U.S. patent application number 09/906411 was filed with the patent office on 2002-05-23 for integrated microvolume device.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Mathies, Richard A., Simpson, Peter C., Williams, Stephen J..
Application Number | 20020060156 09/906411 |
Document ID | / |
Family ID | 22827828 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020060156 |
Kind Code |
A1 |
Mathies, Richard A. ; et
al. |
May 23, 2002 |
Integrated microvolume device
Abstract
A fully integrated monolithic small volume PCR-CE device in
glass, or the like materials, is fabricated using thin film metal
heaters and thermocouples to thermally cycle sub-microliter PCR
volumes. Successful amplification of a PCR fragment is demonstrated
on a PCR-CE chip. The process utilizes a linear polyacrylamide
surface coating coupled with addition of BSA to the amplification
buffer was necessary to obtain amplification efficiencies
comparable to a positive control. The micro-reactor reduced
significantly the time required for amplification and the reaction
volume was in the sub-microliter regime. Likewise addressed are the
known problems connected with reliable microfabrication of metal
coatings and the insulating layers required to shield these layers
from the PCR reaction mix, and the longstanding unresolved issue of
exposed metal regions in the PCR-CE chip resulting in electrolysis
of water and bubble formation whenever a voltage is applied. The
instant teachings employ external heaters and thermocouples and, as
such, have alleviated many of these problems. Heaters and
thermocouples may still be thin film deposited after chip bonding
allowing for easy scale-up to multichannel devices. In addition,
direct deposition of these chip components insures good thermal
contact with the PCR reactor.
Inventors: |
Mathies, Richard A.;
(Berkeley, CA) ; Simpson, Peter C.; (Berkeley,
CA) ; Williams, Stephen J.; (Berkeley, CA) |
Correspondence
Address: |
Richard H. Zaitlen
PILLSBURY WINTHROP LLP
Suite 2800
725 South Figueroa Street
Los Angeles
CA
90017-5406
US
|
Assignee: |
Affymetrix, Inc.
|
Family ID: |
22827828 |
Appl. No.: |
09/906411 |
Filed: |
July 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09906411 |
Jul 16, 2001 |
|
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|
09221436 |
Dec 28, 1998 |
|
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Current U.S.
Class: |
204/601 ;
204/451; 204/453; 204/602; 204/605; 435/286.1; 435/287.2;
435/288.5; 435/303.1 |
Current CPC
Class: |
G01N 27/44743 20130101;
G01N 27/44791 20130101 |
Class at
Publication: |
204/601 ;
204/451; 435/287.2; 435/288.5; 435/303.1; 204/453; 204/602;
204/605; 435/286.1 |
International
Class: |
G01N 027/26; C12M
001/38; G01N 027/447; C12M 001/40 |
Goverment Interests
[0001] This work was supported in part by an NIST ATP grant to
Affymetrix, Incorporated which has been used in conjunction with
the Department of Chemistry of the University of California at
Berkeley, and the Molecular Dynamics Company.
Claims
We claim:
1. In a fully integrated glassine monolithic microvolume PCR-CE
device, the improvement which comprises: utitilizing thin film
metal heating means to maintain a predetermined range of
temperature for desired denaturations; and employing thermocoupling
means for assuring requisite cycling of sub-microliter PCR
volumes.
2. Device as defined in claim 1, further comprising a linear
polyacrylamide surface coating; and a means for passivating the
surface of an involved PCR mix.
3. Device as defined in claim 2, wherein said means for passivating
is BSA added to the amplification buffer.
4. Method of microfabricating the device of claim 3, comprising the
steps of: providing a means for delivering fluid samples a series
of PCR chambers as well as a means for removing the samples from
the chambers; disposing said thin film metal heating means on by
thin film deposition; and, harmonizing thermal cycling and
resistance temperature sensing and concomitant adjustments by means
of utilizing cycling control software further comprising heating
and cooling times as part of a user-set hold time such that the
hold time at each temperature does not begin until the actual
temperature is within a user-defined percentage of a predetermined
set point.
