U.S. patent application number 10/801460 was filed with the patent office on 2004-12-23 for devices and methods for the performance of miniaturized in vitro amplification assays.
Invention is credited to Able, Charles, Arnold, Todd, Carvalho, Bruce L., Kellogg, Gregory J., Kieffer-Higgins, Stephen, Kob, Mikayla, Lin, Hsin-Chiang, Ommert, Shari, Sheppard, Norman F..
Application Number | 20040259237 10/801460 |
Document ID | / |
Family ID | 31949730 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259237 |
Kind Code |
A1 |
Kellogg, Gregory J. ; et
al. |
December 23, 2004 |
Devices and methods for the performance of miniaturized in vitro
amplification assays
Abstract
This invention relates to methods and apparatus for performing
microanalytic and microsynthetic analyses and procedures. The
invention provides a microsystem platform and a micromanipulation
device for manipulating the platform that utilizes the centripetal
force resulting from rotation of the platform to motivate fluid
movement through microchannels. The invention specifically provides
devices and methods for performing miniaturized in vitro
amplification assays such as the polymerase chain reaction. Methods
specific for the apparatus of the invention for performing PCR are
provided.
Inventors: |
Kellogg, Gregory J.;
(Cambridge, MA) ; Able, Charles; (Cambridge,
MA) ; Arnold, Todd; (Glastonbury, CT) ;
Carvalho, Bruce L.; (Watertown, MA) ; Lin,
Hsin-Chiang; (Cambridge, MA) ; Kieffer-Higgins,
Stephen; (Boston, MA) ; Sheppard, Norman F.;
(Bedford, MA) ; Kob, Mikayla; (Somerville, MA)
; Ommert, Shari; (Stoneham, MA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Family ID: |
31949730 |
Appl. No.: |
10/801460 |
Filed: |
March 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10801460 |
Mar 16, 2004 |
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09602394 |
Jun 22, 2000 |
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6706519 |
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60140477 |
Jun 22, 1999 |
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Current U.S.
Class: |
435/287.1 |
Current CPC
Class: |
B01L 2300/0861 20130101;
B01L 2300/0806 20130101; G01N 21/4795 20130101; G01N 2035/00237
20130101; B01F 13/0064 20130101; B01F 13/0094 20130101; B01F
2215/0477 20130101; B01F 15/0233 20130101; B01F 2215/0431 20130101;
G01N 35/00069 20130101; B01L 2400/0409 20130101; B01L 3/50273
20130101; B01L 2200/0621 20130101; B01F 15/0201 20130101; B01L
2300/1827 20130101; B01L 2400/0677 20130101 |
Class at
Publication: |
435/287.1 |
International
Class: |
C12M 001/34 |
Claims
What is claimed is:
1. A centripetally-motivated microsystem platform comprising: a) a
rotatable platform comprising a substrate having a surface
comprising one or a multiplicity of microfluidics structures
embedded in the surface of the platform, wherein each microfluidics
structure comprises: i) a sample input port fluidly connected to
ii) a chamber in thermal contact with a temperature control
element, and iii) a sample outlet port, wherein the temperature
control element changes or maintains the temperature of a fluid in
the chamber at a temperature greater than ambient temperature, and
wherein rotation of the platform maintains or changes the
temperature of a fluid in a chamber to be substantially equal to
ambient temperature.
2. A microsystem platform of claim 1 wherein the temperature
control element is a resistive heater.
3. A microsystem platform of claim 1 wherein the temperature
control element is a Peltier heater.
4. A microsystem platform of claim 1 further comprising a
temperature sensing element in thermal contact with the chamber of
the temperature control element or both.
5. A microsystem platform according to claim 4, wherein the
temperature sensing element is a thermistor.
6. A microsystem platform of claim 1 wherein the platform further
comprises: b) an electric platen comprising a substrate bearing one
or a multiplicity of temperature control elements, wherein each of
the temperature control elements is electrically connected to at
least two electrical leads, and wherein the electrical leads are
connected to a power source through a slip ring wherein the
substrate comprising the sample input port, the chamber and the
sample outlet port is separate from the platen and wherein each
temperature control element is in thermal contact with a chamber in
the substrate of the platform.
7. A microsystem platform according to claim 6, wherein the platen
substrate is a printed circuit board.
8. A microsystem platform of claim 6 wherein the temperature
control element further comprises one or a multiplicity of metal
contact plates in thermal contact with a heating element.
9. A microsystem platform of claim 8 wherein at least one metal
contact plate is in thermal contact with the chamber.
10. A microsystem platform of claim 8 wherein at least one metal
contact plate is in thermal contact with a heat sink.
11. A microsystem platform of claim 8 wherein the metal contact
plate is brass.
12. A microsystem platform of claim 1 wherein the temperature of a
fluid in the chamber can be changed at a rate sufficient for
performing an in vitro amplification reaction.
13. A microsystem platform of claim 12 wherein the in vitro
amplification reaction is polymerase chain reaction.
14. A microsystem platform of claim 1 that is a circular disk.
15. A microsystem platform of claim 1 wherein the sample chamber
further comprises a sample input port.
Description
BACKGROUND OF THE INVENTION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/140,477, filed Jun. 22, 1999, the
disclosure of which is explicitly incorporated by reference
herein.
1. FIELD OF THE INVENTION
[0002] This invention relates to methods and apparatus for
performing microanalytic and microsynthetic analyses and
procedures. In particular, the invention relates to
microminiaturization of genetic, biochemical and bioanalytic
processes. Specifically, the present invention provides devices and
methods for the performance of integrated and miniaturized sample
preparation, nucleic acid amplification, and nucleic acid detection
assays. These assays may be performed for a variety of purposes,
including but not limited to forensics, life sciences research, and
clinical and molecular diagnostics. The invention may be used on a
variety of liquid samples of interest, including bacterial and cell
cultures as well as whole blood and processed tissues. Methods for
performing any of a wide variety of such microanalytical or
microsynthetic processes using the microsystems apparatus of the
invention are also provided.
2. BACKGROUND OF THE RELATED ART
[0003] Extraction and isolation of DNA from host cells is a
cornerstone of modern molecular biology. One type of DNA, bacterial
plasmid DNA has been particularly useful as a convenient vector for
the insertion of genetic material into bacterial, yeast and
mammalian cells. DNA isolated from an organism is inserted by being
contiguously and covalently linked to plasmid DNA and is then
introduced into a cell, such as a bacterial cell, and allowed to
multiply, thereby creating large copy numbers of the plasmid in
each cell. These plasmids may advantageously be harvested to
provide a sufficient amount of DNA (typically on the order of
several micrograms, although up to milligram quantities can be
produced on an industrial scale) for a variety of experimental or
therapeutic purposes. The harvesting of plasmid DNA, defined as its
removal from cells and isolation from the genomic DNA content of
the cells, has growing utility in life sciences research,
diagnostics, therapeutics and other applications.
[0004] Currently, the extraction and isolation of DNA is either
performed manually or through the use of robotic sample preparation
stations. In either case, a variety of technologies and materials
are used (see, for example, QIAamp DNA Mini Kit and QIAamp DNA
Blood Mini Kit Handbook, 1999, Qiagen GmbH, Max-Volmer-Strasse 4,
40724 Hildren, Germany; Bimboim & Doly, 1979, Nucl. Acids Res.
1: 1513-1522). Typically, cells are first incubated in a surfactant
(detergent) solution, in some cases containing protein digesting
enzymes such as Protease or Proteinase K. These lyse the cells,
thereby releasing the DNA into solution. This is frequently
performed under alkaline conditions, to destabilize nucleases and
hydrolyze contaminating RNA. The DNA must then be separated from
other cell constituents, which is performed using a number of
different separation protocols, including, for example, selective
precipitation of proteins and other cell debris, organic chemical
extraction (using phenol and chloroform), and DNA affinity column
chromatography. Plasmid DNA must also be isolated from
contaminating cellular (bacterial genomic DNA). Filtration methods
can produce a plasmid DNA solution, but the solutions required to
solvate DNA are usually inappropriate for the desired final
application of the DNA. As a consequence, plasmid DNA is removed
from these solutions by ethanol precipitation, or solid-phase
separation is used, which often requires further changes in solvent
pH and salt concentration (especially for affinity binding methods
using glass or silica). The technologies required for these steps
include pipetting, pumping, filtration, washing, and
centrifugation, requiring an expensive suite of devices and skilled
operators thereof. The additional requirements of automated systems
include sample transfer and robotics for the handling of sample
containers.
[0005] This discussion illustrates the need in the art for more
efficient, rapid, inexpensive automated methods and devices for
performing DNA sample preparation, particularly plasmid DNA
preparation.
[0006] In the field of integrated genetic analysis, some progress
has been made in the integration of sample preparation, PCR, and
detection via real-time fluorescence or hybridization methods
(Anderson et al., 1998, "Advances in Integrated Genetic Analysis,"
in Proc. Micro Total Analysis '98, Harrison & van den Berg,
eds., Kluwer: Amsterdam, pp.11-16). These systems rely on
macroscopic fluid handling systems such as pumps and valves that
must be interfaced with the microfluidic devices within which
fluids are processed.
[0007] However, there exists a need for devices and methods capable
of processing cell cultures for harvesting DNA, particularly
plasmid DNA.
[0008] In the biological and biochemical arts, analytical
procedures frequently require incubation of biological samples and
reaction mixtures at temperatures greater than ambient temperature.
Moreover, many bioanalytical and biosynthetic techniques require
incubation at more than one temperature, either sequentially or
over the course of a reaction scheme or protocol.
[0009] One example of such a bioanalytical reaction is the
polymerase chain reaction. The polymerase chain reaction (PCR) is a
technique that permits amplification and detection of nucleic acid
sequences. See U.S. Pat. No. 4,683,195 to Mullis et al. and U.S.
Pat. No. 4,683,202 to Mullis. This technique has a wide variety of
biological applications, including for example, DNA sequence
analysis, probe generation, cloning of nucleic acid sequences,
site-directed mutagenesis, detection of genetic mutations,
diagnoses of viral infections, molecular "fingerprinting," and the
monitoring of contaminating microorganisms in biological fluids and
other sources. The polymerase chain reaction comprises repeated
rounds, or cycles, of target denaturation, primer annealing, and
polymerase-mediated extension; the reaction process yields an
exponential amplification of a specific target sequence.
[0010] Methods for miniaturizing and automating PCR are desirable
in a wide variety of analytical contexts, particularly under
conditions where a large multiplicity of samples must be analyzed
simultaneously or when there is a small amount of sample to be
analyzed.
[0011] In addition to PCR, other in vitro amplification procedures,
including ligase chain reaction as disclosed in U.S. Pat. No.
4,988,617 to Landegren and Hood, are known and advantageously used
in the prior art. More generally, several important methods known
in the biotechnology arts, such as nucleic acid hybridization and
sequencing, are dependent upon changing the temperature of
solutions containing sample molecules in a controlled fashion.
Automation and miniaturization of the performance of these methods
are desirable goals in the art.
[0012] Mechanical and automated fluid handling systems and
instruments produced to perform automated PCR have been disclosed
in the prior art.
[0013] U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et
al. teach fluid handling on microscale analytical devices.
[0014] International Application, Publication No. WO93/22053,
published 11 Nov. 1993 to University of Pennsylvania disclose
microfabricated detection structures.
[0015] International Application, Publication No. WO93/22058,
published 11 Nov. 1993 to University of Pennsylvania disclose
microfabricated structures for performing polynucleotide
amplification.
[0016] Wilding et al., 1994, Clin. Chem. 40: 43-47 disclose
manipulation of fluids on straight channels micromachined into
silicon.
[0017] Kopp et al., 1998, Science 21M: 1046 discloses microchips
for performing in vitro amplification reactions using alternating
regions of different temperature.
[0018] One drawback of the synthetic microchips disclosed in the
prior art for performing PCR and other temperature-dependent
bioanalytic reactions has been the difficulty in designing systems
for moving fluids on the microchips through channels and reservoirs
having diameters in the 10-100 .mu.m range. This is due in part to
the need for high-pressure pumping means for moving fluid through
the small sizes of the components of these microchips. These
disabilities of the prior art microchips limits the usefulness of
these devices for miniaturizing and automating PCR and other
bioanalytic processes.
[0019] Thus, there exists a need in the art for devices and methods
that provide integrated sample preparation and analysis,
particularly of DNA samples. This need is particularly acute for
high throughput analyses, which are currently burdened by the high
costs and complexity of automated, typically robotic, systems.
Integration of DNA sample preparation and analysis would be
particularly useful if it reduced the current need in the art for
need for multiple, complex technologies that demand highly-skilled
operators. Importantly, for DNA analysis integration of sample
preparation and in vitro amplification methods would minimize the
possibility of contamination and sample carry-over, which is
particularly important in high-sensitivity techniques such as
various in vitro amplification reactions used in the art.
[0020] Some of the present inventors have developed a microsystem
platform and a micromanipulation device to manipulate said platform
by rotation, thereby utilizing the centripetal forces resulting
from rotation of the platform to motivate fluid movement through
microchannels embedded in the microplatform, as disclosed in
co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned
and co-pending patent applications U.S. Ser. No. 08/761,063, filed
Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No.
08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec.
19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No.
09/579,492, filed May 12, 2000 and 09/filed Jun. 16, 2000 (Attorney
Docket No. 95,1408-XX), the disclosures of each of which are
explicitly incorporated by reference herein.
SUMMARY OF THE INVENTION
[0021] This invention provides Microsystems platforms as disclosed
in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and
co-owned and co-pending patent applications U.S. Ser. No.
08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18,
1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No.
08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19,
1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______,
filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the
disclosures of each of which are explicitly incorporated by
reference herein.
[0022] The invention provides apparatus and methods for performing
microscale processes on a microplatform, whereby fluid is moved on
the platform in defined channels motivated by centripetal force
arising from rotation of the platform. The Microsystems platform is
provided to perform integrated and miniaturized sample preparation,
nucleic acid amplification, and nucleic acid detection assays. A
first element of the apparatus of the invention is a microplatform
that is a rotatable structure, most preferably a disk, the disk
comprising fluid (sample) inlet ports, fluidic microchannels,
reagent reservoirs, collection chambers, detection chambers and
sample outlet ports, generically termed "microfluidic structures".
The disk is rotated at speeds from about 1 to about 30,000 rpm for
generating centripetal acceleration that enables fluid movement
through the microfluidic structures of the platform. The disks of
the invention also preferably comprise air outlet ports and air
displacement channels. The air outlet ports and in particular the
air displacement ports provide a means for fluids to displace air,
thus ensuring uninhibited movement of fluids on the disk. The disk,
and most preferably a face of the platform, may also contain
heating elements for raising the temperature of fluids contained
therein to temperatures greater than ambient temperatures. Specific
sites on the disk also preferably comprise elements that allow
fluids to be analyzed.
[0023] A preferred embodiment of the platforms of the invention is
a platen that rotates with the microfluidics disk. The platen is
most preferably a printed circuit board comprising resistive
heating elements, thermoelectric (Peltier) elements, temperature
sensors, assay optics and microprocessor and other electronic
components. Electrical communication between a rotating platen and
stationary power sources, motor controllers, temperature
controllers, and computers is most preferably accomplished through
a slip-ring assembly. By mounting the microfluidic disk on the
platen and rotating both disk and platen together, the distribution
and flow rate of fluid throughout the microfluidic structures as
well as the temperature of fluid within localized regions of the
microfluidics disc can be controlled.
[0024] In a preferred embodiment, one face of the microfluidics
disk is mounted onto a face of the platen and the temperature of
fluids at particular positions within the microfluidics disk is
controlled through temperature exchange between the platen and
disk. In alternative embodiments, a microfluidic disk is positioned
between two platens, each comprising elements that effect
temperature exchange between the disk and thermal regulation
elements comprising the platens. In a preferred embodiment, the
platen is a printed circuit board with resistive heating elements,
Peltier elements and temperature sensors embedded therein or
affixed thereto. In an alternative embodiment, thermal regulation
within the microfluidic disk is achieved by permanently bonding a
layer comprising resistive heaters directly to the disk; in this
case, fluids within the disk are heated to temperatures greater
than ambient temperature with resistive heating elements and cooled
to temperatures above or equal to ambient temperature by spinning
the disk and through the loss of heat to the environment. As with
the platen, electrical communication between this composite disk
and power supplies, temperature controllers and computers is most
preferably accomplished through a slip-ring assembly.
[0025] In a first aspect, the present invention provides devices
and methods for the performance of integrated and miniaturized
sample preparation for the extraction, isolation, and purification
of DNA from cells. In preferred embodiments, the devices and
methods of the invention are particularly provided to isolate
plasmid DNA from bacterial cells.
[0026] The plasmid DNA sample preparation platforms of the
invention are provided to perform the following functions: sample
processing to free DNA from the bacterial cell; filtration of the
resultant solution to remove bacterial cell fragments; application
of the solution to a binding matrix using solvent conditions that
promote DNA binding to the matrix; washing of bound DNA and
replacement of the original solution by a solution that is
compatible with further analytical methods; and elution of the DNA
from the binding matrix in a suitable solvent. The DNA thus eluted
can be isolated, amplified in vitro or sequenced using methods
known in the art. The platforms of the invention are provided
comprising microfluidic structures that perform plasmid DNA sample
preparation as described in further detail below. These
microstructures are illustrated for clarity with regard to a single
microstructure. However, platforms comprising a multiplicity of
such plasmid DNA preparation microfluidic structures are provided
by the invention, wherein the microfluidics structures are arrayed
on the surface of the platform with a density determined by the
size of the platform and the volumetric capacity of the chambers
and reservoirs comprising the microfluidic structures as disclosed
herein.
[0027] In a second aspect, the invention is provided having
microfluidics structures as described herein for performing an
integrated suite of biochemical processes for accomplishing in
vitro amplification reactions. These include sample processing to
isolate DNA from bacterial or mammalian cells; sample conditioning
to adjust the solution conditions to those appropriate for PCR;
mixing of the conditioned sample with PCR reagents, including
deoxyribosenuclotides, polymerase enzyme, primers, and appropriate
salts, buffers and additives; and thermal cycling to effect
PCR.
[0028] In certain preferred embodiments, the discs of the invention
are provided with a multiplicity of microfluidics structures that
enable to platform to process and amplify several samples
simultaneously. In these embodiments, multiple copies of an
arrangement of microfluidics structures for performing the
biochemical reaction suite are arrayed on the disc, and sample
input ports or reservoirs provided for each copy, thereby
permitting processing of multiple samples. In addition, the portion
of the sample DNA to be amplified can be independently, by the
choice of amplification primers provided in each of the individual
copies of the microfluidics structures arrayed on the disc, thereby
permitting amplification "multiplexing" of a particular sample.
Alternatively, the same primers can be provided to process in
parallel multiple samples for amplification of the same target
fragment in the DNA of each sample. Independent thermal cycling
profiles, including the temperature used for each step of the
amplification cycle, temperature ramp-rates, and hold times, may be
individually programmed into the instrument for each of the
microfluidics structures or for each of the samples processed.
