-fabrication techniques. The use of silicone as the polymer
enables the simple integration of EAP actuators with microfluidic
channels via soft lithography. This allows both rapid prototyping
and the potential for scale-up to manufacturing quantities. EAP
.mu.FACS delivers throughput equivalent to benchtop FACS in a
handheld format.
[0299] We have optimized the performance of the EAP .mu.FACS and
incorporated the improved device into a cell-concentration/sorting
module that integrates with the iMFC system. The module allows the
system to increase the operation envelope by either concentrating a
dilute cell concentration in a large volume (e.g., bacteria present
in environmental samples at a few cells per ml) or sorting out
select cells from a background of many cells (e.g., activated T
cells from a population of peripheral blood mononuclear cells.
Embodiments of the microsorter are further described below.
[0300] Sorter Design.
[0301] Sorters perform three key functions: alignment, detection,
and sorting. Our designs (e.g. FIG. 9) incorporate multiple
innovations, including the combination of hydrodynamic and inertial
focusing for alignment, and the use of EAP actuation for rapid
sorting. In conventional cell sorters, particles or cells are
focused both horizontally and vertically using a coaxial sheath
flow that pinches the sample stream in a tight line. Since most
microfluidic devices are planar, they can only focus particles in
one direction (i.e., horizontal); multilayer devices can focus
particles vertically or laterally as well but are significantly
more complex to manufacture. Without vertical focusing, particles
can be distributed across the height of the channel, leading to
variations in velocity and overlap. In our designs, we align
horizontally in the cross region via hydrodynamic focusing and
vertically in the long "neck" via inertial focusing. Inertial
focusing occurs when the fluid velocity is sufficiently high to
generate lift forces on particles [6]. The combination allows us to
effectively align particles in a single-layer device, which retains
our simple manufacturing approach.
[0302] For sorting, we first detect particles using fluorescence,
where upon detection of a targeted particle, a voltage pulse is
applied to one or more EAP actuators. The actuators create a
transient cross flow that deflects targeted particles onto a new
pathline that leads to the sort outlet, as shown in FIG. 9.
[0303] FIG. 9. depicts (a) EAP microsorter schematic showing the
channel layout of the .mu.EAP FACS. The inset images illustrate the
main functions of the channel. Horizontal alignment is accomplished
by hydrodynamic focusing (left), while vertical alignment is
achieved via inertial focusing (middle). Finally, targeted
particles are sorted via the .mu.EAP actuators (right). The
extended exposure image shows streaks from an unsorted and sorted
particle. The flow rate was 8 .mu.l/min (107 mm/s), and the
actuation pulse was 1 ms at 400 V.
[0304] The use of paired actuators that operate 180.degree. out of
phase significantly improves performance by doubling the force
applied to the fluid and reducing fluidic resistance, which is
proportional to channel length. With two actuators, one "pulls"
while the other "pushes" the fluid, and the fluid is displaced only
along the short distance between the actuators (typically .about.2
mm) In contrast, with a single actuator, the fluid displacement
occurs from the actuator to the device outlets (.about.30 mm).
[0305] To test our .mu.FACS devices, we use a detection and control
system, which consists of an epifluorescent microscope,
charge-coupled device (CCD) camera, photomultiplier tubes,
field-programmable gate array (FPGA)-based data acquisition system,
and voltage amplifiers.
[0306] Initial Particle Separation Demonstration.
[0307] To demonstrate the separation capabilities of the .mu.EAP
sorter, we sorted a mixture of green (7 .mu.m) and red (5 .mu.m)
fluorescent particles by gating on the green fluorescence signal
and applying a 620-V, 500-.mu.s pulse to the EAP actuators. The
fluid flow rate was 10 .mu.l/min, which resulted in a mean linear
velocity of 133 mm/s Unsorted cells followed their default path to
the waste channel, while sorted cells were deflected to a pathline
that exited through the sort channel (FIG. 10). We captured
10-.mu.l fluid volumes from both channel outlets with sorting
disabled and enabled. The volumes were imaged in separate manual
cytometers. Based on the cytometer results, we estimated a purity
of 100% and yield of 93%.
[0308] Actuator Performance Optimization.
[0309] Following our initial proof of concept, we initiated a
design study of the EAP fluidic actuators to improve our sorting
throughput. We developed simplified electromechanical models of the
actuators using COMSOL Multiphysics. Our results indicated that
most actuation occurs at the perimeter of the devices. Based on
these results, we developed a range of actuator designs and
empirically tested their performance. By increasing the applied
voltage and modifying the actuator geometry, we were able to
improve the performance of the EAP actuators. We successfully
sorted particles at a flow rate of 30 .mu.l/min (400 mm/s) with a
25-.rho.s, 800 V pulse, a 20-fold improvement over the sorter used
in our initial particle separation demonstration.
[0310] Cell-Sorting Demonstration.
[0311] To demonstrate cell separation within the .mu.EAP FACS, we
prepared a sample of mouse lymphocytes with fluorescently labeled B
cells. The white blood cells were separated via centrifugation and
then fixed prior to labeling with phycoerythrin-conjugated B220
antibody. The sample was input into the FACS. FIG. 11 shows an
image of a sorted B cell. Sorting was performed with a 100-.mu.s,
800-V pulse, at 11 .mu.l/min (147 mm/s). Note that the fluorescent
streak is brightest in the center. Since the labeled cells are
significantly dimmer than fluorescent particles, we used a 488-nm
diode laser to illuminate the cell in the detection region, while
the dimmer light-emitting diode (LED) lamp provided
full-field-of-view illumination. The bright spots to the right are
cells that were trapped on the wall by a contaminating
filament.
