U.S. patent application number 13/590051 was filed with the patent office on 2012-12-13 for universal sample preparation system and use in an integrated analysis system.
This patent application is currently assigned to IntegenX, Inc.. Invention is credited to luliu I. Blaga, Stevan B. Jovanovich, Michael Van Nguyen, William D. Nielsen, Mattias Vangbo.
Application Number | 20120315635 13/590051 |
Document ID | / |
Family ID | 41016643 |
Filed Date | 2012-12-13 |
United States Patent
Application |
20120315635 |
Kind Code |
A1 |
Vangbo; Mattias ; et
al. |
December 13, 2012 |
Universal Sample Preparation System And Use In An Integrated
Analysis System
Abstract
The invention provides for devices and methods for interfacing
microchips to cartridges and pneumatic manifolds. The cartridges,
microchips, and pneumatic manifolds can be integrated with
downstream preparation devices, such as thermal regulating devices
and separation and analysis devices.
Inventors: |
Vangbo; Mattias; (Fremont,
CA) ; Nielsen; William D.; (San Jose, CA) ;
Blaga; luliu I.; (Fremont, CA) ; Nguyen; Michael
Van; (San Diego, CA) ; Jovanovich; Stevan B.;
(Livermore, CA) |
Assignee: |
IntegenX, Inc.
Pleasanton
CA
|
Family ID: |
41016643 |
Appl. No.: |
13/590051 |
Filed: |
August 20, 2012 |
Related U.S. Patent Documents
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Application
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Patent Number |
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12321594 |
Jan 21, 2009 |
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13590051 |
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61140602 |
Dec 23, 2008 |
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61022722 |
Jan 22, 2008 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
B01L 2300/1822 20130101;
G01N 1/40 20130101; B01L 2200/10 20130101; B01L 7/52 20130101; G01N
35/1095 20130101; B01L 2300/0819 20130101; B01L 2200/0647 20130101;
B01L 2400/043 20130101; B01L 2300/1827 20130101; G01N 27/44791
20130101; B01L 2200/04 20130101; G01N 1/34 20130101; Y10T
436/143333 20150115; G01N 2035/00158 20130101; B01D 17/06 20130101;
B01L 2200/027 20130101; B01L 3/50273 20130101; Y10T 436/25375
20150115; B01L 2300/087 20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
G01N 27/26 20060101
G01N027/26; G01N 21/64 20060101 G01N021/64 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] Aspects of this invention were made with government support
under one or more of Project No. W911SR-04-P-0047 awarded by the
Department of Defense and Grant No. 5R01HG003583 awarded by the NM.
The government may have certain rights in this invention.
Claims
1.-124. (canceled)
125. An integrated biochip system comprising: (a) a biochip
comprising a plurality microfluidic systems, wherein each
microfluidic system comprises a first reaction chamber in
microfluidic communication with a separation chamber, wherein the
first reaction chamber is adapted for: (i) nucleic acid extraction;
(ii) nucleic acid purification; (iii) pre-nucleic acid
amplification cleanup; (iv) nucleic acid amplification; (v)
post-nucleic acid amplification cleanup; (vi) pre-nucleic acid
sequencing cleanup; (vii) nucleic acid sequencing; (viii)
post-nucleic acid sequencing cleanup; (ix) reverse transcription;
(x) pre-reverse transcription cleanup; (xi) post-reverse
transcription cleanup; (xii) nucleic acid ligation; (xiii) nucleic
acid hybridization; or (xiv) quantification; and the separation
chamber comprises a detection position; and (b) a separation and
detection system comprising, (i) a separation element for
simultaneously separating a plurality of target analytes in the
separation chamber; (ii) one or more light sources positioned to
illuminate the detection positions on the biochip; (iii) a mirror
to scan said one or more light sources sequentially between said
detection positions; (iv) one or a plurality of first optical
elements positioned for collecting and directing light emanating
from the detection positions; and (v) a light detector positioned
to accept light directed from the one or plurality of first optical
elements, wherein the light detector comprises a wavelength
dispersive element to disperse the light from the one or plurality
of first optical elements according to light wavelength into at
least 6 wavelength components and, the wavelength dispersive
element is positioned to provide at least a portion of the
dispersed at least 6 wavelength components to at least 6 detection
elements, wherein each of the detection elements is in
communication with a first control element for simultaneously
collecting detection information from each of the detection
elements; and wherein said light detector detects fluorescence from
at least 6 dyes labeled to one or more biological molecules, each
dye having a unique peak wavelength.
126. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for nucleic acid extraction.
127. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for nucleic acid purification.
128. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for nucleic acid amplification.
129. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for cleanup.
130. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for nucleic acid sequencing.
131. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for reverse transcription.
132. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for nucleic acid ligation.
133. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for nucleic acid hybridization.
134. The integrated biochip system of claim 125, wherein the first
reaction chamber is adapted for quantification.
135. An integrated biochip system comprising: (a) a biochip
comprising a plurality microfluidic systems, wherein each
microfluidic system comprises a first reaction chamber in
microfluidic communication with a separation chamber, wherein the
first reaction chamber is adapted for (i) nucleic acid extraction;
(ii) nucleic acid purification; (iii) pre-nucleic acid
amplification cleanup; (iv) nucleic acid amplification; (v)
post-nucleic acid amplification cleanup; (vi) pre-nucleic acid
sequencing cleanup; (vii) nucleic acid sequencing; (viii)
post-nucleic acid sequencing cleanup; (ix) reverse transcription;
(x) pre-reverse transcription cleanup; (xi) post-reverse
transcription cleanup; (xii) nucleic acid ligation; (xiii) nucleic
acid hybridization; or (xiv) quantification; and the separation
chamber comprises a detection position; and (b) a separation and
detection system comprising, (i) a separation element for
simultaneously separating a plurality of biological molecules
comprising DNA sequences, in the separation chamber; (ii) one or
more light sources positioned to illuminate the detection positions
on the biochip; (iii) a mirror to scan said one or more light
sources sequentially between said detection positions; (iv) one or
a plurality of first optical elements positioned for collecting and
directing light emanating from the detection positions; and (v) a
light detector positioned to accept light directed from the one or
plurality of first optical elements, wherein the light detector
comprises a wavelength dispersive element to disperse the light
from the one or plurality of first optical elements according to
light wavelength into at least 6 wavelength components, and the
wavelength dispersive element positioned to provide at least a
portion of the dispersed at least 6 wavelength components to at
least 6 detection elements, wherein each of the detection elements
is in communication with a first control element for simultaneously
collecting detection information from each of the detection
elements; and wherein said light detector detects fluorescence from
at least 8 dyes labeled to one or more DNA sequences, each dye
having a unique peak wavelength, said dyes being members of at
least two 4-dye containing subsets, such that said dye sets are
capable of detecting at least two DNA sequences in a single
channel, wherein the number of dyes is a multiple of four, and the
number of DNA sequences to be detected is equal to one quarter of
the multiple, such that each of the different dyes is present in
only one subset.
136. A system, comprising: (a) a microchip comprising a plurality
microfluidic systems, wherein each microfluidic system comprises a
first reaction chamber in microfluidic communication with a
separation chamber, wherein the first reaction chamber is adapted
for: (i) nucleic acid extraction; (ii) nucleic acid purification;
(iii) pre-nucleic acid amplification cleanup; (iv) nucleic acid
amplification; (v) post-nucleic acid amplification cleanup; (vi)
pre-nucleic acid sequencing cleanup; (vii) nucleic acid sequencing;
(viii) post-nucleic acid sequencing cleanup; (ix) reverse
transcription; (x) pre-reverse transcription cleanup; (xi)
post-reverse transcription cleanup; (xii) nucleic acid ligation;
(xiii) nucleic acid hybridization; or (xiv) quantification; and the
separation chamber comprises a detection position; and (b) a
separation and detection system comprising: (i) a separation
element for simultaneously separating a plurality of target
analytes in the separation chamber; (ii) an optical assembly
positioned to illuminate the detection positions on the microchip,
and for collecting and directing light emanating from the detection
positions; and (iv) a light detector positioned to accept light
directed from the optical assembly, wherein said light detector is
in communication with a processor for simultaneously collecting
detection information from said light detector; and wherein said
light detector detects fluorescence from a plurality of dyes
labeled to one or more biological molecules, each dye having a
unique peak wavelength.
137. The system of claim 136, wherein the first reaction chamber is
adapted for nucleic acid extraction.
138. The system of claim 136, wherein the first reaction chamber is
adapted for nucleic acid purification.
139. The system of claim 136, wherein the first reaction chamber is
adapted for nucleic acid amplification.
140. The system of claim 136, wherein the first reaction chamber is
adapted for cleanup.
141. The system of claim 136, wherein the first reaction chamber is
adapted for nucleic acid sequencing.
142. The system of claim 136, wherein the first reaction chamber is
adapted for reverse transcription.
143. The system of claim 136, wherein the first reaction chamber is
adapted for nucleic acid ligation.
144. The system of claim 136, wherein the first reaction chamber is
adapted for nucleic acid hybridization.
145. The system of claim 136, wherein the first reaction chamber is
adapted for quantification.
146. A system, comprising: (a) a microchip comprising a plurality
microfluidic systems, wherein each microfluidic system comprises a
first reaction chamber in microfluidic communication with a
separation chamber, wherein the first reaction chamber is adapted
for (i) nucleic acid extraction; (ii) nucleic acid purification;
(iii) pre-nucleic acid amplification cleanup; (iv) nucleic acid
amplification; (v) post-nucleic acid amplification cleanup; (vi)
pre-nucleic acid sequencing cleanup; (vii) nucleic acid sequencing;
(viii) post-nucleic acid sequencing cleanup; (ix) reverse
transcription; (x) pre-reverse transcription cleanup; (xi)
post-reverse transcription cleanup; (xii) nucleic acid ligation;
(xiii) nucleic acid hybridization; or (xiv) quantification; and the
separation chamber comprises a detection position; and (b) a
separation and detection system comprising: (i) a separation
element for simultaneously separating a plurality of biological
molecules comprising DNA sequences, in the separation chamber; (ii)
an optical assembly positioned to illuminate the detection
positions on the microchip, and for collecting and directing light
emanating from the detection positions; and (iv) a light detector
positioned to accept light directed from the optical assembly,
wherein the light detector comprises a wavelength dispersive
element to disperse the light from the one or plurality of first
optical elements according to light wavelength into at least 6
wavelength components, and the wavelength dispersive element
positioned to provide at least a portion of the dispersed at least
6 wavelength components to at least 6 detection elements, wherein
said light detector is in communication with a processor for
simultaneously collecting detection information from said light
detector; and wherein said light detector detects fluorescence from
a plurality of dyes labeled to one or more DNA sequences, each dye
having a unique peak wavelength, said dyes being members of at
least two 4-dye containing subsets, such that said dye sets are
capable of detecting at least two DNA sequences in a single
channel, wherein the number of dyes is a multiple of four, and the
number of DNA sequences to be detected is equal to one quarter of
the multiple, such that each of the different dyes is present in
only one subset.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/022,722, filed Jan. 22, 2008 and U.S.
Provisional Application No. 61/140,602, filed Dec. 23, 2008, which
are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] Sample preparation is a ubiquitous problem in biological
analytical systems. The issue of providing sufficiently purified
targets from diverse raw sample types to reliably perform
downstream analytical assays is pervasive and covers cell biology,
genomics, proteomics, metabolomics, food biology, molecular
diagnostics, and many other biological and medical assays. While
many advances in sample preparation have been made the chief
solution has been to develop reagents that are used manually or in
robotic systems that use rectilinear stages or multi-axis arms to
manipulate samples.
[0004] Microfluidics and nanofluidics allow miniaturized sample
volumes to be prepared for analysis. Advantages include the
nanoscale consumption of reagents to reduce operating costs and
full automation to eliminate operator variances. Microfluidic
sample preparation can either interface with existing or future
detection methods or be part of a completely integrated system. In
the present application, methods and apparatuses are disclosed that
integrate full volume sample preparation with volumes over 10 mL
with microliter and smaller volumes for sample preparation and
analysis.
[0005] Starting from the sample, the present invention can be
applied to concentrate, and pre-separate components for further
processing to detect and classify organisms in matrices comprising
aerosol samples, water, liquids, blood, stools, nasal, buccal and
other swabs, bodily fluids, environmental samples with analysis by
ELISA, PCR or other nucleic acid amplification techniques, single
molecule detection, protein arrays, mass spectroscopy, and other
analytical methods well known to one skilled in the art.
[0006] Microfluidic nucleic acid purification can be performed to
prepare the sample for nucleic acid assays. For DNA analysis, PCR
amplification is one current method. Microarray DNA, RNA and
protein analysis also requires extensive sample preparation before
the sample can be applied to the microarray for reaction and
readout.
[0007] Samples can be obtained by a wide variety of substrates and
matrices. The matrix may contain complex mixtures including
inhibitory compounds such as hemes, indigo, humic acids, divalent
cations, and proteins etc that interfere with DNA-based
amplification. Aerosols can contain large amounts of molds, metals,
and soils humic and other acids that all interfere with PCR
amplification--the gold standard.
[0008] Early work showed that as few as three seeded organisms
could be detected from diluted samples of soil extracts followed by
PCR amplification of two 16S ribosomal gene fragments.
Low-melting-temperature agarose has been used to extract DNA from
soil samples for 16S and 18S rDNA PCR amplification using universal
primers. Spun separation gels in column format can be used, such as
Sephadex columns. Multistep purifications such as organic
extractions combined with Sephadex columns were developed. Bead
beating was found to be an effective way to prepare samples for
high numbers of organisms and grinding in liquid nitrogen to detect
low numbers of organisms. While these methods are effective they
were best suited for research laboratory environments.
[0009] Solid phase extractions to columns, beads, and surfaces can
be used to purify DNA before DNA analysis. Proteinase K followed by
a Qiagen QIA Amp silica-gel membrane columns and IsoCode Stix, an
impregnated membrane-based technology, followed by heating, washing
and a brief centrifugation were compared for B. anthracis Sterne
vegetative cells in buffer, serum, and whole blood and spores in
buffer and found to work well.
[0010] A variety of separations can be performed using the devices
and methods of the invention. For example, the devices and methods
of the invention can be used to perform chromatography, phase-based
or magnetic-based separation, electrophoresis, distillation,
extraction, and filtration. For example, a microfluidic channel or
a capillary can be used for chromatography or electrophoresis. As
well, beads, such as magnetic beads can be used for phase-based
separations and magnetic-based separations. The beads, or any other
surfaces described herein, can be functionalized with binding
moieties that exhibit specific or non-specific binding to a target.
The binding can be based on electrostatics, van der Walls
interactions, hydrophobicity, hydrophilicity, hydrogen bonding,
ionic interactions, as well as partially covalent interactions like
those exhibited between gold and sulfur. In preferred embodiments,
the devices and methods of the invention utilize immunomagnetic
separations.
[0011] Immunomagnetic separation (IMS) is a powerful technology
that allows targets to be captured and concentrated in a single
step using a mechanistically simplified format that employs
paramagnetic beads and a magnetic field (see Grodzinski P, Liu R,
Yang J, Ward M D. Microfluidic system integration in sample
preparation microchip-sets--a summary. Conf Proc IEEE Eng Med Biol
Soc. 2004; 4:2615-8., Peoples M C, Karnes H T. Microfluidic
immunoaffinity separations for bioanalysis. J Chromatogr B Analyt
Technol Biomed Life Sci. 2007 Aug. 30., and Stevens K A, Jaykus L
A. Bacterial separation and concentration from complex sample
matrices: a review. Crit Rev Microbiol. 2004; 30(1):7-24.). IMS can
be used to capture, concentrate, and then purify specific target
antigens, proteins, toxins, nucleic acids, cells, and spores. While
IMS as originally used referred to using an antibody, we generalize
its usage to include other specific affinity interactions including
lectins, DNA-DNA, DNA-RNA, biotin-streptavidin, and other affinity
interactions that are coupled to a solid phase. IMS works by
binding a specific affinity reagent, typically an antibody or DNA,
to paramagnetic beads which are only magnetic in the presence of an
external magnetic field. The beads can be added to complex samples
such as aerosols, liquids, bodily fluids, or food. After binding of
the target to the affinity reagent (which itself is bound to the
paramagnetic bead) the bead is captured by application of a
magnetic field. Unbound or loosely bound material is removed by
washing with compatible buffers, which purifies the target from
other, unwanted materials in the original sample. Because beads are
small (nm to um) and bind high levels of target, when the beads are
concentrated by magnetic force they typically form bead beds of
just nL-uL volumes, thus concentrating the target at the same time
it is purified. The purified and concentrated targets can be
conveniently transported, denatured, lysed or analyzed while
on-bead, or eluted off bead for further sample preparation, or
analysis.
[0012] Immunomagnetic separations are widely used for many
applications including the detection of microorganisms in food,
bodily fluids, and other matrices. Paramagnetic beads can be mixed
and manipulated easily, and are adaptable to microscale and
microfluidic applications. This technology provides an excellent
solution to the macroscale-to-microscale interface: beads are an
almost ideal vehicle to purify samples at the macroscale and then
concentrate to the nanoscale (100's of nL) for introduction into
microfluidic or nanofluidic platforms. Immunomagnetic separations
are commonly used as an upstream purification step before real-time
PCR, electrochemiluminescence, and magnetic force
discrimination.
[0013] The ability to move fluids on microchips is a quite
important. This invention describes technologies in sample capture
and purification, micro-separations, micro-valves, -pumps, and
-routers, nanofluidic control, and nano-scale biochemistry. A key
component of the technology is Micro-robotic On-chip Valves (MOVe)
technology (an example of which is shown in FIG. 1) and its
application to miniaturize and automate complex workflows.
Collectively the MOVe valves, pumps, and routers and the
instrumentation to operate them can be referred to as a microchip
fluid processing platform.
[0014] The heart of the microchip fluid processing platform
technology are MOVe pumps, valves, and routers that transport,
process, and enable analysis of samples. These novel externally
actuated, pneumatically-driven, on-chip valves, pumps, and routers,
originally developed in the Mathies laboratory at the University of
California at Berkeley (U. C. Berkeley) (Grover, W. H. A. M.
Skelley, C. N. Liu, E. T. Lagally, and R. M. Mathies. 2003. Sensors
and Actuators B89:315-323; Richard A. Mathies et al., United States
Patent Application, 20040209354 A1 Oct. 21, 2004; all of which are
herein incorporated by reference in their entirety) can control
fluidic flow at manipulate volumes from 20 nL to 10 .mu.L.
[0015] The MOVe valves and pumps (FIG. 1) can combine two glass
microfluidic layers with a polydimethyl siloxane (PDMS) deformable
membrane layer that opens and closes the valve, and a pneumatic
layer to deform the membrane and actuate the valve. The
microfluidic channel etched in the top glass fluidic wafer is
discontinuous and leads to vias through the "via wafer" and
microfluidic channels to a valve seat which is normally closed
(FIG. 1A). When a vacuum is applied to the pneumatic displacement
chamber by conventional-scale vacuum and pressure sources, the
normally closed PDMS membrane lifts from the valve seat to open the
valve (FIG. 1B). The bottom panel of FIG. 1 shows a top view of the
valve a similar scale as the other panels.
[0016] Three microvalves can be used to make a micropump on a
microchip to move fluids from the Input area to the Output area on
Microchip A. The fluids are moved by three or more valves. The
valves can be created actuation of a deformable structure. In some
implementations a valve seat is created and in other embodiments no
valve seat may be needed. FIG. 2 shows MOVe devices from top to
bottom: valve, router, mixer, bead capture. Self-priming MOVe pumps
(FIG. 2, top) are made by coordinating the operation of three
valves and can create flow in either direction. Routers are made
from three or more MOVe valves (FIG. 2, top middle panel). Mixing
has been a holy grail for microfluidics: MOVe mixers (FIG. 2,
bottom middle panel) rapidly mix samples and reagents. MOVe devices
work exquisitely with magnetic beads to pump or trap sets of beads
(FIG. 2, bottom panel).
[0017] The normally closed MOVe valves, pumps, and routers are
durable, easily fabricated at low cost, can operate in dense
arrays, and have low dead volumes. Arrays of MOVe valves, pumps,
and routers are readily fabricated on microchips. Significantly,
all the MOVe valves, pumps, and routers on a microchip are created
at the same time in a simple manufacturing process using a single
sheet of PDMS membrane--it costs the same to make 5 MOVe micropumps
on a microchip as to create 500. This innovative technology offers
for the first time the ability to create complex micro- and
nanofluidic circuits on microchips.
[0018] Patents and applications which discuss the use and design of
microchips include U.S. Pat. No. 7,312,611, issued on Dec. 25,
2007; U.S. Pat. No. 6,190,616, issued on Feb. 20, 2001; U.S. Pat.
No. 6,423,536, issued on Jul. 23, 2002; U.S. patent Ser. No.
10/633,171 Mar. 22, 2005; U.S. Pat. No. 6,870,185, issued on Mar.
22, 2005 US Application No. US 2001-0007641, filed on Jan. 25,
2001; US Application US20020110900, filed on Apr. 18, 2002; US
patent application 20070248958, filed Sep. 15, 2005; US patent
application US 20040209354, filed on Dec. 29, 2003; US patent
application US2006/0073484, filed on Dec. 29, 2003; US20050287572,
filed on May 25, 2005; US patent application US20070237686, filed
on Mar. 21, 2007; US 20050224352 filed on Nov. 24, 2004; US
20070248958, filed on, Sep. 15, 2005; US 20080014576, filed on Feb.
2, 2007; and, US application US20070175756, filed on Jul. 26, 2006;
all of which are herein incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0019] In one aspect this invention provides a device comprising: a
microfluidic microchip comprising at least one port aperture
fluidically connected to at least one microfluidic channel in the
microfluidic microchip, wherein the channel comprises at least one
valve that controls movement of a fluid through the channel; and a
cartridge mated to the microchip and comprising a chamber, wherein
said chamber comprises two chamber apertures that are each aligned
with a port aperture of said microfluidic microchip. In one
embodiment said cartridge is adapted to receive at least one sample
or one reagent. In another embodiment said cartridge is fluidically
connected to another cartridge that is mated to another microchip.
In another embodiment said cartridge comprises at least two
chambers. In another embodiment at least one of said at least two
chambers is adjacent to a movable magnet. In another embodiment at
least one of said at least two chambers is temperature controlled.
In another embodiment at least one of said at least two chambers
comprises a filter. In another embodiment said at least one chamber
comprises a fluidic volume greater than or equal to 5 .mu.L. In
another embodiment said at least one chamber comprises a fluidic
volume greater than or equal to 10 .mu.L. In another embodiment the
fluidic volume of said cartridge is a 100.times. of the fluidic
volume of said microfluidic microchip. In another embodiment one of
said at least one chambers further comprises a filter. In another
embodiment said device further comprises a magnet for applying a
magnetic field to the cartridge or the microfluidic microchip. In
another embodiment the valve is pneumatically actuated. In another
embodiment said cartridge is adapted to be connected to at least
one pressure source for the delivery of said at least one reagent
or said at least one sample. In another embodiment said pressure
source provides a positive or negative pressure to the cartridge.
In another embodiment said at least one pressure source is
controlled by a pneumatic solenoid. In another embodiment said at
least one pressure source is a pneumatic manifold. In another
embodiment the microfluidic microchip comprises a fluidic layer, an
elastomeric layer, and a pneumatic layer. In another embodiment
said cartridge further comprises at least one input port, wherein
said at least one input port is adapted to mate with a delivery
device, wherein said delivery device is fluidically connected to
the fluidic layer of said microfluidic microchip; and wherein one
of said at least one chamber is a closed reaction chamber
fluidically connected to the fluidic layer of said microfluidic
microchip. In another embodiment said delivery device is thermally
coupled to a temperature modulator. In another embodiment said
delivery device is a syringe. In another embodiment said cartridge
is designed to enrich at least one component from said sample and
comprises at least one sample input port, wherein said at least one
chamber is a closed reaction chamber comprising beads, and wherein
said beads bind to said at least one component. In another
embodiment the cartridge further comprises at least one reagent
reservoir comprising reagents for amplifying a nucleic acid,
wherein the at least one reagent reservoir is fluidically connected
to the chamber through the microchip. In another embodiment the
cartridge further comprises at least one bead reservoir comprising
beads for binding an amplified nucleic acid, wherein the at least
one bead reservoir is fluidically connected to the chamber through
the microchip. In another embodiment said beads are paramagnetic
beads or glass beads. In another embodiment said binding of at
least one component to a bead is reversible. In another embodiment
said beads are paramagnetic beads, and wherein said device further
comprises a movable magnet that can attract said paramagnetic beads
to the wall of said closed reaction chamber. In another embodiment
said cartridge is designed to enrich at least one component from a
sample, wherein said at least one component is DNA, RNA, microRNA,
siRNA, protein, lipid, or polysaccharide.