[0029] The invention advantageously permits simultaneous,
independent thermal cycling of a multiplicity of different samples,
independent amplification of different target fragments from a
particular sample, or both. This feature also enables a user to
optimize thermal cycling parameters for a single sample or amplicon
quickly and in a single experiment, by varying reaction parameters
on a plurality of the microfluidics structures arrayed in the disc,
thereby simultaneously performing multiple experiments
simultaneously. Since particular copies of the microfluidics
structures can be arranged in microfluidic isolation from other
copies on the platform, portions comprising less than all of the
microfluidics structures can be discretely used and the remainder
retained for future use.
[0030] In alternative embodiments of the platforms of the
invention, metering structures as disclosed in co-owned U.S. Pat.
No. 6,063,589, issued May 16, 2000 and incorporated by reference
herein, are used to distribute aliquots of reagent to each of a
multiplicity of mixing structures, each mixing structure being
fluidly connected to one of a multiplicity of sample reservoirs,
thereby permitting parallel processing and mixing of the samples
with a common reagent. This reduces the need for automated reagent
distribution mechanisms, reduces the amount of time required for
reagent dispensing (that can be performed in parallel with
distribution of reagent to a multiplicity of reaction chambers),
and permits delivery of small (nL-to-.mu.L) volumes without using
externally-applied electromotive means.
[0031] The assembly of a multiplicity of collection chambers on the
platforms of the invention also permits simplified detectors to be
used, whereby each individual collection/detection chamber can be
scanned using mechanisms well-developed in the art for use with,
for example, CD-ROM technology. Finally, the platforms of the
invention are advantageously provided with sample and reagent entry
ports for filling with samples and reagents, respectively, that can
be adapted to liquid delivery means known in the art (such as
micropipettors).
[0032] The discs of this invention have several advantages over
those that exist in the centrifugal analyzer art. Foremost is the
fact that flow is laminar due to the small dimensions of the fluid
channels; this allows for better control of processes such as
mixing and washing. Secondly, the small dimensions conferred by
microfabrication enable the use of "passive" valving, dependent
upon capillary forces, over much wider range of rotational
velocities and with greater reliability than in more macroscopic
systems. To this are added the already described advantages of
miniaturization.
[0033] The present invention solves problems in the current art
through the use of a microfluidic disc in which centripetal
acceleration is used to move fluids. It is an advantage of the
microfluidics platforms of the present invention that the
fluid-containing components are constructed to contain a small
volume, thus reducing reagent costs, reaction times and the amount
of biological material required to perform an assay. It is also an
advantage that the fluid-containing components are sealed, thus
eliminating experimental error due to differential evaporation of
different fluids and the resulting changes in reagent
concentration. Because the microfluidic devices of the invention
are completely enclosed, both evaporation and optical distortion
are reduced to negligible levels. The platforms of the invention
also advantageously permit "passive" mixing and valving, i.e.,
mixing and valving are performed as a consequence of the structural
arrangements of the components on the platforms (such as shape,
length, position on the platform surface relative to the axis of
rotation, and surface properties of the interior surfaces of the
components, such as wettability as discussed below), and the
dynamics of platform rotation (speed, acceleration, direction and
change-of-direction), and permit control of assay timing and
reagent delivery.
[0034] The devices of the invention also implement simpler, more
robust, and more economical sample preparation for performing in
vitro amplification reactions such as PCR. All mechanical aspects
of sample processing are carried out using a single motor that
rotates the disc at prescribed velocities, thereby driving fluids
on the disc through microchannels and other microfluidics
structures. This is in advantageous over current sample preparation
methods involving robotic pipetting stations or other fluid
transfer mechanisms, automation for the delivery of processing
plates to different "stations," or both.
[0035] The invention advantageously integrates sample preparation
with thermal cycling for PCR, thereby eliminating additional fluid
transfer steps. This minimizes the potential for contamination or
fluid loss.
[0036] The platforms of the invention reduce the demands on
automation in at least three ways. First, the need for precise
metering of delivered fluids is relaxed through the use of on-disc
metering structures, as described more fully in co-owned U.S. Pat.
No. 6,063,589, issued May 16, 2000, and co-owned and co-pending
patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996;
Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726,
filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser.
No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May
12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket
No. 95,1408-XX), the disclosures of each of which are explicitly
incorporated by reference herein. By loading imprecise volumes,
slightly in excess of those needed for the assay, and allowing the
rotation of the disc and use of appropriate microfluidic structures
to meter the fluids, much simpler (and less expensive) fluid
delivery technology may be employed than is the conventionally
required for high-density microtitre plate assays.
[0037] Second, the total number of fluid "delivery" events on the
microfluidic platform is reduced relative to microtiter plates. By
using microfluidic structures that sub-divide and aliquot common
reagents (such as reagent solutions, buffers, and enzyme
substrates) used in all assays performed on the platform, the
number of manual or automated pipetting steps are reduced by at
least half (depending on the complexity of the assay). A reduction
in fluid transfers to the device can reduce total assay time.
Examples of these structures have been disclosed in co-owned U.S.
Pat. No. 6,063,589, issued May 16, 2000, and incorporated by
reference herein.
[0038] The invention also provides on-platform means for mixing
reagents with sample and washing the resulting reaction products,
removing the need for transferring the assay collection chamber(s)
to a separate "wash" station. This also reduces manipulation of the
assay device as well as providing controlled and integrated fluid
processing.
[0039] The invention disclosed herein is flexible as to sample and
source, being capable of isolating nucleic acid from bacteria,
whole animal blood, tissues and cellular sources. It is rapid,
being about 50% more rapid than existing "automated" nucleic acid
preparatory methods. The nucleic acid output of the system is of a
quality higher than or equal to methods known in the art. The
system is simple and easy to use, robust because it is not
dependent on operator variability. In addition, the platforms and
systems disclosed are self-contained and integrated, thereby
minimizing both operator handling and error.
[0040] Certain preferred embodiments of the apparatus of the
invention are described in greater detail in the following sections
of this application and in the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 depicts an exploded, oblique view of a Microsystems
platform of the invention.
[0042] FIG. 2 depicts a plan view of one component of the
Microsystems platform shown in exploded, oblique view in FIG. 1,
the microfluidics layer.
[0043] FIG. 3 is a detail of a section of the microfluidics layer
illustrated in FIG. 2.
[0044] FIG. 4 shows a detail of a region of the structure
illustrated in FIG. 3.
[0045] FIG. 5 is a cross-sectional view of the Microsystems
platform of FIG. 1 in the vicinity of the thermal cycling
chamber.
[0046] FIG. 6 depicts an explode, oblique view of a microfluidics
disc and a printed circuit.
[0047] FIG. 7 illustrates a plan view of the microfluidics disc
shown in FIG. 6.
[0048] FIG. 8 illustrates the velocity profile, rotational rate
(rpm) vs. time, used to effect fluid motion through the
Microsystems platform in Examples 1 and 2.
[0049] FIG. 9 illustrates the sequence of fluid motions motivated
by the velocity profile of FIG. 8.
[0050] FIG. 10 is a photograph of gel electrophoretic analysis of
PCR amplification of a target fragment contained in DNA isolated
from E. coli.
[0051] FIG. 11 is a photograph of gel electrophoretic analysis of
PCR amplification of a target fragment contained in DNA isolated
from bovine blood.
[0052] FIG. 12 depicts an exploded, oblique view of the DNA sample
preparation disk.
[0053] FIG. 13 is a plan view of this disk shown in FIG. 12.
[0054] FIG. 14 depicts a plan view of the microfluidics structure
for a plasmid DNA preparation platform.
[0055] FIG. 15 depicts a plan view of the heating layer for a
plasmid DNA preparation platform.
[0056] FIG. 16 depicts a plan view of a base layer for a plasmid
DNA preparation platform.
[0057] FIGS. 17A through 17K illustrates fluid movement through the
microfluidics structure for a plasmid DNA preparation platform.
[0058] FIG. 18 is a photograph of gel electrophoretic analysis of a
restriction enzyme digestion profile of plasmid DNA prepared
conventionally (control) or using a plasmid DNA preparation
platform of the invention, wherein the outside lanes are size
markers.
[0059] FIG. 19 is a photograph of gel electrophoretic analysis of
an in vitro amplification reaction using primers specific for
plasmid DNA or bacterial genomic DNA, wherein the amount of
template DNA decreases in each set of amplification reactions
moving from left to right, using a plasmid DNA preparation platform
of the invention; the outside lanes are size markers.
[0060] FIG. 20 is a plan view diagram of the electric platen and
controlling elements of the invention.
[0061] FIG. 21 is a plan view diagram of the temperature control
elements on an electric platen of the invention.
[0062] FIG. 22 is a plan view diagram of the electrical contacts
between the electrical leads on the printed circuit board of the
platen and temperature control elements.
[0063] FIG. 23 is a cross-sectional view of the structure of a
temperature control element comprising a Peltier element according
to the invention.
[0064] FIG. 24 depicts a plan view of the electric circuit layer
shown in FIG. 6.
[0065] FIG. 25 shows a cross-sectional view on the disk shown in
FIG. 6.
[0066] FIG. 26 is a photograph of gel electrophoretic analysis of
amplified DNA target from an E. coli sample using the disk pictured
in FIG. 6.
[0067] FIG. 27 depicts an example of multiplexed PCR performed in
the thermal cycling chamber.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0068] This invention provides a microplatform and a
micromanipulation device as disclosed in co-owned U.S. Pat. No.
6,063,589, issued May 16, 2000, and co-owned and co-pending patent
applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No.
08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug.
12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No.
09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12,
2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No.
95,1408-XX), the disclosures of each of which are explicitly
incorporated by reference herein, adapted for performing
microanalytical and microsynthetic assays of biological
samples.
[0069] For the purposes of this invention, the term "sample" will
be understood to encompass any fluid, solution or mixture, either
isolated or detected as a constituent of a more complex mixture, or
synthesized from precursor species. In particular, the term
"sample" will be understood to encompass any biological species of
interest. The term "biological sample" or "biological fluid sample"
will be understood to mean any biologically-derived sample,
including but not limited to blood, plasma, serum, lymph, saliva,
tears, cerebrospinal fluid, urine, sweat, plant and vegetable
extracts, semen, and ascites fluid.
[0070] For the purposes of this invention, the term "a
centripetally motivated fluid micromanipulation apparatus" is
intended to include analytical centrifuges and rotors, microscale
centrifugal separation apparatuses, and most particularly the
microsystems platforms and disk handling apparatuses as described
in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and
co-owned and co-pending patent applications U.S. Ser. No.
08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18,
1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No.
08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19,
1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______,
filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the
disclosures of each of which are explicitly incorporated by
reference herein.
[0071] For the purposes of this invention, the term "microsystems
platform" is intended to include centripetally-motivated
microfluidics arrays as described in co-owned U.S. Pat. No.
6,063,589, issued May 16, 2000, and co-owned and co-pending patent
applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No.
08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug.
12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No.
09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12,
2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No.
95,1408-XX), the disclosures of each of which are explicitly
incorporated by reference herein.
[0072] For the purposes of this invention, the terms "capillary",
"microcapillary" and "microchannel" will be understood to be
interchangeable and to be constructed of either wetting or
non-wetting materials where appropriate.
[0073] For the purposes of this invention, the term "reagent
reservoir," "assay chamber," "fluid holding chamber," "collection
chamber" and "detection chamber" will be understood to mean a
defined volume on a Microsystems platform of the invention
comprising a fluid. The volumetric capacity of these structures as
provided herein is from about 2 nL to about 1000 .mu.L.
[0074] For the purposes of this invention, the terms "entry port"
and "fluid input port" will be understood to mean an opening on a
microsystems platform of the invention comprising a means for
applying a fluid to the platform.
[0075] For the purposes of this invention, the terms "exit port"
and "fluid outlet port" will be understood to mean a defined volume
on a Microsystems platform of the invention comprising a means for
removing a fluid from the platform.
[0076] For the purposes of this invention, the term "capillary
junction" will be understood to mean a region in a capillary or
other flow path where surface or capillary forces are exploited to
retard or promote fluid flow. A capillary junction is provided as a
pocket, depression or chamber in a hydrophilic substrate that has a
greater depth (vertically within the platform layer) and/or a
greater width (horizontally within the platform layer) that the
fluidics component (such as a microchannel) to which it is fluidly
connected. For liquids having a contact angle less than 90.degree.
(such as aqueous solutions on platforms made with most plastics,
glass and silica), flow is impeded as the channel cross-section
increases at the interface of the capillary junction. The force
hindering flow is produced by capillary pressure, that is inversely
proportional to the cross sectional dimensions of the channel and
directly proportional to the surface tension of the liquid,
multiplied by the cosine of the contact angle of the fluid in
contact with the material comprising the channel. The factors
relating to capillarity in microchannels according to this
invention have been discussed in co-owned U.S. Pat. No. 6,063,589,
issued May 12, 2000 and in co-owned and co-pending U.S. patent
application Ser. No. 08/910,726, filed Aug. 12, 1997, incorporated
by reference in its entirety herein.
[0077] Capillary junctions can be constructed in at least three
ways. In one embodiment, a capillary junction is formed at the
junction of two components wherein one or both of the lateral
dimensions of one component is larger than the lateral dimension(s)
of the other component. As an example, in microfluidics components
made from "wetting" or "wettable" materials, such a junction occurs
at an enlargement of a capillary as described in co-owned and
co-pending U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No.
08/768,990, filed Dec. 18, 1996; and Ser. No. 08/910,726, filed
Aug. 12, 1997. Fluid flow through capillaries is inhibited at such
junctions. At junctions of components made from non-wetting or
non-wettable materials, on the other hand, a constriction in the
fluid path, such as the exit from a chamber or reservoir into a
capillary, produces a capillary junction that inhibits flow. In
general, it will be understood that capillary junctions are formed
when the dimensions of the components change from a small diameter
(such as a capillary) to a larger diameter (such as a chamber) in
wetting systems, in contrast to non-wettable systems, where
capillary junctions form when the dimensions of the components
change from a larger diameter (such as a chamber) to a small
diameter (such as a capillary).
[0078] A second embodiment of a capillary junction is formed using
a component having differential surface treatment of a capillary or
flow-path. For example, a channel that is hydrophilic (that is,
wettable) may be treated to have discrete regions of hydrophobicity
(that is, non-wettable). A fluid flowing through such a channel
will do so through the hydrophilic areas, while flow will be
impeded as the fluid-vapor meniscus impinges upon the hydrophobic
zone.
[0079] The third embodiment of a capillary junction according to
the invention is provided for components having changes in both
lateral dimension and surface properties. An example of such a
junction is a microchannel opening into a hydrophobic component
(microchannel or reservoir) having a larger lateral dimension.
Those of ordinary skill will appreciate how capillary junctions
according to the invention can be created at the juncture of
components having different sizes in their lateral dimensions,
different hydrophilic properties, or both.
[0080] For the purposes of this invention, the term "capillary
action" will be understood to mean fluid flow in the absence of
rotational motion or centripetal force applied to a fluid on a
rotor or platform of the invention and is due to a partially or
completely wettable surface.
[0081] For the purposes of this invention, the term "capillary
microvalve" will be understood to mean a capillary microchannel
comprising a capillary junction whereby fluid flow is impeded and
can be motivated by the application of pressure on a fluid,
typically by centripetal force created by rotation of the rotor or
platform of the invention. Capillary microvalves will be understood
to comprise capillary junctions that can be overcome by increasing
the hydrodynamic pressure on the fluid at the junction, most
preferably by increasing the rotational speed of the platform.
[0082] For the purposes of this invention, the term "sacrificial
valve" will be understood to mean a valve preferably made of a
fungible material that can be removed from the fluid flow path. In
preferred embodiments, said sacrificial valves are wax valves and
are removed from the fluid flow path by heating, using any of a
variety of heating means including infrared illumination and most
preferably by activation of heating elements on or embedded in the
platform surface as described in co-owned U.S. Pat. No. 6,063,589,
incorporated by reference.
[0083] For the purposes of this invention, the term "in fluid
communication" or "fluidly connected" is intended to define
components that are operably interconnected to allow fluid flow
between components. In preferred embodiments, the platform
comprises a rotatable platform, more preferably a disk, whereby
fluid movement on the disk is motivated by centripetal force upon
rotation of the disk.
[0084] For the purposes of this invention, the term "air
displacement channels" will be understood to include ports in the
surface of the platform that are contiguous with the components
(such as microchannels, chambers and reservoirs) on the platform,
and that comprise vents and microchannels that permit displacement
of air from components of the platforms and rotors by fluid
movement.
[0085] The microplatforms of the invention (preferably and
hereinafter collectively referred to as "disks"; for the purposes
of this invention, the terms "microplatform", "Microsystems
platform" and "disk" are considered to be interchangeable) are
provided to comprise one or a multiplicity of microsynthetic or
microanalytic systems (termed "microfluidics structures" herein).
Such microfluidics structures in turn comprise combinations of
related components as described in further detail herein that are
operably interconnected to allow fluid flow between components upon
rotation of the disk. These components can be microfabricated as
described below either integral to the disk or as modules attached
to, placed upon, in contact with or embedded in the disk. For the
purposes of this invention, the term "microfabricated" refers to
processes that allow production of these structures on the
sub-millimeter scale. These processes include but are not
restricted to molding, photolithography, etching, stamping and
other means that are familiar to those skilled in the art.
[0086] The invention also comprises a micromanipulation device for
manipulating the disks of the invention, wherein the disk is
rotated within the device to provide centripetal force to effect
fluid flow on the disk. Accordingly, the device provides means for
rotating the disk at a controlled rotational velocity, for stopping
and starting disk rotation, and advantageously for changing the
direction of rotation of the disk. Both electromechanical means and
control means, as further described herein, are provided as
components of the devices of the invention. User interface means
(such as a keypad and a display) are also provided, as further
described in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000,
and co-owned and co-pending patent applications U.S. Ser. No.
08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18,
1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No.
08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19,
1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______,
filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the
disclosures of each of which are explicitly incorporated by
reference herein.
[0087] Temperature control elements are provided to control the
temperature of the platform during incubation of a fluid thereupon.
The invention therefore provides heating elements, including heat
lamps, direct laser heaters, Peltier heat pumps, resistive heaters,
ultrasonication heaters and microwave excitation heaters, and
cooling elements, including Peltier devices and heat sinks,
radiative heat fins and other components to facilitate radiative
heat loss. Thermal devices are preferably arrayed to control the
temperature of the platform over a specific area or multiplicity of
areas. Preferably, heating and cooling elements comprise the
platforms of the invention comprising a thermal regulation layer in
the platform surface that is in thermal contact with the
microfluidics components, most preferably microchannels as
described herein. The temperature of any particular area on the
platform (preferably, the microchannels at any particular thermally
regulated area) is monitored by resistive temperature devices
(RTD), thermistors, liquid crystal birefringence sensors or by
infrared interrogation using IR-specific detectors, and can be
regulated by feedback control systems. Temperature control on the
microsystems platforms of this invention is most preferably
achieved using the methods and devices disclosed in co-owned U.S.
Pat. No. 6,063,589, incorporated by reference herein.