5. An integrated monolithic PCR reactor coupled to a microcapillary
electrophoresis system including an electrophoresis column, the
apparatus comprising in combination: a reaction cavity
microfabricated within two-bonded substrates defining a space
therebetween; a plurality of apertures functioning as access means
for introducing fluids into said reaction cavity; exit channel
means for connecting the reactor to the electrophoresis system and
for injecting material onto the electrophoresis column incorporated
therein; a heating means for regulating temperature disposed on the
outside of one of said bonded substrates; and, a thermocouple
disposed in a least one position selected from the group consisting
of within the space defined by said reaction cavity and between
said heater and said bonded substrate.
6. Apparatus as defined in claim 5, said heating means for
regulating temperature further comprising a thin film resistive
heater.
7. Apparatus as defined in claim 5, said heating means for
regulating temperature further comprising a Minko-type contact
resistance heater.
8. Apparatus as defined in claim 5, wherein said bonded substrate
upon which said means for regulating temperature is disposed is
about 1 mm thick or less and said other substrate is thicker.
9. Apparatus as defined in claim 5, heating means for regulating
temperature further comprising a heater disposed within said
reaction cavity and covered with an insulating layer.
10. Apparatus as defined in claim 5, said thermocouple is Au:Cr
type junction.
11. Apparatus as defined in claim 5, wherein said thermocouple is
disposed between said heater and said bonded substrate.
12. Apparatus as defined in claim 6, wherein said thermocouple is
covered with a dielectric layer.
13. Apparatus as defined in claim 6, wherein said thermocouple is
within the space defined by said reaction cavity.
14. Apparatus as defined in claim 1, each said substrate being at
least one material selected from the group consisting of soda lime
glass, borofloat glass, plastic, PMMA, and the like glassine
substances.
15. Device as defined in claim 1, further comprising an
oligonucleotide array disposed therein, said oligonucleotide array
including a substrate having a plurality of positionally distinct
oligonucleotide probes coupled to a surface of said substrate.
16. Device as defined in claim 1, wherein said fully integrated
glassine monolithic microvolume PCR-CE device further comprises at
least first and second planar members, said first planar member
having a first surface and wells disposed in said first surface,
said second planar member having a second surface, said second
surface being mated to said first surface whereby said wells are
contiguous with a respective plurality of cavities having curved
insides spaces defining said surface and well-mated
arrangement.
17. Device as defined in claim 16, wherein a temperature sensor is
deposited on said second surface wherein when said second surface
is mated with said first surface, said temperature sensor on said
second surface is positioned within said cavity whereby a
temperature at said temperature sensor is substantially the same as
a temperature within said plurality of cavities.
18. Device as defined in claim 5, wherein said supplemental means
for resistance temperature sensing further comprises a thermocouple
having a sensing junction positioned adjacent said cavity, and a
reference junction positioned outside of said cavity, said
thermocouple being electrically connected to a means for measuring
a voltage across said thermocouple.
19. Microfabricated reaction chamber system as defined in claim 5,
said thermocouple further comprising at least a first gold film
adjoined to a chromium film as said sensing junction and said
chromium film adjoined to a second gold film as said reference
junction.
20. Device as defined in claim 3, further comprising an effective
amount of four deoxynucleotide triphosphates, a nucleic acid
polymerase and amplification primer sequences disposed within said
fully integrated glassine monolithic microvolume PCR-CE device.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Area of the Art
[0003] The present invention relates to methods and apparatus for
assaying biomolecular characteristics and structural configurations
effective for use within the context of microfluidic loading and
transfer methods. Particularly, the present invention relates to
novel enhanced techniques for the facilitation of fabrication and
improvement of microapparatus including the development of a
microfabricated PCR reactor that is integrated on a CE chip, inter
alia.
[0004] 2. Description of the Prior Art
[0005] The "DNA Chip" has been heralded as the long awaited union
between contemporary microelectronic technology and the genetic
engineering arts. (Wade, N., "Meeting of Computers and Biology: The
DNA Chip", Apr. 8, 1997, Science Times, THE NEW YORK TIMES, N.Y.
Times New Service). The present invention constitutes another
aspect of the innovative cycle which was both the genesis of
contemporary `gene chip` technology and a prominent candidate for
its most utile application to date.