[0020] In another aspect this invention provides a method for
performing biochemical reactions comprising: (a) providing the
device comprising a microfluidic microchip comprising at least one
port aperture fluidically connected to at least one microfluidic
channel in the microfluidic microchip, wherein the channel
comprises at least one valve that controls movement of a fluid
through the channel; and a cartridge mated to the microchip and
comprising a chamber, wherein said chamber comprises two chamber
apertures that are each aligned with a port aperture of said
microfluidic microchip., and (b) performing at least one enzymatic
reaction within said chamber. In one embodiment said at least one
enzymatic reaction comprises ligating, blunting, nick repairing,
denaturing, polymerizing, hydrolyzing, phosphorylation or any
combination thereof. In another embodiment the method further
comprises separating a product of said enzymatic reaction using
solid-phase particles.
[0021] In another aspect this invention provides a method for
enriching at least one component from a sample comprising: (a)
mating a delivery device to an input port of a device comprising: a
microfluidic microchip comprising at least one port aperture
fluidically connected to at least one microfluidic channel in the
microfluidic microchip, wherein the channel comprises at least one
valve that controls movement of a fluid through the channel; and a
cartridge mated to the microchip and comprising a chamber, wherein
said chamber comprises two chamber apertures that are each aligned
with a port aperture of said microfluidic microchip wherein said
cartridge further comprises at least one input port, wherein said
at least one input port is adapted to mate with a delivery device,
wherein said delivery device is fluidically connected to the
fluidic layer of said microfluidic microchip; and wherein one of
said at least one chamber is a closed reaction chamber fluidically
connected to the fluidic layer of said microfluidic microchip, (b)
treating said sample with at least one reagent to increase the
availability of said at least one component for enrichment, (c)
delivering said at least one component to said at least one
reaction chamber of said cartridge, (d) binding said component to
one or more particles in said at least one closed reaction chamber,
(e) washing said particle bound component to remove waste, and (f)
eluting said particle bound component. In one embodiment said
delivering comprises pumping said at least one component to said at
least one reaction chamber through said at least one valve of the
microfluidic microchip. In another embodiment said binding
comprises pumping said particles from a reagent port in the
cartridge to said at least one reaction chamber through said at
least one valve of the microfluidic microchip. In another
embodiment said particle is a paramagnetic bead, a nanoparticle, a
resin, or a solid-phase particle. In another embodiment said at
least one component is DNA, RNA, microRNA, siRNA, protein, lipid,
or polysaccharide. In another embodiment step (b) further comprises
thermally modulating said delivery device. In another embodiment
step (b) further comprises delivering a lysis reagent or a
component isolation reagent from a reagent port on said cartridge
into said delivery device to increase the availability of said at
least one component for enrichment. In another embodiment the beads
of step (d) are paramagnetic beads. In another embodiment said
washing step (e) comprises attracting said paramagnetic beads with
a movable magnet. In another embodiment said microfluidic microchip
directs the flow of said waste in a second direction. In another
embodiment step (c) comprises using pneumatically actuated valves
in the microfluidic microchip or an external pressure source to
deliver said at least one component to said at least one closed
reaction chamber of said cartridge. In another embodiment the
external pressure source provides a positive or negative pressure
to the microfluidic microchip. In another embodiment said sample
delivery device is a syringe.
[0022] In another aspect this invention provides a device
comprising: (a) a first fluid manipulation module comprising: (i) a
first microfluidic microchip comprising a port aperture fluidically
connected to a microfluidic channel in the microfluidic microchip,
wherein the channel comprises at least one valve that controls
movement of a fluid through the channel; and (ii) a cartridge mated
to the microfluidic microchip and comprising at least one sample
input port, at least one chamber, an exit port, wherein the sample
input port is connected to the port aperture, wherein at least one
of said at least one exit ports is aligned with an exit port
aperture of said first microfluidic microchip, and wherein said at
least one chamber is fluidically connected to the fluidic layer of
said first microfluidic microchip; (b) a reaction channel, wherein
said reaction channel is not contained within said first microchip;
(c) a temperature modulator, wherein said reaction channel is
fluidically connected to a port on said cartridge that is
fluidically connected to said exit port and at least a portion of
said reaction channel is in thermal contact with said temperature
modulator; and (d) a magnet for applying a magnetic field to the
microfluidic microchip, the cartridge, or the reaction channel. In
one embodiment said magnet is adjacent to said reaction channel. In
another embodiment the device further comprises a second
microfluidic microchip that is fluidically connected to said first
microfluidic microchip through said reaction channel. In another
embodiment said temperature modulator is a Peltier device. In
another embodiment the device further comprises a paramagnetic
bead.
[0023] In another aspect this invention provides a method
comprising: delivering a sample containing a nucleic acid to a
device comprising: (a) a first fluid manipulation module
comprising: (i) a first microfluidic microchip comprising a port
aperture fluidically connected to a microfluidic channel in the
microfluidic microchip, wherein the channel comprises at least one
valve that controls movement of a fluid through the channel; and
(ii) a cartridge mated to the microfluidic microchip and comprising
at least one sample input port, at least one chamber, an exit port,
wherein the sample input port is connected to the port aperture,
wherein at least one of said at least one exit ports is aligned
with an exit port aperture of said first microfluidic microchip,
and wherein said at least one chamber is fluidically connected to
the fluidic layer of said first microfluidic microchip; (b) a
reaction channel, wherein said reaction channel is not contained
within said first microchip; (c) a temperature modulator, wherein
said reaction channel is fluidically connected to a port on said
cartridge that is fluidically connected to said exit port and at
least a portion of said reaction channel is in thermal contact with
said temperature modulator; and (d) a magnet for applying a
magnetic field to the microfluidic microchip, the cartridge, or the
reaction channel.; transporting the nucleic acid and an effective
amount of reagents through the portion of the reaction channel in
thermal contact with the temperature modulator one or more times;
and amplifying the nucleic acid; and analyzing the amplified
nucleic acid. In one embodiment the method further comprises using
the temperature modulator to perform thermocycling. In another
embodiment said reagents are reagents for polymerase chain reaction
or cycle sequencing.
[0024] In another aspect this invention provides a device
comprising: (a) a first microfluidic microchip comprising a
fluidics layer, an actuation layer, and a pneumatic layer, wherein
the fluidics layer comprises one or more microfluidic channels,
wherein at least one of said one or more microfluidic channels
comprises an exit aperture, (b) a flexible connector fluidically
connected to the exit aperture at a first end of the flexible
connector; (c) a capillary fluidically connected to said flexible
connector; and (d) a first electrode and a second electrode,
wherein the first electrode and second electrode are configured to
produce an electric field along a path of the capillary. In one
embodiment the flexible connector is surgical,
poly(tetrafluoroethylene) or silicon tubing. In another embodiment
the flexible connector is elastic tubing. In another embodiment the
flexible connector has an outer diameter of about 1.5 to 3 mm and
an inner diameter of about 0.25 to 0.5 mm. In another embodiment
the flexible connector is also fluidically connected to a second
microfluidic microchip or the first microfluidic microchip at a
second end of the flexible connector. In another embodiment the
flexible connector is fluidically connected to the exit aperture by
a cannula, an upfit tubing, a microtubing fitting, or an upchurch
tubing adapter. In another embodiment the capillary has an outer
diameter of about 150-500 microns and an inner diameter of about
10-100 microns. In another embodiment the capillary is polyamide or
poly(tetrafluoroethylene) coated. In another embodiment the
capillary comprises a separation gel. In another embodiment the
capillary is about 10 to 100 cm long. In another embodiment the
first electrode is a forked electrode. In another embodiment said
forked electrode comprises one or more conductive channels or one
or more metallic conductors. In another embodiment the first
electrode and the second electrode produce an electric field that
is about 25 to 500 V/cm.
[0025] In another aspect this invention provides a method
comprising: providing the composition to a microfluidic microchip,
wherein the microfluidic microchip comprises a fluidics layer, a
elastomeric layer, and a pneumatic layer; delivering the
composition to a flexible connector that is fluidically connected
to the microfluidic microchip; providing a electric field to move
the composition into a capillary; and performing capillary
electrophoresis on the composition to separate a component based on
size or charge. In one embodiment the electric field is about 25 to
500 V/cm. In another embodiment said composition in said tube is
adjacent to a first and second bolus of gas, wherein said first
bolus of gas is upstream of said composition and said second bolus
of gas is downstream of said composition in said tube. In another
embodiment said first and second boluses of gas isolates said
composition from other compositions. In another embodiment the
composition comprises at least one component that is a nucleic
acid, protein, fatty acids, or polysaccharides. In another
embodiment the nucleic acid is microRNA, DNA, RNA, or siRNA.
[0026] In another aspect this invention provides a device
comprising: (a) a separation channel fluidically connected to a
loading channel; (b) a forked electrode comprising at least two
electrodes that are electrically connected to the loading channel
and the separation channel through the loading channel, wherein the
fluidic connection between the separation channel and the loading
channel is located between the electrical connections of the two
electrodes to the loading channel; and (c) a pneumatically actuated
valve fluidically connected to the loading channel. In one
embodiment the device further comprises a cannular electrode in
electrical contact with said forked electrode, wherein the inner
diameter of said cannular electrode is at least about 0.2 mm. In
another embodiment said cannular electrode is configured to reduce
injection of gas into said separation channel. In another
embodiment the separation channel is a capillary, and wherein the
capillary is fluidically connected to the pneumatically actuated
valve using a flexible connection. In another embodiment the
separation channel is a microchannel. In another embodiment the
separation channel and pneumatically actuated valve are integrated
on a microfluidic microchip. In another embodiment the loading
channel comprises a loading channel solution and the separation
channel comprises a separation channel solution, and wherein the
sample solution has a lower electrical conductivity than the
separation channel solution. In another embodiment the at least two
electrodes comprise at least two microchannels that are on one end
fluidically connected to the loading channel on either side of the
fluidic connection between the separation channel and the loading
channel and on the other end fluidically connected to a base
channel. In another embodiment each of the at least two electrodes
is a metallic conductors that is electrically connected to a
voltage source and the loading channel.
[0027] In another aspect this invention provides a device
comprising: a first, a second, and a third microfluidic channel
that are joined to form a three-way junction; wherein the first
microfluidic channel is electrically connected to a first electrode
of a forked electrode, wherein the second microfluidic channel is
electrically connected to a second electrode of the forked
electrode, and wherein the first, the second, and the third
microfluidic channel are each fluidically connected to a
pneumatically actuated valve.
[0028] In another aspect this invention provides a method for
performing capillary electrophoresis comprising: providing a device
comprising: (1) a separation channel fluidically connected to a
loading channel; (2) a forked electrode comprising at least two
electrodes that are electrically connected to the loading channel
and the separation channel through the loading channel, wherein the
fluidic connection between the separation channel and the loading
channel is located between the electrical connections of the two
electrodes to the loading channel; and (3) a pneumatically actuated
valve fluidically connected to the loading channel; providing a
separation channel solution to the separation channel; delivering a
composition to the loading channel, wherein the pneumatically
actuated valve is used to control the delivery of the composition
to the loading channel; and applying an electric field along the
separation channel using the forked electrode; performing capillary
electrophoresis on the composition to separate the component based
on size or charge. In another embodiment the composition has a
lower electrical conductivity than the separation channel solution.
In another embodiment the composition is concentrated by said
applying the electric field.
[0029] In another aspect this invention provides a microfluidic
device comprising: (a) a microfluidic microchip comprising: (1) a
first channel comprising a first valve; (2) a second channel that
intersects the first channel on one side of the first valve; (3) a
third channel that intersects the first channel on the other side
of the first valve; wherein at least one of the second or third
channel intersect the first channel at a T-valve or at least one of
the second or third channel comprise a second valve, and wherein
the second and third channel each connect to a port; and (b) a
fluid loop that is removably attached to the ports such that fluid
can flow from the first channel to the fluid loop.
[0030] In another aspect this invention provides a microfluidic
device comprising: (a) a microchip comprising one or more
pneumatically actuated valves; and (b) a sample loop, wherein the
sample loop is fluidically connected to the one or more
pneumatically actuated valves through ports in the microfluidic
microchip, and wherein the sample loop has a fixed volume and the
sample loop is removable. In one embodiment said pneumatically
actuated valves are actuated by one or more pneumatic channels in
the microfluidic device. In another embodiment the sample loop
comprises a capillary tube. In another embodiment the sample loop
is fluidically connected to a pass-through microfluidic channel at
a first junction and a second junction, and wherein a pass-through
microfluidic pneumatically actuated valve is positioned in the
pass-through microfluidic channel between the first and second
junctions. In another embodiment at least one junction comprises a
T-valve, wherein closure of the T-valve does not prevent passage of
fluid through the pass-through microfluidic channel. In another
embodiment the sample loop is connected to the pass-through
microfluidic channel through first and second channels, and wherein
at least one of the first and the second channel comprise a sample
loop valve.
[0031] In another aspect this invention provides a method for
delivering a fixed volume of fluid to a microfluidic device
comprising: configuring a device with a sample loop comprising a
desired volume, wherein the sample loop is removable; using one or
more pneumatically actuated valves on a microfluidic device to fill
the sample loop with the fixed volume of the fluid; and delivering
the fluid to the microfluidic device. In one embodiment the sample
loop and a pass-through microfluidic channel are fluidically
connected at a first junction and a second junction, and wherein at
least one junction comprises a T-valve. In another embodiment the
pass-through microfluidic channel comprises a pass-through
microfluidic valve positioned between the first junction and the
second junction.
[0032] In one aspect, this invention provides a device comprising:
(a) a cartridge; (b) a microfluidic microchip having one or more
microfluidic diaphragm valves, fluidically interfaced with the
cartridge; and (c) a pneumatic manifold interfaced with the
microfluidic microchip on a surface of the microfluidic microchip,
wherein the pneumatic manifold covers all or only a portion of the
surface of the microfluidic microchip. In one embodiment the device
further comprises a magnet configured to generate a magnetic field
in a chamber of the microfluidic microchip. In one embodiment
wherein the pneumatic manifold has an annular space for the
magnetic component. In another embodiment the microfluidic
microchip comprises a fluidics layer comprising fluidics channels,
a pneumatics layer comprising pneumatics channels, and an
activation layer sandwiched there between, wherein the cartridge
comprises a chamber with an opening, wherein the opening mates with
an opening in the fluidics layer that connects to a fluidics
channel, and the pneumatic manifold comprises an opening that mates
with an opening in the pneumatics layer of the microfluidic
microchip that connects with a pneumatic channel.
[0033] In another aspect this invention provides a device
comprising: (a) a cartridge; (b) a microfluidic microchip having
one or more microfluidic diaphragm valves and interfaced with the
cartridge; (c) a pneumatic manifold interfaced with the
microfluidic microchip on a surface of the microfluidic microchip;
and (d) a temperature controlling block in thermal contact with the
cartridge. In one embodiment the microfluidic microchip comprises a
fluidics layer comprising fluidics channels, a pneumatics layer
comprising pneumatics channels, and an activation layer sandwiched
there between, wherein the cartridge comprises a chamber with an
opening, wherein the opening mates with an opening in the fluidics
layer that connects to a fluidics channel, and the pneumatic
manifold comprises an opening that mates with an opening in the
pneumatics layer of the microfluidic microchip that connects with a
pneumatic channel.
[0034] In another aspect this invention provides a device
comprising a microfluidic microchip having one or more microfluidic
diaphragm valves and interfaced with a cartridge; wherein the
microfluidic microchip has a bead rail and a reagent rail.
[0035] In another aspect this invention provides a method for
amplifying mRNA and purifying amplified RNA comprising: (a)
providing device comprising: (i) a cartridge; (ii) a microfluidic
microchip having one or more microfluidic diaphragm valves,
fluidically interfaced with the cartridge; (iii) a pneumatic
manifold interfaced with the microfluidic microchip on a surface of
the microfluidic microchip, wherein the pneumatic manifold covers
all or only a portion of the surface of the microfluidic microchip
and (iv) a magnet configured to generate a magnetic field in a
chamber of the microfluidic microchip, wherein the pneumatic
manifold has an annular space for the magnetic component; (b)
supplying a sample containing mRNA and reagents to the cartridge;
(c) mixing the sample and reagents in a well of the microfluidic
microchip; (d) amplifying the mRNA to form amplified RNA; (e)
capturing the amplified RNA using magnetic beads; and (f)
positioning the magnet in the annular space to capture magnetic
beads in a reservoir of the microfluidic microchip.
[0036] In another aspect this invention provides a method for
amplifying mRNA comprising: (a) providing a device comprising: (i)
a cartridge; (ii) a microfluidic microchip having one or more
microfluidic diaphragm valves and interfaced with the cartridge;
(iii) a pneumatic manifold interfaced with the microfluidic
microchip on a surface of the microfluidic microchip; and (iv) a
temperature controlling block in thermal contact with the
cartridge; (b) supplying a sample containing mRNA and reagents to
the cartridge; (c) mixing the sample and reagents in a well of the
microfluidic microchip to form a mixture; (d) heating the mixture
using the temperature controlling block; and (e) amplifying the
mRNA.
[0037] In another aspect this invention provides a method for
amplifying mRNA and purifying amplified RNA comprising: (a)
providing a device comprising a microfluidic microchip having one
or more microfluidic diaphragm valves and interfaced with a
cartridge; wherein the microfluidic microchip has a bead rail and a
reagent rail; (b) supplying reagents to one or more reagent rail
wells; (c) supplying magnetic bead slurry to a bead rail well; (d)
supplying a sample containing mRNA to a sample well; (e) pumping
the sample and the reagents to an output well of the microfluidic
microchip to form a mixture; (f) amplifying the mRNA to form
amplified RNA; (g) pumping the magnetic bead slurry to a
purification well; (h) contacting the magnetic bead slurry with
amplified RNA by pumping the amplified RNA to the purification
well; and (i) purifying the amplified RNA.
[0038] In another aspect this invention provides a method for
pumping a fluid in a microfluidic device comprising: (a) providing
a microfluidic device comprising a pumping valve, a source well,
and a mixing well, wherein the pumping valve, the source well, and
the mixing well are fluidically connected by a channel; (b) pumping
the fluid in a first direction through the channel from the source
well to the pumping valve; and (c) pumping the fluid in a second
direction through the channel from the pumping valve to the mixing
well, wherein the second direction is opposite of the first
direction.
[0039] Sample preparation is a challenging area of the
bioanalytical process. In one aspect, a method is disclosed for the
preparation of samples from many different sample types. In another
aspect, an apparatus is disclosed that can prepare samples from
many different sample types. In one embodiment, the apparatus
operates a cartridge with microscale valves that direct fluid flow
in a microchip component that can be fabricated separately or as an
integral part of the cartridge. In another embodiment, the
apparatus can move samples into the cartridge using pressure-driven
flow or vacuum modulated by the microvalves on the cartridge. In
another embodiment, the apparatus can manipulate paramagnetic beads
for magnetic separation of components of the sample to purify
desired analytes with fluid flow directed by the microvalves.
[0040] In one embodiment, the apparatus is a universal sample
preparation system that can process biological or chemical samples.
Samples can be loaded in liquid, swabs, swipes, solids, gases, or
other matrices into the cartridge. The apparatus is controlled by
electronics which may include a computer to select the proper
reagents and direct the fluids using the microscale valves to open
and close circuitry that is formed by the cartridge and by the
microchip component. The sample can be processed to extract nucleic
acids, including DNA, RNA, microRNAs, proteins, lipids,
polysaccharides, cell walls, small molecules, and all other
biological components of a sample. Similarly the sample can also be
processed to extract or purify chemical components. For example,
DNA can be processed onto microbeads.
[0041] In one embodiment, a sample can be moved into a reaction
chamber in a cartridge comprising one or more chambers, channels,
tubing, or capillaries that may be permanently attached to the
cartridge or may be reversibly joined to the cartridge. Samples may
be reproducibly positioned in the channels, tubing or capillaries
using vacuum or pressure modulated or created by micropumps that
may be located on the cartridge, on the apparatus, on an external
device, or other configurations.
[0042] In one embodiment, for DNA, the processed sample can be
amplified by PCR, rolling circle, branched DNA, EXPAR, LAMP, and
other DNA amplification methods well known to one skilled in the
art or analyzed by mass spectroscopy or single molecule detection
methods. RNA may be processed by Reverse Transcriptase real
time-PCR, or samples prepared for DNA microarrays, or other
analytical methods. Real time or end point analyzes can be
performed with the apparatus. For proteins, assays may be performed
in the cartridge including enzymatic assays, sandwich immunoassays,
antibody precipitation, protein digestion, protein and peptide
labeling, and other commonly used protein analysis methods.
Similarly, other cellular components or chemicals can be extracted
or purified using standard methods in the apparatus. Molecular
biology methods are readily adapted to the apparatus. Samples can
be completely analyzed on the apparatus in a single cartridge,
moved to a separate cartridge, or analyzed or further processed in
a separate instrument comprising a capillary electrophoresis system
or microchip capillary electrophoresis; multidimensional gel and
capillary electrophoresis; mass spectroscopy, multidimensional mass
spectroscopy with HPLC, ICP, Raman spectroscopy, particle,
nanoparticles, and bead based detection, imaging, comprising
fluorescence, IR, optical, or any other analytical systems well
know to one in the art.
[0043] In one embodiment, the integration of a complete
sample-to-answer instrument incorporating the cartridge to prepare
DNA samples from many inputs and sample types and a microchip-based
capillary electrophoresis device for separation of DNA fragments is
used for analysis, such as DNA sequencing, fragment sizing, and
forensics.
INCORPORATION BY REFERENCE
[0044] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0046] FIG. 1 depicts an example of a microscale on-chip valve
(MOVe).
[0047] FIG. 2 shows a MOVe microvalve, a microrouter, a MOVe mixer,
and bead capture on microchips.
[0048] FIG. 3 shows a fluidic cartridge with MOVe microvalves.
[0049] FIG. 4 shows a fluidic cartridge with ports to a
microfluidic microchip with microvalves.
[0050] FIG. 5 shows a microfluidic microchip with MOVe valves that
controls flows in a cartridge.
[0051] FIG. 6 shows a cartridge connected to reaction chamber and
detector with downstream MOVe pumps and reagents.
[0052] FIG. 7 shows a temperature control device that can thermal
cycle and incorporates magnetic capture, pinch clamps and the
capability of cycling seven reactions simultaneously.
[0053] FIG. 8 shows a temperature control device that can thermal
cycle and incorporates magnetic capture, pinch clamps and the
capability of cycling seven reactions simultaneously.
[0054] FIG. 9 shows PowerPlex16 STR (Single tandem repeat)
amplification reaction performed in a passive, Teflon (PTFE) based
Tube reaction chamber.
[0055] FIG. 10 shows purification of DNA from 25 uL of blood at
69', 23.5', 10.5.degree., and 4.5.degree.; yield in ng is shown on
the bars.
[0056] FIG. 11 shows a schematic of using microvalves to capture
beads on a microchip.
[0057] FIG. 12 shows bead capture from a cartridge on a microchip
using a MOVe microvalve.
[0058] FIG. 13 shows bead capture from a cartridge on a microchip
using a MOVe microvalve.
[0059] FIG. 14 shows a capture and reaction microchip using MOVe
microvalves.
[0060] FIG. 15 shows a capture and reaction microchip using MOVe
microvalves.
[0061] FIG. 16 shows a four cartridge assembly.
[0062] FIG. 17 shows an example of STR reactions on microchips.
[0063] FIG. 18 shows a universal sample preparation workflow to
prepare nucleic acids and toxins.
[0064] FIG. 19 shows purification of samples in a cartridge using
paramagnetic beads.
[0065] FIG. 20 shows an integrated pneumatic manifold to operate
the MOVe microvalves in cartridge.
[0066] FIG. 21 shows a cartridge mounted on a computer controlled
apparatus.
[0067] FIG. 22 shows a cartridge mounted on a computer controlled
apparatus.
[0068] FIG. 23 shows a reagent distribution manifold based on MOVe
technology that can distribute five reagents to five
extraction/isolation or other devices.
[0069] FIG. 24 shows a reagent distribution manifold based on MOVe
technology that can distribute five reagents to five
extraction/isolation or other devices.
[0070] FIG. 25 shows a distribution manifold with sample loops and
MOVe microvalves.
[0071] FIG. 26 shows a pneumatic manifold, top panel shows the top
side and the lower panel the bottom side.
[0072] FIG. 27 shows detection of E. coli by immunomagnetic
separation, followed by alkaline lysis and PEG-facilitated capture
on magnetic beads, and analyzed by real-time PCR.