[0312] Multi-Sorter Integration.
[0313] Due to its straightforward fabrication and compatibility
with soft lithography, the .mu.EAP sorter can be easily integrated
into more complex devices. To illustrate the integration
capabilities of our EAP actuators, we developed additional devices
featuring multiple independent sorters. FIG. 12 shows parallel
sorting in a dual-channel device, wherein extended time exposure
shows two particles independently sorted in parallel channels, and
FIG. 13 shows dual-stage serial sorting into multiple outlets,
wherein extended time exposure shows two particles sorted by two
serial sorters into one of three bins--note: Bin 3 was the default
(unsorted) bin. Higher order multiple channel parallel and
multistage serial sorting configurations are analogously
constructed.
[0314] Our microfluidic fluorescence-activated cell sorter
(.mu.FACS) provides all the functions needed to sort particles:
aligns the particles, detects their fluorescent signals, makes sort
decisions based on fluorescence, and then sorts appropriately.
Demonstrated capabilities include: particle sorting with multiple
actuator pulse lengths as low as 20 .mu.s; sorting with both
multiple- and single-actuator configurations; independent parallel
sorting in a multiple channel sorter; sequential sorting in a
multi-stage sorters. Benefits of micro electroactive polymer
(.mu.EAP) sorters include: (a) Sorting is rapidly triggered via a
simple electrical input (20 .mu.sec sorting demonstrated); (b)
.mu.EAP actuators are compatible with a variety of microfabrication
techniques; and (c) .mu.EAP sorters are easily integrated,
parallelizable, and well-suited for portable-scale devices
REFERENCES
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intersection of flow cytometry with microfluidics and
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[0318] [4] A. K. Price, K. M. Anderson, and C. T. Culberson,
"Demonstration of an integrated electroactive polymer actuator on a
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[0321] Embodiments of our claimed invention include:
[0322] 1. A high performance microfluidic electroactive polymer
(.mu.EAP) actuator configured about a flow channel wherein a
voltage pulse applied to the actuator induces the actuator to
create across the flow channel a transient cross flow that deflects
targeted particles within the flow channel onto a new pathline,
wherein the actuator comprises a dead-end fluid chamber in which a
surface of the chamber comprises an electrode covered with an EAP
layer of dielectric elastomer.
[0323] 2. A plurality of actuators according to claim 1 configured
about the flow channel and out of phase with each other, wherein a
voltage pulse applied to the actuators induces the actuators to
create across the flow channel a transient cross flow that deflects
targeted particles within the flow channel onto a new pathline,
wherein each actuator comprises a dead-end fluid chamber in which a
surface of the chamber comprises an electrode covered with an EAP
layer of dielectric elastomer.
[0324] 3. A pair of actuators according to claim 1 configured about
the flow channel and 180.degree. out of phase with each other,
wherein a voltage pulse applied to the actuators induces the
actuators to create across the flow channel a transient cross flow
that deflects targeted particles within the flow channel onto a new
pathline, wherein each actuator comprises a dead-end fluid chamber
in which a surface of the chamber comprises an electrode covered
with an EAP layer of dielectric elastomer.
[0325] 4. The actuator(s) of a foregoing or subsequent claim
wherein a plurality of surfaces of the chamber(s) comprise an
electrode covered with a EAP layer of dielectric elastomer.
[0326] 5. The actuator(s) of a foregoing or subsequent claim
wherein the flow channel is configured to provide a combination of
hydrodynamic focusing for horizontal alignment and inertial
focusing for vertical alignment of the particles.
[0327] 6. The actuator(s) of a foregoing or subsequent claim
wherein the new pathline leads to a sort outlet.
[0328] 7. The actuator(s) of a foregoing or subsequent claim
wherein the flow channel comprises a sample input channel and
sorted and unsorted output channels and the new pathline leads to
the sorted output channel.
[0329] 8. The actuator(s) of a foregoing or subsequent claim
wherein the flow channel is configured for fluorescence detection,
whereupon detection of the targeted particles, the voltage pulse is
applied to the .mu.EAP actuators.
[0330] 9. The actuator(s) of a foregoing or subsequent claim
wherein the EAP layer is 1-50 (or 2-25, or 5-15 .mu.m thick).
[0331] 10. The actuator(s) of a foregoing or subsequent claim
wherein the elastomer is silicone.
[0332] 11. The actuator(s) of a foregoing or subsequent claim
configured to provide parallel sorting in a multi-channel
device.
[0333] 12. The actuator(s) of a foregoing or subsequent claim
configured to provide multi-stage serial sorting into multiple
outlets.
[0334] 13. The actuator(s) of a foregoing claim functionally
integrated in a fluorescence-activated particle sorter.
[0335] 14. A method using the actuator(s) of a foregoing claim
comprising the step of applying a voltage pulse to induce the
actuator(s) to create across the flow channel a transient cross
flow that deflects targeted particles within the flow channel onto
a new pathline.
[0336] The invention encompasses all combinations of recited
particular and preferred embodiments. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims. All publications, patents, and patent
applications cited herein, including citations therein, are hereby
incorporated by reference in their entirety for all purposes.
* * * * *