[0088] In preferred embodiments, portions of the Microsystems
platform surface are adapted for providing regions of controlled
temperature (termed "thermal regions" or "thermal arrays" herein)
using integral heating elements as disclosed in U.S. Pat. No.
6,063,589, incorporated by reference. In more preferred
embodiments, the portions of the microsystems platform surface are
constituted in arrays of thermal control elements, most preferably
wherein is produced adjacent regions of the platform surface having
different temperatures. In preferred embodiments, the platform also
comprises other components as disclosed in co-owned and co-pending
patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996;
Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726,
filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser.
No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May
12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket
No. 95,1408-XX), the disclosures of each of which are explicitly
incorporated by reference herein, most preferably channels and
microchannels, whereby fluid flow traverses each of the different
regions having different temperatures at least once, or more
preferably, several times. In these embodiments, the amount of time
fluid is within any particular thermal region, and thus at any
particular temperature is dependent on the path length of the
channel in the region, the square of the hydraulic diameter of the
channel, and the square of the rotational speed of the platform. In
preferred embodiments, the arrays comprise at least 2 or 3 regions
of different temperature adjacent to one another. In certain
embodiments, the thermal regions are rectangular in shape, while in
other embodiments the thermal regions are wedge-shaped, having a
broader annular diameter at positions distal to the axis of
rotation than at positions proximal to the axis of rotation.
[0089] In preferred embodiments of the platforms of the invention,
the thermal arrays and regions of elevated temperatures constructed
in the surface of the platforms of the invention comprise a thermal
heating element. In preferred embodiments, the thermal heating
element is a resistive heater element or a thermofoil heater, which
is an etched-foil heating element enclosed in an electrically
insulating plastic (Kapton, obtained from Minco). Resistive heater
elements comprising the platforms of the invention are as described
in co-owned U.S. Pat. No. 6,063,587. Briefly, said resistive heater
elements comprise in combination an electrically inert substrate
capable of being screen printed with a conductive ink and a
resistive ink; a conductive ink screen-printed in a pattern; and a
resistive ink screen-printed in a pattern over the conductive ink
pattern wherein the resistive ink in electrical contact with the
conductive ink and wherein an electrical potential applied across
the conductive ink causes current to flow across the resistive ink
wherein the resistive ink produces heat. Such structures are
defined as "electrically-resistive patches" herein. Preferably, the
conductive ink is a silver conductive ink such as Dupont 5028,
Dupont 5025, Acheson 423SS, Acheson 426SS and Acheson SS24890, and
the resistive ink is, for example, Dupont 7082, Dupont 7102, Dupont
7271, Dupont 7278 or Dupont 7285, or a PTC (positive temperature
coefficient) ink. In alternative embodiments, the resistive heater
element can further comprise a dielectric ink screen-printed over
the resistive ink pattern and conductive ink pattern.
[0090] The invention provides a combination of specifically adapted
microplatforms that are rotatable, analytic/synthetic microvolume
assay platforms, and a micromanipulation device for manipulating
the platform to achieve fluid movement on the platform arising from
centripetal force on the platform as result of rotation. The
platform of the invention is preferably and advantageously a
circular disk; however, any platform capable of being rotated to
impart centripetal for a fluid on the platform is intended to fall
within the scope of the invention. The micromanipulation devices of
the invention are more fully described in co-owned and co-pending
U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990,
filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser.
No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May
19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No.
______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the
disclosures of each of which are explicitly incorporated by
reference herein.
[0091] Fluid (including reagents, samples and other liquid
components) movement is controlled by centripetal acceleration due
to rotation of the platform. The magnitude of centripetal
acceleration required for fluid to flow at a rate and under a
pressure appropriate for a particular microfluidics structure on
the Microsystems platform is determined by factors including but
not limited to the effective radius of the platform, the interior
diameter of microchannels, the position angle of the microchannels
on the platform with respect to the direction of rotation, and the
speed of rotation of the platform. In certain embodiments of the
methods of the invention an unmetered amount of a fluid (either a
sample or reagent solution) is applied to the platform and a
metered amount is transferred from a fluid reservoir to a
microchannel, as described in co-owned U.S. Pat. No. 6,063,589,
issued May 16, 2000, and co-owned and co-pending patent
applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No.
08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug.
12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No.
09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12,
2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No.
95,1408-XX), the disclosures of each of which are explicitly
incorporated by reference herein. In preferred embodiments, the
metered amount of the fluid sample provided on an inventive
platform is from about 1 nL to about 500 .mu.L. In these
embodiments, metering manifolds comprising one or a multiplicity of
metering capillaries are provided to distribute the fluid to a
plurality of components of the microfluidics structure.
[0092] The components of the platforms of the invention are in
fluidic contract with one another. In preferred embodiments,
fluidic contact is provided by microchannels comprising the surface
of the platforms of the invention. Microchannel sizes are optimally
determined by specific applications and by the amount of and
delivery rates of fluids required for each particular embodiment of
the platforms and methods of the invention. Microchannel sizes can
range from 0.1 .mu.m to a value close to the thickness of the disk
(e.g., about 1 mm); in preferred embodiments, the interior
dimension of the microchannel is from 0.5 .mu.m to about 500 .mu.m.
Microchannel and reservoir shapes can be trapezoid, circular or
other geometric shapes as required. Microchannels preferably are
embedded in a microsystem platform having a thickness of about 0.1
to 25 mm, wherein the cross-sectional dimension of the
microchannels across the thickness dimension of the platform is
less than 1 mm, and can be from 1 to 90 percent of said
cross-sectional dimension of the platform. Sample reservoirs,
reagent reservoirs, reaction chambers, collection chambers,
detections chambers and sample inlet and outlet ports preferably
are embedded in a microsystem platform having a thickness of about
0.1 to 25 mm, wherein the cross-sectional dimension of the
microchannels across the thickness dimension of the platform is
from 1 to 75 percent of said cross-sectional dimension of the
platform. In preferred embodiments, delivery of fluids through such
channels is achieved by the coincident rotation of the platform for
a time and at a rotational velocity sufficient to motivate fluid
movement between the desired components.
[0093] Input and output (entry and exit) ports are components of
the microplatforms of the invention that are used for the
introduction or removal of fluid components. Entry ports are
provided to allow samples and reagents to be placed on or injected
onto the disk; these types of ports are generally located towards
the center of the disk. Exit ports are also provided to allow
products to be removed from the disk. Port shape and design vary
according specific applications. For example, sample input ports
are designed, inter alia, to allow capillary action to efficiently
draw the sample into the disk. In addition, ports can be configured
to enable automated sample/reagent loading or product removal.
Entry and exit ports are most advantageously provided in arrays,
whereby multiple samples are applied to the disk or to effect
product removal from the microplatform.
[0094] In some embodiments of the platforms of the invention, the
inlet and outlet ports are adapted to the use of manual pipettors
and other means of delivering fluids to the reservoirs of the
platform. In alternative, advantageous embodiments, the platform is
adapted to the use of automated fluid loading devices. One example
of such an automated device is a single pipette head located on a
robotic arm that moves in a direction radially along the surface of
the platform. In this embodiment, the platform could be indexed
upon the spindle of the rotary motor in the azimuthal direction
beneath the pipette head, which would travel in the radial
direction to address the appropriate reservoir.
[0095] Also included in air handling systems on the disk are air
displacement channels, whereby the movement of fluids displaces air
through channels that connect to the fluid-containing microchannels
retrograde to the direction of movement of the fluid, thereby
providing a positive pressure to further motivate movement of the
fluid.
[0096] Platforms of the invention such as disks and the
microfluidics components comprising such platforms are
advantageously provided having a variety of composition and surface
coatings appropriate for particular applications. Platform
composition will be a function of structural requirements,
manufacturing processes, and reagent compatibility/chemical
resistance properties. Specifically, platforms are provided that
are made from inorganic crystalline or amorphous materials, e.g.
silicon, silica, quartz, inert metals, or from organic materials
such as plastics, for example, poly(methyl methacrylate) (PMMA),
acetonitrile-butadiene-styrene (ABS), polycarbonate, polyethylene,
polystyrene, polyolefins, polypropylene and metallocene. These may
be used with unmodified or modified surfaces as described below.
The platforms may also be made from thermoset materials such as
polyurethane and poly(dimethyl siloxane) (PDMS). Also provided by
the invention are platforms made of composites or combinations of
these materials; for example, platforms manufactures of a plastic
material having embedded therein an optically transparent glass
surface comprising the detection chamber of the platform.
Alternately, platforms composed of layers made from different
materials may be made. The surface properties of these materials
may be modified for specific applications, as disclosed in co-owned
U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and
co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec.
5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No.
08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec.
19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No.
09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16,
2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of
which are explicitly incorporated by reference herein.
[0097] Preferably, the disk incorporates microfabricated
mechanical, optical, and fluidic control components on platforms
made from, for example, plastic, silica, quartz, metal or ceramic.
These structures are constructed on a sub-millimeter scale by
molding, photolithography, etching, stamping or other appropriate
means, as described in more detail below. It will also be
recognized that platforms comprising a multiplicity of the
microfluidics structures are also encompassed by the invention,
wherein individual combinations of microfluidics and reservoirs, or
such reservoirs shared in common, are provided fluidly connected
thereto. An example of such a platform is shown in FIG. 1.
[0098] Platform Manufacture and Assembly
[0099] Microfluidics structures are provided embedded in a
substrate comprising the microsystems platform of the invention.
The platform is preferably manufactured and assembled as layers
containing separate components that are bonded together. As
illustrated in FIG. 1, the exemplified embodiment of the platforms
of the invention comprise two layers, a reservoir layer and a
microfluidics layer. Platforms having additional layers are also
within the scope of the invention.
[0100] The reservoir layer of the platform is manufactured from a
thermoplastic material such as acrylic, polystyrene, polycarbonate,
or polyethylene. For such materials, fabrication methods include
machining and conventional injection molding. For injection
molding, the mold inserts that are used to define the features of
the platform can be created using standard methods of machining,
electrical discharge machining, and other means known in the
art.
[0101] The reservoir layer of the platform can also be manufactured
from a thermoset material or other material that exists in a liquid
form until subjected to heat, radiation, or other energy sources.
Examples of thermoset materials include poly(dimethyl siloxane)
(PDMS), polyurethane, or epoxy.
[0102] Typically, these materials are obtained from the
manufacturer in two parts; the two parts are mixed together in a
prescribed ratio, injected into or poured over a mold and subjected
to heat to initiate and complete cross-linking of the monomers
present in the pre-polymer fluid. The process of rapidly injecting
a pre-polymer fluid into a mold and then cross-linking or curing
the part is often referred to as reaction injection molding (RIM).
The process of pouring a pre-polymer fluid over a mold and then
allowing the part to cross-link or cure is often referred to as
casting. Mold inserts for RIM or casting can be fabricated using
standard methods of machining, electrical discharge machining, and
other means known in the art.
[0103] The microfluidics layer of the platform can also be
manufactured from a thermoplastic material such as acrylic,
polystyrene, polycarbonate, or polyethylene. Because the dimensions
of the channels and cuvettes may be much smaller than those found
in the reservoir layer, typical fabrication methods with these
materials may include not only machining and conventional injection
molding but also compression/injection molding, and embossing or
coining. For injection molding, the mold inserts that are used to
define the features of this layer of the platform can be created
using conventional methods such as machining or electrical
discharge machining. For mold inserts with features too fine to be
created in conventional ways, various microfabrication techniques
are used. These include silicon micromachining, in which patterns
are created on a silicon wafer substrate through the use of a
photoresist and a photomask (Madou, 1997, Fundaments of
Microfabrication, CRC Press: Boca Raton, Fla.). When the silicon
wafer is subjected to an etching agent, the photoresist prevents
penetration of the agent into the silicon beneath the photoresist,
while allowing etching to occur in the exposed areas of the
silicon. In this way patterns are etched into the silicon and can
be used to create microfabricated plastic parts directly through
embossing. In this process, the etched silicon is brought into
contact with a flat thermoplastic sheet under high pressure and at
a temperature near the glass transition temperature of the plastic.
As a result, the pattern is transferred in negative into the
plastic.
[0104] Etched silicon may also be used to create a metal mold
insert through electroplating using, for example, metallic nickel.
Silicon etched using any one of a variety of techniques such as
anisotropoic or isotropic wet etching or deep reactive ion etching
(DRIE) may serve as a basis for a metal mold. A seed layer of
nickel is deposited through evaporation on the silicon; once such
an electrically-conductie seed layer is formed, conventional
electroplating techniques may be used to build a thick nickel
layer. Typically, the silicon is then removed (Larsson, 1997, Micro
Structure Bull. 1: 3). The insert is then used in conventional
injection molding or compression/injection molding.
[0105] In addition to silicon micromachining for mold inserts,
molds can alternatively be created using photolithography without
etching the silicon. Photoresist patterns are created on silicon or
other appropriate substrates. Rather than etching the silicon wafer
as in silicon micromachining, the photoresist pattern and silicon
are metallized through electroplating, thermal vapor deposition, or
other means known in the art. The metal relief pattern then serves
as a mold for coining, injection molding, or compression/injection
molding as described above.
[0106] The microfluidic layer of the platform can also be
manufactured using a thermoset material as described above for
production of the reservoir layer, wherein the mold pattern for
thermosets of the microfluidics layer is prepared as described
above. Because reaction-injection molding and casting do not
require the high pressures and temperatures of injection molding, a
wider variety of mold patterns may be used. In addition to the use
of a silicon or metal mold insert, the photoresist pattern as
described can also be used as a mold relief itself. While the
photoresist would not withstand the high pressures and temperatures
of injection molding, the milder conditions of casting or RIM
create no significant damage.
[0107] The assembly of the platform involves registration and
attachment of the microfluidic layer to the reservoir layer. In
order for the microfluidics structures on the platform to be useful
for performing assays as described herein, certain microfluidics
pathways in the reservoir layer must be connected to certain
microfluidics pathways in the microfluidics layer. Registration of
these microfluidics pathways may be accomplished through optical
alignment of fiducial marks on the microfluidic and reservoir
layers or through mechanical alignment of holes or depressions on
the microfluidic layer with pins or raised features on the
reservoir layer. The required registration tolerances may be
relaxed by designing the microfluidics pathway in the reservoir
layer to be much larger than the microfluidics pathway in the
microfluidics layer, or vice versa to Attachment may be
accomplished in a number of ways, including conformal sealing, heat
sealing or fusion bonding, bonding with a double-sided adhesive
tape or heat-sealable film, bonding with a ultraviolet (UV) curable
adhesive or a heat-curable glue, chemical bonding or bonding with a
solvent.
[0108] A requirement for conformal sealing is that one or both of
the layers are made of an elastomeric material and that the
surfaces to be bonded are free of dust or debris that could limit
the physical contact of the two layers. In a preferred assembly
approach, an elastomeric microfluidics layer is registered with
respect to and then pressed onto a rigid reservoir layer. The
elastomeric microfluidics layer may be advantageously made of
silicone and the rigid reservoir layer may be advantageously made
of acrylic or polycarbonate. Hand pressure allows the layers to
adhere through van der Waals forces.
[0109] A requirement for heat sealing or fusion bonding is that
both the reservoir and microfluidics layers are made of
thermoplastic materials and that the sealing occurs at temperatures
above the glass transition temperatures, in the case of amorphous
polymers, or melting temperatures, in the case of semi-crystalline
polymers, of both of the layer materials. In a preferred assembly
approach, the microfluidics layer is registered with respect to and
pressed onto the reservoir layer, this composite disk is then
placed between two flat heated blocks and pressure is applied to
the composite through the heated blocks. By adjusting the
temperature versus time profile at each of the faces of the
composite disk and by adjusting the pressure versus time profile
that is applied to the composite system, one can determine the
time-temperature-pressure profile that allows for bonding of the
two layers yet minimizes variation of the features within each of
the layers. For example, heating two acrylic disks from room
temperature to a temperature just above the glass transition
temperature of acrylic at a constant pressure of 250 psi over one
hour is a recipe that allows for minimal variation of 250 .mu.m
wide fluidic channels. In another assembly approach, the bond
surfaces of the microfluidics and reservoir layers are separately
heated in a non-contact fashion with radiative lamp and when the
bond surfaces have reached their glass transition temperatures the
microfluidics layer is registered with respect to and pressed onto
the reservoir layer.
[0110] A double-sided adhesive tape or heat sealable film may be
used to bond the microfluidics and reservoir layers. Before
bonding, holes are first cut into the tape (or film) to allow for
fluid communication between the two layers, the tape (or film) is
registered with respect to and applied onto the reservoir layer,
and the microfluidics layer is registered with respect to and
applied onto the tape (or film)/reservoir layer composite. In order
to bond a heat-sealable film to a surface, it is necessary to raise
the temperature of the film to above the glass transition
temperature, in the case of an amorphous polymer, or the melting
temperature, in the case of a semicrystalline polymer, of the
film's adherent polymer material. For bonding with an adhesive tape
or a heat-sealable film, an adequate bond can typically be achieved
with hand pressure.
[0111] A photopolymerizable polymer (for example, a UV-curable
glue) or a heat-curable polymer may be used to adhere the
microfluidics and reservoir layers. In one approach, this glue is
applied to one or both of the layers. Application methods include
painting, spraying, dip-coating or spin coating. After the
application of the glue the layers are assembled and exposed to
ultraviolet radiation or heat to allow for the initiation and
completion of cross-linking or setting of the glue. In another
approach, the microfluidics and reservoir layers are each
fabricated with a set of fluid channels that are to be used only
for the glue. These channels may, for example, encircle the fluid
channels and cuvettes used for the assay. The microfluidics layer
is registered with respect to and pressed onto the reservoir layer.
The glue is pipetted into the various designated channels and after
the glue has filled these channels, the assembled system is exposed
to ultraviolet radiation or heat to allow for the cross-linking or
setting of the glue.
[0112] When polydimethylsiloxane (PDMS) or silicone is first
exposed to an oxygen plasma and then pressed onto a similarly
treated silicone surface in an ambient environment, the two
surfaces adhere. It is thought that the plasma treatment converts
the silicone surface to a silanol surface and that the silanol
groups are converted to siloxane bonds when the surfaces are
brought together (Duffy et al., 1998, Anal. Chem. 1: 4974-4984).
This chemical bonding approach is used to adhere the silicone
microfluidics and reservoir layer.
[0113] A requirement for solvent bonding is that the bond surfaces
of both the microfluidics and reservoir layers can be solvated or
plasticized with a volatile solvent. For solvent bonding, the bond
surfaces are each painted with the appropriate solvating fluid or
each exposed to the appropriate solvating vapor and then registered
and pressed together. Plasticization allows the polymer molecules
to become more mobile and when the surfaces are brought in contact
the polymer molecules become entangled; once the solvent has
evaporated the polymer molecules are no longer mobile and the
molecules remain entangled, thereby allowing for a physical bond
between the two surfaces. In another approach, the microfluidics
and reservoir layers are each fabricated with a set of fluid
channels that are to be used only for the solvent and the layers
are bonding much like they are with the UV-curable or heat-curable
glue as described above.