[0073] FIG. 28 shows application of a cartridge with three chambers
that can be used to construct genomic libraries and other
applications.
[0074] FIG. 29 shows the workflow to prepare genomic libraries
using the cartridge.
[0075] FIG. 30 shows a forked injector for microchip based
electrophoresis.
[0076] FIG. 31 shows sample stacking with a forked injector.
[0077] FIG. 32 shows a forked injector coupled to MOVe
microvalves.
[0078] FIG. 33 shows a forked cathode injector coupled with a MOVe
microchip.
[0079] FIG. 34 shows a photograph of a microchip with the forked
injector.
[0080] FIG. 35 shows a photograph of a microchip with the forked
injector.
[0081] FIG. 36 shows an electropherogram of a single color from a
DNA sequencing trace from a forked cathode injector.
[0082] FIG. 37 shows STR separations on a forked cathode injection
system.
[0083] FIG. 38 shows a forked cathode with MOVe microfluidics for
shuttle loading.
[0084] FIG. 39 shows an integrated system for nucleic acid
isolation, amplification(s), separation and detection.
[0085] FIG. 40 depicts a device with a cartridge, microfluidic
microchip, and a magnet.
[0086] FIG. 41 depicts a microfluidic microchip with a fluidics
layer, an elastomeric layer, and a pneumatics layer.
[0087] FIG. 42 depicts a fluidics layer made of two layers of
material.
[0088] FIG. 43 depicts a fluidics layer made of a single layer of
material.
[0089] FIG. 44 depicts a reaction scheme for amplifying mRNA.
[0090] FIG. 45 depicts an expanded view of a heat block 509, a
cartridge 50, a microfluidic microchip 519 and a pneumatics
manifold 507.
[0091] FIG. 46 depicts a heat block, a cartridge, a microfluidic
microchip and a pneumatics manifold in an assembled form.
[0092] FIG. 47 depicts fluidics and pneumatic layers of a
microfluidic microchip with four sets of pumps.
[0093] FIG. 48 depicts a cartridge for interfacing with a fluidics
layer of a microfluidic microchip.
[0094] FIG. 49 depicts a block for holding tips that interface with
a cartridge.
[0095] FIG. 50 depicts results of reverse transcription reactions
of an mRNA amplification scheme.
[0096] FIG. 51 depicts an expanded view of a heat block with a heat
distributing element, a cartridge, a microfluidic microchip and a
pneumatics manifold.
[0097] FIG. 52 depicts a heat block with a heat distributing
element, a cartridge, a microfluidic microchip and a pneumatics
manifold in an assembled form.
[0098] FIG. 53 depicts a pneumatics manifold.
[0099] FIG. 54 depicts a pneumatics manifold.
[0100] FIG. 55 depicts a pneumatics manifold.
[0101] FIG. 56 depicts a pneumatics manifold.
[0102] FIG. 57 depicts fluidics and pneumatic layers of a
microfluidic microchip with a reagent and bead rail with the
fluidic layer shown in solid lines and the pneumatics layer shown
in dashed lines.
[0103] FIG. 58 depicts fluidics layers of a microfluidic microchip
with a reagent and bead rail.
[0104] FIG. 59 shows a microfluidic microchip with MOVe valves that
controls flows in a cartridge.
[0105] FIG. 60 shows a forked electrode.
[0106] FIG. 61 shows a forked electrode, a forked electrode with a
wire run electrode, and a forked electrode with a cannular
electrode.
[0107] FIG. 62 shows sample injection into a separation
channel.
[0108] FIG. 63 shows a device for mating a separation capillary
with an injection tubing.
[0109] FIG. 64 shows a device for mating separation capillaries
with four injection tubings.
[0110] FIG. 65 shows a thermocycler with an Ultem pinch clamp.
[0111] FIG. 66 shows a diagram indicating movement of reagents
between components of a four channel parallel processing
device.
[0112] FIG. 67 shows a four-channel parallel reagent delivery
device: the Chip C microchip design is shown on the top left, a
fluidic manifold is shown on the bottom left, and the fabricated
and assembled device is shown on the right.
[0113] FIG. 68 shows a four-channel sample preparation device on
the left and a four-channel sample preparation device mounted on a
monolithic pneumatic manifold on the right.
[0114] FIG. 69 shows MOVe microchip designs of the four-channel
sample preparation device: the Chip D microchip design is shown on
the left with flow through valves that form a T-junction between
two bisecting channels shown in top panel; the Chip D microchip
design with flow-through valves is shown on the right top; the Chip
F microchip design with in-line valves that have only one channel
passing through the middle of the valve is shown on the right
bottom.
[0115] FIG. 70 shows IdentiFiler STR profiles of DNA samples
prepared on the four-channel sample preparation device, where STR
amplifications were performed using fast protocols (1.5 hrs) on a
STR Reaction subsystem thermocycler.
[0116] FIG. 71 shows a four-channel post amplification device
combined with an Chip A microchip with a fluidics manifold: the
Chip A microchip design is shown on the left, the fabricated
microchip is shown in the center, and the assembled fluidic
manifold and microchip is shown on the right.
[0117] FIG. 72 shows a post-amplification STR clean-up subsystem
with the post-amplification device.
[0118] FIG. 73 shows the Chip E microchip design, which can be used
a post-amplification device.
[0119] FIG. 74 shows a diagram of a mixer.
[0120] FIG. 75 shows a diagram of a mixer.
[0121] FIG. 76 shows results of using a mixer to lyse cells.
DETAILED DESCRIPTION OF THE INVENTION
[0122] This invention includes devices that incorporate valves,
such as microvalves (including but not limited to pneumatically
actuated valves and microscale on-chip valves), into their design
in order to control the movement of fluid. These devices can be
used for the enrichment of a component, for sample preparation,
and/or for analysis of one or more components in or from a
sample.
[0123] The invention also provides devices for fluid and analyte
processing and methods of use thereof. The devices of the invention
can be used to perform a variety of actions on the fluid and
analyte. These actions can include moving, mixing, separating,
heating, cooling, and analyzing. The devices can include multiple
components, such as a cartridge, a microfluidic microchip, and a
pneumatic manifold. FIG. 40 shows an exemplary device having a
cartridge (101), microfluidic microchip (103), and pneumatic
manifold (113).
I. Sample Preparation Device
[0124] In one aspect a sample preparation device as shown in device
800 in FIG. 16, device 1000 in FIG. 21 and FIG. 22, and device 1000
in FIG. 68 comprises a cartridge integrated with a microfluidic
microchip that controls movement of the fluid in the cartridge
through microvalves and the components to operate the cartridge.
The cartridge and/or the compartments therein can be of sufficient
size to process one or more milliliter of an input sample in an
automated device. The cartridge can process a sample to output a
component that can be moved using pressure-driven flow or vacuum
modulated by microvalves. The cartridge can provide an interface
with a delivery device comprising macroscale samples, such as
blood, aerosol liquids, swabs, bodily fluids, swipes, and other
liquid, solid, and gas samples. The cartridge can process
macroscale sample volumes using microscale sample preparation and
analysis. The cartridge can allow for processing of macroscale or
large volume samples using microfluidic devices and components have
reduced void volumes that allow for reduced loss of materials.
A. Cartridges
[0125] A cartridge, also referred to as a fluidic manifold herein,
can be used for a number of purposes. In general, a cartridge can
have ports that are sized to interface with large scale devices as
well as microfluidic devices. Cartridges or fluidic manifolds have
been described in U.S. Patent Application No. 61/022,722, which is
hereby incorporated by reference in its entirety. The cartridge can
be used to receive materials, such as samples, reagents, or solid
particles, from a source and deliver them to the microfluidic
microchip. The materials can be transferred between the cartridge
and the microfluidic microchip through mated openings of the
cartridge and the microfluidic microchip. For example, a pipette
can be used to transfer materials to the cartridge, which in turn,
can then deliver the materials to the microfluidic device. In
another embodiment, tubing can transfer the materials to the
cartridge. In another embodiment, a syringe can transfer material
to the cartridge. In addition, a cartridge can have reservoirs with
volumes capable of holding nanoliters, microliters, milliliters, or
liters of fluid. The reservoirs can be used as holding chambers,
reaction chambers (e.g., that comprise reagents for carrying out a
reaction), chambers for providing heating or cooling (e.g., that
contain thermal control elements or that are thermally connected to
thermal control devices), or separation chambers (e.g. paramagnetic
bead separations, affinity capture matrices, and chromatography).
Any type of chamber can be used in the devices described herein,
e.g. those described in U.S. Patent Publication Number
2007/0248958, which is hereby incorporated by reference. A
reservoir can be used to provide heating or cooling by having
inlets and outlets for the movement of temperature controlled
fluids in and out of the cartridge, which then can provide
temperature control to the microfluidic microchip. Alternatively, a
reservoir can house Peltier elements, or any other heating or
cooling elements known to those skilled in the art, that provide a
heat sink or heat source. A cartridge reservoir or chamber can have
a volume of at least about 0.1, 0.5, 1, 5, 10, 50, 100, 150, 200,
250, 300, 400, 500, 750, 1000, 2000, 3000, 4000, 5000 or more
.mu.L. The relative volume of a chamber or reservoir can be about
1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000 or more
greater than a channel or valve within the microfluidic microchip.
The size of the chambers and reservoirs of the cartridge, which can
be mated to the microfluidic microchip, can be chosen such that a
large volume of sample, such as a sample greater than about 1, 5,
10, 50, 100, 500, 1000, 5000, 10000, 50000 or more .mu.L, can be
processed, wherein the flow of fluids for processing the sample is
controlled by valves in the microfluidic microchip. This can allow
for a reduced amount of sample and reagent loss due to the reduced
void volumes in the microfluidic microchip compared to other flow
control devices, such as pipettes and large scale valves. The void
volume within a microfluidic microchip can be less than 1000, 500,
100, 50, 10, 5, 1, 0.5, 0.1, or 0.05 .mu.L. This can allow for the
amount of sample or reagent loss during processing of a sample to
be less than 20, 15, 10, 7, 5, 3, 2, 1, 0.5, 0.05 percent.
[0126] For example, FIG. 40 shows cartridge (101) with a reservoir
with a port (115) opening to a side of the cartridge that can be
used to receive materials from a pipette or any other large scale
device. The port can also be adapted with fitting to receive tubing
or a capillary to connect the cartridge to upstream fluidics. The
reservoir can taper down to form a cartridge reservoir opening
(117) that interfaces, aligns, or mates with an opening 105 in the
fluidics layer of the microfluidic microchip.
[0127] A cartridge can be constructed of any material known to
those skilled in the art. For example, the cartridge can be
constructed of a plastic, glass, or metal. A plastic material may
include any plastic known to those skilled in the art, such as
polypropylene, polystyrene, polyethylene, polyethylene
terephthalate, polyester, polyamide, poly(vinylchloride),
polycarbonate, polyurethane, polyvinyldiene chloride, cyclic olefin
copolymer, or any combination thereof. The cartridge can be formed
using any technique known to those skilled in the art, such as
soft-lithography, hard-lithography, milling, embossing, ablating,
drilling, etching, injection molding, or any combination
thereof.
[0128] As exemplified in FIG. 3 and FIG. 4, a cartridge (1) can
comprise a rectilinear, configuration with flat sides. In another
embodiment, a cartridge comprises a surface that is curved,
rounded, indented or comprises a protrusion. In one embodiment a
cartridge has at least one substantially flat surface which is
adjacent to a microfluidic microchip. The cartridge is adapted to
be fluidically connected with ports in the microchip. For example,
openings in the surface of the cartridge can be aligned with ports
in the microchip. When the cartridge and microchip are mated to one
another, the openings align to create the fluidic connections
allowing liquids to pass from the cartridge into the ports of the
microchip, which are connected to channels typically having valves
that form fluidic circuits. Another embodiment of a cartridge is
shown in FIG. 59. FIG. 59 shows a cartridge with multiple ports
that mate with a microchip and external components, such as
syringes. The compartment in the cartridge can be shaped to allow
insertion of the syringe and its protrusion. The cartridge can also
include a vent port to vent gases in chambers of the cartridge or
chambers of the microchip. As well, FIG. 59 shows the position
where an actuated magnet can be used to apply a magnetic field to a
mix chamber. Additionally, FIG. 59 shows a cap that can be used to
close the mix chamber.
[0129] In one embodiment a cartridge contains one or more features,
including but not limited to a chamber, a port, a channel or a
magnet. In one embodiment, microvalves, such as pneumatically
actuated valves are combined with the microfluidic cartridge. In
some embodiments the microvalves are active mechanical microvalves
(such as magnetic, electrical, or piezoelectric thermal
microvalves), non-mechanical microvalves (such as bistable
electromechanical phase change or rheological microvalves),
external microvalves (such as modular or pneumatic), passive
mechanical (such as check microvalves or passive non-mechanical
(such as capillary microvalves) (Oh et al., A review of
microvalves, J. Micromech Microeng. 16 (2006) R13-R39, herein
incorporated by reference in its entirety)).
[0130] In another embodiment, pneumatically actuated valves, such
as MOVe valves modulate the flow of air pressure, vacuum, or fluids
in a microfluidic microchip 2 or multiple microfluidic microchips.
MOVe valves can be microscale on-chip valves, microfluidic on-chip
valves or micro-robotic on-chip valves. In one embodiment the flow
of air pressure, vacuum, or fluids is regulated by one or more
variable pressure pumps, such as solenoid valves or solenoid pumps.
In one embodiment, a microfluidic microchip is a structure that
contains microchannels and/or microtrenches, where a microchannel
is a closed structure and a microtrench is an open structure. In
one embodiment a microfluidic microchip is a planarr structure. In
a related embodiment a microfluidic device comprises a microfluidic
microchip with microvalves clustered on one side of a cartridge. In
one embodiment (FIG. 3 and FIG. 4) the cartridge (1) can comprise
one or more ports (4, 5, 6, 7, 8, 9) to external fluids, air, or
vacuum. Functions of the ports can be for waste (4), reagent entry
(5), vent (6), sample input (7), product output (8). The cartridge
(1) can contain one or more sample input or reaction chambers, (7)
and (3).
[0131] A single chamber within the cartridge, such as a reaction
chamber, can have one or more, or at least one, two, or three
fluidic connections to a microchip. For example, reaction chamber
(3) can have a fluidic connection to the microchip through
connection 120, which is at the base of the chamber, and another
fluidic connection to the microchip through port (9), which is
connected to chamber (3) through a passageway located at the top of
the chamber. The top of chamber (3), port (9), and the passageway
between chamber (3) and port (9) can be closed from the exterior
environment such that fluids in chamber (3) necessarily are pumped
into port (9) when chamber (3) is full and vice versa. Such a
chamber or combination or chamber and port can be referred to as a
closed chamber. The positioning of the fluidic connections need not
necessarily be at the base and top of the chamber, however, fluidic
connections at the base and top positions of the chamber allow for
reduced trapping of gas in the chamber. Alternatively, reaction
chamber (3) can be viewed as a combination of two chambers that are
fluidically connected to each other at a top position, which can be
within the cartridge, and, where each chamber also has an opening
at a base location. The openings at the base locations, also called
chamber apertures, can be fluidically connected to port apertures
on the microchip. The two fluidic connections can allow for fluids
to be directed into and out of the chamber through the microfluidic
microchip.
[0132] In another embodiment a device comprises a cartridge
comprising at least one pneumatically actuated valve, such as a
MOVe valve, located on one or more surfaces or structures in a
non-linear manner. A cartridge can comprise one or more
pneumatically actuated valves that are located within the
cartridge, in a location other than the base of the cartridge.
[0133] Functional elements of a cartridge can include ports,
channels, chambers, filters, magnets, or vents, chambers can be
collectively referred to as functional elements. In one embodiment,
FIG. 4, the functional elements connect to the microfluidic
microchip containing microvalves at junctions 100, 120, 140, 160,
and 230. The functional elements can connect with tubing or
capillaries inserted into the ports, by a flush connection, or by
fittings. In one embodiment a flush connection can comprise a port
of a cartridge aligned directly with an aperture of a microfluidic
microchip. In one embodiment the cartridge and microfluidic
microchip form an integrated module. In another embodiment the
cartridge and microfluidic microchip are two separate pieces which
are attached together, prior to use.
[0134] A cartridge can comprise at least one chamber, a sample
input port, a reagent port, an exit port, a waste port and a
magnet. The magnet can be located adjacent to the chamber, so that
the magnet force generated by the magnet can attract paramagnetic
particles in said chamber to a wall of the chamber. In one
embodiment the paramagnetic particles are beads or cells rendered
magnetically responsive (e.g., cells comprising hemoglobin that are
treated with sodium nitrate). The magnet can be an electric magnet
or a permanent magnet, such as a rare earth metal magnet.
[0135] In one embodiment, FIG. 4, connections or ports (4, 5, 6, 7,
8, and 9) lead to channels in the cartridge (14, 15, 16, 17, 18,
and 19) respectively. Ports (4, 5, 6, 7, and 8) show indents to
reliably attach a connector or tubing to the indent, such as the
indent shown for connection (7) (see the difference in diameter of
connection (7) with channel (17)). In one embodiment, the ports or
ports can interface with a variety of connector or tubes, such as
the capillaries as described in U.S. Pat. No. 6,190,616, U.S. Pat.
No. 6,423,536, U.S. application Ser. No. 09/770,412, Jan. 25, 2001,
U.S. Application No. 60/402,959 or one or more microchips with
modular microfluidic ports as described in U.S. Pat. No. 6,870,185
and U.S. application Ser. No. 11/229,065; all of which are herein
incorporated by reference in their entirety. In one embodiment, the
modular microfluidic ports enable microchips or capillaries to be
reversibly joined without dead volumes or leakage.
[0136] In another embodiment chamber (3) is connected to passageway
(9) and to cone (13), leading to junction (120). Chamber (3) can be
used for reactions as may any of the channels. In FIG. 4 the
cartridge channels lead directly to the apertures of ports on the
microchip (2). The channels of the cartridge can interconnect with
each other as needed. In some embodiments, at least one channel in
a cartridge does not physically connect to a microfluidic
microchip. In another embodiment at least one channel in a
cartridge is fluidically connected to at least one microchannel in
a microfluidic microchip. The connection may or may not utilize an
aperture on the microfluidic microchip. An aperture can be an
opening or a fitting designed to mate between the microchip and the
cartridge. In some embodiments of the invention, the fitting
comprises a seal such as a gasket or an o-ring.
B. Microchips
[0137] In one embodiment a cartridge and a microfluidic microchip
are integrated together to form a single modular device. The
cartridge and a microfluidic microchip can be attached by a fluid
or solid adhesive or mechanically. In one embodiment the adhesive
is a polyacrylate, adhesive tape, double-sided tape, or any other
adhesive known to one skilled in the art. A cartridge can comprise
a feature (12) that is capable of wicking a fluid-based adhesive
into the junction between a microfluidic microchip and a cartridge.
In another embodiment a cartridge is attached to a microfluidic
microchip with a non-fluidic adhesive layer. Alternatively, the
cartridge and microchip can be held together by clips, clamps, or
another holding device. The cartridge and microchip can be aligned
prior to integration by visual cues, with or without a microscope,
or by physical guiding features. Visual cues can include lines or
features that are drawn, etched, or otherwise present on the
cartridge, the microchip, or both. Physical guiding features
include indentations, protrusions, and edges that can be `keyed` to
aid or insure proper assembly.
[0138] In some instances, the microfluidic microchip has diaphragm
valves for the control of fluid flow. Microfluidic devices with
diaphragm valves that control fluid flow have been described in
U.S. Pat. No. 7,445,926, U.S. Patent Publication Nos. 2006/0073484,
2006/0073484, 2007/0248958, and 2008/0014576, and PCT Publication
No. WO 2008/115626, which are hereby incorporated by reference in
their entirety. The valves can be controlled by applying positive
or negative pressure to a pneumatics layer of the microchip through
a pneumatic manifold.
[0139] In one embodiment, the microchip is a "MOVe" microchip. Such
microchips comprise three functional layers--a fluidics layer that
comprises microfluidic channels; a pneumatics layer that comprises
pneumatics channels and an actuation layer sandwiched between the
two other layers. In certain embodiments, the fluidics layer is
comprised of two layers. One layer can comprise grooves that
provide the microfluidics channels, and vias, or holes that pass
from the outside surface to a fluidics channel. A second layer can
comprise vias that pass from a surface that is in contact with the
actuation layer to the surface in contact with the pneumatic
channels on the other layer. When contacted together, these two
layers from a single fluidics layer that comprises internal
channels and vias that open out to connect a channel with the
fluidics manifold or in to connect a channel with the activation
layer, to form a valve, chamber or other functional item. The
actuation layer typically is formed of a deformable substance,
e.g., an elastomeric substance, that can deform when vacuum or
pressure is exerted on it. At points where the fluidic channels or
pneumatic channels open onto or are otherwise in contact with the
actuation layer, functional devices such as valves can be formed.
Such a valve is depicted in cross section in FIG. 1. Both the
fluidics layer and the pneumatics layer can comprise ports that
connect channels to the outside surface as ports. Such ports can be
adapted to engage fluidics manifolds, e.g., cartridges, or
pneumatics manifolds.
[0140] As shown in FIG. 40, the microfluidic microchip (103) can be
interfaced with the cartridge (101). The microfluidic microchip can
have a chamber (105) with an opening that is mated to an opening
(117) of the cartridge (101). The chamber can be used for a variety
of purposes. For example, the chamber can be used as a reaction
chamber, a mixing chamber, or a capture chamber. The chamber can be
used to capture magnetic particles such as magnetic beads,
paramagnetic beads, solid phase extraction material, monoliths, or
chromatography matrices.
[0141] A magnetic component (109) can be positioned such that
magnetic particles in the cartridge reservoir (107) and/or the
microfluidic chamber (105) are captured against a surface of the
microfluidic chamber (105). The magnetic component can generate a
magnetic and/or electromagnetic field using a permanent magnet
and/or an electromagnet. If a permanent magnet is used, the magnet
can be actuated in one or more directions to bring the magnet into
proximity of the microfluidic microchip to apply a magnetic field
to the microfluidic chamber. In some embodiments of the invention,
the magnet is actuated in the direction (111) indicated in FIG.
40.
[0142] Alternatively, any of a variety of devices can be interfaced
with the microfluidic microchip. For example detectors, separation
devices (e.g. gas chromatographs, liquid chromatographs, capillary
electrophoresis, mass spectrometers, etc), light sources, or
temperature control devices can be positioned next to the
microfluidic microchip or used in conjunction with the microfluidic
microchip. These devices can allow for detection of analytes by
detecting resistance, capacitance, light absorbance or emission,
fluorescence, or temperature or other chemical or physical
measurements. Alternatively, these devices can allow for light to
be introduced to a region or area of the microfluidic
microchip.
[0143] A microfluidic device can be designed with multiple chambers
that are configured for capture of magnetic particles. The multiple
chambers and magnetic component can be arranged such that a
magnetic field can be applied simultaneously to all chambers, or be
applied to each or some chambers independent of other chambers. The
arrangement of chambers and magnetic components can facilitate
faster or more efficient recovery of magnetic particles. In
particular, the arrangement can facilitate recovery of magnetic
particles in multiple chambers.
[0144] As shown in FIG. 41, the microfluidic microchip (103) can be
formed of a fluidics layer (203), an elastomeric layer (205), and a
pneumatic layer (207). The fluidics layer can contain features such
as a chamber (105), as well as channels, valves, and ports. The
channels can be microfluidic channels used for the transfer of
fluids between chambers and/or ports. The valves can be any type of
valve used in microfluidic devices. In preferred embodiments of the
invention, a valve includes a microscale on-chip valve (MOVe), also
referred to as a microfluidic diaphragm valve herein. A series of
three MOVes can form a MOVe pump. The MOVes and MOVe pumps can be
actuated using pneumatics. Pneumatic sources can be internal or
external to the microfluidic microchip.
[0145] An example of a MOVe valve is shown in FIG. 1. A
cross-sectional view of a closed MOVe valve is shown in FIG. 1A. A
cross-sectional view of an open MOVe valve is shown in FIG. 1B.
FIG. 1C shows a top-down view of the MOVe valve. A channel (251)
that originates from a fluidic layer can interface with an
elastomeric layer (259) by one or more vias (257). The channel can
have one or more seats (255) to obstruct flow through the channel
when the elastomeric layer (259) is in contact with the seat (255).