[0006] The Polymerase Chain Reaction ("PCR") is a powerful
procedure for amplifying and labeling long stretches of DNA using
chromosomal or plasmid DNA as well as labeled nucleotides, those
skilled in the art define the same as an in vitro technique for
rapidly synthesizing large quantities of a given DNA segment that
involves separating the DNA into its two complementary strands,
binding a primer to each single strand at the end of the given DNA
segment where synthesis will start, using DNA polymerase to
synthesize two-stranded DNA from each single strand, and repeating
the process.
[0007] Likewise, Capillary Electrophoresis ("CE") is a method of
using silica capillaries to separate a wide variety of solutes,
both charged and uncharged, and having particularly effective uses
for the separation of small peptides, proteins and the like
biomolecular moities, as in the case of the instant teachings.
[0008] By way of background, attention is called to co-pending U.S.
Ser. No. 08/535,875; filed Sep. 28, 1995, which is assigned a
common assignee, and was likewise invented by one of the present
inventors. The '875 app. is incorporated in its entirety herein by
reference, and provides valuable insight into the state of the
art.
[0009] Generic schemata which likewise comprise basic reactor
designs, including miniature temperature controlled reaction
chambers for carrying out a variety of synthetic and diagnostic
applications, for tasks from sizing of nucleic acids for
hybridization, chemical labeling, thermal cycling, nucleic acid
fragmentation, and transcription all of the way to rudimentary
sequencing were disclosed by the '875 app.
[0010] A large number of diagnostic and synthetic chemical
reactions require repeated cycling through a number of specific
temperatures to carry out the melting, annealing, and ligation or
extension steps which are part of the respective processes. By
reducing reaction volumes, the amount of time required for thermal
cycling may also be reduced, thereby accelerating the amplification
process. Further, this reduction in volume also results in a
reduction of the amounts of reagents and sample used, thereby
decreasing costs and facilitating analyses of increasingly smaller
amounts of material.
[0011] Similarly, in hybridization applications, precise
temperature controls likewise are used to obtain optimal
hybridization conditions. Finally, a number of other pre- and
post-hybridization treatments also require some degree of precise
temperature control, such as fragmentation, transcription, chain
extension for sequencing, labeling, ligation reactions, and the
like.
[0012] A number of researchers have attempted to miniaturize and
integrate reaction vessels for carrying out a variety of chemical
reactions, including nucleic acid manipulation. For example,
published PCT Application No. WO 94/05414, to Northrup and White
reports an integrated micro-PCR apparatus fabricated from thin
silicon wafers, for collection and amplification of nucleic acids
from a specimen. Similarly, U.S. Pat. Nos. 5,304,487 to Wilding, et
al., and U.S. Pat. No. 5,296,374 to Kricka, et al. discuss chambers
and flow channels micromachined from silicon substrates for use in
conjunction with the collection and analysis of cell samples.
However, neither of these references address the added constraints
of PCR, and how to protect adequately against the same.
[0013] The increased desire for automated chemical processes in
both analytical and synthetic applications has led to a need for
further and further miniaturization and integration of existing
processes and equipment for carrying out such processes.
[0014] For miniaturized DNA analysis the successful and reliable
coupling of PCR amplification and electrophoretic DNA separation
constitutes a particularly noteworthy and certainly laudable
aspiration. Integration of such techniques offers numerous
potential advantages in terms of speed, cost and automation.
Multiple reactors and separation channels have been envisaged by
artisans as part of a high throughput genetic analysis system. The
first effort which has become generally accepted among the
technical community as a reasonable attempt at a substantially
integrated PCR reactor on a CE chip was the hybrid device reported
by Woolley and coworkers in 1996 (Woolley, A. T., Hadley, D.,
Landre, P., deMello, A. J., Mathies, R. A. and Northrup, M A.,
1996, 68 Analytical Chemistry 4081-4086) as a part of a
collaboration likewise initiated by an NIST ATP Project.
[0015] Woolley's team integrated a silicon sandwiched PCR type of
reactor fabricated by Northrup and coworkers at LLNL with a glass
CE chip fabricated at UC Berkeley. The Si reactor was mated to a
side channel through a polypropylene sleeve. An HEC sieving matrix
was used as an electrophoretic valve to separate the PCR solutions
from the CE channels.
[0016] Much later, Ramsey and coworkers simply placed the PCR mix
in a plastic reservoir on the chip and thermally cycled the entire
chip on a conventional cycler. This was followed by injection as
done earlier. This cycler was much slower than the hybrid device
developed in the Berkeley-LLNL collaboration but did lead to
ostensibly successful amplifications (Waters, L. C., Jacobson, S.