[0114] Once assembled, the internal surfaces of the microfluidic
manifold may be passivated with a parylene coating. Parylene is a
vapor-deposited conformal polymer coating that forms a barrier
layer on the internal, fluid-contacting surfaces of a microfluidic
device following construction. The coating forms an impermeable
layer that prevents any exchange of matter between the fluids and
materials used to construct the device. The use of a low
temperature, vapor deposition method allows the device to be
manufactured and then passivated in its final form. This
passivation approach can be used to improve the performance of
assays. In particular, when an adhesive is used in the disk
construction, there is a potential for contamination of the fluids
by the adhesive material (or the plastic substrate or cover).
Interfering substances leaching from the adhesive, or adsorption
and binding of substances by the adhesive, can interfere with
chemical or biochemical reactions. This can be more of a problem at
elevated temperatures or if solvents, strong acids or bases are
required.
[0115] Construction of Electric or Electronic Platen Comprising
Temperature Control Elements
[0116] The invention provides an electric or electronic platen
containing temperature control elements positioned on the platen to
correspond to microfluidics structures such as thermal cycling
chambers and sacrificial valves. The platen and microfluidics
structures are aligned using fiducials or other registers for
proper positioning the components on each platform layer with each
other.
[0117] The invention also provides a micromanipulation apparatus
for rotating the platen and microfluidics platform, including most
preferably a slip ring feature on a rotational spindle or axis that
permits electrical contact to be maintained between the device and
the rotating platen. Temperature controlling elements are provided
in the device to maintain any particular temperature at a specific
position on the disc surface using thermistors and heating
elements, including resistive heaters and Peltier elements. The
device controls rotation of the microfluidics disc and distributes
and receives electrical signals to the platen rotating with the
microfluidics disc in real time.
[0118] The relationship between the device and platen is
illustrated in FIG. 20. With regard to the Figure, platen 509 is
inserted on a spindle containing 24-channel slip ring 510,
commercially available from Litton, (Part No. AC6023-24). Rotation
of the platen about the spindle is controlled by drive motor 507,
preferably also comprising an encoder such as one commercially
available from Micromo, (Part No. 3557K012CR), via drive belt 508.
Drive motor 507 is controlled by the device through drive motor
power line 505 and where application encoder signal line 506.
[0119] The device is controlled by microprocessor 501, most
preferably comprising a computer such as a PC. Platform rotation is
controlled by servomotor controller 503, for example as
commercially available from J. R. Kerr (Part No. PIC-SERVO). Servo
motor 503 is equipped with a power supply 504, commercially
available from Skynet Electronic (Part No. ARC-2133). The servo
motor is controlled by the PC through an interface, for example,
using a serial port converter connected to the COM port of the PC
(Part No. Z238485, J. R. Kerr).
[0120] The device is also provided having a control system for
controlling electric power to the platen. A multiline cable 511
connects the slip ring to a breakout board 517, which is connected
to a proportional integral derivative (PID) circuit connected to a
commercially-available AJD board in the PC (Computer Boards, Part
No. CIO-DAS1600) by temperature sensor line 512. This circuit
receives temperature data from thermistors on the platen surface,
disclosed more extensively below, and controls current delivery to
temperature control elements by programmable current source 515 and
power source 516.
[0121] The platen itself is shown in plan view in FIG. 21. The
platen most preferably is constructed from printed circuit board
551 onto which electronic elements (including electrical leads,
thermistors, Peltier elements, brass blocks for providing thermal
contact with the microfluidics disc, and radiative fins for heat
dissipation have been affixed.
[0122] FIG. 21 shows the layout of the temperature control elements
on the platen, illustrated in the Figure with Peltier elements 554.
The platen has brass thermal contacts 552 and 553 positioned on the
platen surface to correspond to microfluidics structure on the
microfluidics disc. Brass contact 552 has a groove 555 embedded
therein to accommodate a temperature sensing element. Positioned in
between the thermal contacts in each combination is Peltier element
554. Also illustrated in the Figure is a second temperature control
element, comprising brass thermal contacts 557 and 558, groove 559,
and Peltier element 558. The positioning of these elements permits
temperature control and heating of multiple components of a
particular microfluidics structure (such as control of thermal
cycling chambers and lysis chambers, for example).
[0123] Electric leads controlling the temperature control elements
on the platen are more specifically depicted in FIG. 22. Peltier
element 554 is controlled by leads 601 and 605 connected through
607 and 608. Thermistor 606 contained in groove 555 is controlled
(that is, the temperature information in the form of changes in
resistance to current flow in the thermistor upon heating or
cooling is transmitted to the temperature control elements in the
PC) through leads 600 and 602 connected through 609 and 610.
Similarly, Peltier element 558 is controlled by leads 603 and 605
connected through 612 and 613. Thermistor 611 contained in groove
559 is controlled (that is, the temperature information in the form
of changes in resistance to current flow in the thermistor upon
heating or cooling is transmitted to the temperature control
elements in the PC) through leads 602 and 604 connected through 614
and 615. In construction of the electric connections between the
elements on the platen and the slip ring, the use of the same lead
as a "ground" (see, for example, the common connection to lead 602
between thermistor 606 and thermistor 611) conserves the number of
connections used per element and permits control of up to 8
elements per platen.
[0124] The structure of the temperature control element is
displayed in cross section in FIG. 23. Peltier element 554 is
positioned on platen surface 551 between brass contacts 552 and 553
and held together with bolts 653 and 654. Brass contacts 552 and
553 act as heat sources and sinks to transfer heat to and from the
Peltier element 554. The microfluidic disk sits on brass contact
552. When heating the disk, the top surface of the Peltier element
554 heats brass contact 552, and brass contact 553 is cooled. When
cooling the disk, the top surface of the Peltier element 554 cools
brass contact 554 and heats brass contact 553. An additional
aluminum heat sink 652 is positioned in thermal contact with brass
contact 553, providing additional heat sink capacity, enhancing
Peltier element 554 performance. Aluminum heat sink 652 is mounted
to the platen 551 using screws 651 and 655. Brass contact 552
contains thermistor 606 in a cavity containing alumina-filled epoxy
650 that increases the temperature sensitivity of the thermistor.
Thermal grease is applied between pieces 552, 554, 553, and 652 to
increase thermal contact between the parts.
[0125] In the use of the platen of the invention, the platform of
the invention is assembled using thermal grease between the brass
contacts and the plastic microfluidics layer. Alternatively, the
brass contact is provided as a convex layer that is mechanically
clamped to the flexible plastic microfluidics layer.
[0126] Alternative embodiments of the platens of the invention
include so-called "intelligent" platens comprising one or a
multiplicity of microprocessors, thereby permitting a reduced set
of connections between the slip ring and the printed circuit board
comprising the substrate of the platen. Integrated circuit packages
such as a BASIC STAMP II embedded controller can be preprogrammed
by the PC to control the distribution of signals through the platen
circuitry, thereby requiring only an input power and ground
connection between the slip ring and the platen.
[0127] DNA Sample Preparation Platform
[0128] The invention provides a DNA sample application platform for
preparing plasmid and genomic DNA from bacteria or eukaryotic, most
preferably mammalian cells. This aspect of the invention is
described herein for a single microfluidics structure. However,
platforms comprising a multiplicity of these microfluidics
structures are provided and are encompassed by the invention,
wherein a multiplicity of the microfluidics structures described
herein are provided on the platform.
[0129] Referring now to the Figures for a more thorough description
of the invention, FIG. 14 illustrates one embodiment of sample
processing structure of the microfluidics disc in close-up. For
orientation, the center of the disc is beyond the top of the
Figure. Chamber 1005 has a depth in the platform surface of from
about 0.1 cm to about 0.25 cm, about 0.7 cm to 1.5 cm in width and
about 0.4 cm to 0.8 cm in length, and has a volumetric capacity of
from about 50/L to about 11.0 mL (depth 0.2286 cm, width 1.4315 cm,
length 0.7879 cm) is a combination sample input chamber and mixing
chamber. Within the chamber are mixing baffles 1006 that are from
about 0.05 cm to about 0.1 cm wide and from about 0.2 cm to about
0.4 cm long (width 0.1155 cm, length 0.3720 cm) for producing
turbulent fluid motion in the chamber upon disc rotation,
particularly disc rotation that changes direction rapidly and/or
repeatedly. In a position in chamber 1005 most radially most distal
from the center of rotation is slot 1008 having depth in the
platform surface of from about 0.1 cm to about 0.25 cm (depth
0.2286 cm, width 1.0537 cm, length 0.0794 cm), which contains frit
material in which a filter 1009 is placed. The frit material is
manufactured by Porex, X-4588, 70 .mu.m pore size, and the filter
is a Whatman filter paper #54 which is placed further out radially
on the disc from the frit material. The frit material acts in this
application as a filter that, as a porous membrane, allows the
liquid to flow through but retains the precipitate. The filter
paper in this case is used as a final filtration step; the frit has
filtered the majority of the precipitate, but the filter paper has
a finer pore size that allows little to no precipitate through.
Chamber 1005 is also equipped with an inclined portion 1007 where
the depth of the platform decreases in a radially-outward direction
and rises from the chamber floor to a depth from about 0.1 cm to
about 0.25 cm (depth 0.2286 cm) to form the inner edge of filter
slot 1008; the depth of the inner portion of the slot (depth 0.1524
cm) is intermediate between the floor of the chamber 1005 and the
top surface of the disc, and thus forms a gap through which fluids
flow upon disc rotation at sufficient speed. Entry port 1015 is
fluidly connected to chamber 1005 through microchannel 1014, having
dimensions of from about 0.001 cm to 0.2 cm in depth, about 0.0125
cm to about 0.025 cm in length and cross-sectional dimension of
from about 0.001 cm to about 0.2 cm (depth 0.0254 cm, width 0.0254
cm, length 0.2118 cm). Entry port 1015 is preferably adapted to
fluidics loading devices such as pipettors and automated
embodiments thereof.
[0130] On the radially-distal side of filter 1009, the
radially-outward exit from chamber 1005 is fluidly-connected to
microchannel 1010 having a depth in the platform surface of from
about 0.001 cm to about 0.2 cm, length of from about 0.01 cm to
about 0.025 cm and cross-sectional dimension of from about 0.001 cm
to about 0.2 cm (depth 0.0508 cm, width 0.0508 cm, length, 0.2631
cm). Microchannel 1014 is fluidly connected to pocket 1011 that
defines the edge of sacrificial valve 1012. This pocket 1011 has a
depth in the platform surface of from about 0.05 cm to about 0.1
cm, length of from about 0.04 cm to about 0.08 cm in length and
cross-sectional dimension of from about 0.05 cm to about 0.1 cm
(depth 0.1016 cm, width 0.1060 cm, length 0.0762 cm), on the
radially-distal extent of which is sacrificial valve 1012 wherein
the sacrificial valve is preferably made using wax or other
material as set forth more fully in co-owned. U.S. Pat. No.
6,063,589, issued May 16, 2000, incorporated by reference, and
recrystallization chamber 1013, where melted wax resolidifies
without blocking the flow path. Sacrificial valve 1012 has a depth
in the platform surface of from about 0.001 cm to about 0.2 cm,
length of from about 0.05 cm to about 0.1 cm in length and
cross-sectional dimension of from about 0.001 cm to about 0.2 cm
(depth 0.0254 cm, width 0.0254 cm, length 0.1056 cm), and
recrystallization chamber 1013 has dimensions of from about 0.75 cm
to about 0.15 cm deep, from about 0.1 cm to about 0.25 cm long and
from about 0.1 cm to about 0.2 cm wide (depth 0.1524 cm, width
0.2031 cm, length 0.2351 cm). As is disclosed more fully herein,
sacrificial valve 1012 is in thermal contact with a heating
element, most preferably a resistive heater or Peltier heater,
wherein the wax comprising the valve is melted by operation of the
heater. In preferred embodiments, the heater is constructed in a
platform layer beneath, most preferably immediately beneath, the
valve, or alternatively comprises a separate platen positioned to
have the heater be above or below, most preferably immediately
above or below, the valve. Microchannel 1011 and recrystallization
chamber 1013 serve to define the length of sacrificial valve 1012,
and wax when deposited in the molten state is naturally confined to
this short length of channel by the openings of 1011 and 1013.
[0131] Lysis solution reservoir 1016 is positioned radially on the
disc substantially at the same distance from the axis of rotation
as chamber 1005. Lysis solution reservoir 1016 has a depth in the
platform surface of from about 0.1 cm to about 0.25 cm, about 0.18
cm to 0.36 cm in width and about 0.3 cm to 0.8 cm in length, and
has a volumetric capacity of from about 25/L to about 300 .mu.L
(depth 0.2286 cm, width 0.3579 cm at the top and 0.5536 cm at the
bottom, length 0.7292 cm) and is fluidly connected to chamber 1005
through microchannel 1017. Lysis solution is most preferably
applied fresh to the platform before use using entry port 1019 that
is fluidly connected to lysis solution reservoir 1016 through
microchannel 1017. In these structures, entry port 1019 is
preferably adapted to fluidics loading devices such as pipettors
and automated embodiments thereof, and microchannel 1017 has
dimensions of from about 0.001 cm to about 0.2 cm deep, from about
0.25 cm to about 0.5 cm in length and cross-sectional dimension of
from about 0.001 cm to about 0.2 cm (depth 0.0508 cm, width 0.0508
cm, length 0.4845 cm).
[0132] Precipitant buffer reservoir 1020 is also positioned
radially on the disc substantially at the same distance from the
axis of rotation as chamber 1005. Precipitant buffer reservoir 1020
has a depth in the platform surface of from about 0.1 cm to about
0.25 cm, about 0.4 cm to 0.8 cm in width and about 0.25 cm to 0.50
cm in length, and has a volumetric capacity of from about 35 .mu.L
to about 400 .mu.L (depth 0.2286 cm, width 0.7349 cm, length 0.4960
cm) and is fluidly connected to chamber 1005 through microchannel
1023. Between precipitant buffer reservoir 1020 and microchannel
1023 is sacrificial valve 1021 and recrystallization chamber 1022,
arrayed substantially as described above for sacrificial valve 1012
and recrystallization chamber 1013. Precipitant buffer solution is
most preferably applied fresh to the platform before use using
entry port 1025 that is fluidly connected to precipitant buffer
reservoir 1020 through microchannel 1024. In these structures,
entry port 1020 is preferably adapted to fluidics loading devices
such as pipettors and automated embodiments thereof, and
microchannel 1024 has dimensions of from about 0.001 cm to about
0.2 cm deep, from about 0.25 cm to about 0.5 cm in length and
cross-sectional dimension of from about 0.001 cm to about 0.2 cm
(depth 0.0508 cm, width 0.0508 cm, length 0.4845 cm).
[0133] Radially distal to sacrificial valve 1012 is microchannel
1026, which is thereby fluidly connected to chamber 1005.
Microchannel 1026 has dimensions of from about 0.001 cm to about
0.2 cm deep, from about 0.025 cm to about 0.05 cm in length and
cross-sectional dimension of from about 0.001 cm to about 0.2 cm
(depth 0.1524 cm, width 0.0508 cm, length 0.7060 cm). Microchannel
1026 is fluidly connected to binding column 1027, which further
comprises a DNA affinity matrix. Binding column 1027 has dimensions
of from about 0.10 cm to about 0.2 cm deep, from about 0.3 cm to
about 0.65 cm in length and cross-sectional dimension of from about
0.2 cm to about 0.4 cm, and has a volumetric capacity of from about
5 to about 20 .mu.L (depth 0.1875 cm, width 0.3511 cm at the
bottom, 0.04 cm at the top, length 0.5727 cm). Binding column 1027
also comprises matrix material slot 1028 having a depth in the
platform surface of from about 0.1 cm to about 0.25 cm and is from
about 0.05 cm to about 0.1 cm in length and cross-sectional
dimension of from about 0.25 cm to about 0.5 cm, and has a
volumetric capacity of from about 5 .mu.L to about 15 .mu.L (depth
0.2383 cm, width 0.5082 cm, length 0.0921 cm), that when assembled
contains matrix material 1029, structurally supported by a frit.
The binding material consists of Whatman Glass Fiber filter (GF-F)
that is a glass fiber that binds the DNA when the DNA is in a
chaotropic salt solution where the hydration shell of the DNA is
disrupted. The frit material is a solid porous material (70 cm size
pores) that is used in this case to provide a solid support backing
for the glass fiber that is too flexible to support itself within
the structure. Binding column 1027 is also equipped with an air
displacement channel 1051, having a depth in the platform surface
of from about 0.001 cm to about 0.2 cm and is from about 1.75 cm to
about 3.5 cm in length and cross-sectional dimension of from about
0.001 cm to about 0.2 cm (depth 0.1524 cm, width 0.0508 cm, length
3.1955 cm) and air vent 1052, which are constructed to permit air
to be displaced from the binding column upon fluid flow
therethrough without permitting fluid to flow into air displacement
channel 1051.
[0134] Microchannel 1030, having a depth in the platform surface of
from about 0.001 cm to about 0.2 cm, cross-sectional dimension of
from about 0.001 cm to about 0.2 cm, and being from about 0.1 cm to
about 0.2 cm in length (depth 0.0508 cm, width 0.0508 cm, length
0.2041 cm), is fluidly connected to pocket 1031 which defines the
edge of sacrificial valve 1032. This pocket has a depth in the
platform surface of from about 0.05 cm to about 0.1 cm, length of
from about -0.04 cm to about 0.08 cm in length and cross-sectional
dimension of from about 0.06 cm to about 0.12 cm (depth 0.1016 cm,
width 0.1121 cm, length 0.0762 cm). Pocket 1031 is separated from
sample collection reservoir 1053 by sacrificial-valve channel 1032
having a depth in the platform surface of from about 0.001 cm to
about 0.2 cm, cross-sectional dimension of from about 0.001 cm to
about 0.2 cm, and being from about 0.05 cm to about 0.1 cm in
length (depth 0.0254 cm, width 0.0254 cm, length 0.0876 cm), which
is constructed as described above for sacrificial valves 1012 and
1021. Sample collection reservoir 1053 has dimensions of from about
0.1 cm to about 0.25 cm deep, from about 0.3 cm to about 0.6 cm in
length and cross-sectional dimension of from about 0.2 cm to about
0.4 cm, and has a volumetric capacity of from about 20 .mu.L to
about 250 .mu.L (depth 0.2286 cm, width 0.6132 cm, length 0.3748
cm). Sample collection reservoir is equipped with air displacement
channel 1054 and air vent 1055. Air displacement channel 1054 has a
depth in the platform surface of from about 0.001 cm to about 0.2
cm and is from about 0.2 cm to about 0.4 cm in length and
cross-sectional dimension of from about 0.001 cm to about 0.2 cm
(depth 0.0254 cm, width 0.0254 cm, length 0.3316 cm). These
structures are constructed to permit air to be displaced from the
binding column upon fluid flow therethrough without permitting
fluid to flow into air displacement channel 1054.