The elastomeric layer can either be normally in contact with the
seat, or normally not in contact with the seat. Application of
positive or negative pressure through a pneumatic line (261) to
increase or decrease the pressure in a pneumatic chamber (253)
relative to the fluidic channel (251) can deform the elastomeric
layer, such that the elastomeric layer is pushed against the seat
or pulled away from the seat. In some embodiments of the invention,
a MOVe does not have a seat, and fluid flow through the fluidic
channel is not completely obstructed under application of positive
or negative pressure. The vacuum that can be applied include
extremely high vacuum, medium vacuum, low vacuum, house vacuum, and
pressures such as 5 psi, 10 psi, 15 psi, 25 psi, 30 psi, 40 psi, 45
psi, and 50 psi.
[0146] Three MOVe valves in series can form a pump through the use
of a first MOVe as an inlet valve, a second MOVe as a pumping
valve, and a third MOVe as an outlet valve. Fluid can be moved
through the series of MOVes by sequential opening and closing of
the MOVes. For a fluid being supplied to an inlet valve, an
exemplary sequence can include, starting from a state where all
three MOVes are closed, (a) opening the inlet valve, (b) opening
the pumping valve, (c) closing the inlet valve and opening the
outlet valve, (d) closing the pumping valve, and (e) closing the
outlet valve. Since the inlet and outlet valve can have the same
structure, a MOVe pump can move fluids in either direction by
reprogramming of the sequence of opening inlet or outlet
valves.
[0147] The fluidic layer (203) can be constructed of one or more
layers of material. As shown in FIG. 42, the fluidic layer (203)
can be constructed of two layers of material. Channels (301, 303,
305) can be formed at the interface between the two layers of
material, and a chamber (105) can be formed by complete removal of
a portion of one layer of material. The channels can have any
shape, e.g., rounded and on one side (301), rectangular (303), or
circular (305). The channel can be formed by recesses in only one
layer (301, 303) or by recesses in both layers (305). The channels
and chambers can be connected by fluidic channels that traverse the
channels and chambers shown. Multidimensional microchips are also
within the scope of the instant invention where fluidic channels
and connections are made between multiple fluidic layers.
[0148] The thickness (307) of the second layer of material can be
of any thickness. In some embodiments of the invention, the second
layer has a thickness that minimizes reduction of a magnetic field
in the chamber (105) that is applied across the second layer from
an external magnetic component or minimizes reductions in heat
transfer.
[0149] As shown in FIG. 43, the fluidic layer (203) can be
constructed of a single layer of material. The single layer is then
interfaced with an elastomeric layer, such that channels (305, 303)
and chambers (305) are formed between the fluidic layer and the
elastomeric layer (205).
[0150] The microfluidic microchip can be constructed from any
material known to those skilled in the art. In some embodiments of
the invention, the fluidics and pneumatic layer are constructed
from glass and the elastomeric layer is formed from PDMS. In
alternative embodiments, the elastomer can be replaced by a thin
membrane of deformable material such as Teflon (PTFE), silicon, or
other membrane. The features of the fluidics and pneumatic layer
can be formed using any microfabrication technique known to those
skilled in the art, such as patterning, etching, milling, molding,
embossing, screen printing, laser ablation, substrate deposition,
chemical vapor deposition, or any combination thereof.
[0151] In one embodiment, microchannel circuits are formed on a
microfluidic microchip 2, as shown in FIG. 5, linking sets of
microvalves with microchannels. In one embodiment the microvalves
are pneumatically actuated valves. In one embodiment the
pneumatically actuated valves are MOVe microvalves. In one
embodiment, the fluidic path between a cartridge and a microfluidic
microchip, such as between chambers, ports, channels,
microchannels, and other functional elements can be controlled by
opening or closing at least one microvalve. In one embodiment the
microvalve is controlled by a microprocessor control such as a
computer. A computer can include an input/output controller, or any
other components known to one skilled in the art such as memory
storage and a processor. In one embodiment, a microvalve is a MOVe
valve that is actuated by a pneumatic source, such as through
pneumatic ports 10, 20, 30, 40, 50, 60, or 70. In one embodiment
the pneumatic source is controlled by at least one solenoid. In one
embodiment the solenoid is miniaturized and can be connected to
vacuum or pressure sources. In one embodiment the pneumatic source
is connected to a pneumatic port using a force such as clamping,
springs, pneumatics, or a screw force, optionally with sealing
provided by an o-ring.
[0152] In one embodiment FIG. 5 shows a view of the top of a
microfluidic microchip (2), this side makes contact with the bottom
of cartridge (1). A microvalve 110 controls the fluidic path
between microchannels 101 and 121. A microvalve 130 controls the
fluidic path between microchannels 131 and 141. Microvalve (150)
controls the fluidic path between microchannels 151 and 152.
Microvalve 180 controls the fluidic path between microchannels 181
and 191. Microvalve 200 controls the fluidic path between
microchannels 201 and 212. Microvalve 220 controls the fluidic path
between microchannels 221 and 231.
[0153] In one embodiment junctions can connect one or more
microchannels. FIG. 5 shows the schematic for a microchip that can
be mated with the cartridge shown in FIG. 4. In FIG. 5, junction
100 connects to single microchannel 101, junction 140 connects to
single microchannel 141, junction 160 connects to single
microchannel 161, and junction 230 connects to single microchannel
231. Junction 190 connects to two microchannels 191 and 201.
Junction 120 connects to three microchannels 121, 131, and 151. In
one embodiment more than three microchannels can be connected to a
single junction.
[0154] The microchannels can be fabricated by one or more
techniques such as photolithography, molding, embossing, casting,
or milling. The microchannels can be manufactured in a material
such as glass, plastic, polymer, ceramic, gel, metal, or another
suitable solid.
[0155] In another embodiment a device comprises a cartridge
comprising at least three chambers, more than one input port and
more than one output port (FIG. 28). The cartridge can be adapted
to process a nucleic acid sample for analysis. The cartridge can be
adapted to receive one or more reagents from an external reagent
source. The reagents can be a paramagnetic bead, a non-paramagnetic
bead, an enzyme, a dNTP, a buffer solution, a salt solution, an
alcohol solution, an solution comprising EDTA or an oligonucleotide
or other reagents. The enzymes can be a ligase, a restriction
enzyme, a polymerase, or a kinase or any other enzyme or catalytic
biomaterials including RNAs. The device can comprise a magnet which
can attract paramagnetic beads to a wall of one or more chambers.
In another embodiment at least one chamber comprises a filter to
capture beads, such as non-paramagnetic beads.
[0156] In one embodiment the cartridge is used in a method of
sample enrichment comprising: delivery of a sample to a chamber by
a sample port and delivery of paramagnetic particles to a chamber
by a reagent port. The paramagnetic particles (e.g. paramagnetic
beads) bind to at least one component in the sample (such as DNA,
RNA, micro RNA, a protein, a lipid, a polysaccharide or other
ligand). The paramagnetic particles are attracted to a wall of a
chamber by virtue of the Magnetic force exerted by a magnet located
outside the chamber. The paramagnetic particles are washed with a
wash solution delivered to the chamber comprising the paramagnetic
particles by a reagent port, and the wash solution is removed by a
waste port. A reagent can be added to elute the component of the
sample from the paramagnetic particles and output the sample
component to another device for further processing or analysis. A
preferred embodiment is to output the component of the sample on
the paramagnetic particles.
[0157] In one embodiment a device comprising a microfluidic
microchip is used in a method of diagnosis. In one embodiment the
diagnosis comprises the detection of an infectious agent in a
sample. In one embodiment the infectious agent is a bacteria,
virus, fungi, mycoplasm or prion. In another embodiment a device
comprising a microfluidic microchip is used in a method of
diagnosis of a hereditary disease. In one embodiment the hereditary
disease is caused by one or more DNA mutations, such mutations
include but are not limited, triplet base expansions, base
substitution mutations, deletion mutations, addition mutations,
nonsense mutations, premature stop codons, chromosomal deletions,
chromosomal duplications, aneuploidy, partial aneuploidy or
monosomy. In another embodiment a device comprising a microfluidic
microchip is used in a method to diagnose cancer or a
predisposition to cancer. In another embodiment a device comprising
a microfluidic microchip is used in a method to diagnose a
hereditary disease such as autism, downs syndrome, trisomy,
Tay-sachs, or other hereditary diseases. In some embodiments a
sample used for diagnosis in a device comprising a microfluidic
microchip is a blood sample, a mucus sample, a lung lavage sample,
a urine sample, a fecal sample, a skin sample, a hair sample, a
semen sample, a vaginal sample, or an amniotic sample.
[0158] In another embodiment a device comprising a microfluidic
microchip is used to identify the presence of environmental
contamination of an agent. In one embodiment the agent is a
biological agent such as bacteria, virus, fungi, or mycoplasm in an
environmental sample. In another embodiment the agent is a
contaminant agent, such as a pesticide, an herbicide, or a
fertilizer. In one embodiment the environmental sample is a soil
sample, a water sample, an air sample, a meat sample, a vegetable
sample or a fruit sample. In another embodiment, the agent is a
genetically modified organism.
[0159] In another embodiment a device comprising a microfluidic
microchip is used for genotyping, identification of an individual
mammal (such as a human), forensics, gene expression, gene
modification, microRNA analysis, or ribotyping.
[0160] In another embodiment a microfluidic microchip is used in a
method comprising molecular biological analysis, including but not
limited to polymerase chain reaction (PCR) amplification of nucleic
acids in a sample (such as Allele-specific PCR, Assembly PCR,
Asymmetric PCR, Colony PCR, Helicase-dependent amplification,
Hot-start PCR, Intersequence-specific (ISSR) PCR, Inverse PCR,
Ligation-mediated PCR, Methylation-specific PCR Multiplex
Ligation-dependent Probe Amplification, Multiplex-PCR, Nested PCR,
Overlap-extension PCR, Quantitative PCR Reverse Transcription
PCR-PCR, Thermal asymmetric interlaced--PCR, Touchdown PCR, or
PAN-AC PCR), isothermal nucleic acid amplifications, (such as
Loop-mediated Isothermal Amplification (LAMP); nick displacement
amplification; Helicase Dependant Amplification platform (HDA); and
the primase-based Whole Genome Amplification platform (pWGA);
single primer isothermal amplification (SPIA) and Ribo-SPIA for
RNA; strand displacement amplification (SDA); EXPAR [Van Ness J,
Van Ness L K, Galas D J. (2003) Isothermal reactions for the
amplification of oligonucleotides. Proc Natl Acad Sci USA.
100:4504-9.]; rolling circle amplification (RCA);
transcription-based amplification system (TAS) and its derivatives
include self-sustaining sequence replication (3SR), isothermal
nucleic acid sequence-based amplification (NASBA), and
transcription-mediated amplification (TMA); ligase chain reaction
(LCR)), sequencing reactions of DNA or RNA (such as Maxam-Gilbert
sequencing, Sanger chain-termination method, Dye-terminator
sequencing Emulsion PCR sequencing, massively parallel sequencing,
polony sequencing, sequencing by ligation, sequencing by synthesis,
or sequencing by hybridization), restriction fragment length
polymorphism (RFLP) analysis, single nucleotide polymorphism (SNP)
analysis, short tandem repeat (STR) analysis, microsatellite
analysis, DNA fingerprint analysis, DNA footprint analysis, or DNA
methylation analysis.
[0161] In one embodiment a cartridge employs beads coupled to a
binding moiety, including but not limited to a binding receptor,
transferrin, an antibody or a fragment thereof (such as an Fc
fragment or an Fab fragment), a lectin, or a DNA or RNA sequence.
In another embodiment a cartridge comprises a reagent such as an
anti-coagulant, a fixative, a stabilization reagent, a preservative
or precipitation reagent.
C. Pneumatic Manifold
[0162] A pneumatic manifold can be integrated with any microchip
and/or cartridge described herein to facilitate distribution of air
pressure or vacuum. The air pressure or vacuum can be used to
actuate valves on the microchip. Alternatively, air pressure or
vacuum can be supplied to a cartridge such that air pressure or
vacuum is provided to microchannels within the fluidics layer of a
microchip which can be used to move fluids or gases within the
fluidics layer. A pneumatic manifold provides the air pressure or
vacuum to operate microvalves on microchip (2) on cartridge (1) of
FIG. 3 or operate microvalves in other devices.
[0163] A pneumatic manifold can be used to mate the pneumatic lines
of a microfluidic microchip to external pressure sources. The
pneumatic manifold can have ports that align with ports on the
pneumatics layer of the microfluidic microchip and ports that can
be connected to tubing that connect to the external pressure
sources. The ports can be connected by one or more channels that
allow for fluid communication of a liquid or gas, or other material
between the ports.
[0164] The pneumatic manifold can be interfaced with the
microfluidic microchip on any surface of the microchip. The
pneumatic manifold can be on the same or different side of the
microfluidic microchip as the cartridge. As shown in FIG. 40, a
pneumatic manifold (113) can be placed on a surface of the
microfluidic microchip opposite to the cartridge. As well, the
pneumatic manifold can be designed such that it only occupies a
portion of the surface of microfluidic microchip. The positioning,
design, and/or shape of the pneumatic manifold can allow access of
other components to the microfluidic microchip. The pneumatic
manifold can have a cut-out or annular space that allows other
components to be positioned adjacent or proximal to the
microfluidic microchip. This can allow, for example, a magnetic
component (109) to be placed in proximity of a chamber within the
microfluidic microchip.
[0165] A pneumatic manifold can be constructed of any material
known to those skilled in the art. For example, the cartridge can
be constructed of a plastic, glass, or metal. A plastic material
includes any plastic known to those skilled in the art, such as
polypropylene, polystyrene, polyethylene, polyethylene
terephthalate, polyester, polyamide, poly(vinylchloride),
polycarbonate, polyurethane, polyvinyldiene chloride, cyclic olefin
copolymer, or any combination thereof. The pneumatic manifold can
be formed using any technique known to those skilled in the art,
such as soft-lithography, conventional lithography, milling,
molding, embossing, drilling, etching, or any combination
thereof.
[0166] A pneumatic manifold (370) was designed (FIG. 20) that
eliminates over twenty tubing ports and provides a robust,
reproducible interface between the control system, the pneumatic
solenoids and the MOVe input ports. The manifold (370) has a gasket
(380) and a bottom plate (390) that are fastened together. The
cartridge (1) is held on the plate (370) by a bracket in position
to align the pneumatic ports 10, 20, 30, 40, 50, 60, and 70 on
microfluidic microchip (2), shown in FIG. 5, with the pneumatic
lines shown of the reverse side of 370 in the FIG. 20 insert. The
external pneumatics are controlled by a solenoid valve bank that
can be miniaturized and can be connected to vacuum or pressure
sources.
[0167] The apparatus shown in FIG. 21 and FIG. 22 can incorporate
the pneumatic manifold shown in FIG. 20. The apparatus can be used
for sample preparation, as described herein, and can incorporate a
cartridge. Cartridge (1), labeled `cube`, is attached to manifold
(370) with solenoids (1819). The assembly of the cartridge and
manifold is mounted on a base plate of the apparatus. The pneumatic
manifold can be controlled by an IO controller (1803).
[0168] A gas supply, such as a reservoir that can be maintained at
a desired pressure or vacuum, can supply gas to the manifold. The
gas supply can be connected to an outside pressure or vacuum
source. The gas supply feeding the gas supply manifold can have a
pressure gauge to monitor the inlet pressure. The gas supply can
supply gas to multiple components of the system through a gas
supply manifold (1821). The gas supply manifold can supply gas to
the pneumatic manifold (370) and to individual reagent containers,
(1809) and (1807). The line supplying the distribution valve (390)
with gas can be regulated by a regulator (1815).
[0169] Reagents and/or sample can also be supplied to the cartridge
through the reagent distribution valve (390) that is connected to
containers (1809) in a reagent storage region (380) and a bead
solution container (1807) that is mounted on a bead mixer (1805).
Adapter (1817) can be mounted and/or aligned with the cartridge
such that a delivery device, such as a syringe, can deliver a
material to the cartridge. The adapter (1817) can be thermally
regulated by a heater control (1801). The adapter can have a
thermal conductor, such as brass, to distribute heat generated by
heater coil or a Peltier device. The adapter can maintain
temperature between about 20 to 100, 20 to 75, or 50 to 60 degrees
Celsius.
[0170] A magnet assembly (1811) can be positioned adjacent to the
cartridge. A magnet (300) of the magnet assembly can be positioned
adjacent to the cartridge (1) and moved by an actuator, such that
the magnet can exert a magnetic field within the cartridge, or a
microchip integrated, mated, or interfaced with the cartridge. The
magnetic field can be used to capture paramagnetic or magnetic
particles, such as beads, within the cartridge or microchip and
separate material bound to the particles from waste materials.
Waste from the cartridge and/or microchip can be delivered to a
waste container (1813).
[0171] The apparatus shown in FIG. 21 and FIG. 22 can use seven
solenoid valves to operate the cartridge (1). The size and
complexity of the apparatus can be further reduced with MOVe
microvalves. FIG. 23 and FIG. 24 shows a reagent distribution
device that contains microfluidic microchip 600, which is
approximately two inches wide. Solenoid banks 680 and 684 provide
connection to full scale external vacuum and pressure through
connectors 681, 682, 685, and 686. The solenoids are controlled
through electrical junctions 689 and 687. The microfluidic
microchip 600, which has MOVe valves, is held in contact with the
manifold 700 by attachment 711 using clamp 710. Other methods known
to one skilled in the art can be used to connect the microchip to
the pneumatics manifold 700.
[0172] As shown in FIG. 25, microchip 600 connects five reagent
sources, 621, 622, 623, 624, and 625 with two sample loops 630 and
631 and five devices 634, 635, 636, 637, and 638 which may be
microfluidic devices such as cartridge (1). Sample loops 630 and
631 can be configured to have predetermined volumes. The sample
loops can have a portion which is removable. Thus, they can be
removably connectable to ports in the fluidic manipulation module.
The sample loop can be removed to allow for adjustment of the
volume of the sample loop. A portion of the sample loop can be
capillary tubing, any other type of tubing, or a microfluidic
channel. The sample loop can be connected to the microchip using
any type of junction described herein. For example, a junction can
connect to a cannula, an upfit tubing, a microtubing fitting, an
Upchurch tubing adapter, or a FROLC connector [Jovanovich, S. B.,
G. Ronan, D. Roach and R. Johnston. Capillary valve, connector, and
router. Feb. 20, 2001. U.S. Pat. No. 6,190,616]. It is apparent
that the number of reagent sources, microfluidic devices, and
sample loops can be increased or decreased. Each microfluidic
device can perform the same function, different, or complementary
functions. The devices can be connected through modular
microfluidic ports.
[0173] In an aspect of this invention shown in FIG. 25, a microchip
(600) comprises a main microfluidic channel that intersects with
two other second microfluidic channels. The main microfluidic
channel can be a channel that connects the reagent source 625 with
device 634, as shown in FIG. 25. The second channels can be the
channel that connect valve 606 to the sample loop 630 and the
channel that connects valve 608 to sample 630. At least one, and
optionally both, of these second channels connects with the main
channel through a flow-through valve (606 and 608) that allow a
fluid to flow through the main channel but only into or out of the
second channel with the flow through valve is open. The
flow-through valve can be redesigned as an in-line valve. The main
channel also comprises an intermediate valve (674) between the
points of intersection of the two second channels. Each second
channel opens from the microchip at an entry port. A sample loop
(630) having a channel of defined volume is removably attached to
each of the entry ports. Thus, a specific volume of fluid in the
sample loop can be injected into the main channel by closing the
intermediate valve (674), opening the flow through valves (606 and
608) and applying pressure to the main channel. The sample loop can
also be referred to as a fluid loop or reagent loop.
[0174] The microchip 600 of the distribution manifold uses eighteen
microvalves 601 to 618 to direct flow through the manifold. The
microvalves are operated through pneumatic ports with o-rings or
other connectors including modular microfluidic ports to pneumatic
manifold 700. For example, connection 671 provides pressure or
vacuum to microvalve 641 and connection 673 provides pressure or
vacuum to microvalve 642. The flow of reagents from reagent
sources, 621, 622, 623, 624, and 625 can be directed to fill the
sample loops individually or to move samples to devices 634, 635,
636, 637, and 638.
[0175] The pneumatics on manifold 700, as shown in FIG. 26,
connects pressure sources 685 and vacuum sources 686 through
solenoid banks 680 and 681 to pneumatic channels 683 leading to the
array of ports 684 that includes ports 671 and 673. The top portion
of FIG. 26 shows the pneumatic lines of the pneumatic manifold that
lead into the pneumatic layer of a microchip from the solenoids.
The bottom portion of FIG. 26 shows the solenoids and vacuum (685)
and pressure source (686) that are connected to each solenoid. The
solenoid banks are controlled by electronics to open and close each
individual solenoid to the common vacuum or pressure sources. The
individual vacuum or pressure control is also envisioned.
[0176] The pneumatic manifold 700, shown in FIG. 23, can operate
the microvalves 601 to 618 on microfluidic microchip 600, shown in
FIG. 25. For example, to move a reagent from reagent source 622 to
sample loop 630, microvalves 602, 606, and 608 are opened and
pass-through valve 658 is closed. Alternatively, reagent can
by-pass sample loop 630 by closure of T-valves 606 and 608 and
opening pass-through valve 658. The valves can be controlled by
pneumatic lines that are fluidically connected to the pneumatic
manifold. For example, pneumatic line 672 is controlled by a
solenoid on pneumatic manifold 700, shown in FIG. 23, to open or
close the microvalve 642. Valves 603, 604, 605, 609, 610, are
always open to flow through the microfluidic circuit containing
microchannels 641 to 644 and 656 to 668. The reagent is moved into
sample loop 630.
[0177] The circuit between 622 and 612 can be overfilled if desired
or precisely controlled by MOVe microvalves to modulate flow or
control of timing. Once the sample loop 630 is filled, a defined
volume has been selected. The microfluidic circuit can be cleaned
by flushing cleaning solutions or air or gas through the main
channel to further define the reagent volume. If reagent source 621
was a compressed air or gas source (pressure and vacuum are types
of reagents in a pressurized flow system), opening microvalve 601
and microvalves 606, 610, and 616 creates the circuit to move the
measured reagent in sample loop 630 to device 636. In one
embodiment a means to connect any number of reagent sources to a
microdevice such as cartridge (1) is provided.
II. Parallel Processing of Samples
[0178] In some embodiments of the invention, one or more cartridges
can be operated simultaneously to allow for parallel processing of
samples. FIG. 16 illustrates parallel or ganged operation of
multiple cartridges with microvalves on a single pneumatic manifold
in swab extraction assembly (800). The manifold (370) distributes
regulated vacuum and pressure to operate four cartridges (1),
indicated in the figure, using solenoids (680). Solenoids (680)
control pressure to the pneumatic layer of a microchip integrated
with each cartridge through the pneumatic manifold (370, 380, 390).
The pneumatic manifold is formed by a top plate (370), a gasket
(380) and a bottom plate (390). The top plate can have channels
etched into it. The channels can be sealed by the gasket, which is
sandwiched against the top plate by the bottom plate (390).
Actuator 310 moves rod 810 to move magnets (320) close to or away
from the cartridges (1). Clamps 805 hold cartridges (1) in
place.
[0179] In other embodiments of the invention, a single cartridge
integrated with a microchip can process multiple samples at one
time using parallel channels. FIG. 14 and FIG. 15 shows an
assembled capture and reaction microchip with capillary feed and
magnets. This microchip can capture bead solutions and perform four
STR-PCR reactions simultaneously. FIG. 14 shows a microchip (1201)
with a cartridge (1203) adhered to the microchip and tubes (1205,
1207, 1209, 1211, 1213, 1215, 1217, and 1219) leading into and out
of the microchip. A total of eight tubes are shown and two tubes
are used per parallel reaction. For example, one unit of the
parallel processing device is served by tubes 1205 and 1213.
[0180] In another embodiment, a four-channel sample preparation
device (FIG. 66) combines a four-channel parallel reagent delivery
device (FIG. 67) that meters and delivers reagents simultaneously
to all four channels of a single integrated cartridge (FIG. 68)
enabling four samples to be processed simultaneously and
rapidly.
[0181] The four-channel parallel reagent delivery device combines
an Chip C microchip (see FIG. 67) with a fluidics manifold mounted
on a pneumatics control manifold. Reagents are metered, using one
of the two different size reagent loops, which can be similar to
the sample loops described herein, for each channel, and delivered
in parallel to all four channels of the sample preparation device.
Delivering reagents simultaneously to all four channels of the
sample preparation device using the parallel reagent delivery
device can takes <4 minutes, representing a process time saving
of >11 minutes as compared to the first generation serial
reagent delivery device that took .about.15 minutes per four
samples processed.
[0182] Bonded pneumatics manifolds can be used to control both the
reagent delivery and sample preparation devices by fabricating the
manifolds using an adhesive bonding approach; however, these may be
prone to delamination over time due to the pneumatic pressures used
in the subsystem, and the size and complexity of the manifold.