C., Kroutchinina, N., Khandurina, J., Foote, R. S. and Ramsey, J.
M., 1998, 70 (1) Anal. Chem. 158-162).
[0017] By way of further background, attention is likewise called
to the following U.S. Pat Nos.:
1 4,821,997; 5,241,363; 5,554,276; 5,585,069; 5,593,838; 5,603,351;
5,632,876; 5,643,738; and, 5,681,484.
[0018] It is respectfully submitted that each of the cited
references merely defines the state of the art, or highlights
aspects of the problems addressed and ameliorated according to the
teachings of the present invention. The same are also fully
referenced, and upon review each is clearly distinguished, as will
be seen from the IDS filed concurrently herewith. Accordingly,
further discussions of these references has been omitted at this
time due to the fact that each of the same is readily
distinguishable from the instant teachings to one having a modicum
of skill in the art, as shall be denoued by the claims which are
appended hereto.
[0019] To date the present inventors are not aware of any
successful efforts to fabricate a fully integrated monolithic small
volume PCR-CE device in glass using thin film metal heaters and
thermocouples to thermally cycle sub-microliter PCR volumes.
Accordingly it is the longstanding need to address and ameliorate
this concern which is a primary focus of the teachings of the
present invention.
OBJECTS AND SUMMARY OF THE INVENTION
[0020] A submicroliter PCR type of a reaction chamber is taught for
the amplification of specific diagnostic targets using PCR, among
other things. Subject amplicons are then directly injected into
microfabricated CE channels for fragment size analysis, or use with
related biomolecular assays.
[0021] According to a preferred embodiment, PCR chambers and CE
channels are etched into a thickened glass substrate using chemical
etching. A thin (for example 0.20 mm) glass substrate is bonded to
the etched surface defining the chambers and channels. The thin
substrate utilizes a thermocouple or platinum resistance
temperature sensing device on its interior surface in combination
with a platinum or Peltier heater on the external surface for
driving thermal cycling.
[0022] Briefly stated, A fully integrated monolithic small volume
PCR-CE device in glass, or the like materials, is fabricated using
thin film metal heaters and thermocouples to thermally cycle
sub-microliter PCR volumes. Successful amplification of a PCR
fragment is demonstrated on a PCR-CE chip. The process utilizes a
linear polyacrylamide surface coating coupled with addition of BSA
to the amplification buffer was necessary to obtain amplification
efficiencies comparable to a positive control. The micro-reactor
reduces significantly the time required for amplification and the
reaction volume within the context of a sub-microliter regime.
[0023] Likewise addressed are the known problems connected with
reliable microfabrication of metal coatings and the insulating
layers required to shield these layers from the PCR reaction
itself, and the longstanding unresolved issue of exposed metal
regions in the PCR-CE chip resulting in electrolysis of water and
bubble formation whenever a voltage is applied. The instant
teachings employ external heaters and thermocouples and, as such,
have alleviated many of these problems. Heaters and thermocouples
may still be thin film deposited after chip bonding allowing for
easy scale-up to multichannel devices. In addition, direct
deposition of these chip components insures good thermal contact
with, for example, a PCR reactor.
[0024] According to a feature of the invention there is provided in
a fully integrated glassine monolithic small volume PCR-CE device,
the improvement which comprises; thin metal heating means, and
thermocoupling means for cycling sub-microliter PCR volumes
[0025] According to a further feature of the invention there is
provided a method for microfabrication of an integrated PCR-type of
device by positioning a series of chambers, including a
microcapillary electrophoresis system which further comprises a
multiplicity of fluidly communicant microtubules, on a chip-based
substrate, the improvement comprising; providing a means for
delivering fluid samples to the chambers as well as a means for
removing the samples from the chambers, disposing resistive heating
means on the chip-based substrate by thin film deposition, and
harmonizing thermal cycling and resistance temperature sensing and
concomitant adjustments by means of utilizing cycling control
software further comprising heating and cooling times as part of a
user-set hold time such that the hold time at each temperature does
not begin until the actual temperature is within a user-defined
percentage of a predetermined set point.