[0135] Leading radially inward from pocket 1031 is microchannel
1033 having dimensions of from about 0.001 cm to about 0.2 cm deep,
from about 0.025 cm to about 0.50 cm in length and cross-sectional
dimension of from about 0.001 cm to about 0.2 cm (depth 0.0254 cm,
width 0.0254 cm, length 0.4195 cm). In some embodiments,
microchannel 1033 is treated to present a hydrophobic surface
resistant to wetting by fluids. Along the extent of this
microchannel is a bend that turns the microchannel direction
radially outward and leads to waste reservoir 1034. Waste reservoir
1034 has dimensions of from about 0.15 cm to about 0.3 cm deep,
from about -0.3 cm to about 0.6 cm in length and cross-sectional
dimension of from about 2.5 cm to about 5.0 cm, and has a
volumetric capacity of from about 350 .mu.L to about 1.5 mL (depth
0.2794 cm, width 4.4783 cm, length 0.5687 cm). The waste reservoir
is equipped with air displacement channel 1035 and air vent 1099.
Air displacement channel 1035 has a depth in the platform surface
of from about 0.001 cm to about 0.2 cm and is from about 0.5 cm to
about 1.0 cm in length and cross-sectional dimension of from about
0.001 cm to about 0.2 cm (depth 0.0254 cm, width 0.0254 cm, length
0.9240 cm). These structures are constructed to permit air to be
displaced from the binding column upon fluid flow therethrough
without permitting fluid to flow into air displacement channel
1035.
[0136] The sample preparation microstructures of the platform also
comprise first and second wash reservoirs 1036 and 1047. First wash
reservoir 1036 has dimensions of from about 0.1 cm to about 0.25 cm
deep, from about 0.23 cm to about 0.46 cm in length and
cross-sectional dimension of from about 0.75 cm to about 1.5 cm,
and has a volumetric capacity of from about 50 .mu.L to about 600
.mu.L (depth 0.2286 cm, width 1.3357 cm, length 0.4600 cm). This
reservoir is fluidly connected to microchannel 1026 by microchannel
1039, which is interrupted by sacrificial valve 1037 and
recrystallization chamber 1038, arrayed substantially as described
above for sacrificial valve 1012 and recrystallization chamber
1013. Sacrificial valve 1037 has dimensions of from about 0.01 cm
to about 0.03 cm, length of from about 0.08 cm to about 0.12 cm in
length and cross-sectional dimension of from about 0.01 cm to about
0.03 cm (depth 0.0254 cm, width 0.0254 cm, length 0.1007 cm).
Recrystallization chamber 1038 has dimensions of from about 0.1 cm
to about 0.2 cm deep, from about 0.15 cm to about 0.3 cm in length
and cross-sectional dimension of from about 0.15 cm to about 0.3 cm
(depth 0.1524 cm, width 0.2126 cm, length 0.2302 cm) and
microchannel 1039 has dimensions of from about 0.01 cm to about 0.2
cm deep, from about 0.3 cm to about 0.6 cm in length and
cross-sectional dimension of from about 0.001 cm to about 0.2 cm
(depth 0.1524 cm, width 0.0508 cm, length 0.5910 cm. First wash
solution is most preferably applied fresh to the platform before
use using entry port 1040 that is fluidly connected to precipitant
buffer reservoir 1036 through microchannel 1041. In these
structures, entry port 1040 is preferably adapted to fluidics
loading devices such as pipettors and automated embodiments
thereof, and microchannel 1041 has dimensions of from about 0.001
cm to about 0.2 cm deep, from about 0.15 cm to about 0.3 cm in
length and cross-sectional dimension of from about 0.001 cm to
about 0.2 cm (depth 0.0254 cm, width 0.0508 cm, length 0.2521
cm).
[0137] First wash reservoir 1047 has dimensions of from about 0.1
cm to about 0.25 cm deep, from about 0.25 cm to about 0.5 cm in
length and cross-sectional dimension of from about 0.75 cm to about
1.5 cm, and has a volumetric capacity of from about 75 .mu.L to
about 850 .mu.L (depth 0.2388 cm, width 1.4180 cm, length 0.5065
cm). This reservoir is fluidly connected to microchannel 1026 by
microchannel 1050, which is interrupted by sacrificial valve 1048
and recrystallization chamber 1049, arrayed substantially as
described above for sacrificial valve 1012 and recrystallization
chamber 1013. Sacrificial valve 1048 has dimensions of from about
0.001 cm to about 0.2 cm deep, from about 0.05 cm to about 0.1 cm
in length and cross-sectional dimension of from about 0.001 cm to
about 0.2 cm (depth 0.0254 cm, width 0.0254 cm, length 0.1023 cm).
Recrystallization chamber 1049 has dimensions of from about 0.75 cm
to about 1.5 cm deep, from about 0.15 cm to about 0.3 cm in length
and cross-sectional dimension of from about 0.15 cm to about 0.3 cm
(depth 0.1524 cm, width 0.2179 cm, length 0.2435 cm) and
microchannel 1050 has dimensions of from about 0.001 cm to about
0.2 cm deep, from about 0.2 cm to about 0.4 cm in length and
cross-sectional dimension of from about 0.001 cm to about 0.2 cm
(depth 0.1524 cm, width 0.0508 cm, length 0.3724 cm) Second wash
solution is most preferably applied fresh to the platform before
use using entry port 1057 that is fluidly connected to precipitant
buffer reservoir 1047 through microchannel 1056. In these
structures, entry port 1057 is preferably adapted to fluidics
loading devices such as pipettors and automated embodiments
thereof, and microchannel 1056 has dimensions of from about 0.001
cm to about 0.2 cm deep, from about -0.25 cm to about 0.5 cm in
length and cross-sectional dimension of from about 0.001 cm to
about 0.2 cm (depth 0.0254 cm, width 0.0508 cm, length 0.4347
cm).
[0138] Elution buffer reservoir 1042 is positioned radially more
distal from the center of rotation than first or second wash
reservoirs 1036 and 1047. Elution buffer reservoir 1042 has
dimensions of from about 0.1 cm to about 0.25 cm deep, from about
0.15 cm to about 0.3 cm in length and cross-sectional dimension of
from about 0.4 cm to about 0.8 cm, and has a volumetric capacity of
from about 20 .mu.L to about 250 .mu.L (depth 0.2286 cm, width
0.7308 cm, length 0.2787 cm) and is fluidly connected to
microchannel 1026 by microchannel 1044, which is interrupted by
sacrificial valve 1058 and recrystallization chamber 1043, arrayed
substantially as described above for sacrificial valve 1012 and
recrystallization chamber 1013. Sacrificial valve 1058 has
dimensions of from about 0.001 cm to about 0.2 cm deep, from about
0.05 cm to about 0.1 cm in length and cross-sectional dimension of
from about 0.001 cm to about 0.2 cm (depth 0.0254 cm, width 0.0254
cm, length 0.1012 cm). Recrystallization chamber 1043 has
dimensions of from about 0.075 cm to about 0.15 cm deep, from about
0.15 cm to about 0.3 cm in length and cross-sectional dimension of
from about 0.15 cm to about 0.3 cm (depth 0.1524 cm, width 0.2126
cm, length 0.2302 cm) and microchannel 1044 has dimensions of from
about 0.001 cm to about 0.2 cm deep, from about 0.025 cm to about
0.05 cm in length and cross-sectional dimension of from about 0.001
cm to about 0.2 cm (depth 0.1524 cm, width 0.0508 cm, length 0.8586
cm). Second wash solution is most preferably applied fresh to the
platform before use using entry port 1057 that is fluidly connected
to precipitant buffer reservoir 1047 through microchannel 1056. In
these structures, entry port 1057 is preferably adapted to fluidics
loading devices such as pipettors and automated embodiments
thereof, and microchannel 1056 has dimensions of from about 0.001
cm to about 0.2 cm deep, from about 1.0 cm to about 2.0 cm in
length and cross-sectional dimension of from about 0.001 cm to
about 0.2 cm (depth 0.0254 cm, width 0.0254 cm, length 1.6623
cm).
[0139] The microfluidics structure as disclosed herein are
preferably operated in thermal contact with a heater layer, which
can be another layer of the platform or provided as a separate
platen element as disclosed herein.
[0140] In embodiments of the platform wherein the heater layer
comprises a separate layer of the platform itself, the heater layer
serves both to seal the reservoir layer, thereby forming enclosed
chambers out of the pockets in the surface of that disc, and to
provide localized heating through an array of electrical leads and
heaters. Heater layer 1101 and component structures thereof is
illustrated in FIG. 15. Heater layer 1101 consists of a flexible
plastic sheet 1102 (most preferably a Mylar sheet, commercially
available from ICI, and having a thickness of about 0.00762 cm)
onto which are printed or otherwise deposited electrical leads 1104
having a width of from about 0.05 cm to about 0.07 cm (width 0.0508
cm) and a length that from about 1 to 10 cm arrayed on the surface
of the heater layer. Heaters 1108 are also printed or otherwise
deposited on flexible plastic sheet 1102 and have dimensions of
from about 0.1 to about 1 cm wide (width 0.6891 cm to 0.2032 cm)
and from about 0.1 to about 0.5 cm long (length 0.2032 to 0.2067
cm). Electrical leads 1104 are preferably constructed of a
conductive material, such as a silver-based ink, while heaters 1108
are preferably constructed of a resistive material, such as a
carbon-based ink (having a thickness of printed material on Mylar
sheet of about 10 .mu.m). The electrical circuit consists of
contact pads 1105 having a radius of about 0.1 to 0.2 cm (radius
0.1397 cm) arrayed near the center of the heater layer which may be
contacted by a pin assembly on the micromanipulation instrument;
when contacting these pins, closed electrical circuits may be
formed in which the instrument forms one part and the heater layer
the other part. These types of electrical connections between the
heater layer and the instrument are more fully disclosed in
co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000 and
incorporated by reference herein. Lead 1106 forms a common source
or sink of current. Leads 1107 branch from this common lead through
the heater elements 1108, and are then connected to the various
pads 1105. When a source of high voltage is applied to pads
corresponding to individual heaters and the contact pad associated
with 1106 is grounded, electrical current flows through the lead
and the resistive element, which dissipates energy and generates
heat.
[0141] Finally, in some embodiments the platform also comprises a
base layer 1201 that serves to insulate the platform for more
efficient heating using the heater later. The structure of the base
layer 1201 is simply a thermally-insulating material having a
center hole 1202 (having a radius of about 1 to 1.5 cm; radius
1.4351 cm) for mating with the rotor or spindle of the 10
micromanipulation device of the invention.
[0142] In the use of the DNA preparation platforms of the
invention, cell cultures, particularly bacterial cell cultures, are
applied to the platform and plasmid DNA is isolated by cell lysis,
separation of plasmid DNA from bacterial cell debris, cell proteins
and cell genomic DNA, plasmid DNA capture on an affinity matrix
that is washed to effect a buffer change, thereby removing
components of the isolation solutions that are incompatible with
down-stream uses of the isolated plasmid DNA; and plasmid DNA
elution and recovery.
[0143] The sequence of fluid movements through the microfluidics
structures of the plasmid DNA preparation platforms of the
invention is illustrated in FIG. 17. In the description of the use
of the platform, the sizes and amounts of the microfluidics
structures, reagents and samples are provided in exemplary
embodiments parenthetically.
[0144] To prepare the platform for use, reagent solutions are
applied as follows. An amount of the following solutions are added
to the platform: alkaline cell lysis solution from about 25 .mu.L
to about 300 .mu.L (50 .mu.L) is loaded into reservoir 1016 using
entry port 1019 and microchannel 1018; elution buffer from about 20
.mu.L to about 250 .mu.L (404L) is loaded into reservoir 1042 using
entry port 1046 and microchannel 1045; first wash solution from
about 50 .mu.L to about 600 .mu.L (100 .mu.L) is loaded into
reservoir 1036 using entry port 1040 and microchannel 1041; second
wash solution from about 75 .mu.L to about 850 .mu.L (150 .mu.L) is
loaded into reservoir 1047 using entry port 1057 and microchannel
1056; and precipitating solution from about 35 .mu.L to about 400
.mu.L (70 .mu.L) is loaded into reservoir 1020 using entry port
1025 and microchannel 1024.
[0145] A sample in an amount from about 25 .mu.L to about 300 .mu.L
(50 .mu.L) of bacterial culture is then added to chamber 1005 via
entry port 1015 and channel 1014.
[0146] The platform is then placed into the micromanipulation
device, preferably onto a rotor comprising a slip-ring as described
herein and in co-owned U.S. Pat. No. 6,063,589, issued May 16,
2000, incorporated by reference, to enable activation of heaters as
described herein. For embodiments with a heater layer, the pins of
a slip-ring assembly are brought into contact with the pads 1105 of
the heater layer. In these embodiments, alignment groove 1003
serves to set the orientation of the slip-ring assembly relative to
the disc, as shown in FIG. 15. The heater layer is aligned with the
fluidics layer in such a way that the slip ring pin lines up the
pin marks on the heater layer.
[0147] The disc is then accelerated to the first rotational speed
from about 500 to about 1500 rpm sufficient to motivate fluid flow
of alkaline cell lysis solution from reservoir 1016 through
microchannel 1017 into chamber 1005. The platform is then agitated
by being subjected to rapid, positive and negative (i.e., forward
and backward) angular accelerations, thereby increasing and
decreasing its angular velocity to effect mixing. Mixing baffles
1006 serve to create a circular motion within the fluid,
"laminating" the fluid by drawing it into long portions which are
folded back onto the main mass of the fluid, as illustrated in the
Figure. As the fluid is repeatedly laminated, it is homogenized.
For large volumes and systems which are not microfabricated, and
with sufficient angular acceleration, the flow within the chamber
1005 is also turbulent, which aids in mixing.
[0148] After time sufficient to achieve homogeneous mixing (which
will be dependent on the amount of sample and the size of the
chamber 1005 and thus empirically determined), bacteria within the
sample will have undergone lysis, releasing DNA into the solution.
The disc's rotational velocity is then maintained at a second
rotational speed from about 500 to about 1500 rpm (???) and the wax
valve within capillary 1021 is melted through application of
voltage to the corresponding heater pad in thermal contact with the
valve. Heat is applied for time sufficient to melt the wax and for
the flowing fluid to drive the molten wax out of the capillary and
into recrystallization chamber 1022. The angular velocity is
maintained until all of the precipitating solution is driven into
the mixing chamber.
[0149] The disc is then again agitated as described above to effect
mixing. The effect of the precipitating solution is to precipitate
genomic DNA, proteins and cellular debris components of the
homogenous lysis mixture. This precipitation results in aggregates
that can be trapped by filter 1009. Described above and in assembly
portion of the text--comprised of Porex frit material X-4588, 70
.mu.m pore size and Whatman Filter paper #54.
[0150] After sufficient mixing, the disc's rotational velocity is
maintained at a third rotational speed from about 500 to about 1500
rpm and the wax valve within capillary 1012 is then melted through
application of voltage to the corresponding heater pad in thermal
contact with the valve. Heat is applied for time sufficient to melt
the wax and for the flowing fluid to drive the molten wax out of
the capillary and into recrystallization chamber 1013. The angular
velocity is maintained until all of fluid is driven from the mixing
chamber through capillaries 1010, 1012, and 1026 into binding
column 1027. The cell debris and unwanted precipitates are trapped
on the filter 1009.
[0151] The platform is rotated at this angular velocity as the
fluid is driven via centripetal acceleration through the binding
matrix. Because microcapillary 1032 is blocked by a sacrificial
valve, fluid is driven into microchannel 1033, with the
centrifugally-induced pressure overcoming the hydrophobicity of the
surface treatment of the channel. The fluid is then delivered to
the waste chamber 1034.
[0152] Platform rotation is maintained until all fluid passes
through the matrix 1028 in binding chamber 1027, which is typically
a porous material. Because the end of microchannel 1033 that is
fluidly connected to waste chamber 1034 is more distant from the
platform's axis of rotation than the opposite end of the channel,
it is possible to draw all fluid out of binding chamber 1027,
microchannel 1030, and pocket 1031 by siphoning action. This has
the advantage of reducing contamination or backflow between various
steps.
[0153] Once all of the fluid has been driven through the matrix
1028, plasmid DNA is bound thereto. Typically, these matrix
materials present positive charges that attract the negative
charges of the DNA. There are several different types of affinity
matrices that have been implemented on the disc that utilize the
differences in charge between the DNA and the matrix. Both ion
exchange resins as well as silicas and glass fibers including
diatomaceous earth, refined silica resins, DEAE as well as glass
fiber filters have been used to demonstrate the capabilities on the
disc to use charge differences to bind DNA. The platform is then
rotated at a fourth rotational speed from about 500 to about 1500
rpm while heat is applied to the wax valve within capillary 1037,
releasing the first wash solution through microchannels 1039 and
then 1026, fluidly connected to binding column 1027. This fluid is
driven through the binding matrix and into the waste chamber as
previously described. This wash is intended to remove the
components of the lysis and precipitating solutions trapped within
the matrix material.
[0154] The second wash solution contained within reservoir 1047 is
then released through melting of its sacrificial valve 1048 and
fluid flow through microchannels 1050 and 1026 and into binding
chamber 1027 where it is washed through matrix 1028 and into waste
chamber 1034.
[0155] After these treatments, the bound plasmid DNA on the matrix
contains only trace amounts of other fluids and materials used in
the sample processing steps. The platform is then rotated at fifth
rotational speed from about 500 to about 1500 rpm and sacrificial
valve in capillary 1032 is opened through application of heat,
opening the fluid connection between matrix 1028 in binding chamber
1027 with sample collection reservoir 1053. Heat is also applied to
the sacrificial valve within capillary 1058. Elution buffer is
driven through microchannels 1044 and 1026 and binding column 1027
and most particularly through matrix 1028. Because microchannel
1032 is open, fluid preferentially flows into sample collection
reservoir 1053. Under these conditions, the topography of
microchannel 1033, the hydrophobic coating thereof or both
advantageously prevents the fluid from flowing into waste reservoir
1034. The hydrophobic coating also advantageously prevents
back-flowing of waste solution into sample collection chamber 1053
through microchannel 1033.
[0156] The platform is then brought to a halt and the fluid sample
containing isolated plasmid DNA is recovered through port 1055 and
microchannel 1054.
[0157] Integrated Sample Preparation and In Vitro Amplification
Platform
[0158] The invention also provides platforms having microfluidics
structures that are able to perform an integrated suite of
biochemical reactions that include DNA sample preparation, in vitro
amplification, and product recovery or analysis. This aspect of the
invention is described herein for a single microfluidics structure.
However, platforms comprising a multiplicity of these microfluidics
structures are provided and are encompassed by the invention,
wherein a multiplicity of the microfluidics structures described
herein are provided on the platform.
[0159] Referring now to the Figures for a more thorough description
of the invention, FIG. 1 shows an exploded view of an example of a
disc for performing a multiplicity of m vitro amplification
reactions. The illustrated embodiment of this platform is capable
of performing six independent sample prep and PCR operations on six
individual samples. Alternative embodiments of the invention,
extending the capacity of the platform to larger sample numbers, as
well as subdivision and/or combination of samples, is
straightforward and is discussed below.