Thermally bonded manifolds can mitigate delamination issues, but
may only be a viable approach for relatively small and low
complexity manifold designs such as the reagent delivery device. A
monolithic manifold made from a single piece of polycarbonate with
tubing connecting pneumatic ports to the solenoid control valves
can operate the four-channel sample preparation cartridge and has
proved to be a viable alternative to bonded pneumatic manifolds,
see FIG. 45 and FIG. 46 for examples. This pneumatic manifold
design concept is also being utilized for control of the Chip A
microchip on the Post-amplification STR (Single Tandem Repeat)
clean-up subsystem.
[0183] Assembly processes for the microchip and fluidic manifold of
the four-channel sample preparation cartridge have also been
improved. Historically, silicon epoxy can be used to attach the
cartridge to its associated MOVe microchip by wicking the adhesive
between the microchip and the cube. An inherent lack of control of
the movement of the epoxy can allow it to occasionally wick into
the ports on either the microchip or the cube creating a blockage
in the fluidic pathway rendering the device unusable. This process
has been improved by using a double-sided adhesive tape (Adhesives
Research ARcare90106) to assemble the fluidic cubes and microchips;
this is now the preferred assembly method used for the four-channel
reagent delivery cartridge, the sample preparation device, and the
post amplification device in the Post-amplification STR clean-up
subsystem described below.
[0184] The integrated four-channel sample preparation cartridge
with the Chip D microchip (see FIG. 69) was tested. The Chip D
microchip, shown in FIG. 69 on the left panel, highlighted an issue
with the design wherein the PDMS membrane inadvertently closed off
fluidic channels adjacent to flow through valves on the MOVe
microchip. Without being limited to theory or conjecture, it is
thought that this effect is due to a combination of variables
including minor differences in alignment during microchip assembly,
the etch depth of the microchip's fluidic layer, and the pneumatic
pressure used to operate the microchip on the sample preparation
device. A microchip, Chip E, shown in FIG. 69 right panel, was been
designed to convert all flow through valves that form a T-junction
between two bisecting channels in the Chip D microchip, to in-line
channel that have only one channel passing through the middle of
the channel. The Chip E microchip can reduce the occurrence of
inadvertent channel closure during valve closure. In FIG. 69, left
panel, the four circles in that are positioned along the middle of
the microchip can be operated independently and are each
fluidically connected to a swab extraction device that can be used
to extract analytes from a swab. Other ports in the microchip can
connect to chambers in a cartridge (similar to the ports and
chambers described for FIG. 3 and FIG. 4) that are mated to the
microchip (shown in FIG. 68) for performing reactions, such as
nucleotide binding. Operation of the integrated four-channel sample
preparation cartridge with microchip is similar to the operation of
the device shown in FIG. 3 and FIG. 4.
[0185] Microchip blockages due to the inadvertent introduction of
fibers into the systems and devices described herein can be
problematic in microfluidics. To minimize blockages, all reagents
with the exception of paramagnetic bead solutions, can be filtered
prior to loading and in-line filters used to minimize microchip
blockages.
[0186] Subsystem testing of the second-generation sample
preparation device focused on characterization of system
reproducibility and failure modes. A total of 80 samples were
processed on the subsystem with a success rate of 63%. Failure
modes included Reagents line accidentally becoming disconnected
(2.5%), chip blockages (3.75%), no STR profiling observed (9%), and
DNA yield <0.08 ng (22%), which is the limit of detection of the
downstream system. The average yield of purified DNA yield was
found to be 0.26 ng. Approximately half of the samples tested in
STR reactions gave full profiles and half gave partial profiles
(see FIG. 70). A number of blank samples were processed on the
system using cleaned and recycled cartridges, and run-to-run cross
contamination was found to be negligible.
III. Integrated Sample Preparation and Polymerase Chain
Reaction
[0187] In some embodiments of the invention, a cartridge can be
integrated with devices for performing polymerase chain reaction
and product analysis. Such a device is shown in FIG. 6. FIG. 6
shows a cartridge with integrated microchip (1), temperature
modulating device (400), and downstream analysis device (500). In
certain embodiments the device comprises a fluid preparation module
comprising a cartridge mated or otherwise fluidically connected to
a microchip; an off-chip thermal modulation module connected to the
fluid preparation module through a fluid transporter with a fluidic
channel, such as a tube, through the cartridge, and configured to
modulate the temperature in the fluid transporter, wherein the
fluid transporter is further fluidically connected to a second
microchip with valves and fluidic channels that can selectively
route fluid to one or more subsequent devices. This device can be
used for thermal cycling or isothermal reactions.
[0188] The cartridge with integrated microchip can be formed of any
cartridge and microchip described herein. For example, the
cartridge and microchip shown in FIG. 3, FIG. 4, and FIG. 5. A
movable magnet (300) can be positioned adjacent to the cartridge.
The movable magnet can be moved by an actuator (310). The movable
magnet can be used to apply a magnetic field within the cartridge
or the microchip. In some embodiments, the movable magnet can be
used to facilitate gathering or collecting of beads against a wall
of a chamber within the cartridge or the microchip.
[0189] A temperature modulator can be fluidically connected to the
cartridge and microchip through reaction channel (250). The
reaction chamber (250) can be connected at an end (251) to the
cartridge. The temperature modulator can be used for thermal
cycling the temperature of a reaction channel (250) containing a
reaction mixture and a nucleic acid enriched from a sample
(collectively referred to as the PCR reaction sample). A control
mechanism can be used for controlling the operation of the
temperature modulator. An optical assembly can be used to monitor
or control the reaction. The optical assembly can introduce or
detect light. For example, an optical assembly 410 can be used for
performing Real-time PCR or other real-time or end point
measurements. In certain embodiments the temperature modulator
employs a thermo-coupled Peltier thermoelectric module, a
conventional thermoelectric module, hot air, infrared light or
microwave. In one embodiment the temperature modulator uses a
Peltier thermoelectric module external to the reaction channel to
heat and cool the PCR reaction sample as desired. The heating and
cooling of the thermoelectric module can be distributed over a
region 350. Additional views of the temperature modulator 400 are
shown in FIG. 7 and FIG. 8. FIG. 7 shows the reaction channel 250
in contact with a temperature controlled region 350. The
temperature modulator can also include a movable magnet 320 that is
positioned by an actuator 330. The movable magnet can be used to
capture magnetic particles at position 340, as shown in FIG. 6. In
some embodiments of the invention, the temperature controlled
region comprises two parts. The two parts can be parts of a
clamshell that are clamped, locked, or held together to maintain
thermal contact with the reaction channel 250. One portion of the
temperature controlled region, portion 711 of FIG. 8, can be hinged
to the second portion of the temperature controlled region. The
temperature controlled regions can have grooved channels for
positioning of one or more reaction channels, as shown on the right
side of FIG. 7 and in FIG. 8. The left side of FIG. 7 shows the
temperature controlled region in a closed configuration.
Additionally, the temperature controlled region can comprise one or
more constriction components, shown as 709 and 701 in FIG. 8. The
constricting points can pinch the reaction channel such that a
portion of the reaction channel is isolated from another portion of
the reaction channel. In some embodiments of the invention, the
reaction channel is pinched in two locations such that a body of
fluid, such as a reaction mixture, is isolated. Constriction
components 709 and 701 can mate with additional constriction
components 707 and 705 to facilitate pinching of the reaction
channel.
[0190] Alternatively the temperature modulator can constrict the
reaction tubing using an pinch clamp, as shown in FIG. 65. Use of
the pinch clamp, which can be formed of a plastic such as Ultem,
can reduce heat transfer to the reaction channel. The reduction in
heat transfer can reduce the likelihood that the reaction channel
has for being welded closed during thermocycling or temperature
regulation. Alternatively, different material tubing can be used as
the reaction channel to ensure that the reaction channel can
maintains its shape before and after the thermocycling or
temperature regulation process. Different material tubing can also
be used to reduce rate of evaporation during the temperature
modulating process. Example materials include ethylvinyl acetate,
silicone, and silanized c-flex tubing.
[0191] The temperature modulating device can modulate temperatures
at a rate of 0.5 to over 3 degrees Celsius per second. The heater
can utilize about 25 to 100 Watts and a fan, which can be used to
cool the temperature modulating device, can produce an air flow
rate of at least about 75, 100, 130, 150, 200, 250, or 300 cfm.
[0192] In one embodiment a sample preparation device comprising a
cartridge integrated with a microfluidic microchip 1, which can be
used to control the movement of fluid in the cartridge, can be used
in conjunction with a temperature modulator 400 as a flow-through
PCR thermal cycler. Driving force for moving the fluid can be an
external pressure source or an internal pressure source, such as a
MOVe valves within the microchip. A flow-through PCR thermal cycler
can be used when highly sensitive or high throughput PCR is
desired. There are many situations in which one might want to
sample air, blood, water, saliva, a cellular sample, or other
medium in a sensitive PCR assay. This can be used to look for a
variety of biological contaminants including influenza, bacterial
pathogens, and any number of viral or bacterial pathogens.
Flow-through PCR can allow PCR to be practiced in an automated
manner without the need for human interaction. A flow-through PCR
system can also serve as an early warning system in HVAC systems of
buildings, airplanes, busses, and other vehicles, and can be used
in the monitoring of blood, water, or other sample sources for the
presence of an infectious agent or a contaminant.
[0193] As shown in FIG. 6, the flow-through PCR device takes a
sample from a collection device, such as a buccal swab, a syringe,
an air sampler, fluid sampler or other sampler and delivers it to a
sample preparation device 1. FIG. 6 is not necessarily drawn to
scale. The sample is prepared in the preparation device 1, which in
some embodiments may include cell lysis, DNA, RNA, or micro RNA
enrichment or purification, filtration, or reverse transcription.
In one embodiment at least one nucleic acid is enriched. In another
embodiment at least one enriched nucleic acid is prepared for PCR
by adding the nucleic acid to PCR reagents (such as at least one
DNA polymerase, RNA polymerase, dNTPs, buffer or a salt) and
primers, (such as assay-specific primers or broadly applicable
primer sets for multiple target pathogens). These primers may be
chosen to selectively amplify at least one nucleic acid isolated
from a specific pathogen (such as a mold, virus, bacteria, parasite
or amoeba), gene, other desired nucleic acid, or any combination
thereof. The composition comprising at least one nucleic acid
enriched from a sample, PCR reagents and primers is called a PCR
reaction sample. In one embodiment, the flowthrough PCR can be used
as a continuous flow device while in other embodiments samples are
moved into the thermal cycling region and stopped.
[0194] The PCR reaction sample then flows through a reaction
channel (250) to a temperature controlled device or region (350).
In some embodiments the reaction channel is clear or transparent.
In another embodiment the reaction channel is opaque. In one
embodiment the reaction channel is a cylinder. In another
embodiment the reaction channel's cross section comprises one or
more planes forming a shape such as a triangle, square, rectangle,
pentagon, hexagon, heptagon, octagon, nonagon, decagon, or other
polygon. In one embodiment the volume of PCR reaction sample is
such that it takes up a small discrete length of space in the
reaction channel, the rest of which is occupied by air, gas, or a
non-reactive liquid, such as mineral oil. Air, gas, or a
non-reactive liquid can be used to separate individual PCR reaction
samples from each other. In one embodiment the temperature
controlled region (350) is thermally modulated by one or more
modules, including but not limited to thermo-coupled Peltier
thermoelectric module, a conventional thermoelectric module, hot
air, microwave, or infrared light. In one embodiment the thermal
cycler uses Peltier thermoelectric modules external to the tube to
heat and cool the sample as desired. In one embodiment a detection
module (410) measures fluorescence, luminescence, absorbance or
other optical properties to detect a signal emitted from a PCR
reaction sample while it is located with a temperature control
region, or after it has left a temperature control region. A
detection module can comprise a light source (such as a coherent
light source or incoherent light source) used to excite a
fluorescent dye (such as an intercalating dye, including but not
limited to ethidium bromide or Syber green) in a PCR reaction
sample, and the excitation light is sensed with a photodetector
(such as a CCD, CMOS, PMT, or other optical detector). Detection
electronics can evaluate the signal sent from the detection module
(410).
[0195] In one embodiment, after the desired number of thermal
cycles are complete, the PCR reaction sample is pumped or pushed
further down the reaction channel, using pressure or vacuum,
exiting the temperature controlled region and passing into a second
microfluidic microchip (500). The second microchip (500) can be
attached at end (252) to the reaction channel (250). Microfluidic
microchip (500) can comprise microvalves (510, 520, 530, and 545).
Any three microvalves such as 510, 520, and 530 or 510, 520, and
545 can form a pump. Microchannels 505, 515, 525, and 540 can
connect the pumps on the microchip. Downstream devices 535 and 550
can be connected to the microchip. Flow of material to devices (535
and 550) can be controlled by the microvalves, for example, by
keeping either valve 530 or 545 closed while pumping or moving
fluid. In one preferred embodiment, the downstream device are
analytical devices that can be used for performing electrophoresis,
mass spectroscopy, or other analytical techniques known to one
skilled in the art.
[0196] In one embodiment the second microfluidic microchip can
deliver the PCR reaction sample to a module or region for further
processing or analysis. In another embodiment multiple reaction
channels may be used in parallel to increase sample throughput. In
yet another embodiment the system may alert the user when
amplification has occurred (a positive result), indicating that the
target sequence is present. In one embodiment a reaction channel is
used for a single use only, then disposed of. In an alternative
embodiment a reaction channels can be used to amplify and detect
the presence or absence of PCR amplification products in multiple
samples. More than one PCR reaction samples can be loaded at
intervals and interspaced with a barrier bolus of gas or liquid to
prevent intermixing. In one embodiment samples are spaced apart in
a manner so that as one is undergoing thermal cycling another
sample is in the detection region undergoing interrogation. It will
be obvious to one skilled in the art that the PCR amplification can
be replaced by other nucleic acid amplification technologies which
may use thermal cycling or be isothermal reactions.
[0197] In other embodiments, the device can perform isothermal
reactions such as sandwich assays using affinity reagents such as
antibodies or aptamers to determine if cells, proteins, toxins, or
other targets are present with the detection module (410) providing
a reading of the amount of target present. In these applications,
the cartridge 1 may perform an affinity purification such as an IMS
purification and then add a secondary antibody that may have a
fluorescent label attached. The sample can then move into region
350 where the thermal control is set to optimize the reaction.
Detection module (410) can then monitor the reaction. In one
embodiment, a plurality of cartridges are ganged to reaction
channel (250) and a series of boluses can be readout with detector
410.
IV. Device for Capillary Electrophoresis
[0198] In one embodiment a complete sample-to-answer system is
used, which can comprises microfluidics, requiring coupling all
steps together to match volumes and concentrations. Sample analysis
using capillary electrophoresis is a standard analytical method
that can be used with microfluidic sample preparation methods as
described above. Capillary electrophoresis is readily adaptable to
microfluidic microchips. In the instant invention, capillary
electrophoresis on microchips is combined with MOVe valves to
provide control of samples, process beads to concentrate the
samples, and improve the loading and separations.
[0199] In one embodiment the Twin-T injection system is used in the
design of the microfluidic injector for separations. In an
alternative embodiment a design is used for the Forked Cathode
injector (FIG. 30). The layout is similar to the Twin-T in that the
sample plug is described by a section of channel adjacent to the
separation channel but key differences exist. First, the cathode
channel is divided into two parts, this splits the injection
electrically into two parts and thus doubles the quantity of
material injected for a given sample plug dimension. Second, the
sample channel and separation channel are at right angles to one
another. This allows the sample channel to be straight and filled
with buffer (rather than separation polymer), which facilitates
manipulating the contents of this channel with pumps and fluid
flows, and allows the separation polymer interface to be sharp.
Lastly, the injector can be run in a mode that allows Field
Amplified Sample Stacking (FASS).
[0200] FIG. 30 shows an example of a forked cathode injector that
utilizes microchannels as the forked cathode. As shown in FIG. 30,
a sample is moved electrokinetically across a sample loading
channel (shown in the drawing on the lower left with the arrow
through it). Then the sample is driven into the separation channel
(the vertical channel) by applying a field between it and the
cathode arms (the two channel dropping down) while pull back is
applied to the sample and waste. The initial sample plug dimension
is defined by the distance between the cathode arms. The
configuration of the channels allows for a more reproducible plug
and better integration with MOVe microfluidic systems.
[0201] In an aspect of the invention shown in FIG. 30, fluidic
channel 3003 is in electrical contact with forked electrodes 3001
and 3002. The points of contact of the electrodes with the channel
are spaced apart, thereby creating a segment in the channel in
which there is an electric field. Separation channel 3004
intersects fluidic channel 3003 at a point in the segment between
the points of contact of the forked electrodes. Another electrode
of opposite charge is put in electrical contact with the separation
channel. In this way, a voltage is applied through the separation
channel.
[0202] FIG. 62 show a sample source 6009 connected to a sample
channel 6005, also referred to as a loading channel, that is mated
with a separation channel 6011. Two electrodes, 6003 and 6001, can
be used to apply an electric field to the separation channel. In
some embodiments of the invention, the sample source can pass
through a MOVe pump in a microchip used to drive fluid flow within
the sample channel. The sample channel can be a microfluidic
channel or an injection tubing. The injection tubing can be
flexible tubing or another flexible connector. Examples of flexible
tubing include polytetrafluoroethylene tubing or silicon tubing.
The flexible connector can also connect to another cartridge
interfaced with a microchip. Alternatively, the flexible connector
can return to the cartridge that it originated from. The separation
channel can be a microfluidic channel, capillary tubing, or
capillary electrophoresis tubing. The capillary tubing can have an
outer diameter of about 150 to 500 microns and an inner diameter of
about 10 to 100 microns. The capillary can be polyimide or
polytetrafluoroethylene clad. The capillary can be about 2 to 100
cm long. The capillary can be mated to the injection tubing or
flexible tubing by first drilling a hole into the injection tubing
and then inserting the capillary into the flexible tubing.
Alternatively, the capillary can be inserted into the flexible
tubing without having to pre-drill the flexible tubing.
[0203] One of the two electrodes, for example electrode 6003, can
be a cathode and the other electrode, for example 6001, can be an
anode. The cathode can be any cathode, such as a forked cathode,
described herein. The anode can be connected to the separation
channel using any devices known to those skilled in the art. For
example, the separation channel can be joined to a reservoir by an
Upchurch fitting, which is in electrical contact with the anode,
which can be a metallic electrode.
[0204] In some embodiments of the invention, a stabilizing
component, shown at the intersection of a separation capillary and
injection tubing in FIG. 63, can be used to align, seal, and/or
protect the connection between the separation capillary and the
injection tubing. In some embodiments of the invention, multiple
injection tubings are aligned with multiple separation capillaries
using a stabilizing component. As shown in FIG. 64, the stabilizing
component can hold four injection tubings, shown as the vertical
tubings in the figure, and stabilize the connection with four
separation capillaries (not shown).
[0205] Panels 1-6 of FIG. 62 show a process for injecting a sample
into a separation channel. In panel 1, no sample is present in the
sample channel 6005. In panel 2, sample entering the sample channel
from the sample source (6009) is shown. As sample is moved down the
sample channel, the sample intersects the separation capillary, as
shown in panel 3. The sample can be isolated by boluses of gas
upstream and downstream to the sample. Once sample is adjacent to
the separation channel, an electric field, which can be between 25
and 500 V/cm, is applied between a first electrode 6003, which can
be a cathode or a forked cathode, and a second electrode 6001,
which can be an anode. Electrophoresis buffer, shown entering into
the sample channel from the sample source, can also enter the
sample channel, as shown in panel 3. The voltage potential and/or
current between the anode and cathode can drop when an air bolus
passes by the junction between the sample channel and the
separation channel, reducing or preventing the injection of air
into the separation channel. The voltage potential and/or current
drop can be detected to ascertain when the sample and/or
electrophoresis buffer is adjacent to the separation channel. Once
the electrophoresis buffer is adjacent to the separation channel,
as shown in panel 5, the current and/or voltage drop between the
anode and cathode can be increased. This can allow for the
separation of the analyte in the separation channel, as shown in
panel 6, as the electrophoresis buffer provides ions for a high
performance separation.
[0206] FASS is a chromatographic technique that uses the increase
in the electric field caused by areas of low conductivity to
increase analyte mobility in the sample area and concentrate the
analyte at an interface of an area of lower mobility, i.e., at the
separation matrix. The net effect of running the injector in this
manner can be seen in FIG. 31. Significant decrease of the sample
plug length, herein referred to as stacking, can be observed.
[0207] The injector is filled with buffer (dashed line) then the
separation polymer is loaded (solid line) while the interface is
swept. The sample channel (horizontal channel) is filled with
sample reaction products in low ionic strength media. This allows
sample stacking and significantly decreases the sample injection
plug size. This is shown in the five frames on the right of FIG. 31
versus the all polymer injection in the left panel. The effect of
ionic strength and stacking is seen in the images from second left
to right as the buffer dilution increases and the ionic strength
decreases. The sample plug narrows from approximately 300 microns
to less than 100 microns.
[0208] In one embodiment for STR analysis the injection process is
as follows: [0209] The microfluidic channels can be filled with
buffer. [0210] The separation channel can be filled with gel while
buffer is pulled across the sample channel, thus sweeping the
separation polymer from the cross section formed by the separation
and sample channels. [0211] The STR amplified sample (desalted and
captured on beads) can be captured on microchip 500, eluted in a
low conductivity fluid (water) containing the size standard, and
pumped into the sample channel with MOVe technology. [0212] A field
can be applied across the cathode and anode, with "pull back"
voltage on the sample and waste arms, to drive the sample into the
separation channel where it stacks at the head of the separation
polymer. [0213] As the sample is injected the conductivity of the
sample channel can quickly equilibrate with the buffer in the
cathode arms providing a single step injection.
[0214] The MOVe controlled Forked Cathode injector design (FIG. 32)
can be optimized for DNA separations in microchip channels. In
addition to the FASS described above, the unique integrated
injector design also incorporates the MOVe pumping system which
facilitates the use of magnetic bead technology to desalt and
concentrate samples.
[0215] Purified STR amplification products are eluted from magnetic
beads, heat denatured and pumped through the loading channel of the
Forked Cathode injector. A voltage regime is applied to facilitate
an FASS injection at the head of the polymer column, and DNA
separation is performed in a polymer filled micro-channel (FIG.
33). In FIG. 33, the photos show the movement of dye in the
injector in order to illustrate the STR sample injection mechanism.
Field amplified stacking occurs at the polymer head when injection
is initiated.
[0216] Alternatively, the forked electrode or cathode can be two
metallic conductors, as shown in FIG. 60. The fluid path for a
sample to be analyzed, as shown in FIG. 60, can be along a loading
channel. When the location of the sample is adjacent to the
separation channel, the forked electrode can be used to inject the
sample into the separation channel, as described herein. The
conductance of the material in the sample channel can be lower than
the conductance of the material in the separation channel, which
can be a separation polymer. The difference in conductance can
cause sample stacking when an electric field is applied through the
forked electrode, which can be a cathode, and a downstream
electrode, which can be an anode. The polarity of the forked
electrode and the downstream electrode can be reversed such that
the forked cathode is the anode and the downstream electrode is the
cathode.
[0217] In some embodiments of the invention, an additional
electrode can be used to reduce injection of gas into the
separation channel or formation of bubbles within the sample
loading channel which can lead to loss of the applied field on the
separation channel. Injection of gas into the separation channel or
formation of bubbles within the sample loading channel can cause
inconsistent separation of analytes and can be detected by
inconsistent current between the anode and cathode used to apply an
electric field to the separation channel. Use of an additional
electrode to circumvent or reduce injection of gas or bubbles into
the separation channel is shown in FIG. 61. The additional
electrode can be a single wire run electrode or a cannular run
electrode. The increased surface area and/or larger internal
diameter of the cannular run electrode can allow for a significant
reduction in bubble formation or blockage and/or injection into the
separation channel. In some embodiments of the invention, the
cannula used for the cannular run electrode and has an inner
diameter of at least about 1/64, 1/32, 1/16, 1/8, or 1/4
inches.
V. mRNA Amplification
[0218] The devices of the invention can be utilized for microarray
sample preparation processes. Gene expression microarrays monitor
cellular messenger RNA (mRNA) levels. However, mRNA can constitute
only 1-3% of cellular total RNA. The vast majority of cellular RNA
is ribosomal RNA (rRNA), and these molecules may interfere with
mRNA analysis by competing with mRNA for hybridization to
microarray probes. Any mRNA amplification method can be performed
by the devices described herein, for example LAMP, TLAD (Eberwine),
and MDA. In some embodiments of the invention, isothermal mRNA
amplification methods can be performed using the devices described
herein. In other embodiments, thermal cycling can be performed to
accomplish PCR or cycle sequencing.