[0026] According to yet another feature of the invention there is
provided a microfabricated reaction chamber system, comprising, in
combination; a substrate member having at least a plurality of
cavities disposed thereon; a means for driving thermal cycling; a
supplemental means for resistance temperature sensing, an
electrical means for connecting with a power source for applying a
desired voltage, at least a software means for integrating said
means for driving thermal cycling, said supplemental means for
resistance temperature sensing, and said electrical means for
connecting whereby a desired power level flowing through said
electrical means for connecting and a desired temperature level is
maintained with respect to a range defined by the difference in
temperature of said at least one cavity and said supplemental means
for resistance temperature sensing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The above-mentioned and other features of this invention and
the manner of obtaining them will become more apparent, taken in
conjunction with the accompanying drawings. These drawings depict
only a typical embodiment of the invention and do not therefore
limit its scope. They serve to add specificity and detail, in
which:
[0028] FIG. 1 is a prior art example of a generic design for a
chip-based reaction chamber assembly;
[0029] FIG. 2 is a series of schematic illustrations showing the
evolution of two preferred embodiments of chip designs according to
the teachings of the present invention;
[0030] FIG. 3 is a series of three graphical representations
plotting time (abscissa) against fluorescence (ordinate) in an
analysis of an injection cross plug during a thermal cycling
experiment according to teachings of the embodiments of the present
invention;
[0031] FIG. 4 is a graphical representation of temperature profile
as a function of time during on-chip amplification of a 136 bp M13
fragment, and an expanded or detailed view of two cycles;
[0032] FIG. 5 is a series of electropherograms of plasmids
amplified with M13 primers;
[0033] FIG. 6 shows on chip amplification of a 487 bp fragment with
off-chip separation, the bottom view being a successful chip
amplification and the top view a positive control;
[0034] FIG. 7 graphically depicts on chip amplification of a 487 bp
pUC19 vector +insert; and,
[0035] FIG. 8 shows a chip amplified reaction flushed from the
reaction chamber with water sandwiched between a positive and a
negative control depiction.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present inventors, realizing that one of the most
desirable features of microfabricated analysis systems is the
ability to integrate multiple reaction processes onto a single
monolithic substrate (Woolley, A. T. and Mathies, R. A., 1994, 91
PNAS 11348-11352; Simpson. P. C., Woolley, A. T. and Mathies, R.
A., 1998 95 Proc. Natl. Acad. Sci. USA. 2256-2261) have developed
the teachings of the present invention. In this way the benefits of
miniaturization are imparted to all steps in an assay, and the
often time consuming and error prone manual transfer of samples
between the various steps can be eliminated.
[0037] Miniature reaction chambers are needed for carrying out a
variety of chemical reactions in fluid samples. In particular, the
present invention provides devices incorporating miniature reaction
chambers wherein the temperature of the chamber can be monitored
and controlled. Further, as miniaturized devices, the devices of
the invention provide the benefits of low volume reactions (e.g,
low sample and reagent volume requirements), high thermal transfer
rates, flexibility of applications and integratability of
additional functions, reproducible standardized mass production of
the devices, ability to perform multiple simultaneous
analyses/reactions in small spaces leading to greater automability,
and a variety of other advantages.
[0038] By "low volume" it is meant the reaction chambers of the
present invention will typically have a volume of from about 0.001
.mu.l to about 10 .mu.l. Preferably, the devices of the present
invention will have a volume of from about 0.01 .mu.l to about 1
.mu.l and more preferably, about 0.02 .mu.l to about 0.5 .mu.l.
[0039] The reaction chambers and devices of the present invention
have a wide variety of uses in chemical and biotechnology
applications where controllable and monitorable temperatures are
desirable, such as nucleic acid manipulation, e.g., amplification
by PCR, extension by polymerase, thermal cycling, labeling
reactions, sizing and hybridization and fragmentation reactions. In
particularly preferred embodiments, the reaction chambers and
devices herein described, can be used for PCR amplification, which
is extremely temperature dependent. PCR amplification generally
involves the use of one strand of the target nucleic acid sequence
as a template for producing a large number of complements to that
sequence.
[0040] Generally, two primer sequences complementary to different
ends of a segment of the complementary strands of the target
sequence hybridize with their respective strands of the target
sequence, and in the presence of polymerase enzymes and nucleoside
triphosphates, the primers are extended along the target sequence.