[0160] The disc shown here performs 96 assays of the general form:
mix first fluid A with second fluid B,
[0161] This disc illustrates that identical assays may be made by
repeating microfluidics structures around the disc at a given
radius as well as modifying the structures for placement at
different radial positions. In this way, it is possible to fully
cover the surface of the disc with microfluidics structures for
performing assays. The maximum number of assays that may be
performed will depend upon the volume of fluid that may be
manipulated reproducibly, i.e., the minimum reproducible dimensions
with which the disc may be fabricated, and the amount of
hydrodynamic pressure required to drive small volumes of fluid
through microchannels at convenient rotational rates. Taking these
considerations into account, it is estimated that greater than
10,000 assays having volumes of 1-5 nL can be created in a circular
platform having a 6 cm radius.
[0162] In FIG. 1, the disc 100 comprises at least three components:
a microfluidics disc 201, a sealing layer 301, and one or more
thermal sealing layers 401. In certain embodiments, microfluidics
disc 201 is further provided as a combination of at least two
component layers, wherein a reservoir layer 501 is bonded to a
microfluidics layer 601. In these embodiments, the bottom face of
the reservoir layer, when mated with the microfluidic layer
described below, forms a complete network of enclosed channels and
reservoirs through which fluids flow under the impetus of
centripetal force created by rotation of the platform about a
central axis. In all embodiments, fluid flow permits mixing of
various component fluids in the assay and movement of the fluids
from sample and reagent reservoirs through mixing structures and
into assay collection chambers. In addition, fluid flow can be
effectuated to include incubation and wash steps, using structures
disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000
and incorporated by reference herein. Fluid flow rates of from
about 1 nL/s to about 1000 .mu.L/s are achieved at rotational
speeds of from about 4 to about 30,000 rpm. "Passive" or capillary
valves are preferably used to control fluid flow in the platform as
described in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000,
and co-owned and co-pending patent applications U.S. Ser. No.
08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18,
1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No.
08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19,
1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______,
filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the
disclosures of each of which are explicitly incorporated by
reference herein. In the operation of the platforms of the
invention, competition between rotationally-induced hydrostatic
pressure and the capillary pressure exerted in small channels and
orifices are exploited to provide a rotation-depending gating or
valving system. After fluids are deposited in detection chambers
positioned towards the outer edge of the platform, a signal, most
preferably an optical signal, is detected.
[0163] Platform 100 is preferably provided in the shape of a disc,
a circular planar platform having a diameter of from about 10 mm to
about 50 mm and a thickness of from about 0.1 mm to about 25 mm.
The structure of microfluidics disc 201 is shown in FIG. 2, which
depicts the "bottom" face to more clearly illustrate this
embodiment of the platforms of the invention.
[0164] Microfluidics disc 201 is preferably provided in the shape
of a disc, a circular planar platform having a diameter of from
about 10 mm to about 50 mm and a thickness of from about 0.1 mm to
about 25 mm. The disc preferably comprises a center hole 202 for
mounting on a spindle, having a diameter of from about 1 mm to
about 20 mm. Center hole 202 can be replaced by an extruded fitting
for connection to a spindle, or may be absent entirely, in which
case registry and connection to the spindle is accomplished using
another portion of the surface of the platform. Microfluidics disc
201 can also include registry features such as the groove 203 that
permits a clamping fixture above the platform to be brought in
proximity with, but not in contact with, the top surface of the
platform when the platform is loaded into the spindle. In
embodiments having this feature, a pin on the clamping fixture,
preferably spring-loaded, slips into the groove as the disc is spun
at low rpm, and captures the clamping fixture, thus determining the
platform's orientation with respect thereto. In other embodiments,
the platform comprises "home-flag" 204, that is a reflective or
absorbing stripe that can be positioned on the surface of the
platform and sensed by an emitter/photodiode pair as the disc is
spun, thus permitting the orientation of the disc with respect to
the instrument to be determined.
[0165] FIG. 3 illustrates an expanded view of a section of the
microfluidics disc, with the center of the disc being beyond the
top of the figure. Reservoirs 204, 205, and 206 are designed to
contain the fluid sample, cell alkaline lysis solution, and
neutralizing buffer, respectively. Reservoirs 204 and 205 are
designed such their radial position on the disc and extent are
identical, and with cross-sectional areas (i.e., depth in the
platform.times.lateral dimension) which form a ratio equal to the
desired ratio of fluids to be placed within the reservoirs. Each
reservoir has a loading hole (207, 208 and 209, respectively)
located in the reservoir at a position proximal to the center of
the disc. Each reservoir has dimensions of from about 0.05 mm to
about 5 mm wide, from about 0.05 mm to about 20 mm long, and from
about 0.05 mm to about 5 mm thick, and has a volumetric capacity of
from about 0.1 nL to about 500 .mu.L. Loading holes 207, 208 and
209 preferably have dimensions adapted to automated loading devices
such as micropipettors, for example, a standard 200 .mu.L plastic
pipette tip having a tip diameter of 1.5 mm; micropipette tips of
diameter 1 mm; piezoelectric or ceramic drop delivery systems (such
as are sold by the IVEK Corp., Springfield, Vt.); and inkjet-based
fluid delivery systems. For non-contact delivery systems such as
piezoelectric or inkjet delivery, the dimensions of the ports must
be a few times greater than the size of the droplets, e.g., 0.2 mm
for a 1 nL drop.
[0166] The opposite end of reservoir 204 is fluidly connected to
microchannel 210 that preferably is constructed at a different
depth in the platform surface than the reservoir. This microchannel
ends at capillary 211 that preferably is constructed at a different
depth in the platform surface than the microchannel 210. Capillary
211 ends at air-vent 212 located more proximally to the center of
the disc than the inner ends of the reservoirs. Reservoir 205 is
fluidly connected to microchannel 213 at a position in the
reservoir distal to the axis of rotation. Microchannel 213 also
ends at capillary 211 as described for microchannel 210. The
construction of the microchannels 210 and 213 and the connection of
these microchannels to capillary 211 permits air to be displaced by
fluid flow from reservoirs 204 and 205, but the cross-sectional
area of capillary 211 is constructed to be too small to permit
liquid fluid flow therethrough. Reservoir 206 is fluidly connected
to microchannel 214 that ends at capillary junction 215.
[0167] Microchannels 210 and 213 are fluidly connected to mixing
microchannel 216, and permit mixing of the contents of reservoirs
204 (herein illustrated to contain sample) and 205 (herein
illustrated to contain alkaline cell lysis solution). Mixing
microchannels are configured to provide mixing of different
solutions as the mixture traverses the longitudinal extent of the
microchannel. The degree of mixing is dependent on the flow rate of
the fluids and the longitudinal extent of the mixing microchannel,
which is proportional to the amount of time the two fluids are in
contact and are mixed together. The degree of mixing is also
dependent on the lateral extent of the mixing microchannel, and is
further dependent on the diffusion constants of the fluids to be
mixed. In order to accommodate mixing microchannels having
sufficient lengths for mixing fluids having a useful range of
viscosities, the mixing microchannels are provided as shown. Mixing
is promoted by configuring the microchannel to bend several times
as it traverses a path on the platform surface that is
perpendicular to the direction of rotation, but extends radially on
the surface of the platform from a position more proximal to a
position more distal to the axis of rotation. Mixing microchannel
216 has a length of from about 1 mm to about 100 mm, its length in
some cases achieved through the use of bends.
[0168] Mixing in the device is promoted through diffusion. If two
small volumes A and B are added to a single container, diffusion of
A into B and/or B into. A will effect mixing. The amount of time
required for this mixing will depend upon the diffusion constants
of the molecules within the solutions whose mixing is desired and
the distances over which the molecules must diffuse. For example,
0.5 microliter of solution A comprising a molecule with diffusion
constant D is added to a reservoir 1 mm on a side. Solution B
comprising a molecule whose diffusion constant is also D is added.
The solutions will initially occupy the volume with an interface
partitioning them. Even if the fluids are highly miscible, the
diffusion times to create a completely homogeneous solution will be
approximately t=2.times..sup.2/D. For x=0.05 cm (0.5 mm) and
D=10.sup.-5 cm.sup.2/s, the mixing time is 500 seconds, an
unacceptably long time for most reactions. This mixing time may be
reduced by mechanical stirring, for example, but stirring is
difficult to obtain in fluids confined in small structures because
the flow of the fluid is laminar and does not contain turbulent
eddies that are known to promote mixing. If, instead of placing
fluids A and then B in a 1 mm.sup.3 container, fluids A and B were
placed side-by-side in a long, thin capillary of lateral dimension
d, the relevant time for mixing is much shorter. If, for example, d
is 100 microns, mixing time t is 20 seconds. The mixing channels of
the device simulate the placement of fluid in a long capillary by
co-injecting fluid streams A and B into a capillary microchannel.
These fluids flow side-by-side down the channel initially. As the
fluid is pushed through the microchannel due to centrifugal force
produced by rotation of the platform, diffusion occurs between the
fluids. By choosing a capillary of sufficiently narrow diameter,
sufficient length, and a pumping rate that is sufficiently low, the
portion of A and B of the total volumes of A and B present in the
channel during pumping can be caused to mix.
[0169] These choices may be determined by setting the required time
for mixing equal to the amount of time necessary for the fluid to
traverse the channel. The required time for diffusion is 1 t m 2 w
2 D
[0170] where w is the lateral size of the channel. The amount of
time necessary to traverse the channel is simply the length of the
channel divided by the fluid velocity, the velocity being
calculated as described in co-owned and co-pending U.S. Ser. No.
08/910,726, filed Aug. 12, 1997, and Duffy et al. (1999, Anal.
Chem. 71: 4669-4678): 2 t t = l U = l ( 2 R R ( d H ) 2 32 l ) = 32
l 2 2 R R ( d H ) 2
[0171] where the fluid properties are the density .rho. and
viscosity .eta., .DELTA.R and <R> are the extent along the
radius and average radial position of the fluid subject to
centripetal acceleration, and l and d.sup.H are the length and
hydraulic diameter of the channel. By choosing variables such that
t.sub.t is at least equal to or greater than t.sub.m, mixing in the
microchannels is assured.
[0172] Mixing microchannel is fluidly connected at its end distal
to the axis of rotation to reservoir 217. Reservoir 217 has
dimensions of from about 0.05 mm to about 5 mm wide, from about
0.05 mm to about 20 mm long, and from about 0.05 mm to about 5 mm
thick, and has a volumetric capacity of from about 0.1 nL to about
500 .mu.L, and is equipped at the end of the reservoir proximal to
the axis of rotation with air displacement capillary 220 and air
vent 221. This permits air to be displaced from the reservoir upon
fluid flow through mixing microchannel 216 and delivery to
reservoir 217.
[0173] Mixing microchannel is fluidly connected at its end distal
to the axis of rotation to reservoir 217. Reservoir 217 has
dimensions of from about 0.05 mm to about 5 mm wide, from about
0.05 mm to about 20 mm long, and from about 0.05 mm to about 5 mm
thick, and has a volumetric capacity of from about 0.1 nL to about
500 .mu.L, and is equipped at the end of the reservoir proximal to
the axis of rotation with air displacement capillary 220 and air
vent 221. This permits air to be displaced from the reservoir upon
fluid flow through mixing microchannel 216 and delivery to
reservoir 217.
[0174] Capillary junction 215 is fluidly connected to microchannel
218. As illustrated in FIG. 3, this microchannel is preferably
configured to have the same length and diameter as mixing
microchannel 216 in order to most easily permit delivery of fluid
from reservoirs 204 and 205 into reservoir 217, and from reservoir
207 to reservoir 219, to be accomplished simultaneously and
coordinately. In alternate embodiments, microchannel 218 is
provided having a different length than mixing microchannel 216,
since no mixing occurs in microchannel 218. In other alternative
embodiments, fluid is loaded directed into reservoir 219. Reservoir
219 has dimensions of from about 0.05 mm to about 5 mm wide, from
about 0.05 mm to about 20 mm long, and from about 0.05 mm to about
5 mm thick, and has a volumetric capacity of from about 0.1 nL to
about 500 .mu.L, and is equipped at the end of the reservoir
proximal to the axis of rotation with air displacement capillary
220 and air vent 221. This permits air to be displaced from the
reservoir upon fluid flow through mixing microchannel 218 and
delivery to reservoir 219. Reservoirs 217 and 219 are designed such
their radial position on the disc are identical, and with
cross-sectional areas (depth in the platform.times.lateral
dimension) that form a ratio equal to the desired ratio of fluids
to be placed within the reservoirs.
[0175] Reservoir 217 is fluidly connected to microchannel 222
having a length of from about 0.15 cm to about 0.30 cm (length
0.2552 cm) and terminates at capillary junction 223. On the other
side of the capillary junction is microchannel 224 having a length
of from about 0.015 cm to about 0.030 cm (length 0.0218 cm) that
terminates at air displacement capillary 225. Air displacement
capillary 225 is connected by capillary 227 to expansion volume
228; alternatively in some embodiments expansion volume 228 is
replaced by air vent 228. The construction of microchannels 222 and
224 and the connection of these microchannels to capillary 227
permits air to be displaced by fluid flow from reservoirs 217 and
219, but the cross-sectional area of capillary 227 is constructed
to be too small to permit liquid fluid flow therethrough. These
structures are more fully illustrated in FIG. 4.
[0176] Reservoir 219 is fluidly connected to microchannel 226
having a length of from about 0.20 cm to about 0.40 cm (length
0.3205 cm) and terminates at air displacement capillary 225,
forming a capillary junction therewith. The depth and width of
microchannel 226 where it joints capillary 225 is preferably
different than the depth and width of microchannels 222 and
224.
[0177] Microchannels 224 and 226 are fluidly connected to mixing
microchannel 229, designed similarly to mixing microchannel 216 to
insure mixing of fluids from reservoirs 217 and 219. Mixing
microchannel 229 has a length of from about 1 mm to about 100 mm,
its length in some cases achieved through the use of bends, and
terminates at reservoir 230, which also contains air vent 231.
Reservoir 230 has dimensions of from about 0.05 mm to about 5 mm
wide, from about 0.05 mm to about 20 mm long, and from about 0.05
mm to about 5 mm thick, and has a volumetric capacity of from about
0.1 nL to about 500 .mu.L Reservoir 230 is fluidly connected to
microchannel 232, which is arrayed on the surface of the platform
in a direction radially-inward and terminating at air displacement
capillary 233. The junction 299 between microchannel 232 and
capillary 233 is designed with a smoothly-widening enlargement to
substantially inhibit capillary-pinning (valving) action, as
described more fully below. This junction 299 is fluidly connected
to mixing microchannel 234, the radially-inward end of which is
terminated in an expansion volume 235. Reservoir 236 is positioned
at the same radial distance from the axis of rotation as reservoir
230. Reservoir 236 has dimensions of from about 0.05 mm to about 5
mm wide, from about 0.05 mm to about 20 mm long, and from about
0.05 mm to about 5 mm thick, and has a volumetric capacity of from
about 0.1 nL to about 500 .mu.L. In preferred embodiments, a
portion 237 of reservoir 236 radially more distal from the axis of
rotation has a depth in the surface of the platform that is more
shallow than the depth in the surface of the platform of the
portion 238 radially more proximal to the axis of rotation.
Reservoir 236 also comprises air vent 239, which also serves as a
loading port.
[0178] Microchannel 240 having dimensions of from about 0.15 cm to
0.3 cm long (length 0.25 cm) and a cross sectional area of from
about 0.001 cm to about 0.2 cm fluidly connects reservoir 236 with
air displacement capillary 233 at capillary junction 298. In
contrast to capillary junction 299, the joint of the microchannel,
is designed to provide a "pinning" action, that is, the junction
prevents fluid flow. Microchannel 240 is designed to contain a
restriction 297 followed by a flared opening in the retrograde
direction (i.e., back towards the axis of rotation), connected to
capillary 233. Alternative embodiments of capillary junctions, as
disclosed more fully in co-owned U.S. Pat. No. 6,063,589 issued May
16, 2000 and in co-owned and co-pending patent applications U.S.
Ser. No. 08/910,726, filed Aug. 12, 1997, incorporated by reference
herein.
[0179] Mixing microchannel 234 having a length of from about 1 mm
to about 100 mm, most preferably wherein its length in some cases
achieved through the use of bends, terminates at thermal cycling
chamber 241. The depth in the platform surface of thermal cycling
chamber 241 is determined by the thermal requirements of cycling,
as disclosed more fully below. As shown in FIG. 3, the portion of
thermal cycling chamber 241 more proximal to the axis of rotation
is connected to air channel 242 that terminates in an air-vent 243.
The chamber also comprises an insulating air pocket 245 that is a
depression having a diameter greater than the diameter of thermal
cycling chamber 241 in the other face of the disc 201.
[0180] FIG. 5 illustrates the assembled disc in the region of the
thermal cycling chamber in cross-section. The outward radial
direction is indicated by the arrow. The thermal cycling chamber
241 is a depression in the face of the disc 201, with channel 234
entering at a position radially distal from the axis of rotation,
as shown. Sealing layer 301 is provided as described below to cover
thermal cycling chamber 241. Insulating air pocket 245 is shown in
he surface of platform 201, covered by sealing layer 301.
[0181] The microsystems platforms of the invention are provided to
perform in vitro amplification reactions, most preferably in an
integrated suite of biochemical reactions including DNA isolation,
amplification most preferably using the polymerase chain reaction
(PCR) and isolation of the amplified fragment, for detection
on-disc or off disc (for example, using conventional gel
electrophoresis).
[0182] This use of the platforms of the invention is shown in FIGS.
1 through 5; although this use will be understood by those with
skill in the art from the disclosure and examples, the functioning
of the platform is explicitly described as follows.
[0183] The platforms of the invention most preferably accept raw,
or at most pre-diluted, biological fluids containing bacteria or
eukaryotic cells, or sample of bacterial or eukaryotic, most
preferably mammalian cell culture, or biological fluids such as
blood comprising mammalian cells, and to process these fluids
through the steps of DNA release from the cells and amplification,
most preferably via PCR.
[0184] The fluid sample is added to reservoir 204 through entry
port 207, an alkaline cell lysis solution, in one instance, NaOH,
is added to reservoir 205 through entry port 208, and a
conditioning or neutralization buffer solution, in one example,
Tris HCl, is added to reservoir 206 through entry port 209. An
amplification solution comprising a balanced mixture of
deoxyribonucleotide triphosphates (dNTPs) at a concentration of
about 200 .mu.M, 1-10 Units of a polymerase, most preferably a
thermostable polymerase such as Taq polymerase from Thermus
aquaticus, and buffers and salts, particularly MgCl.sub.2,
appropriate for the amount of target DNA in the sample and the
enzyme (the amount of magnesium chloride is typically determined
empirically due to the sensitivity of amplification reactions to
the concentration of this salt) is added through entry port 239
into reservoir 237. This is shown in FIG. 9a. The platform is then
placed on a micromanipulation device described herein and more
fully in co-owned U.S. Pat. No. 6,063,589, and co-owned and
co-pending U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No.