[0219] The Eberwine mRNA amplification procedure specifically
targets polyadenylated mRNA (polyA+ mRNA) for amplification,
virtually eliminating rRNA interference. This characteristic
removes any need to pre-purify mRNA from total RNA, which can be an
inefficient, time-consuming, and expensive process. In addition, by
greatly increasing the amount of target RNA (that is, amplified
mRNA or aRNA) available for microarray hybridization, mRNA
amplification can allow much smaller samples (fewer numbers of
cells) to be analyzed. This can be helpful because the relatively
large amount of target RNA required for microarray analysis
(typically 15 ug) is frequently difficult to obtain. Moreover, it
can be essential in many important clinical diagnostic applications
analyzing samples containing few cells, for example, samples
derived from fine needle aspirates (FNA) or laser capture
microdissection (LCM).
[0220] Any process that alters relative mRNA abundance levels may
potentially interfere with accurate gene expression profiling. An
important aspect of the Eberwine amplification procedure is that it
employs a linear amplification reaction that can be less prone to
bias mRNA populations than exponential amplification methods such
as PCR.
[0221] The original Eberwine protocol has been streamlined and
simplified by commercial vendors such as Ambion. As shown in FIG.
44, the Ambion procedure comprises three binary (two component)
additions followed by an RNA purification process. Each binary
addition can be followed by incubation(s) at specific temperatures,
as indicated in FIG. 44. The initial reverse transcription (RT)
reaction can have three inputs (primer, total RNA, and reverse
transcriptase [RT] Mix); however, total RNA and primer can
conveniently be premixed. Typical volumes for this first reaction
can be 5 ul RNA+Primer 5 ul RT Mix. Only mRNA hybridizes to the
oligo dT primer and is transcribed into DNA. The second-strand
reaction can be initiated by addition of 20 ul of a Second-Strand
Mix, and the final T7 amplification reaction can be initiated by
addition of 30 ul of a T7 Mix. Synthesized RNA can be labeled at
this stage by incorporation of biotin-labeled ribonucleotides.
Mixes contain buffers (Tris), monovalent and divalent salts (KCl,
NaCl, MgCl.sub.2), nucleotides, and DU, along with enzymes as
indicated. Typically, enzymes can be premixed with concentrated
mixes just prior to use.
[0222] After synthesis, aRNA can be purified to remove enzymes,
buffers, salts, unincorporated nucleotides, pyrophosphate, etc.
Purification typically relies on commercial kits exploiting the
association of aRNA with silica membranes or beads in the presence
of chaotropic salts such as guanidinium hydrochloride (GuHCl) or
thiocyanate (GuSCN). After binding, the silica is washed with 70%
ethanol (EtOH), dried, and aRNA is eluted with water.
[0223] Each of these steps can be carried out on the devices
described herein (See U.S. Provisional Patent Application No.
61/140,602). For example, reagents and sample can be supplied
through ports in the cartridge and then delivered to the
microfluidic microchip. The on-chip valves can be used to pump the
reagents and samples to chambers and reservoirs in the cartridge
and the microfluidic microchip through channels. Temperature
control can be accomplished using internal or external heating and
cooling devices. The reaction products can be moved to product
outlet ports of the cartridge for further handling. Alternatively,
the reaction products can be purified or separated using the
devices of the invention.
VI. Separation and Cleanup
[0224] A variety of separations can be performed using the devices
described herein. These separations include chromatographic,
affinity, electrostatic, hydrophobic, ion-exchange, magnetic,
drag-based, and density-based separations. In some embodiments of
the invention, affinity or ion-exchange interactions are utilized
to bind materials to solid-phase materials, such as beads. The
beads can be separated from fluid solutions using any method known
to those skilled in the art.
[0225] Magnetic separation can be used to capture and concentrate
materials in a single step using a mechanistically simplified
format that employs paramagnetic beads and a magnetic field. The
beads can be used to capture, concentrate, and then purify specific
target antigens, proteins, carbohydrates, toxins, nucleic acids,
cells, viruses, and spores. The beads can have a specific affinity
reagent, typically an antibody, aptamer, or DNA that binds to a
target. Alternatively electrostatic or ion-pairing or salt-bridge
interactions can bind to a target. The beads can be paramagnetic
beads that are only magnetic in the presence of an external
magnetic field. Alternatively, the beads can contain permanent
magnets. The beads can be added to complex samples such as
aerosols, liquids, bodily fluids, extracts, or food. After (or
before) binding of a target material, such as DNA, the bead can be
captured by application of a magnetic field. Unbound or loosely
bound material is removed by washing with compatible buffers, which
purifies the target from other, unwanted materials in the original
sample. Beads can be small (nm to um) and can bind high amounts of
target. When the beads are concentrated by magnetic force they can
form bead beds of just nL-.mu.L volumes, thus concentrating the
target at the same time it is purified. The purified and
concentrated targets can be conveniently transported, denatured,
lysed or analyzed while on-bead, or eluted off the bead for further
sample preparation, or analysis.
[0226] Separations are widely used for many applications including
the detection of microorganisms in food, bodily fluids, and other
matrices. Paramagnetic beads can be mixed and manipulated easily,
and are adaptable to microscale and microfluidic applications. This
technology provides an excellent solution to the
macroscale-to-microscale interface: beads can purify samples at the
macroscale and then concentrate to the nanoscale (100's of nL) for
introduction into microfluidic or nanofluidic platforms. Magnetic
separations can be used as an upstream purification step before
real-time PCR, electrochemiluminescence, magnetic force
discrimination, magnetophoretic, capillary electrophoresis,
field-flow separations, or other separation methods well known to
one skilled in the art.
[0227] The devices of the invention can accommodate the use of
magnetic beads. For example, beads or bead slurry can be supplied
to a port of a cartridge. The beads can be mixed or suspended in
solution within the cartridge using pumping, magnetic fields, or
external mixers. The beads can then be pumped to desired chambers
or reservoirs within the microfluidic device of cartridge. Beads
can be captured within a chamber using a magnetic field. Beads in a
solution can be captured as the solution travels through the
magnetic field, or beads can be captured in a stagnant
solution.
[0228] To illustrate methods of use of the cartridge, several
examples are described below. The first example describes
processing of nucleic acid from a buccal swab with paramagnetic
beads to purify the sample followed by PCR amplification and bead
purification of the PCR products. A second example describes
performing immunomagnetic separations to purify cells, proteins, or
other antigenic material using a binding moiety coupled to beads. A
third example describes performing molecular biology to prepare
samples for sequencing technologies such as sequencing by
synthesis, sequencing by hybridization, or sequencing by ligation.
It would be known to one skilled in the art that many different
chemistries and biochemistries can be used with the instant
invention. These include, but are not limited to, enzymatic
reactions, purifications on gels, monoliths, beads, packed beds,
surface reactions, molecular biology, and other chemical and
biochemical reactions.
EXAMPLES
Example 1
Operation of a Cartridge for Nucleic Acid Purification
[0229] This example refers to the use of a device comprising a
cartridge mated to a microchip. The numbers refer to the cartridge
of FIG. 3 and FIG. 4 mated to a microchip with the circuit
architecture of FIG. 5. This sub-assembly also can be fluidically
connected other sub-assemblies in the instrument of FIG. 6. For
reference, a cartridge mated with a microchip also is shown in FIG.
40 and FIG. 59.
[0230] Nucleic acids can be purified from a wide variety of
matrices for many purposes including, but not limited to,
genotyping, identification, forensics, gene expression, gene
modification, microRNA analysis, ribotyping, diagnostics, or
therapeutics. The input sample can be a solid, swab, liquid,
slurry, aerosol or a gas.
[0231] For molecular diagnostics and forensics, swabs are commonly
used. A buccal swab can be taken using a swab with an ejectable tip
and the swab ejected into a syringe attached to connection 7 of
FIG. 4. Connection 5 of FIG. 4 leads by tubing or capillary to a
reagent manifold that can select a single reagent from multiple
reagents by opening a full scale valve or by opening a MOVe valve
with the reagents either under pressure or moved by vacuum. MOVe or
other micropumps on microchip 2 of FIG. 4 can also move the fluids
or gases.
[0232] In one embodiment, human and other cells in a swab are first
lysed using a buffer with a heated chaotrophic agent and/or other
commercial-off-the shelf (COTS) chemistries in a syringe inserted
into port 7. The lysate is transported to a DNA isolation chamber
(FIG. 4 #3) where paramagnetic beads have been added from a
reservoir to adsorb nucleic acids onto the beads. A moveable magnet
is then actuated to capture the beads onto the side of the
isolation chamber where they are washed automatically using a
buffer. The purified DNA, still bound to beads, is then pumped
through a small diameter tube 250 where multiplexed PCR is
performed. Pre-scripted DevLink.TM. software automates the complete
process. The DevLink software defines a set of communication and
command protocols in a standardized automation architecture that is
simpler, more flexible, and quicker to implement than other
software development approaches. The DevLink implementation
framework is based on core technologies that span multiple
operating systems, development languages, and communication
protocols. Software drivers wrap individual smart components of the
system, greatly reducing the time needed for typical de novo system
software development. This makes it relatively straightforward to
integrate the operation of multiple system modules (pumps, valves,
temperature controllers, I/O controllers, etc.) that are either
COM- or .NET-based. DevLink provides a professional quality
software development system for prototyping through product release
and maintenance.
[0233] While DNA amplification is useful for positive
identification of microorganisms, samples can be obtained from a
wide variety of substrates and matrices that contain compounds that
are inhibitory to DNA amplification reactions. Raw samples are most
often complex mixtures that can include inhibitors such as hemes,
metal ions, humic and fulvic acids, chelators, DNases, proteases,
and molds. While the initial isolation of target organisms and
toxins from the sample matrix by IMS should remove most of these
inhibitors, lysed cell components and lysis agents can also need to
be removed or diluted from nucleic acid samples so that they do not
interfere with successful amplification.
[0234] In one embodiment, a small volume nucleic acid purification
is used. These purification methods can be used with a wide range
of samples, such as blood, to aerosols, to buccal swabs.
Paramagnetic beads can be used in a disclosed device to purify DNA
from various sample sources. In one embodiment a microfluidic
microchip can be used to sequence a nucleic acid using magnetic
beads and reagents to purify nucleic acid products for sequencing
in microscale reactions. In one embodiment, the microfluidic
microchip is a 24-channel microfluidic microchip.
[0235] In one embodiment, polyethylene glycol (PEG)-based nucleic
acid purification is used on carboxylated magnetic beads. This
PEG-facilitated process can produce yields of over 80% from
upstream immunomagnetic separations (IMS) captured samples.
Development of a universal sample preparation module (USPM) can
partly involve porting the PEG-based nucleic acid purification onto
a device containing a cartridge such as the devices shown in FIG.
21 or FIG. 16. In another embodiment, Agencourt Orapure or Promega
DNA IQ chemistries are used in conjunction with a device of the
present invention.
[0236] Bead Dispensation and Delivery.
[0237] To purify nucleic acids, paramagnetic beads with different
surface chemistries can be mixed in a reagent container. Pressure
is then applied to send the reagents to connection 5. MOVe
microvalves or other valves may be closed unless referred to as
open. To move the paramagnetic beads into the reaction chamber (3),
microvalves 180 and 150 are opened. The beads are moved through
connection 5 into channel 15 which leads to junction 190 and
microchannel 191. Because microvalves 180 and 150 are open and
microvalves 200 and 170, and the other microvalves, are closed, an
open microfluidic connection is from microchannel 191 through
microvalve 180 to microchannel 181 through microchip 152 to open
microvalve 150 and microchip 151 to junction 120. Junction 120
leads to cone 13 and chamber 3, which can be filled with beads. The
volume of beads supplied to chamber 3 can be controlled by timing
the opening of the reagent valves and the microvalves or by filling
and emptying a sample loop connected to the microchip or the
cartridge.
[0238] Commercial bead based chemistries can be used in the
disclosed system, including but not limited to Orapure from
Agencourt (Waltham Mass.) and DNA IQ from Promega (Madison, Wis.).
Orapure uses a carboxylated bead surface and SPRI chemistry while
DNA IQ is an example of a silica bead and chaotrophic chemistry.
Other embodiments of paramagnetic beads or chemistries to process
nucleic acids can be used in conjunction with the disclosed system,
including but not limited to beads with oligonucleotides, locked
nucleic acids, degenerate bases, synthetic bases, conformation,
nucleic acid structures, or other hybridization and specific
capture methods.
[0239] Filling Chamber (3) with Beads.
[0240] For Orapure or DNA IQ beads, 450 microliters can be moved
into chamber (3) using three fills of a 150 microliter sample loop
630 or 631. A movable magnet 300 attached to actuator 310 can then
be moved towards cartridge (1) near the side of 3 to pull the beads
to the side of chamber (3). Magnet size and orientation can be
adjusted to generate magnetic fields appropriate to specific
applications. Pressurized air can then be applied through the
reagent manifold with microvalve 180, 150, and 110 open. The
opening of microvalve 110 connects from junction 190 which connects
to the reagent manifold through junction 120 and microchannels 121
and 101 to connection 100 which leads through channel 14 to
connection (4) and to waste. The air can move any remaining liquid
through the circuit. Air or other gases can also be used to dry
beads, volatilize solvents, or for bubble-enabled mixing (described
herein).
[0241] Bubbling of Gas Through Chamber (3).
[0242] If microvalves 180, 150, and 220 are open, and all other
microvalves closed, the pressure can force air through chamber (3)
to channel 9 and down channel 19 to junction 210 through
microchannels 211 and 221, through open microvalve 220 and
microchannel 231 to junction 230, through channel 16 to connection
6 which can be a vent. This sequence can bubble air or other gases
through chamber (3) and can be used to mix reactions in chamber (3)
or to change the gas phase.
[0243] Moving Liquids and Beads from Chamber (3) to Waste.
[0244] Liquids and beads can be moved from reaction chamber (3) or
any other location to waste. This can be used to wash beads, flush
channels, move liquids or beads to waste. When pressure is applied
to connection 6 with microvalves 220 and 110 open, and all other
microvalves closed, the pressure can force air through channel 16
to junction 230 to microchannel 231, through open microvalve 220
and microchannels 222 and 221, though junction 210, and channels 19
and 9 into reaction chamber (3) and through junction 120 through
microchannel 121, open microvalve 110, microchannel 101, channel 14
and to connection 4.
[0245] The equivalent effect can be obtained by applying vacuum to
connection (4) if connection 6 is a vent without any additional
control of air pressure. The air pressure or vacuum can move any
liquids in chamber (3) to the waste connection 4. When magnet 300
is close to chamber (3), paramagnetic beads can remain on the side
of chamber (3) and the result is that the liquid is removed. When
magnet 300 is far enough from chamber (3), paramagnetic beads can
not remain on the side of chamber (3) and the result is that the
liquid and beads are removed.
[0246] To clean paramagnetic beads, the beads are pulled to the
side of chamber (3) with magnet 300 (see FIG. 6) and the liquid
removed to waste. 450 microliters of buffer can be dispensed from
the reagent manifold and added to chamber (3) by opening
microvalves 180 and 150. The beads can be released if desired and
then recaptured by moving the magnet 300 and the liquid then
removed. This is repeated for a total of three times to produce
beads ready to process samples.
[0247] Lysis and Extraction of Nucleic Acids from Cells on the
Swab.
[0248] A swab can be loaded into a syringe barrel inserted into
connection 7 and then be lysed by addition of lysis buffer through
reagent connection 5 with microvalves 180 and 170 opened. In some
embodiments Orapure or DNA IQ chemistries are used.
[0249] Movement of the Lysed Cellular Material to Chamber (3) and
Mixing with Beads.
[0250] The material in the syringe connected to connection 7 can be
moved into chamber (3) by applying pressure to the syringe or by
applying vacuum to vent 6. When vacuum is used, microvalves 170,
150, and 220 are opened. The vacuum connects through microchannels
231, 221, 211, and channels 9 and 19 through chamber (3),
microchannels 151, 152, 171, and 161 to pull material from
connection 7 into chamber (3). When paramagnetic beads are loaded
and cleaned in chamber (3), the lysed sample material mixes with
the beads in chamber (3) with the magnet is the far position.
[0251] Purification of Nucleic Acids on the Beads.
[0252] The paramagnetic beads are then incubated with the lysed
sample. Continued air or gas flow can aid mixing. The magnet 300 is
then moved to the closed position and the beads are captured on the
wall of chamber (3). The sample lysate can then be removed from
chamber (3) to waste and multiple volumes of wash solution added
according to manufacturers' specifications for the Orapure
chemistry or DNA IQ chemistry. The sample components on the beads
have now been purified and are ready for reactions in the cartridge
or exporting to the sample product connection. In one embodiment
the beads are used to enrich a nucleic acid component from a
sample.
[0253] Exporting Samples Through the Sample Product Connection
8.
[0254] The purified sample components on the beads can be moved to
connection 8 by applying pressures on reagent connection 5 with
microvalves 180, 150, and 130 open. In one embodiment, connection 8
is connected with reaction channel 250 such as C-flex tubing (Cole
Parmer) and additional reactions are performed in the reaction
channel.
[0255] Multiplexed PCR Amplification of STR Markers.
[0256] DNA amplification can be performed by PCR amplification. The
present invention enables PCR reactions as well as many other DNA
amplification and modification reactions. The reactions can be
performed in chamber (3), in reaction channel 250 attached to
connection 8 which can be a tube 250 (FIG. 3, FIG. 4, FIG. 6), or
in another device or microdevice connected to tube 250. This
demonstrates the utility of the sample preparation for DNA
reactions including thermal cycling.
[0257] Capture of Nucleic Acid Containing Beads in a Reaction
Channel.
[0258] The purified DNA output through the sample product
connection 8 is moved into a reaction channel 250 at end 251 by
applied pressure or alternatively through vacuum applied to end
252. An actuator 330 moves a magnet 320 under software control into
a position close to bead capture region 340. Fixed magnets of
different sizes and shapes (such as rare earth magnets) as well as
electromagnets or superconducting magnets can be used. As the
solution containing the beads moves through region 340, the
magnetic field attracts the beads to the side of the reaction
channel and holds them in place. The fluid is then followed by air
pressure through reagent connection 5 leaving the beads region 340
in air.
[0259] Addition of Reagents and Movement of Samples into Reaction
Region.
[0260] Reagents can be added from the reagent manifold as
described. In one embodiment, reagents are added from end 252 of
reaction channel 250. End 252 is attached to a microfluidic
microchip 500 comprising microvalves 510, 520, 530, and 540. Any
three microvalves such as 510, 520, and 530 or 510, 520, and 540
can form a pump. Microvalve 530 connects through a microchannel to
a downstream device 535, which can connect to tubing leading to a
reagent reservoir. Microvalve 540 connects through a microchannel
to downstream device 545, which can connect to tubing that leads to
a reagent reservoir.
[0261] Reaction mixes (such as at least one DNA polymerase, dNTPs,
buffer and a salt) including but not limited to master mixes and
primers, (such as assay-specific primers or broadly applicable
primer sets for multiple target pathogens), or complete PCR master
mixes such as PowerPlex 16 from Promega (Madison, Wis.) or
IdentiFiler or MiniFiler from Applied Biosystems (Foster City,
Calif.) in reagent reservoir 600 can be delivered by a micropump
formed by microvalves 530, 520, and 510 through tubing 610 and
microchannels 531, 521, 511, and 512, into end 252 of reaction
channel 250, as shown in FIG. 6. MOVe microvalves can precisely
position fluids and move the fluid to region 340 where the reaction
mix encounters the beads comprising nucleic acids. Magnet 320 is
moved away from reaction channel 250 by actuator 330 which releases
the beads from the inner surface of the reaction channel 250. The
MOVe microvalves on microchip 500 pump the beads into device 400
with an area of reaction channel 250 forming temperature controlled
region 350. The region 350 can be held at isothermal temperatures
or thermal cycled or other varied as is well known to one skilled
in the art. The region 350 can be a temperature modulator or
thermally coupled to a temperature modulator.
[0262] FIG. 7 shows a temperature control device 400 that is
capable of thermal modulation using a temperature modulator for
heating and cooling to thermocycle the reaction channel. In one
embodiment the temperature modulator comprises a Peltier module,
infra-red module, microwave module, a hot air module or a light
module. In another embodiment a PCR reaction sample is moved inside
the reaction channel past one or more constant temperature
zones.
[0263] FIG. 9 shows the amplification of PowerPlex 16 STR reactions
that have been prepared in a cartridge (1) from buccal swab samples
and processed in reaction channel 250 using the temperature control
device 400 in FIG. 7. The STR markers are amplified from standard
conditions with Mg optimized for the apparatus 1000.
[0264] The temperature control device 400 can also have a detector
410. The detector can detect optical detection such as absorbance,
fluorescence, chemiluminescence, imaging, and other modalities well
known to one skilled in the art or measurement such as IR, NMR, or
Raman spectroscopy. The detector can comprise a light source is
used to excite a fluorescent or luminescent dye in the PCR reaction
sample, and the excitation light is sensed with a photodetector
(such as a CCD, CMOS, PMT, or other optical detector). In one
embodiment the light source is a coherent light source, such as a
laser or a laser diode. In another embodiment the light source is
not a coherent light source, such as a light emitting diode (LED)
or a halogen light source or mercury lamp.
[0265] For nucleic acid amplification, real-time PCR is one example
of a nucleic acid assay method that can be performed in tube 250 in
temperature controlled region 350 and detected with detector
410.
[0266] On Microchip Reactions
[0267] In addition to transfer to tubing, apparatus 1000, as shown
in FIG. 6, can transfer material to microchips. To facilitate the
movement of this solution onto a microfluidic device for processing
a microchip was specifically designed with large MOVe valves for
high volume pumping and bead capture, stepped ports for interface
with input and output capillaries, side ports for reagent
introduction and a 1 .mu.L reaction chamber. Refer to FIG. 11, FIG.
12 and FIG. 14 for microchip details.
[0268] FIG. 11 shows a microchip schematic. The left depiction in
FIG. 11 diagrams the introduction and capture of beads from the
cartridge and apparatus device. The large pump 101 and the magnetic
bead capture chamber 103 are fed by a capillary from the cartridge.
Input capillary indicates where sample is added through the
cartridge. Output capillary indicates where sample is removed
through the cartridge. Beads can be placed in the input capillary
and then moved into the magnetic bead capture chamber by pumping of
valves between the input capillary and the magnetic bead capture
chamber 103. Valves are indicated by the dark circles and opposing
triangles. A movable magnet can be positioned adjacent to the
magnetic bead capture chamber to capture magnetic beads as solution
carrying the beads flow through the magnetic bead capture chamber.
The diagram on the right illustrates the resuspension of the beads
and DNA in the STR pre-mix as the sample is moved into the reaction
chamber. The two arrows in the diagram on the right indicate where
STR pre-mix and DNA can be added to the microchip. Valves between
the location indicated by the arrows and the reaction chamber can
be used to pump the DNA or STR pre-mix into the magnetic bead
capture chamber for resuspension of beads, and then into the
reaction chamber.
[0269] FIG. 12 and FIG. 13 show bead capture on microchip after
transfer from cartridge device. The bead capture can be performed
by using a magnet positioned adjacent to the microchip such that a
magnetic field is applied within a chamber of the microchip. Shown
in FIG. 12 and FIG. 13 is a large capacity (500 nL) MOVe valve,
which can be utilized for pumping and capture of beads. The capture
of beads is shown in the inset with a captured bead bed. As shown
in FIG. 13, the magnet 1103 is positioned over valve 1101 of the
microchip. Beads, which can flow into or out of the valve through
fluidic channels 1111 and 1107, are captured against a wall of the
valve due to the magnetic field exerted by the magnet. The valve
can be actuated by a pneumatic channel 1109 that can deliver a
positive pressure or negative pressure, relative to the fluidic
chamber of the valve, causing the elastomeric layer of the valve to
raise or lower.
[0270] One microliter on microchip reactions has been successfully
run with good signal strength and relatively good loci balance.
FIG. 17 shows the results of reactions with using one microliter on
microchip reactions as compared to equivalent off microchip
reactions and no template controls (NTC) that contain only size
standards. Peaks represent detection of nucleotide base pairs.
[0271] Purification of Reaction Products on Beads.
[0272] In one embodiment, the reaction products on the beads can
then be moved into cartridge (1) using vacuum applied to the
reagent connection 5 with microvalves 200 and 130 open with the
path connecting through 201, 212, 211, to junction 210 and channel
19 and 9 to chamber (3) through 131, 141, and 140 to reaction
channel 250. The microvalves on microchip 500 can modulate the
vacuum and flow. The reaction products can be moved into chamber
(3) which can be loaded with beads that are cleaned in place as
described above.