The extensions are melted from the target sequence and the process
is repeated, this time with the additional copies of the target
sequence synthesized in the preceding steps. PCR amplification
typically involves repeated cycles of denaturation, hybridization
and extension reactions to produce sufficient amounts of the target
nucleic acid. The first step of each cycle of the PCR involves the
separation of the nucleic acid duplex formed by the primer
extension.
[0041] Once the strands are separated, the next step in PCR
involves hybridizing the separated strands with primers that flank
the target sequence. The primers are then extended to form
complementary copies of the target strands. For successful PCR
amplification, the primers are designed so that the position at
which each primer hybridizes along a duplex sequence is such that
an extension product synthesized from one primer, when separated
from the template (complement), serves as a template for the
extension of the other primer. The cycle of denaturation,
hybridization, and extension is repeated as many times as necessary
to obtain the desired amount of amplified nucleic acid.
[0042] Typically, strand separation is achieved by heating the
reaction to a sufficiently high temperature for a sufficient time
to cause the denaturation of the duplex but not to cause an
irreversible denaturation of the polymerase enzyme (For example,
see U.S. Pat. No. 4,965,188, incorporated herein by reference).
Typical heat denaturation involves temperatures ranging from about
80.degree. C. to 105.degree. C. for times ranging from seconds to
minutes. Strand separation, however, can be accomplished by any
suitable denaturing method including physical, chemical, or
enzymatic means. Strand separation may be induced by a helicase,
for example, or an enzyme capable of exhibiting helicase activity.
For example, the enzyme RecA has helicase activity in the presence
of ATP. The reaction conditions suitable for strand separation by
helicases are known in the art.
[0043] Template-dependent extension of primers in PCR is catalyzed
by a polymerizing agent in the presence of adequate amounts of four
deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and
dTT) in a reaction medium which comprises the appropriate salts,
metal cations, and pH buffering system. Reaction components and
conditions are likewise well known in the art. Suitable
polymerizing agents are enzymes known to catalyze
template-dependent DNA synthesis.
[0044] The reaction chambers of the present invention are
particularly suited for use as a micro-PCR devices. In particular,
the precise temperature monitoring and control within the reaction
chamber allows the use of these chambers in the complex thermal
cycling programs used in PCR based operations. Accordingly, in a
specific aspect, the reaction chambers described herein may
incorporate effective amounts of the reagents used in PCR
reactions, as described above. By effective amount is meant that
the reagents are provided within the reaction chamber in sufficient
quantity and concentration, i.e., non-limiting amounts, so that
amplification of a target nucleic acid may be carried out. Such
amounts can be readily determined by those of ordinary skill in the
art and may be extrapolated from readily available sources in the
literature without undue experimentation.
[0045] The reaction chambers and/or devices of the present
invention can be designed and reproducibly fabricated in large
quantities from a solid substrate material using a variety of known
methods and materials. For example, injection molding techniques
may be used to fabricate all or a portion of the reaction chamber.
Materials suitable for injection moldings include a variety of
plastics and other polymers, e.g., polystyrene, polypropylene, etc.
Generally, the material from which the reaction chambers and
devices are to be fabricated will be selected so as to provide
maximum resistance to the full range of conditions to which the
device will be exposed, e.g., extremes of temperature, salt, pH,
application of electric fields, e.g., in electrophoretic analysis
embodiments, as well as compatibility with reagents and other
materials used in fabricating the devices. The teachings of the
present invention likewise contemplate, and have incorporated the
use of photoresistive plastics.
[0046] In preferred embodiments, the devices of the invention which
incorporate the miniature reaction chambers herein described are
made using microfabrication techniques generally used in the
semiconductor and microelectronics industries. These techniques
include film deposition processes such as spin coating,
electrodeposition, low-pressure vapor deposition, laser fabrication
processes, photolithographic methods such as UV or X-ray processes,
or etching processes which may be performed by either wet chemical
processes or plasma processes.