08/768,990, filed Dec. 18, 1996; and Ser. No. 08/910,726, filed
Aug. 12, 1997 incorporated by reference. The platform is rotated to
motivate fluid flow on the disc in a manner determined by the
placement and dimensions of the microfluidics structures on the
disc. An exemplary rotational profile is shown in FIG. 8. As shown,
the platform is first accelerated gently at angular acceleration
.alpha..sub.1 to an initial rotational rate .omega..sub.1 (slightly
higher than 400 rpm in FIG. 8). Rotation is maintained for a time
(about 10 seconds in FIG. 8) sufficient to allow the sample and
alkaline cell lysis solutions to enter microchannels 210 and 213,
flowing until they are stopped ("pinned," as used herein) at the
capillary junction with microchannel 211. Similarly, fluid in
reservoir 206 flows into microchannel 214 until it is pinned at
capillary junction 215. The disc is then accelerated rapidly at
angular acceleration .alpha..sub.2 to speed .omega..sub.2 (about
1800 rpm in FIG. 8), which may be maintained for a period (as shown
in FIG. 8). The pressure induced by rotation at this speed forces
one or both fluids pinned at microchannel 211 to cross the narrow
gap of the microchannel and to come in contact with or "wet" the
other fluid; this permits the fluids, now in contact, to drain into
mixing microchannel 216 under the impetus of rotational speed
.omega..sub.3 (about 500 rpm in FIG. 8).
[0185] As shown in FIG. 3, microchannel 210 has a larger
cross-sectional area than microchannel 213, and as a result
supports a lower capillary pressure than this microchannel.
Consequently, the sample fluid bridges the gap across microchannel
211 first.
[0186] Concurrently, the velocity increase that motivates fluid
from reservoirs 204 and 205 into mixing microchannel 216 also
overcomes capillary junction 215, thereby motivating fluid flow
from reservoir 206 into mixing microchannel 218.
[0187] Once the capillary junctions impeding fluid flow out of the
reservoirs 204, 205 and 206 have been overcome, the rotational rate
is reduced at acceleration .alpha..sub.3 to velocity .omega..sub.3
(about 500 rpm in FIG. 3); in preferred embodiments, acceleration
rate .alpha..sub.3 is substantially equivalent to (but has the
opposite sign, since this is deceleration) acceleration rate
.alpha..sub.2; this is shown in the Figure. At this lower speed,
the overall pressure driving flow into mixing microchannel 216 is
reduced. Because mixing microchannel 216 is narrow and long, it
presents considerable hydraulic resistance to flow. At this low
speed, pressure differences between the fluids in reservoirs 204
and 205 (as may be occasioned, for example, by these reservoirs
having different volumes, for example) are "evened out" as fluids
flow into mixing microchannel 216. For example, if the fluid from
reservoir 204 initially flows more rapidly than the fluid from
reservoir 205 flows, the extent of fluid in the radial direction in
reservoir 204 will be smaller than that in reservoir 205 at a
larger time; hence the pressure exerted at microchannel 211 will be
lower than that exerted by the fluid in reservoir 205, resulting in
an increase in flow rate of the fluid in reservoir 205 relative to
that in reservoir 204, resulting in an "evening out" of the fluid
extents or heads in the radial direction. As a result, the fluids
enter in strict ratios equal to the ratios of the cross-sectional
areas of the reservoirs. Air displaced by the fluids is vented
through channels 220 to air-vent 221. FIG. 9b illustrates the
situation at some time at rotational speed .omega..sub.3.
[0188] The concurrent fluid flow from reservoir 206 through mixing
microchannel 219 is accomplished at rotational speed .omega..sub.3.
Since no actual mixing usually occurs, the shape of microchannel
218 is unimportant as long as the fluid pumped through it does not
fill chamber 219 at too high a rotational rate (i.e., before the
velocity drops to .omega..sub.3).
[0189] Rotation at speed rotational speed .omega..sub.3 motivates
fluid flow from reservoirs 204, 205 and 206 and into reservoirs 217
and 219. In addition to preventing changes in the mixing ratio of
fluids from reservoirs 204 and 205, the lower velocity means that
the pressure exerted at the outer ends of reservoirs 217 and 219 is
low enough not to force the fluids past the capillary junctions
designed to retain them.
[0190] When sufficient time has passed to pump the fluids from
reservoirs 204, 205 and 206 and into reservoirs 217 and 219, the
rotational rate is decreased further at acceleration .alpha..sub.4
to .omega..sub.4; which is shown to be slightly less than 400 rpm,
in FIG. 8. This disposition of the fluids on the disc is shown in
FIG. 9c. Heat is then applied to reservoir 217, containing the
mixture of sample and alkaline cell lysis buffer This heating step,
performed at between 85.degree. C. and 95.degree. C. is applied for
between 60 and 120s, and is sufficient to disrupt the bacterial
cell walls or mammalian cell plasma membranes, thereby releasing
DNA into the solution. The alkaline cell lysis solution also has
the effect of denaturing proteins, such as hemoglobin found in
blood samples that can interfere with the activity of the
polymerase enzyme.
[0191] The velocity is then increased at .alpha..sub.5 to
.omega..sub.5 (about 800 rpm) rapidly, as shown in FIG. 8, causing
the fluids retained at capillary junctions 223 and 226 to come into
contact as capillary pressure is overcome. The microfluidics
structures are provided wherein the cross-sectional area of 223 is
larger than that of 226, insuring that the lysate is the first to
flow. The fluids, once in contact, flow into mixing microchannel
229 and mix, as described above; the velocity is reduced to
.omega..sub.6 (about 500 rpm in FIG. 8) at acceleration
.alpha..sub.6 for controlled mixing of the lysate and
neutralization fluids. FIG. 9d illustrates the movement of the
fluid mixture into reservoir 230; FIG. 9e illustrates the
disposition of fluids after they have been pumped.
[0192] When the mixture of the cell lysate and neutralizing buffer
have been transferred into reservoir 230, platform velocity is
increased at .alpha..sub.7 to velocity .omega..sub.7 (about 1200
rpm in FIG. 8). This acceleration is typically gentle compared with
other accelerations (as depicted by the more lower slope of the
acceleration profile shown in FIG. 8); this is because the design
of chambers 230 and 237 and capillary junctions 233 and 240 present
a very small "pressure head" as shown in FIG. 9f. The pressure at
the capillary junction due to centrifugation is given by
P=.rho..omega..sup.2<R>.DELTA.R
[0193] where .rho. is the fluid density, .omega. is the angular
velocity=2.pi..times.speed in rpm.times.60, <R> is the
average position relative to the center of rotation of the fluid,
and .DELTA.R is the extent of the fluid in the radial direction
inward of the radial position of the capillary junction. Because
the geometry is designed to make .DELTA.R small relative to a
channel emptying radially-outward from the bottom of the reservoir,
for example, the pressure at a given rotational rate can be made
quite low. This has the advantage of making the rotational rate
required to drive the PCR reaction mixture in reservoir 236 beyond
the capillary junction with microchannel 233 very high and allows
the fluid to be retained even during the rapid accelerations sand
decelerations of earlier steps in the velocity profile. In
contrast, the exit of microchannel 232 into microchannel 233 flares
open and presents at least one smooth edge, to prevent pinning of
the advancing meniscus. As a result, fluid is not retained, but
moves into microchannel 233 and wets the retained PCR reaction
mixture. As the rotational velocity is gradually increased, fluids
are pumped into mixing microchannel 234. As shown in FIG. 9g, the
configuration of the junction of microchannel 233 and microchannel
240 relative to the fluid position in reservoirs 230 and 236
results in the fluids being drawn into mixing microchannel 234 by,
in part, a siphoning action. The neutralized cell lysate and PCR
reaction mixture are in contact for sufficient time in the mixing
microchannel 234 to effect mixing by diffusion; as shown in FIG. 8,
this mixing is performed at relatively high speed (about 1200
rpm).
[0194] FIG. 9h illustrates the final microfluidics state of the
disc, with all fluid having been delivered to thermal cycling
chamber 241. The platform speed is reduced to rotational speed
.omega..sub.8 (about 500 rpm in FIG. 8), in a slow deceleration
profile .alpha..sub.8 that is similar to the gradual
acceleration
[0195] Thermal cycling is effected in thermal cycling chamber 241
using a variety of thermal cycling protocols and temperature
profiles. Examples of such temperature profiles include:
[0196] 1. Hold the reaction mixture at high temperature (e.g.,
95.degree. C.) to denature double-stranded DNA
[0197] 2. Perform a cycle of step, wherein for n cycles, the
following steps are repeated identically n-1 times:
[0198] a) drop the temperature to an annealing temperature (e.g.,
45.degree. C.-75.degree. C.), either transiently or for an
annealing period to allow annealing of primers to single-stranded
DNA;
[0199] b) raise the temperature an extension temperature (e.g.,
60.degree. C.-70.degree. C.), either transiently or more preferably
with a primer extension period that allows extension of the
amplification primers; and
[0200] c) raise the temperature to the denature temperature of the
amplified fragment.
[0201] Optionally, the final reaction step comprises dropping the
mixture to the annealing temperature and then raising the
temperature of the thermal cycling chamber to the extension
temperature for a time sufficient to substantially complete the
extension reactions on all extended products.
[0202] The temperature of the sample is then usually reduced to
room temperature or below to stop the reaction.
[0203] NOW, AS I UNDERSTAND IT THE THERMAL CYCLING CHAMBER IS
SIMPLY A BIG RESERVOIR COVERED BY A THERMAL INSULATING LAYER,
RIGHT?
[0204] The following Examples are intended to further illustrate
certain preferred embodiments of the invention and are not limiting
in nature.
EXAMPLE 1
Sample Preparation and PCR of Genomic DNA from E. coli
[0205] A microfluidics platform as depicted in FIGS. 1 through 4
was used to prepare and amplify a DNA target from samples of E.
coli. Aspects of the instrument used for controlling the rotational
profile and thermal cycling are described in co-owned U.S. Pat. No.
6,063,589, issued May 16, 2000, and co-owned and co-pending patent
applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No.
08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug.
12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No.
09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12,
2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No.
95,1408-XX), the disclosures of each of which are explicitly
incorporated by reference herein. The instrument used in this
example is described above in the Detailed Description of Preferred
Embodiments.
[0206] The microfluidics structures were manufactured through
machining of acrylic using computer/numerical code machining using
a Light Machines VMC5000 milling machine running Light Machines
"Benchman" software (Light Machines Corporation, Manchester, N.H.).
The disc was then vapor polished by exposure to vapor from boiling
methylene chloride to remove machine marks and smooth the machined
surface. A layer of double-sided tape (such as 7953 MP
(1.5-0.5-1.5)) was applied to the machined face of the disc. The
tape was either die-cut prior to application or the tape was
razored out from the openings of the thermal cycling chamber after
application. This was done to minimize possible exposure to known
inhibitors of PCR in the adhesive of the tape. A layer of
Mylar(ISI) was then applied to the double-sided tape, completing
the sealing of the fluid structures of the disc. In many cases, the
discs were then passivated by exposure to parylene using methods
disclosed more fully in co-owned and co-pending U.S. patent
application Ser. No. 09/579,492, filed May 12, 2000, incorporated
by reference.
[0207] The solutions used for sample preparation and PCR were
alkaline cell lysis solution, neutralization buffer; and PCR
reaction mixture containing oligonucleotide primers for amplifying
a specific target sequence.
[0208] The E. coli suspension was provided in Luria-Bertani media
and had a cell concentration of between 2.7.times.10.sup.9 cells/mL
and 3.3.times.10.sup.9 cells/mL as calculated using absorbance at
600 nm in a standard laboratory spectrophotometer; dilutions of the
culture to achieve desired cell numbers were made using
Luria-Bertani broth and then diluted 40-fold with deionized
water.
[0209] The alkaline cell lysis solution used was 10 mM NaOH, and
the neutralization buffer was 16 mM Tris-HCl, pH 7.5. The PCR
reaction mixture was either the Amersham Pharmacia Ready-to-Go bead
or Stratagene Taq 2000. The Ready-to-Go bead was resuspended and
brought to a final volume of 25 .mu.L. Both systems give a final
concentration of 200 .mu.M of each dNTP and 1.5 mM MgCl.sub.2,
along with appropriate salts and stabilizers. To this mix were
added the primers of interest at a concentration of 20 pmol of each
primer in the reaction. These primers were EcoCtl, a primer pair
which defines a randomly-selected 300 base-pair (bp) non-coding
sequence of the E. coli genome, or lac I, a pair which defines a
422 bp codon for the lac I repressor protein.
1 EcoCtl-F sequence: 5'-AGTACCGCAAATCGCCATCAAAAGT- AATGC- (SEQ ID
No.: 1) 3'; EcoCtl-R sequence: 5'-GTCAGTTCGCCTTTCAGAGGAATAACCGC-
(SEQ ID No.: 2) 3'; LacI-F sequence: 5'-CCGAGACAGAACTTAATGGGCCC-3';
(SEQ ID No.: 3) LacI-R sequence: 5'ACAACAACTGGCGGGCAAACA-3'. (SEQ
ID No.: 4)
[0210] All primers were obtained from Research Genetics, Inc.,
Huntsville, Ala.
[0211] The PCR protocol was adapted from Rudbeck & Dissing
(1998, BioTechniques 25: 588-592). It consists of the following
steps: mixing 5 .mu.L of the sample with 5 .mu.L of NaOH; heating
the mixture to about 95.degree. C. for 120s to lyse the cells;
mixing the lysate with 5 .mu.L of Tris-HCl neutralization buffer;
and mixing of neutralized lysate with 10 .mu.L of PCR reaction
mixture containing selected primers. This final solution was then
subjected to thermal cycling.
[0212] Control reactions were performed conventionally on the
benchtop by manually carrying out the sample preparation steps
above. Thermal cycling was carried out in an MJ Research Model
PTC-100 with hot bonnet thermal cycler. The cycling parameters for
this reaction were as follows:
2 E. coli Step Temperature Time(min:sec) 1: initial acclimation
25.degree. C. 00:15 2: ramp 1 +70.degree. C. 1.5.degree. C. per
0:01 3: initial dehybridization 95.degree. C. 2:00 4: denature
95.degree. C. 0:15 5: ramp 2 -35.degree. C. 1.4.degree. C. per 0:01
6: anneal 60.degree. C. 0:15 7: ramp 3 +12.degree. C. 1.2.degree.
C. per0:01 8: extend 72.degree. C. 0:15 9: ramp 4 +23.degree. C.
1.5.degree. C. per 0:01 10: denature 95.degree. C. 0:15 11: ramp 2
-35.degree. C. 1.4.degree. C. per 0:01 12: anneal 60.degree. C.
0:15 13: ramp 3 +12.degree. C. 1.2.degree. C. per 0:01 14: extend
72.degree. C. 0:15 15: repeat command Goto step 9 33 times 16:
final extension 72.degree. C. 3:00
[0213] A similar profile was employed using the platform. The ramp
rates or rates of temperature change used were allowed to be "as
fast as possible" given the thermoelectric elements used for
cycling. The constant temperature times for dehybridization,
annealing, and extension were approximately equivalent to those
used for the conventionally-performed controls, although it is
recognized that the response of the fluid in the disc is slower
than that of the temperature sensors embedded in the metal blocks
used to interface the disc with the thermoelectric components.
[0214] After loading the appropriate volumes into chambers 204,
205, 206 and 236 shown in FIG. 3, the disc was placed on the platen
of the instrument, with the center hole of the disc being placed
over a threaded screw on the axis of the platen. Thermal grease was
used to insure good thermal contact between the disc and the
thermoelectric components. A retaining screw was used to hold the
disc in place.
[0215] The rotational profile used consisted of the following
steps:
3 Accele- starting ending ration time time (rpm/ speed Step (sec)
(sec) sec) (rpm) 1. initial acceleration 0 10 45 2. spin liquids to
bottom of chamber 10 20 0 450 3. release NaOH and Tris 20 22 125 4.
decelerate to prevent further 22 24 -100 releases 5. pump the
liquids through to lysis 24 50 0 500 chambers 6. decelerate for
lysis heating 50 52 -100 7. hold for lysis heating 52 140 0 300 8.
release of lysate and Tris to 140 142 250 neutralize sample 9.
decelerate to prevent further 142 145 -100 releases 10. pump the
liquids through to 145 160 0 500 neutralization 11. release of
neutralized lysate and 160 200 25 PCR reagents 12. pump the liquids
through to 200 210 0 1500 cycling chamber 13. decelerate for
thermal cycling 210 245 -25 14. thermal cycling 245 3600 0 500
[0216] The thermal cycling profile as described above was then used
while the platen and disc rotated at 500 rpm.
[0217] The results of a representative assay are shown in FIG. 10.
This Figure depicts a conventional gel electrophoretic analysis (as
described in Sambrook et al, 1989, MOLECULAR CLONING: A LABORATORY
MANUAL, 2.sup.nd ed., Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, N.Y.) of DNA fragments produced by the platforms of
the invention compared with a conventional PCR apparatus. These
results demonstrate that PCR performance on the platform of the
invention produced an amplified target fragment having the correct
size as determined by comparison with the conventionally-amplified
fragment, and with a yield equivalent to the yield of the fragment
amplified using a conventional thermocycling apparatus.
[0218] PCR product fragment yields were quantitatively evaluated
through the use of an epifluorescence microscope with an
illumination source (Nikon, Super high pressure Mercury Lamp Power
Supply, Model HB-10101AF). The typical yields are 60-100% of the
yields compared to the benchtop method (35-40 ng with 20000 target
copies starting material).
[0219] In cycling with the above described instrument, volume loss
due mostly to evaporation is typically around 20% of the total
input volume, so for a 25 .mu.L reaction, typical recovery is
approximately 20 .mu.L. This volume loss occurs gradually over the
course of the amplification, and the majority of the evaporation
condenses in the channel just above the cycling chamber and
collects until enough liquid has collected to spin back down into
the cycling chamber.
[0220] Multiplexing by thermal cycling the sample on the disc was
also demonstrated with E. coli samples. 20000 cells were lysed and
mixed with the PCR reagents on the bench according the protocol
described above. The primers used were the EcoCtl and lacI primers
described above. The sample was pipetted directly into the cycling
chamber and cycled using the parameters outlined above. The
velocity profile was also as above for the cycling portion of the
profile: 500 rpm for the duration of the temperature cycling.
Typical results for heat-cycled, multiplexed samples can be found
in FIG. 27. The PCR product fragment yields were quantitatively
evaluated through the use of an epifluorescence microscope with an
illumination source as described above. The yield of PCR fragments
recovered from the disc was generally about 70% of benchtop yields,
and is within the range of yields of the single-amplicon
samples.
EXAMPLE 2
Sample Preparation and PCR of Genomic DNA from Bovine Blood
[0221] The experiment set forth in Example 1 was repeated using
whole blood samples. In these experiments, heparinized bovine blood
was diluted 1:40 in deionized water. While the precise number of
white blood cells (WBCs) of the bovine blood was not determined, it
is known that the average value is around 5.times.10.sup.6
cells/mL.