[0273] The beads can capture many types of biomolecules using
affinity or other interactions well known to one skilled in the art
using bead purifications, immunomagnetic separations, and reactions
with beads, nanoparticles, quantum dots or other types of
particles.
[0274] Continuing the STR example, after STR amplification is
complete, the reaction products are transferred back to the
cartridge (1) using vacuum. Amplified STR products are purified,
desalted and concentrated prior to injection using the same Orapure
magnetic bead beads present on the device for the isolation of DNA
from the buccal swab. This time the beads are used with only
ethanol; no PEG/NaCl solution as described previously for the swab
extraction is used.
[0275] The beads are loaded into the cube mix chamber, captured and
cleaned with 70% ethanol. Then the 5-10 .mu.L of STR reaction is
pulled back into the chamber from the cycling zone and into contact
with the beads. A 20 .mu.L chase solution of electrophoresis run
buffer (chosen because of its availability on the separation
subsystem) is pulled through to scavenge any remaining STR reaction
solution in the reaction channel and 100% ethanol is added to take
the solution up to a 95% total ethanol concentration.
[0276] FIG. 19 shows data for standard material prepared in this
manner on the swab extractor and analyzed on a MegaBACE. The
products were cleaned with Orapure beads in the sample preparation
device configured as a swab extractor using cartridge (1) and
compared to products that were cleaned using a manually prepared
control that had been processed with CleanSeq (Agencourt). About
.about.60% recovery was observed in comparison with the same
process performed off device. It should be noted that the swab
extractor cleaned material yields significantly more efficient
injections than the commonly used process which dilutes the sample
by 1:50 to 1:100.
[0277] In another embodiment, a Post-amplification STR clean-up
device delivers the STR reaction premix to the thermocycler; meters
the sample during the isolated DNA bead capture; performs a bead
cleanup on the STR amplified products; delivers the eluted products
to the cathode; and provides reagents to the cathode assembly
during preparation of the separation and detection device and
sample injection processes.
[0278] The four-channel Post-amplification STR clean-up device
combines an Chip A microchip, shown in FIG. 47, with an enlarged
fluidics manifold with cleanup chambers (FIG. 71) and mounted on a
pneumatics control manifold (FIG. 72). Agencourt CleanSeq beads are
delivered to the clean-up chamber, the sample is pumped through the
reaction tubing 250 from the thermocycler to the clean-up chamber
and ethanol is added. The sample is mixed by air bubbling to
facilitate DNA capture onto the beads. A magnet is actuated at the
base of the device cause the DNA and beads to be captured against
the bottom of the clean-up chamber; the remaining liquid is then
pumped to waste. The magnet is moved away from the device and
eluent containing fluorescently labeled DNA size standard in a
formamide solution is pumped into the clean-up chamber. The STR
amplification products are eluted in this solution and the magnet
is once again actuated to capture the beads before the purified and
concentrated sample and size standard are pumped to the cathode,
ready for injection into a separation capillary.
[0279] Testing of the post amplification device using Chip A
highlighted issues with priming and pumping ethanol through the
Chip A microchip due to the high level of resistance in the reagent
pathway of this microchip. The microchip design, Chip E, (FIG. 73)
significantly improves functionality and robustness of the post
amplification device by widening channels and replacing three way
MOVe routers with a pair of MOVe microvalves.
Example 2
Universal Sample Preparation
[0280] The previous example illustrated one embodiment in which the
disclosed apparatus can be used to prepare samples for analysis and
showed one example of STR amplification. Another embodiment
involves the use of a Universal Sample Preparation Module (USPM).
The USPM device can consist of a sample processing cartridge (1),
accompanying apparatus to operate the cartridge, a microprocessor,
and software that can readily be interfaced to downstream
analytical devices. In one embodiment the USPM can be tightly
integrated with analytical devices to form a modular
sample-to-answer system. The cartridge can be configured as a
disposable single-use device that can process swabs or liquids
(including aerosol samples) for field monitoring processes, or as a
reusable, flow-through format for remote operations with rare
positives. Target specificity of the USPM is imparted through the
use of specific antibodies (that bind selected targets) attached to
paramagnetic beads; different cartridges can be supplied with
various mixtures of targets.
[0281] A USPM can use a multistep fully automated process to
prepare biological samples for downstream analysis. One example in
FIG. 18 can use swabs or liquids; the operator can select the
sample type and then insert samples into input port(s). The first
step can apply immunomagnetic separations (IMS) to capture,
concentrate, and purify target molecules from solution onto
paramagnetic beads. Targets already tested include cells, spores,
viruses, proteins, or toxins. For toxin and protein detection, or
for use as a triggering device, the captured targets from the IMS
can be exported directly to the downstream analytical device. For
nucleic acid detection, the second step can lyse the cells or
spores to release the DNA and/or RNA using mechanical or other
lysis techniques. The third step, nucleic acid purification, can
adsorb, concentrate, and purify the nucleic acids onto a second set
of paramagnetic beads and output the beads with nucleic acid, or
purified desorbed nucleic acid, for downstream analysis.
[0282] Referring to cartridge (1), the immunomagnetic separation
can be performed by using reagent beads that have antibodies or
other immunomagnetic, affinity magnetic, or surface chemistry
magnetic separations. For example, immunomagnetic beads with
antibodies can be added to cartridge (1) to capture, purify, and
concentrate cells, viruses, spores, toxins and other biomolecules
onto bead.
[0283] Upstream sample processing for the USPM can be done in the
sample preparation devices, which can process samples over 0.6 mL
in a microfluidic cartridge (1) (FIG. 21). The sample processing
cartridge, about 1 in cubed dimension, (FIG. 3, FIG. 21) was
developed to automatically remove collected buccal cells from a
swab, lyses the cells, and purifies released cellular DNA on
magnetic beads. The bead beds are typically 100 nL and can be used
for downstream STR analysis with microfluidics devices or full
scale qPCR reactions.
[0284] The sample preparation device uses a MOVe microvalve
microchip interfaced with the bottom of the cube (FIG. 3, arrow
labeled 2) to direct pressure-driven flows consisting of fluids,
beads, and samples among the reagent and reaction reservoirs. The
MOVe microvalves replace conventional valves and tubing between the
reservoirs, thereby providing a non-leakable, directable fluid
transport and enable miniaturization of the entire cube and sample
preparation device.
[0285] This sample preparation device technology has been used to
automate DNA extraction from buccal swabs as described above. FIG.
10 shows automated preparation of DNA from 25 uL of blood in the
automated sample preparation device using pressure driven flows,
vibrational mixing, MOVe valves, actuated magnets, and magnetic
beads. The fully automated process produced DNA ready for STR
analysis in less than five minutes.
[0286] We have developed an automated system for capturing,
concentrating, and purifying cells, viruses, and toxins from liquid
samples (1-10 mL) using magnetic beads coated with antibodies
specific to targets of interest. Thus, a variety of targets have
been concentrated and purified with this automated system. Using
this approach, E. coli cells were captured and detected at cell
concentrations as low as 15 cells/mL/sample (FIG. 27). Similar
results of greater than 90% capture efficiency were obtained using
Bacillus spores, Gm.sup.+ and Gm.sup.- vegetative cells, a model
virus (bacteriophage fd), SEB, and ovalbumin as targets. Purified
samples can be further processed in the sample preparation device
(e.g., lysis and nucleic acid purification), moved onto a microchip
for analysis, or used with an off-chip PCR/qPCR device.
[0287] We have shown that IMS capture works well in complex samples
such as aerosols and in the presence of biological clutter (See
U.S. Patent Publication No. 20080014576, herein incorporated by
reference in its entirety). For clutter, we showed that up to
10.sup.5-fold levels of added bacteria produced only a two-fold
reduction in capture efficiency. For complex samples, add-back
experiments using many different aerosol samples established that
aerosol samples reduce the binding of B. cereus spores to IMS beads
by less than 50%. Therefore, there is less than a two-fold loss of
sensitivity in complex, real-world samples.
[0288] We have used IMS to capture, concentrate, and detect toxins.
We have developed IMS assays for ovalbumin and SEB, multiplexed the
assays, and developed two generations of completely integrated
microfluidic systems that automate the IMS assays. Less than 10 ng
of SEB can be reliably detected in a one mL samples with no
interference from closely related Staphylococcal enterotoxins.
[0289] We have shown that IMS can: [0290] Select target organisms
from samples with high backgrounds of interferents (selectivity),
[0291] Discriminate between two different strains or species of
bacteria (specificity), [0292] Effectively capture cells and toxins
across a wide range of concentrations from a wide range of samples
(sensitivity, robustness) [0293] Reduce target sample volume
significantly, from mL to nL volume
[0294] The instant invention and the apparatus and methods are
capable of implementing IMS and coupling it to nucleic acid
extractions.
[0295] The next step in the USPM is the lysis of the captured
target when it is a cell, virus, prion, or spore. Lysis of spores
is particularly challenging. A MagMill or magnetically driven lysis
or homogenizing device has been developed for efficient lysis of
Bacillus and other spores, as well as vegetative cells. The MagMill
consists of a rapidly rotating magnet 2000 actuated by a motor 2001
(FIG. 74) that drives rotation of another magnet 2002 contained
within a sample-containing vessel 2003 or compartment (FIG. 75).
The magnet 2002 contained within the sample-containing vessel can
have any shape. For example, the magnet can have a bar, spherical,
cylindrical, rectangular, oval, hexagonal, or propeller shape.
Alternatively, the magnet can have holes through it, such that
liquid may be forced through the holes and increase the shear force
applied to the sample when the magnet is rotated by a magnetic
field. The same basic components can be miniaturized, incorporated
into a microfluidic format, or connected to a microfluidic format.
The overall effect is analogous to a magnetic stir plate, with the
sample being rapidly vortexed within the sample tube. Using
magnetically driven sample agitation by MagMill treatment, spore
lysis is achieved without silica, zirconia or other beads. Lysis
may be accomplished by shear forces generated as the spore passes
between the magnet and the vessel walls. The magnet can rotate at a
rate of greater than about 10, 50, 100, 150; 200, 250, 500, 750,
1000, 1500, 2000, 2500, 5000, or 10000 rpm.
[0296] This device disrupts spores with similar efficiency as
traditional bead beating that employs silica/zirconia beads (FIG.
76). Spores (3.2.times.10.sup.7) were lysed in a volume of 1 ml
with viability was determined by plating on Tryptic Soy Agar;
results are an average of two separate experiments each run with
duplicate samples (total n=4). The non-viability of
magnetically-driven spore lysates was 93% compared to traditional
bead beating (BioSpec beater) lysates using either Zirconia/silica
which was 96% or silica beads which was 80%. The same pattern was
confirmed by qPCR
[0297] The advantage of using the MagMill (versus traditional bead
beating) is that the design is more mechanically robust and thus
able to withstand many cycles of use without failure, and samples
can be lysed using just the agitation of the magnet in the sample,
without the need for inclusion of silica/zirconia beads that have
been shown to bind released DNA causing a loss in follow-on
detection sensitivity. The basic features of the MagMill can be
reconfigured in a miniaturized format that can be integrated into a
sample preparation device. The system can potentially be down-sized
to fit into a microfluidic microchip. Despite changes in
configuration, however, the principle driving lysis, that of a
rapidly rotating magnet contained within a sample vessel, remains
the same.
Example 3
Sample Preparation for Library Construction
[0298] The cartridge (1) technology and the sample preparation
device can be further used to perform a series of complex molecular
biology reactions in small volumes with bead-based manipulation of
DNA samples. The DNA can be processed to prepare genomic libraries
for next generation sequencing systems (i.e., Roche 454 system, or
the Applied Biosystems SOLiD), Real-time PCR, or other DNA assay
systems.
[0299] By incorporating a reverse transcriptase step, RNA libraries
can also be converted into DNA libraries by essentially the same
method. The advantage of building an RNA library is that the
representation in the final amplified library will directly mirror
the original starting material since the amplification will be
based upon single molecule amplification.
[0300] The design concept for the Library Construction Module (LCM)
is an instrument that holds bulk reagents and provides fluidic
control to the small disposable Library Construction Module
cartridges that processes individual libraries. The LCM instrument
can control fluid flow, mixing, temperature, and bead manipulation
in LCM cartridges through DevLink software. An array of reagents
stored in temperature controlled reservoirs can be accessible
through a MOVe block of valves (actuated by computer-directed
pneumatics 700). Workflow for producing beads with amplified DNA
attached from single molecules is shown in FIG. 29. The Library
Construction Module uses the cartridge as a preferred
implementation.
[0301] MOVe valves are extremely durable, compact, inexpensive
compared to conventional valves, have dead volumes of about 10 nL,
and are compatible with dispensing volumes as low as 100 nL. The
instrument can use pressure-driven flows to move fluids from the
reagent reservoirs through the MOVe valve block, to the reaction
chambers in the LCM disposable cartridge, and out to final sample
output vessels (FIG. 25). Reagents and substrates can be mixed in
sample preparation devices using pressurized flow, vibrational
mixing, and pneumatic-driven MOVe valves; magnetic beads are
captured and released using DevLink-controlled actuated
magnets.
[0302] Each disposable LCM cartridge can contain embedded MOVe
valves (FIG. 28). The MOVe valves direct fluids from reagent
reservoirs and connect the three reaction chambers. One chamber,
the processing chamber A, can carry out the sequential
solution-based molecular biology reactions. The second, the
purification chamber B, can be for bead-based purification of
intermediate products. The third chamber, the annealing chamber C,
can perform the final annealing to glass beads. The annealing
chamber can comprise a filter to separate the beads from
surrounding solution. Pneumatic lines (P1, P2, P3, P4, P5, P6, P7,
and P8) can control the valves of the microchip that control and/or
force flow to the chambers.
[0303] Pressure driven flow through the MOVe valves can move fluids
between chambers. The processing and annealing chambers can be
capable of mixing contents and incorporate thermal control.
[0304] In the next sections, the workflow to prepare DNA library
samples using the LCM is described as an example. It is readily
apparent that RNA libraries can be prepared after a reverse
transcriptase step.
[0305] Demonstration of Microfluidic Reactions Preceding emPCR
[0306] The starting input for the Library Construction Module can
be nebulized, sheared DNA that has been size fractionated to
800-1,000 bp, purified, and has already had small fragments
(<500 bp) removed. Size fractionation can also be carried out on
the LCM by selective binding to magnetic beads, e.g., AMPure,
(Agencourt). Input DNA can be assessed for size and concentration
on a BioAnalyzer 2100 (Agilent) and by dye binding (Pico-Green,
Quant-it, Invitrogen). The sequence of reactions is shown
schematically in FIG. 29 in the Library Construction Module
box.
[0307] Fragment End Polishing
[0308] In one embodiment DNA nebulization generates fragments with
a preponderance of frayed ends that require filling in/blunt-ending
before further manipulations can take place (Bankier 1987).
Phosphorylation of 5' hydroxyl termini is also required for
subsequent ligation of adapters. This can be accomplished through
the successive activities of T4 DNA polymerase and T4
polynucleotide kinase. Substrates are combined with reaction
buffer, BSA, ATP, dNTPs and the two enzymes in a small reaction
volume, initially 25 uL, incubated first at 12 degrees Celsius for
15 min., then at 25 degrees Celsius for 15 min. Temperature control
can be through Peltier devices mounted on the hard instrument
interface. Controls for the polishing reactions can rely on
incorporation of fluorescently labeled dNTPs, assayed using
fluorescent imaging and quantification or using radiolabeled
dNTPs.
[0309] Microfluidic Bead-Based Purification
[0310] Following polishing, the fragments are purified by
precipitation onto beads. The LCM instrument can move fluids to the
LCM cartridge (see FIG. 28) to force DNA onto beads, actuate a
magnet to concentrate beads onto a wall of the purification
chamber, and then wash the beads. As needed the DNA can be eluted
from the beads, the DNA can be moved to the processing chamber A
and the beads discarded, or the beads moved into another chamber
for further processing. This method can be reused multiple times
throughout the process. Column-based purifications are thus
replaced with bead-based purification. The sample preparation
device routinely purifies DNA in cartridges using magnetic SPRI
beads (Agencourt Biosciences), DNA IQ (Promega), and carboxylated
beads (Invitrogen) for DNA sequencing, forensics, and biodefense
applications respectively. Purified products can be assayed on a
BioAnalyzer (Agilent) and by dye binding (Pico-Green, Quant-it,
Invitrogen).
[0311] Ligation of Adaptors
[0312] Next, adaptors are ligated to the purified DNA. The DNA in
the purification chamber can first be eluted from the beads into
ligation buffer, which is moved to the reaction chamber by pressure
driven flow, and adaptors and ligase added. The adaptors can
contain nested PCR and sequencing priming sites, blunt 3' ends and
5' overhangs; one adaptor of each pair can have a biotin on the 5'
end. Each adaptor can incorporate a nucleotide-based `key` that
serves to identify the sample and the processing steps and a
`sequencing key` to assist in base-calling. Ligation and further
selection selects for fragments with different adapter (one with
biotin, one without) on each end. At this point before being bound
to streptavidin beads the ligation products again can need to be
purified on beads in the purification chamber and the library moved
back to the processing chamber.
[0313] Ligations can be conducted with ligase such as quick ligase
(New England Biolabs), buffer and adaptors and incubated for 15 min
at 25 degrees Celsius. Products can be assayed for the ligation of
adaptors by Q-PCR, using the PCR priming sites and 5' sequencing
priming site for probe. Efficiencies of ligation and recovery of
target can be calculated by comparing the amount of starting
materials and yields after relevant steps. Also, pre-binding one
adaptor via a biotin-streptavidin linkage to a paramagnetic bead
will result in bead-bound fragments that can then be subjected to a
second round of ligation with the second adapter. This approach can
prevent the generation of fragments with no biotinylated adapters
and those with two biotinylated adapters, which are both lost
during processing. This could significantly improve the final yield
of template. After ligation, the products are again
bead-purified.
[0314] Library Immobilization
[0315] Pre-washed streptavidin-coated beads resuspended in Library
Binding Buffer can be added from a reagent reservoir to the
purified ligation reaction products that have been moved back to
the processing chamber and incubated for 20 min with mixing at RT.
The material is then bead purified on the streptavidin bead in the
purification chamber to remove adaptor dimers.
[0316] Nick Repair
[0317] Ligation results in 3' nicks, which can be repaired by Bst
DNA polymerase (Large Fragment). Fill-in Buffer, and dNTPs, can be
added directly to the beads and incubated at 65 degrees Celsius for
30 min. The beads are then purified in the purification
chamber.
[0318] u
[0319] 25 uL of premixed Melt Solution (125 mM NaOH) can be added
directly to washed beads in the purification chamber, mixed, the
bead pelleted using a magnet, and the resulting supernatant moved
to the processing chamber where 62.5 uL Neutralization Solution
(0.15% acetic acid) can be added. A second round of treating the
beads with denaturant results in a total volume of 113 uL of single
stranded template. These would again be purified before eluting in
a volume of 10 uL.
[0320] Automation, upstream normalization of the input material, or
using limiting amounts of beads are various steps that can greatly
aid in uniform production and amplification of libraries, which can
lead to the elimination of the quality check at this point.
[0321] In one embodiment, continued quality assessment and
functional quantification can be performed. The quality of the SS
library can be assessed off-line, if required, using an Agilent
2100 Bioanalyzer, which can provide the size range of fragments;
yields can also be assessed using Q-PCR, or dye binding (RiboGreen,
Quant-iT, Invitrogen). The library can further be functionally
tested by forming a dilution series, performing emulsion PCR
(emPCR), and pyrosequencing to determine the working dilution for
the emPCR. Automated fragment separation can also be incorporated
into the device.
[0322] Binding to Capture Beads
[0323] The SS DNA prepared in prior steps can be annealed to
controlled pore glass (CPG) or styrene beads with bound DNA capture
primers complementary to the ends of the ligated adapters. Handling
of non-magnetic beads, such as these, can require the use of pumped
filtration technology (to avoid the use of centrifugation). Because
the reagents used can be purified of any particulates and the beads
can be pre-washed and quantified using a Coulter Counter (Beckman
Coulter), filter clogging should not be an issue.
[0324] The beads can be added to the template library, now single
stranded, and mixed to favor annealing of a single template/bead in
hybridization solution in the annealing chamber. Initially, the
bead-template mixture can be divided into aliquots for subsequent
emulsion generation and annealed by ramping from 80 degrees Celsius
with holds at 10 degrees Celsius intervals in a standard thermal
cycler. Temperature-ramping capabilities can also be incorporated
into the LCM device. Resuspension of filtrates (i.e., washed beads)
can be accomplished by back washing the filters. Hybridization of
the SS library can be assessed by assaying for unhybridized SS DNA
in the supernatant by Q-PCR on aliquots before and after bead
hybridization. At this stage of processing the single stranded
template on beads can be transferred to the emPCR Production Module
or similar device for further processing.
Example 4
Coupling of a Sample Preparation Device with a Microchip-Based
Sample Cleanup and Separation
[0325] FIG. 34 and FIG. 35 show a device with a cartridge (2907)
and microchip (2901) that was designed to incorporate the Forked
Injector design, as shown in FIG. 32, a gel filling manifold
(2903), and associated components. The cartridge is fluidically
connected to a pneumatics manifold and tubing (2905). Different
configurations of the injector design, separation channel length
and separation polymer were tested. FIG. 36 show an
electropherogram of an M13 T track injected and separated on a
microchip channel using the Forked Cathode injector, with sample
detection on a confocal microscope breadboard system. The sample
was injected uniformly with short and long DNA fragments
represented equally. The results show that an M13 T track DNA
ladder can be uniformly injected and single base pair resolution
can be obtained out to approximately 330 base pairs in less than 20
minutes. Higher sample signal strengths were obtained compared to
injections using a conventional twin T design. When integrated with
a detection system, the microchip is held at a constant 50.degree.
C. in order to obtain separations with good resolution.
[0326] Using these processes, excellent results were obtained for
MOVe integrated, field amplified stacking injections of liquid
samples (FIG. 37). This data was generated with all sample loading,
manipulation and injection processes carried out under software
control using MOVe microvalves. The data has been minimally
processed, color corrected from a detector that uses eight diode
channels to four dye traces.
[0327] One embodiment of a microchip that combines the forked
cathode with a MOVe sample preparation device is shown in FIG. 38.
This device comprised additional processes that enable integration
with the rest of the system, i.e., the sample preparation device
(1000 shown in FIG. 22), the reaction channel (250 shown in FIG.
6), and the output of the STR purification as described in the STR
example. FIG. 38 shows a forked cathode with MOVe fluidics and
shuttle sample loading for integration with post amplification STR
purification system. The parts are: 1--Reagent input port,
2--Reagent pump head, 3--Sample input port, 4--Size Standard/eluent
input port, 5--Capture valve, 6--Waste port, 7--Elution valve,
8--Sample waste port, 9--Cathode, 10--Cathode port, 11--Sample
valve, 12--Sample port, and 13--Separation channel. The anode port,
which is downstream of the channel, is not shown.
[0328] The sample to be separated is introduced as a bead solution
in ethanol. This can be the purified reaction products on beads
output as described above. In one embodiment, the sample is an STR
reaction. In other embodiments, the sample can be nucleic acid
fragments of different lengths produced by other reaction
chemistries including DNA sequencing by Sanger chemistry. The
solution containing the sample is flowed from the Sample input port
to the Sample waste port with the Capture valve and other
intervening valves open. The open Capture valve facilitates a
slowing of the stream flow and bead capture by a fixed magnet
placed above or below the valve. The ethanol solution is completely
run through the system followed by air yielding a relatively dry
and clean bead bed, with purified products, in the valve. At this
point the valve is closed and reopened (in coordination with other
valves) to fill it eluent solution from the associated port. For an
STR analysis or other analyzes where an internal size standard is
needed, the eluent can contain a size standard. The solution is
moved between the Elution valve and the Capture valve to facilitate
mixing, ending with the solution in the Elution valve. The Sample
valve is then opened in coordination with the Elution valve closing
to "shuttle" the sample through the sample channel leaving it
filled. The sample FASS injection is carried out as previously
described. An additional noteworthy function of the device is that
in one embodiment the Reagent input port and Reagent pump are used
to provide metered STR reaction premix to the reaction channel (250
shown in FIG. 6) after the swab extraction of DNA on the sample
preparation device; in other embodiments, the device can provide
other nucleic acid reaction reagents such as cycle sequencing
mixture or provide PCR reagents to perform a PCR amplification
followed by providing cycle sequencing reagents to perform cycle
sequencing with bead-based cleanup reactions integrated as needed.
Other chemistries will be apparent to one skilled in the art.