[0047] Where these microfabrication methods are used, it will
generally be desirable to fabricate the reaction chamber of the
invention from materials similar to those used in the semiconductor
industry, i.e., silica, silicon or gallium arsenide substrates. For
example, U.S. Pat. No. 5,252,294, to Kroy, et al., incorporated
herein by reference in its entirety for all purposes, reports the
microfabrication of a silicon based multiwell apparatus for sample
handling in biotechnology applications using the above described
methods.
[0048] Methods of etching substrates are well known in the art. For
example, the first sheet of a substrate, e.g., silica, may be
overlaid with a photoresist. A photolithographic mask may expose
the photoresist in a pattern which reflects the pattern of wells to
be etched on the surface of the sheet. After removing the exposed
photoresist, the exposed substrate may be etched to produce the
desired wells and the like. Generally preferred photoresists
include those used extensively in the semiconductor industry. Such
materials include polymethyl methacrylate (PMMA) and its
derivatives, and electron beam resists such as poly (olefin
sulfones) and the like
[0049] The miniature reaction chambers and devices of the present
invention are generally characterized by their relatively small
size and ability to be used for extremely small volumes of
reactants. As discussed further below, it has been discovered that
non-square reaction chambers are preferred to avoid the formation
of air pockets within the chamber itself. For example, round or
elliptical reaction chambers having the above-described volumes are
preferred according to the teachings of the present invention.
[0050] According to the instant teachings, the reaction chambers
described herein are fluidly connected to one or more analytical
devices or chambers, and comprise a preparative step for the
particular analysis to be performed. For example, the reaction
chamber may be fluidly connected to a chamber which includes an
oligonucleotide array as one surface of this latter chamber. The
chamber may have been used for preparation fragmentation,
amplification or labeling of nucleic acid fragments in a sample,
prior to introduction to the oligonucleotide array.
[0051] Oligonucleotide arrays generally include a substrate having
a large number of postitionally distinct oligonucleotide probes
attached to the substrate. These oligonucleotide arrays, also
described as "Genechips.TM.," have been generally described in the
art, for example, U.S. Pat. No. 5,143,854 and PCT patent
publication Nos. WO 90/15070 and 92/10092. These arrays may
generally be produced using mechanical synthesis methods or light
directed synthesis methods which incorporate a combination of
photolithographic methods and solid phase oligonucleotide synthesis
methods. See Fodor et al., Science, 251:767-777 (1991), Pirrung et
al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO
90/15070) and Fodor et al., PCT Publication No. WO 92/10092, all
incorporated herein by reference.
[0052] These references disclose methods of forming vast arrays of
peptides, oligonucleotides and other polymer sequences using, for
example, light-directed synthesis techniques. Techniques for the
synthesis of these arrays using mechanical synthesis strategies are
described in, e.g., U.S. Pat. No. 5,384,261, incorporated herein by
reference in its entirety for all purposes. Oligonucleotide arrays
may generally be used to identify a particular mutation, or the
presence of nucleic acids from an infectious agent, such as a virus
or bacteria.
[0053] In another aspect, the reaction chamber described herein may
be fluidly connected to a microcapillary electrophoresis device or
array, for carrying out a size based electrophoresis of a sample.
Microcapillary array electrophoresis generally involves the use of
a thin capillary which may or may not be filled with a particular
separation medium. Electrophoresis of a sample through the
capillary provides a size based separation profile for the
sample.
[0054] The use of microcapillary electrophoresis in size separation
of nucleic acids has been reported in, e.g., Woolley and Mathies,
Proc. Nat'l Acad Sci. USA (1994) 91:11348-11352, incorporated
herein by reference in its entirety for all purposes.
Microcapillary array electrophoresis generally provides a rapid
method for size based sequencing, PCR product analysis and
restriction fragment sizing. The high surface to volume ratio of
these capillaries allows for the application of higher electric
fields across the capillary without substantial heating,
consequently allowing for more rapid separations. Furthermore, when
combined with confocal imaging methods, these methods provide
sensitivity in the range of attomoles, which is comparable to the
sensitivity of radioactive sequencing methods.
[0055] Microfabrication of capillary electrophoretic devices has
been discussed in e.g., Jacobsen, et al., Anal. Chem. (1994)
66:1114-1118, Effenhauser, et al., Anal. Chem. (1994) 66:2949-2953,
Harrison, et al. Science (1993) 261:895-897, Effenhauser, et al.