[0222] The alkaline cell lysis solution, neutralization solution,
and PCR reagents used were as described above. To the PCR reaction
mixture was added the primer pair of interest, those defining the
289 bp codon for .beta.-actin.
4 .beta.-actin-F sequence: 5'-ACCCACACTGTGCCCATCTA-3' (SEQ ID No.:
5) .beta.-actin-R sequence: 5'-CGGAACCGCTCATTGCC-3'. (SEQ ID No.:
6)
[0223] The general protocol used was similar to that described
above. Because white blood cells are less robust than bacteria, the
lysis temperature chosen was 91.degree. C.
[0224] The rotational profile used is as described in Example
1.
[0225] The thermal cycling parameters were:
5 Bovine blood Step Temperature Time(min:sec) 1: initial
acclimation 25.degree. C. 0:15 2: ramp 1 +66.degree. C. 1.5.degree.
C. per 0:01 3: initial 91.degree. C. 2:00 dehybridization 4:
denature 92.degree. C. 0:30 5: ramp 2 -36.degree. C. 1.5.degree. C.
per 0:01 6: anneal 56.degree. C. 0:30 7: ramp 3 +13.degree. C.
1.5.degree. C. per 0:01 8: extend 69.degree. C. 0:30 9: ramp 4
+23.degree. C. 1.5.degree. C. per 0:01 10: denature 92.degree. C.
0:30 11: ramp 2 -36.degree. C. 1.5.degree. C. per 0:01 12: anneal
56.degree. C. 0:30 13: ramp 3 +13.degree. C. 1.5.degree. C. per
0:01 14: extend 69.degree. C. 0:30 15: repeat command Go to step 9
33 times 16: final extension 69.degree. C. 3:00
[0226] Typical results are shown in FIG. 11, showing the results of
conventional gel electrophoretic analysis (as described in Sambrook
et al., ibid.). These results demonstrate that PCR performance on
the platform of the invention produced an amplified target fragment
having the correct size as determined by comparison with the
conventionally-amplified fragment, and with a yield equivalent to
the yield of the fragment amplified using a conventional
thermocycling-apparatus.
EXAMPLE 3
Sample Preparation and PCR of pTrcHis a Plasmid DNA from E.
coli
[0227] The platform shown in FIG. 14 was used for the processing of
samples of E. coli and the isolation and purification of plasmid
DNA. The instrument used for control of the rotational profile and
thermal cycling was as described The instrument used for
controlling the rotational profile and thermal cycling consisted of
a personal computer, interface electronics between the PC and a
servo-controlled drive motor and interface electronics between the
PC and the screen-printed circuit. For this example, the spindle is
driven by a servo-controlled DC motor with encoder (Micromo part #
3557K012CR). A serial port converter U. R. Kerr part # Z232-485)
and motor control board U. R. Kerr, PIC-SERVO) provide a
communication interface between the PC and motor. A slip-ring
(Litton part # AC6023-24) provided the electrical connection
between the rotating platform and the stationary control
system.
[0228] The microfluidics disc was manufactured through machining of
acrylic using computer/numerical code machining as described above.
The disc was then vapor polished by exposure to vapor from boiling
methylene chloride to remove machine marks and smooth the machined
surface.
[0229] Paraffin wax valves with a melting point of about 54.degree.
C. were inserted by pipetting a small amount of melted wax into the
sacrificial valve capillary and quickly pressing the wax flat with
a flat edge before it solidified. The excess was wiped away from
the edges of the channel, and extra wax on either end of the
channel was cut off using an Exacto blade. After the disc was
completely assembled (as described below), 5V was applied across
each of the leads corresponding to a wax valve, and the wax was
allowed to melt and re-crystallize within the channel, allowing the
wax to continuously cover the cross-section of the channel.
[0230] A piece of Whatman filter paper #54 and piece of frit
material (obtained from Porex, #X-4588, having a 70 .mu.m pore
size) were cut to the width of the slot at the bottom of chamber
1005. The filter paper and frit material, with the "shiny" side
facing toward the filter paper, were inserted into the slot. The
frit material was placed on the side of the slot closest to the
center of the disc. The orientation of the filter paper was
unimportant. The height of the filter paper and frit material were
then cut to be equal to the height of the disc.
[0231] A piece of glass fiber filter (GF-F obtained from Whatman,
ADDRESS) and a piece of frit material, as above, were cut to the
width of slot 1029 in binding chamber 1027. The glass fiber filter
was placed into the slot, and then the frit material was inserted
behind the glass fiber, on the side closest to the edge of the
disc, with the shiny side facing the glass fiber filter. The height
of both materials was then cut to be equal to the height of the
disc.
[0232] A fluorinated coating (Cytonix part # ME00) Perfluorocoat
was painted into the channels X, Y, Z. The excess perfluorocoat was
wiped away from the surrounding areas of the disc, and then a sharp
object such as an Exacto blade was run through the channel to
ensure that it was not blocked. The disc was then cured at
approximately 70.degree. C. for 1 hour to allow the fluorinated
coating to set.
[0233] A piece of adhesive (3M part # 7953 MP) was then placed
across the entire surface of the disc and carefully sealed around
the edges of all of the chambers. A Mylar sheet with silver
conductive and carbon resistive inks corresponding to the positions
of the wax valves and the binding column was aligned with the disc
and adhered with the side containing the ink facing the fluidic
structures. Finally, another sheet of the same 3M tape was placed
across the back of the Mylar sheet surface and a non-machined
acrylic disc (comprising the insulating base layer) was placed
against this layer of tape to provide structural integrity to the
Mylar layer.
[0234] Plasmid DNA was isolated from about 40 .mu.L of a suspension
of an E. coli bacterial culture that carried the pTrcHis vector
containing the CRP insert was acquired from an overnight culture of
transfected E. coli grown up in Luria Bertani broth; the volume of
the suspension used contained between about 1.times.10.sup.8 to
about 1.5.times.10.sup.8 cells in the 40 microliter sample. The
reagent solutions used were adapted from a Qiagen MiniPrep kit
(QIAprep Miniprep Handbook, Qiagen GmbH, Max-Volmer-Strasse 4,
40724 Hildren, Germany) or made from raw materials. The alkaline
cell lysis solution was 200 mM NaOH with 1% sodium dodecyl sulfate
(SDS; weight to volume) corresponding to solution P2 in the
Miniprep kit. The precipitating solution was 1.0M potassium
acetate, pH5.5 and 3M guanidine hydrochloride, corresponding to
solution N3 in the Miniprep kit; this solution adjusts the pH of
the lysis mixture and precipitates large genomic DNA fragments,
SDS, and proteins; in addition, the chaotropic salt breaks the
hydration shell of DNA, allowing it to bind to silaceous materials.
The first and second wash solutions were 70% ethanol in water (v/v)
and corresponds to PE solution in the Miniprep kit; this solution
removed residual salts from the glass fiber matrix. The elution
buffer was 0.1.times.TE (where TE is 10 mM Tris-HCl, pH8 and 1 mM
EDTA) and it eluted bound plasmid DNA from the glass fiber
matrix.
[0235] The process steps for both benchtop controls and using the
platform of the invention were:
[0236] (1) mixing sample (about 50 microliters) with alkaline cell
lysis solution (about 50 microliters) to effect lysis
[0237] (2) mixing the resulting solution with the precipitating
solution (about 70 microliters)
[0238] (3) filtration of the fluid to remove precipitated materials
and cell debris
[0239] (4) addition to the binding matrix (glass fiber filter)
[0240] (5) two sequential washes of ethanol/water solution (70%
v/v) (using about 100 microliters for the first wash and about 150
microliters for the second wash step)
[0241] (6) elution of plasmid DNA using about 40 microliters of
0.1.times. TE solution.
[0242] Steps (1)-(6) are modified from the Qiagen Miniprep
protocol; the only alterations from the published protocol are in
ratios of volumes of the solutions. The volumes listed above were
used both for the bench controls and on the disc. The materials and
procedures for the bench controls are as in the Miniprep kit; the
discs of the invention were run after loading fluids through the
following steps:
[0243] 1) accelerate to 1000 RPM. Alkaline cell lysis solution
flowed from reservoir 1016 into mixing chamber 1005 (shown in FIGS.
17a-c; the arrow marked co shows the rotational direction
(arbitrarily chosen to be counter-clockwise)).
[0244] 2) agitate 5 times, using complete stops and accelerations
of 500 rpm/s, to mix the solution and the sample (shown in FIGS.
17d-e).
[0245] 3) accelerate to 1000 RPM.
[0246] 4) apply voltage to leads corresponding to sacrificial valve
at 1021. Precipitating solution driven into 1005 (FIG. 17f).
[0247] 5) mix as above in step (2)
[0248] 6) accelerate to 1000 RPM. Apply voltage to leads
corresponding to sacrificial valve at 1012. Solution released from
1005 onto binding column 1027 (FIG. 17g). Maintain at 1000 RPM for
a time sufficient to completely move the precipitated mixture
through the glass filter and into waste chamber 1034.
[0249] 7) apply voltage to leads corresponding to sacrificial valve
at 1037, releasing first wash solution. Maintain rotational speed
at 1000 RPM until first wash solution passed completely through
glass fiber into waste chamber 1034 (shown in FIG. 17g)
[0250] 8) apply voltage to leads corresponding to sacrificial valve
1048, releasing second wash solution. Maintain at 1000 RPM until
second wash solution completely passed through glass fiber into
waste chamber 1034. (shown in FIG. 17h)
[0251] 9) apply voltage to leads corresponding to sacrificial valve
at 1032, opening the sample collection chamber.
[0252] 10) 5 seconds later, apply voltage to leads corresponding to
sacrificial valve 1058, releasing elution buffer.
[0253] 11) Maintain platform rotation at 1000 rpm until all elution
buffer washed through glass fiber filter into sample collection
chamber. Maintain rotational speed for at least an additional
minute after no additional elution buffer appears to be flowing
through microchannel 1032. (shown in FIGS. 17j-k).
[0254] 12) Remove fluid sample from platform using port 1055.
[0255] For both bench controls and samples run on the discs of the
invention, the recovered fluid was dried in a Speedvac (Savant
CorporationDrying serves to reduce the volume of liquid (and
increase the concentration) to be analyzed; it also removes
residual ethanol. The dried sample was then resuspended in 10
microliters of deionized water. In some cases a restriction enzyme
such as AlwN1 (New England Biolabs, Beverley, Mass.), a 9-base
cutter, was used to digest the sample. This enzyme is sensitive to
residual salt and as a result serves as a test of sample
cleanliness. In all cases, either all or some of the undigested
sample was resolved using ethidium bromide gel electrophoresis on a
1% agarose gel in 1.times. TBE buffer. The resulting bands when
compared to a size ladder, are indicative of the size of the
plasmid DNA in the sample. The larger genomic DNA, if contaminating
the sample, would be visible in the wells of the gel. For digested
samples, complete cutting of the sample, with no residual uncut
band, indicates the cleanliness of the sample.
[0256] FIG. 18 shows a gel that illustrates the purity of the
sample by using several different restriction enzymes. First, the
uncut sample run alone on the gel shows the plasmid in several
different conformers with very little salt in the sample itself.
The plasmid was cut with EcoRV enzyme, which linearizes the plasmid
and causes all of the conformers to run at one size against the
ladder. This demonstrates that all the conformers can be cut. The
second digestion was a double digestion using both the XhoI and
HindIII sites. These sites fall to either side of the CRP insert
and results in two long fragments and the 653 base insert fragment.
The final as digest was AlwN1. This is a nine-base cutter and is
highly sensitive to salt contamination in the product. The results
of these assays demonstrates that plasmid DNA obtained using the
platforms of the invention is comparable in quality and purity to
plasmid DNA isolated using conventional benchtop methods.
[0257] FIG. 19 shows the results of an assay for genomic DNA
contamination. PCR is performed using plasmid-specific primers as
well as primers specific to a fragment of the E. coli genomic DNA.
The negative and positive controls used genomic DNA and genomic
primers. The first amplification series show the expected amplified
fragment at decreasing amounts of plasmid template DNA using
plasmid-specific primers. The second amplification series show
amplification (or lack thereof) of a genomic DNA fragment using
genomic DNA primers and plasmid DNA template.
[0258] As can be seen, successive 10-fold dilutions yield
significant PCR product using plasmid-specific primers through
1:1000 dilution; only at 1:10000 dilution was little amplification
obtained. By contract, genomic DNA-specific primer shows
amplification only for the neat sample and 1:10 dilution. These
observations indicate an approximately 1000-fold excess of plasmid
to genomic DNA.
EXAMPLE 4
PCR of Genomic DNA from E. coli
[0259] A microfluidics platform as depicted in FIGS. 6, 7, 24, 25
and 26 was used to amplify a DNA target from samples of E.
coli.
[0260] FIG. 6 gives an exploded view of the two main components of
the microfluidics platform. A machined fluidics disk 701 is bonded
to a screen printed electrical circuit disk 710.
[0261] FIG. 7 shows an array of eight cycling chambers 703 within
the fluidics disk. The thermal cycling chambers are circular with a
diameter of 7 mm and depth of 0.5 mm. Each chamber has a reaction
mixture loading channel 704 and an air channel 702. Both channels
are 0.5 mm wide by 0.5 mm deep. The air channel helps to prevent
liquid loss upon heating, as vapors cool and condense along its
walk before spinning back down to the reaction chamber.
[0262] FIG. 24 shows how resistive heaters are arrayed on the
electrical circuit disk. The resistive heater patches 713 are
squares, 10 mm on a side. The heater dimensions were chosen to be
larger than the cycling chamber dimension to minimize thermal
gradients across the cycling chamber. Electrical current is
supplied to the heaters through positive 712 and ground 711
leads.
[0263] FIG. 25 is a cross-sectional view of a cycling chamber, bond
layers, and resistive heater. The fluidics disk 701 is mated to a
0.1 mm thick mylar sheet 726 (ICI part # ST505) using double sided
tape 727 (3M part # 7953 MP). The double sided tape is removed from
the area under the reaction chamber to minimize reaction
contamination from the tape. The mylar layer 726 is mated to the
electrical circuit layer 710 using double sided tape 723. These two
layers are radially aligned to ensure that the reaction chamber
array lines up with the resistive heater array. A thermally
conductive elastomer 724 (Bergquist part # CPU Pad) is inserted
between the resistive heater 713 and mylar sheet 726 to minimize
possible thermal gradients across the cycling chamber. A 0.1 mm
thermocouple 725 (Omega part # CHAL 005--type K), to be used for
temperature cycling control, is inserted between the mylar sheet
726 and conductive elastomer 724. The electrical circuit layer 710
is mated to a bottom polycarbonate support disk 721 using double
sided tape 722.
[0264] The microfluidic structures were manufactured through
machining of polycarbonate disks using a Light Machines VMC5000
milling machine running "Benchman" software (Light Machines
Corporation, Manchester, N.H.). Structures were designed using a
computer drafting program and converted to computer machine code.
The disc was cleaned with ethanol and then air, then polished by
exposure to vapor from boiling methylene chloride to remove surface
imperfections.
[0265] The electrical circuit disk was fabricated with
screen-printing techniques known by those skilled in the art and
also specifically disclosed in U.S. Pat. No. 6,063,589.
Carbon-based resistive ink (Dupont, 7102/7082 blend) was used to
print the resistive heaters, and silver-based ink (Dupont, 5028)
was used to print the conductive leads onto 0.1 mm thick mylar
sheet.
[0266] The instrument used for controlling the rotational profile
and thermal cycling consisted of a personal computer, interface
electronics between the PC and a servo-controlled drive motor and
interface electronics between the PC and the screen-printed
circuit. For this example, the spindle is driven by a
servo-controlled DC motor with encoder (Micromo, 3557K012CR). A
serial port converter G. R. Kerr, Z232-485) and motor control board
G. R. Kerr, PIC-SERVO) provide a communication interface between
the PC and motor. A slip-ring (Litton, AC6023-24) provided the
electrical connection between the rotating platform and the
stationary control system. The temperature-dependent voltage
measured by the thermocouple was converted to current using a
miniature transmitter (Omega part # TX91A) and output through the
slip ring. This current was converted to a voltage and read with a
commercially available analog/digital board (Computer Boards part #
CIO-DAS1600) within the PC. The voltage across the resistive heater
was also applied through the slip-ring and was varied to drive the
temperature to the desired temperature.
[0267] A constant power control loop was used to control reaction
chamber temperature. Empirical data showed that to maintain
temperatures from 60 C to 100 C required power from 0.4 W to 1.2 W.
In this example, the PC software control program read the
thermocouple temperature and output a control voltage proportional
to the setpoint temperature. This voltage was input to a constant
power circuit, which was in series with the resistive heater. As
temperature increased, heater resistance increased. To maintain
constant power, the circuit decreased its output current. Cooling
at zero power was provided by convection from the exposed surfaces
of the platform and was aided by rotating the disk at a constant
speed of 500 rpm. Heating rates were as high as 1.5 C/sec and
cooling rates were 1.0 C/sec.
[0268] The disk was mounted on to the motor spindle through a
center hole in the disc 705. The slip ring was secured to the top
of the disk using a screw. The slip ring lined up with the electric
circuit layer so that the appropriate power control and temperature
measurement leads were connected.
[0269] The solutions PCR consisted of deionized water, E. coli
genomic template (Sigma, St. Louis, Mo.), primer set EBGA that
amplifies a 215 base pair portion of the beta-galactosidase codon
of the E. coli genomic DNA (Research Genetics) and Ready-to-Go
beads (Amersham Pharmacia). The EBGA forward sequence is given by
5'-ACCTGCATCACCAGCTGCTT-3' (SEQ ID No.: 7) and the EBGA reverse
sequence is given by 5'-CGATGATCCTCATTGCTTATTCTC-- 3' (SEQ ID No.:
8). Denaturation, annealing, extension temperatures were chosen to
be 95.degree. C., 60.degree. C. and 72.degree. C., respectively.
The PC software setpoints were 100.degree. C., 60.degree. C., and
72.degree. C. The difference between setpoint and obtained
temperatures is expected based on the location of the thermocouple
beneath the reaction chamber and the temperature gradient through
the disk.
[0270] FIG. 26 shows the gel electrophoresis output after running
30 cycles in this system. The results compare favorably with
amplification performed on a commercial thermal cycler (MJ Research
model # PTC-100), also shown in this gel.
[0271] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
ail modifications or alternatives equivalent thereto are within the
spirit and scope of the invention.
Sequence CWU 1
1
8 1 30 DNA Escherichia coli 1 agtaccgcaa atcgccatca aaagtaatgc 30 2
29 DNA Escherichia coli 2 gtcagttcgc ctttcagagg aataaccgc 29 3 23
DNA Escherichia coli 3 ccgagacaga acttaatggg ccc 23 4 21 DNA
Escherichia coli 4 acaacaactg gcgggcaaac a 21 5 20 DNA Bos sp. 5
acccacactg tgcccatcta 20 6 17 DNA Bos sp. 6 cggaaccgct cattgcc 17 7
20 DNA Escherichia coli 7 acctgcatca ccagctgctt 20 8 24 DNA
Escherichia coli 8 cgatgatcct cattgcttat tctc 24
* * * * *