Example 5
Integrated Nucleic Acid Isolation, Amplification, Separation and
Detection System
[0329] A sample preparation device with cartridge(s) and thermal
regulating device can be integrated with downstream detection
systems to produce a sample-to-answer fully integrated system. The
system can be fashioned into a compact format that is compatible
with laboratory, clinical, and field operation as a benchtop or
portable device, as shown in FIG. 39. FIG. 39 shows one embodiment
of a system that can extract swabs or other materials using a five
channel swab extraction assembly 800 using cartridges (1) to purify
nucleic acids from input buccal swabs, liquids, solids, and other
materials. The purified nucleic acids on beads is PCR amplified in
Thermal Cycling Module 400. In one embodiment, the samples are bead
purified in swab extraction assembly 800 using cartridges (1) to
purify the desired products onto beads. The beads are moved to
Separation and Detection Module 900 to receive the products on
beads, elute the sample and separate it by capillary
electrophoresis or microchip capillary electrophoresis or other
separation methods with detection methods such as Laser Induced
Fluorescence or mass spectroscopy. For capillary electrophoresis or
microchip capillary electrophoresis, Gel Injection Module 850 pumps
separation matrix or gels or other materials to provide the
separation columns and regenerate them. In another embodiment, the
Gel Injection Module 850 could pump chromatography media if the
separation was by HPLC or other liquid chromatography methods.
Electronics and Power Supply Module 860 provides control and power
function. Pneumatics Module 870 supplies regulated air and vacuum
to operate the swab extraction assembly 800. Reagents are stored in
reagent storage 880. Reagents can be stored in solution or
dehydrated or stabilized forms such as Ready-to-Go (GE Healthcare)
and lyophilized forms.
[0330] In one embodiment, the system is configured to perform STR
analysis of buccal swabs. Buccal swabs are extracted in assembly
800 and the extracted samples amplified in Thermal Cycling Module
400 using reagents for STR amplification. The amplified samples are
purified using nucleic acid extractions onto beads, for example
using Orapure or DNA IQ chemistries and beads. The purified STR
products on beads are then moved to Separation and Detection Module
900 and the beads are captured and the DNA eluted, preferably on a
microchip with MOVe microvalves. The samples are then injected
preferably into a Forked Cathode Injector on a microchip or a
capillary electrophoresis capillary coupled to a microchip and
using capillary gel electrophoresis separations with gels such as
dynamic coating gels, V2E (GE Healthcare), polydimethyl acrylamide,
the POP family of matrices (Applied Biosystems),
hydroxymethylcellulose, guarin, and linear polyacrylamide. The
detected products with fluorescent labels pass a Laser Induced
Fluorescent detector which detects the peaks as they move by.
[0331] In another embodiment, the system is configured as an
integrated DNA sequencer. The sample is extracted in assembly 800
and the extracted samples amplified in Thermal Cycling Module 400
using reagents for PCR amplification. The samples can be whole
organisms, tissues, cell, viruses, air, liquid, or solid without
limitation. The DNA extraction can be non-specific using a
bead-based purification method such as Orapure (Agencourt) or can
use hybridization or other methods to select one or more regions
from the input sample to produce a less complex sample. It will be
apparent to one skilled in the art that the USPM workflow with IMS
followed by the nucleic acid purification can also be adapted to
the sample preparation device 800 and the swab extraction replaced
by many other initial purification workflows. The PCR amplification
can be of a single region or multiplexed to target multiple
regions. The amplified PCR samples are purified using nucleic acid
extractions onto beads, for example using Orapure or DNA IQ
chemistries and beads. The purified PCR products on beads are then
moved to the Thermal Cycling Module 400 and cycle sequencing master
mix added and the samples cycle sequenced. This can be with
fluorescent labels or as four sets of unlabeled primers for
label-less detection by UV or other methods. The cycle sequenced
samples are then moved to assembly 800 and bead purified to remove
unwanted ions and labels, and other material. The beads are then
moved to Separation and Detection Module 900 and the beads are
captured and the DNA eluted, preferably on a microchip with MOVe
microvalves or into capillaries. The samples are then injected
preferably into a Forked Cathode Injector on a microchip or a
capillary electrophoresis capillary coupled to a microchip and
using capillary gel electrophoresis separations with gels such as
dynamic coating gels, V2E (GE Healthcare), polydimethyl acrylamide,
the POP family of matrices (Applied
Biosystems)hydroxymethylcellulose, guarin, and linear
polyacrylamide. The detected products with fluorescent labels pass
a Laser Induced Fluorescent detector which detects the peaks as
they move by.
[0332] In other embodiments the protein, carbohydrate, or other
assays are performed and the detection is by mass spectrometry,
imaging, HPLC, GC, or other analytic methods well known to one
skilled in the art.
[0333] For DNA, the processed sample can be amplified by PCR,
rolling circle, branched DNA, EXPAR, and other DNA amplification
methods well known to one skilled in the art or analyzed by mass
spectroscopy or single molecule detection methods. RNA can be
processed by Reverse Transcriptase real time-PCR, or samples
prepared for DNA microarrays, or other analytical methods. Real
time or end point analyzes can be performed with the apparatus. For
proteins, assays can be performed in the cartridge including
enzymatic assays, sandwich immunoassays, antibody precipitation,
protein digestion, protein and peptide labeling, and other commonly
used protein analysis methods. Similarly, other cellular components
or chemicals can be extracted or purified using standard methods in
the apparatus. Molecular biology methods are readily adapted to the
apparatus. Samples can be completely analyzed on the apparatus in a
single cartridge, moved to a separate cartridge, or analyzed or
further processed in a separate instrument comprising a capillary
electrophoresis system or microchip capillary electrophoresis;
multidimensional gel and capillary electrophoresis; mass
spectroscopy, multidimensional mass spectroscopy with HPLC, ICP,
Raman spectroscopy, particle, nanoparticles, and bead based
detection, imaging, comprising fluorescence, IR, optical, or any
other analytical systems well know to one in the art.
[0334] The integration of a complete sample-to-answer instrument
incorporating the cartridge to prepare DNA samples from many inputs
and sample types and a microchip-based capillary electrophoresis
device for separation of DNA fragments is taught for DNA
sequencing, fragment sizing, and forensics.
Example 6
Device with Four Processing Channels
[0335] A microchip or microfluidic microchip can be used to amplify
mRNA, concentrate nucleic acids on magnetic beads and inject
purified samples into electrophoretic separation capillaries. As
shown in FIG. 45 and FIG. 46, a microchip (505) can be interfaced
with a cartridge (503) and a pneumatic manifold (507). FIG. 45
shows an expanded view of the microchip, cartridge, and pneumatic
manifold. FIG. 46 shows a view of the cartridge interfaced with the
microchip, which is interfaced with the pneumatic manifold. The
cartridge can completely cover the surface of the microchip.
Additionally, a block (501) has holes (509) that help to hold for
incubation materials delivered to or from ports (511) of the
cartridge (503). The block can be a heat block or a temperature
controlling block. The holes (509) can be used to hold to hold
pipette tips or be used as a large volume reactor or processor. The
block (509) can be heated or cooled to control the temperature of
material being delivered to cartridge and microchip or removed from
the cartridge and microchip. The block can be in thermal contact
with the cartridge. The ports of the cartridge can lead to
reservoirs that are fluidically connected to ports that mate with
ports (515) on the microchip (505). The microchip (505) can have
pneumatic line ports (519) that mate with ports (517) of the
pneumatic manifold. The ports of the pneumatic manifold can have
o-ring gaskets that seal the pneumatic manifold to the microchip,
allowing for high and low pressures to be delivered without leaking
or with a reduced loss of pressure or vacuum. The cartridge,
microchip, and pneumatic manifold can be held together using bolts
or other securing objects that pass through openings (513, 501) of
the cartridge and the pneumatic manifold.
[0336] A diagram of the Chip A microchip is shown in FIG. 47. The
microchip comprises three layers: (i) a top fluidics layer (e.g.,
glass) carrying fluidic channels and wells, (ii) a bottom
pneumatics layer (e.g., glass) carrying pneumatic channels and
wells, and (iii) a middle flexible membrane, (e.g., 250 um thick,
PDMS layer) (not shown). The PDMS membrane can be featureless. The
PDMS membrane can deflect in response to positive or negative
pressure applied to localized areas defined by the pneumatic
channel system (dashed lines in FIG. 47). Pneumatic and fluidic
channel etch depths are typically 50 um and can be designed to
offer minimal hydraulic resistance. Valves in the microfluidic
system can include pump valves. Pump valves can be larger than
other valves, depending on the desired pump capacity, and can lack
a valve seat. Removal of the valve seat can increase the pumping
capacity, or the volume of fluid that is pumped per pump stroke.
Other valves can be smaller, and have a valve seat that allows them
to close firmly. Such reduced volume on/off valves can reduce the
overall void space within the microfluidic microchip. Inlet and
outlet valves can be such on/off valves.
[0337] As shown in FIG. 47, the microchip has four identical
processing channels, each of which can control reagent flows for
off-microchip or on-microchip magnetic-bead-based nucleic acid
concentration and capillary electrophoresis sample injection. The
four processing channels are fed by a common "reagent rail" which
can select input reagents from four input wells (the fifth well can
be used as a waste port). Individual samples can be fed into each
processing channel from channel-specific sample wells, and can be
processed in parallel (the microchip shown is a 4-plex processing
device). Each processing channel can have a 0.8 .mu.L pump, sample
input well, two output wells, and a waste well. Beads, such as
magnetic beads, can be captured in any of the wells, e.g. one or
both of the output wells. This output well can interface with the
cartridge or fluidic manifold.
[0338] A photograph of the Chip A microchip, is shown in FIG. 48.
The microchip can be mounted between a top fluidic manifold, mating
fluid reservoirs to wells on the top surface of the microchip, and
a bottom pneumatic manifold, mating pneumatic control lines to
wells on the bottom surface of the microchip via o-ring seals.
Microchip valves and pumps can be actuated by a pneumatic control
system driven by computer scripts, such as DevLink scripts, or
other software. The system can supply positive pressure
(approximately +10 psi) to close valves, and/or negative pressure
(approximately -20 psi or vacuum) to open them. The same pneumatic
system can operate microchip pumps, with negative pressure acting
to fill the pump bodies (on the fluidic channel side of an
elastomeric layer), and positive pressure acting to empty them. As
described herein, pumping action depends on the coordinated actions
of pumps and flanking inlet and outlet valves.
Example 7
mRNA Amplification Using Device with Four Processing Channels
[0339] Chip A was used to perform the first, reverse transcription,
reaction of the Eberwine protocol. Effective mixing and incubation
methods were developed.
[0340] In order to provide temperature control for the
approximately 10 ul reaction, a simple heated aluminum block (509),
carrying eight 200 ul pipette tips, was fabricated and mated to the
fluidic manifold as shown in FIG. 45, FIG. 46, and FIG. 49. Each
pipette tip connected to a microchip output. In the experiment,
only 4 tips, connected to microchip Output-1 wells (as indicated in
FIG. 47), were actually used. Heating (50.degree. C.) was
accomplished with a thin-film heater attached to the outer surface
of the block with adhesive. A thermocouple was inserted into the
center of the block, and the heater was controlled with a DevLink
PID control loop.
[0341] For mixing, a DevLink script was developed to mix samples
and a reagent in a 1:1 ratio by alternately pumping approximately
0.6 ul from each respective reservoir into a pipette tip mated to
Output-1. Experiments with food dye confirmed that alternate
pumping effectively mixed the two components in the tip.
[0342] To perform the reaction, a mixture of total RNA and T7
Promoter-Primer (in water) was pipetted into sample well
reservoirs, and a two-fold concentrated RT Mix, containing
Superscript III reverse transcriptase, was pipetted into a reagent
reservoir. After 15 ul had been loaded into the tips (12 pump
cycles), the script terminated, and the reactions continued
incubating for 15 minutes at 50.degree. C. At this point the tips
were removed, emptied into 0.2 ml PCR tubes, and the reactions
terminated by incubation at 85.degree. C. for 5 minutes in a
thermocycler.
[0343] Positive control reactions were treated identically, except
that these reactions were performed entirely at the bench and
incubated 15 min at 50.degree. C. in a thermocycler. Negative
control reactions were mixed (at the bench) and immediately
terminated by incubation for 5 min at 85.degree. C.
[0344] Reactions were analyzed by TaqMan real-time quantitative PCR
using a Gusb primer and probe set (ABI). The results are shown in
FIG. 50 for 15 .mu.L reactions containing approximately 500 ng
total RNA (Rat Liver) and 10 U/.mu.L Superscript III RT. Results
from channels 1-4 are shown with results on the performed entirely
at the bench indicated by iK. In FIG. 50, the pair of bars shown
for each of channels 1, 2, 3, 4, and iK represent replicate
reactions. The results showed that microchip and bench reactions
were identical in their yield of first-strand Gusb cDNA. Both
reactions produced Ct's between 26 and 27. Negative control
reactions produced Ct >35 (data not shown).
Example 8
Device with Bead Clean-Up Chambers
[0345] A microchip or microfluidic microchip can be used to amplify
mRNA, and to concentrate and purify nucleic acids on magnetic
beads. FIG. 51 shows an expanded view of a device that has a
microchip 1307 that can be interfaced with a cartridge made of two
pieces 1313, 1315 and a pneumatic manifold 1301. The cartridge is
made of a first piece that has serpentine channels 1311, wells,
reservoirs, and chambers, and a second piece (1315) that has wells
and chambers. The serpentine channels can be used to increase heat
transfer between the heat distributing piece and a fluid contained
within the serpentine channels. The second piece and first piece
can be bonded together such that the serpentine channels are
enclosed on a top side. The cartridge can be overlayed or in
thermal contact with a heat distributing piece (1317) that
distributes heat from thermal control blocks (1323, 1321). The
thermal control blocks can be thermo electric coolers (TECs or
Peltier devices), thin-film heaters, or other thermal control
devices. The heat distributing piece and heat blocks may or may not
be bonded or secured to the cartridge. Screws, bolts, and/or hinges
may facilitate the securing of the heating distributing piece
and/or heat blocks to the cartridge. FIG. 52 shows a view of the
cartridge interfaced with the microchip, which is interfaced with
the pneumatic manifold. The pneumatic block may have annular spaces
(1305, 1309) for bolts and/or screws and ports (1303) that
interface between pneumatic lines and the pneumatic layer of the
microfluidic microchip. Additional views of the pneumatic layer are
shown in FIG. 53, FIG. 54, FIG. 55, and FIG. 56. FIG. 53 and FIG.
55 show views with dashed lines indicating edges that are hidden
from view. FIG. 53 and FIG. 54 show three dimensional views of the
pneumatic layer. FIG. 55 and FIG. 56 show top views of the
pneumatic layer.
[0346] FIG. 57 and FIG. 58 show diagrams of a microfluidic
microchip, the Chip B microchip, with bead clean-up chambers.
Referring to FIG. 58, the microchip has four main sections: Reagent
Rail, Bead Rail, Processor 1, and Processor 2. The two rails and
the two processors have mirrored geometries. The microchip is
configured so that either reagent rail may feed either processor.
Access to the processors is controlled by valves Vr and Vb for the
top processor and by valves Vrb and Vbb for the bottom processor.
During reagent processing and enzymatic reactions of the top
processor, Vr is opened and Vb is closed. During reagent processing
and enzymatic reactions of the bottom processor, Vrb is opened and
Vbb is closed. During clean-up, the reverse applies, that is, for
the top processor Vr is closed and Vb is opened and for the bottom
processor Vrb is closed and Vbb opened. It can be seen that the top
and bottom processor can operate either in parallel or separately.
Reagent and Bead Rail design closely follows the Chip A design.
Each rail can access four different reagents (R1-4 and B1-4), via
valves Vr1-4 and Vb1-4 respectively, and each rail has waste wells
(RW and BW), accessed by valves Vrw and Vbw, respectively. Each
processor has a sample input well (S), two output wells (Oa, Ob),
and a bead side channel accessed by valves Vs, Voa, Vob, Vbs
respectively. The bead side channel has a bead reservoir (R), and
two valves (Vw and Ve) accessing waste (W) and elution (E) wells,
respectively. Pneumatic lines and ports for control of the valves
are shown as dashed in FIG. 57.
[0347] For purposes of explanation below, it is assumed that
Processor 1 and Processor 2 are operated identically in parallel,
and that Chip B is a duplex device, processing two samples
simultaneously. For clarity, the operation of only the top
processor is detailed. However, non-parallel operation is also
possible. It is also assumed that wells Oa and Ob are connected to
appropriate capacity reservoirs in a fluidic manifold or tubing.
Reservoirs can be pipette tips, a reservoir in a cartridge, or
connected by tubing to larger volumes The microfluidic microchip
can have 75 .mu.m channel depth, 250 gm (final) fluid channel
width, and 0.6 .mu.l (estimated) pumping stroke volume.
[0348] Referring to FIG. 58, a reaction comprising Reagent 1 and
Sample may be assembled in well Oa by alternate 4-cycle pumping (A,
B, C, D). Assume all valves are initially closed. In cycle A,
valves Vr1 and Vr open, allowing pump P to draw Reagent 1 from well
R1 with a down-stroke (negative pressure applied to pump P to open
the valve). In cycle B, valves Vr1 and Vr close and valve Voa
opens, allowing pump P to expel its contents (from R1 in this
example) into well Oa with an up-stroke (positive pressure applied
to pump P to close it). In cycle C, valve Vs opens and valve Voa
closes, allowing pump P to draw Sample from well S with a
down-stroke. In cycle D, valve Vs closes and Voa opens, allowing
pump P to expel its contents into well Oa with an up-stroke. These
four cycles are repeated until a sufficient volume has been pushed
into Oa. The mixing ratio between Sample and Reagent 1 is
determined by the ratio of cycles AB:CD. In the process described
above, the mixing ratio is 1:1, but it can in principle be any
value. Finally, similar procedures can be used to mix any of the
reagents (R1-4) with sample S, by substituting the appropriate
valve for Vr1. Referring to FIG. 58, in 4-cycle pumping, the fluid
can be pumped in a first direction from a first source well to a
space within a pumping valve in the first step. In the second step,
the fluid can be pumped in a direction opposite to the first
direction by moving the fluid from the pumping valve to a mixing
well. The third and fourth steps can be repeated with a second
source well instead of the first source well. The pumping in
opposite directions to obtain mixing in the mixing well can be a
result of having the source well, mixing well, and pumping valve
positioned along a channel such that the pumping valve is not
located between the source well and the mixing well. This
configuration can reduce the dead space within the microfluidic
microchip, improve mixing, or improve uniformity of reagent and
sample handling. As well, this configuration can allow for a
central pump to move liquid between many different wells on a
microfluidic microchip through the opening and closing of
appropriate valves.
[0349] The valves shown in FIG. 57 and FIG. 58 and any other valve
shown herein sometimes are placed at T-shaped junctions. The valves
can close off flow from one channel of the T to the other two
channels leading into the T, while continuing to allow flow between
the other two channels. For example, closing valve Voa prevents
fluid from flowing from pump P to Oa, but does not prevent fluid
from flowing from pump P to S if valve Vs is open. Alternatively, a
valve can obstruct flow between all channels leading into the T.
The same can be applied to valves that are placed at junctions of
4, 5, 6, or more channels. The valves can also be replaced by
valves that are only in the reagent or bead channel as needed.
Example 9
mRNA Amplification Using Device with Bead Clean-Up Chambers
[0350] As described above, the Eberwine mRNA amplification
procedure is a cascade of three binary additions. To execute the
Eberwine sequence, R1 contains RT Mix, R2 contains 2S
(second-strand) Mix, and R3 contains T7 Mix, as shown in FIG. 58. A
two-fold (2.times.) volume of 2S Mix can be added to the RT
reaction, and a one-fold volume of T7 Mix can be added to the 2S
reaction, as shown in FIG. 44. This requires a 2:1 pumping ratio
(AB:CD) for the 2S Mix addition, and a 1:1 ratio for the T7 Mix
addition.
[0351] The first (RT) reaction with a 1:1 mixture of total
RNA+Primer, from well S, and 2.times.RT Mix, from well R1, can be
formed using the methods described herein. After an appropriate
incubation period, the second-strand reaction may be assembled in
well Ob by drawing from well Oa (rather than well S), and drawing
from well R2 (rather than from well R1). A four-cycle pumping
scheme (A, B, C, D) similar to that described in Example 8 can be
used. In cycle A, Vr2 opens rather than Vr1; in cycle B, Vob opens
rather than Voa; in cycle C, Voa opens rather than Vs; and in cycle
D, Vob opens rather than Voa. To obtain the required 2:1 mixing
ratio, two cycles can draw from R2 for every cycle drawing from
Oa.
[0352] After another appropriate incubation period, the third (T7)
reaction may be assembled in well Oa with a similar process
(drawing from R3 and Ob, 1:1 ratio). Thus the final T7 reaction can
reside in Oa. After an appropriate incubation period, aRNA can be
ready for purification.
[0353] Purification involves operation of the Bead Rail rather than
the Reagent Rail. Thus, during this phase of microchip operation,
valve Vr remains closed and Vb can open.
[0354] To purify aRNA, reservoir R must first be loaded with
magnetic beads. This may be accomplished with a 2-cycle procedure,
similar to that of cycles A and B of Example 8, except that input
to pump P can be via valves Vb4 and Vb (cycle A), and output from
pump P can be via valves Vbs and Vw (cycle B). This sequence can
draw bead slurry from well B4 into pump P, and expel bead slurry
from pump P through reservoir R into waste well W. As the slurry
passes through reservoir R, beads can be captured by a magnet
placed below reservoir R.
[0355] Before aRNA (in well Oa) can be captured, it must be mixed
with Binding Buffer. This can be accomplished with another 4-cycle
procedure, similar to that in Example 8, except that Binding Buffer
can be drawn from well B1 in the Bead Rail, and the mixture can
accumulate in well Ob.
[0356] aRNA in well Ob can then be captured by beads in reservoir R
by pumping the contents of well Ob through reservoir R out into
waste well W. This can be accomplished with a 2-cycle procedure in
which pump P is filled via valve Vob (cycle A) and emptied via
valves Vbs and Vw (cycle B).
[0357] Loaded beads can then be washed with ethanol pumped from
well B2. This can be accomplished with a 2-cycle procedure in which
pump P is filled via valves Vb2 and Vb (cycle A) and emptied via
valves Vbs and Vw (cycle B). After ethanol from well B2 has been
exhausted, pumping can continue to draw air over the beads to dry
them.
[0358] Finally, aRNA can be eluted from the beads into well E by
pumping water through reservoir R with a 2-cycle procedure in which
pump P is filled via valves Vb3 and Vb (cycle A) and emptied via
valves Vbs and Ve (cycle B).
Example 10
Short RNA Amplification Using Device with Real-Time PCR
Detection
[0359] MicroRNA (miRNA) are short (19-25 nucleotide)
single-stranded RNAs that are produced by processing larger RNAs,
while siRNA are short (20-25 nucleotide) double-stranded RNAs that
are also produced by processing larger RNAs. miRNAs have been
implicated in regulating translation of mRNA while siRNAs can
silence or activate transcription of genes. Both miRNA and siRNA,
collectively small RNAs, can be assayed using the devices described
herein. To assay either small RNA, the device in FIG. 58 would be
reconfigured with 1) R3 containing a mixture to polyadenylate the
short RNA, 2) R4 containing real-time PCR primers and real-time
master mix, and 3) the RT mixture in R1 can contain a polyT
sequence with a 5' sequence for real-time PCR amplification instead
of a T7 promoter. The reaction would proceed as described in
Example 9 except first a polyA tail would be added to the small
RNAs to produce polyadenylated small RNAs using R3 as the reagent
source. The reverse transcription and second strand synthesis can
operate as described in Example 9. The final step of T7
transcription is then replaced by mixing the real time PCR primers
and master mix from R4 with the second strand product. The real
time PCR primers are then amplified using PCR with real time
detection. The amplification can occur off-microchip or a detector
and thermal cycling can be incorporated on the microchip or in
heating block (509). Real time PCR on microchips was previously
described, e.g. Jovanovich, S., I. Blaga, and D. Rank. Microfluidic
Devices. US Patent Publication No. 2007/0248958 and PCT Publication
No. WO/2006/032044, which are hereby incorporated by reference. It
will be obvious to one skilled in the art that other mRNAs can be
processed as described in this example by omitting the
polyadenylation step.
[0360] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. For example, any MOVe valve, pump, router, or other MOVe
device described herein can be replaced with any pneumatically
actuated valve, pump router or other device. It should be
understood that various alternatives to the embodiments of the
invention described herein can be employed in practicing the
invention. It is intended that the following claims define the
scope of the invention and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
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