Anal. Chem. (1993) 65:2637-2642, and Manz, et al., J. Chromatog.
(1992) 593:253-258. Typically, these methods comprise
photolithographic etching of micron scale capillaries on a silica
or other crystalline substrate or chip.
[0056] In many capillary electrophoresis methods, silica
capillaries are filled with an appropriate separation medium.
Typically, a variety of separation media known in the art may be
used in the microcapillary arrays. Examples of such media include,
e.g., hydroxyethyl cellulose, polyacrylamide and the like.
Generally, the specific gel matrix, running buffers and running
conditions are selected to maximize the separation characteristics
of the particular application, e.g., the size of the nucleic acid
fragments, the required resolution, and the presence of native or
denatured nucleic acid molecules.
[0057] In addition to its use in nucleic acid "fingerprinting" and
other sized based analyses, the capillary arrays may be used in
sequencing applications. In particular, gel based sequencing
techniques may be readily adapted for capillary array
electrophoresis. For example, capillary electrophoresis may be
combined with the Sanger dideoxy chain termination sequencing
methods, which would be known to those skilled in the art. (See
also Brenner, et al., Proc. Nat'l Acad. Sci. (1989) 86:8902-8906).
In these methods, the sample nucleic acid is amplified in the
presence of fluorescent dideoxy nucleoside triphosphates in an
extension reaction. The random incorporation of the dideoxy
nucleotides terminates transcription of the nucleic acid. This
results in a range of transcription products differing from another
member by a single base. Size based separation then allows the
sequence of the nucleic acid to be determined.
[0058] Referring now to FIG. 1, prior art example of a reaction for
use with respect to a microcapillary electrophoresis device is
shown. (See also Brenner, et al., Proc. Nat'l Acad Sci. (1989)
86:8902-8906). Artisans will readily perceive the required the
layout of the capillary channels, reaction chamber and deposited
electrical leads on a planar member. Likewise, those skilled in the
art will readily understand how the orientation whereby a second
illustrated planar member mates with the first. FIG. 1 generally
shows an overlaying of two planar members which form the overall
body of the device which incorporates the reaction chamber
connected to a microcapillary array.
[0059] In addition to including one or more of the above described
elements, the reaction chambers of the present invention include
one or more additional elements which aid in the particular
reaction/analytical operation of the reaction chamber, including,
e.g., mixers, pumps, valves, vents and the like.
[0060] Often, the convective forces resulting from the heating of a
fluid sample within a reaction chamber will be sufficient to
adequately mix that sample. However, in some cases it may be
desirable to provide additional mixing elements. A variety of
methods and devices may be employed for mixing the contents of a
particular reaction chamber. For example, mixing may be carried out
by applying external agitation to the reaction chamber. Typically,
however, the reaction chambers of the present invention have
incorporated therein, devices for mixing the contents of the
reaction vessel.
[0061] Examples of particularly suitable mixing methods include
electroosmotic mixing, wherein the application of an electric field
across the sample results in a movement of charged components
within the sample and thus the mixing of the sample. Alternative
suitable mixers include lamb-wave transducers which may be
incorporated into the reaction chambers. See, Published PCT
Application No. WO 94/05414.
[0062] The reaction chambers described herein will also typically
include a means for delivering a fluid sample to the reaction
chamber as well as a means for removing the sample from the
chamber. This may include a simple sample introduction and removal
port whereby the sample is manually introduced and/or removed from
the reaction chamber, as described above. However, as the reaction
chambers of the invention are typically integrated within devices
which include additional reaction/analysis chambers, it will
typically be desirable to include one or more micropumps for
transporting a fluid sample from one chamber to another.
[0063] A number of positive displacement micropumps have been
described for micron/submicron scale fluid transport including
lamb-wave devices, see for example, U.S. Pat. No. 5,006,749,
electrokinetic pumps, diaphragm pumps, applied pressure
differentials and the like. In particularly preferred embodiments,
applied pressure differentials are used to affect fluid transport
within the device, i.e., between two or more reaction chambers. In
particular, the device may be provided with a pressure or vacuum
manifold which can be used to selectively apply a pressure
differential between two reaction chambers, forcing a sample to
move from a high pressure chamber to a low pressure chamber.
[0064] Selective application of the pressure differentials can be
carried out manually, i.e., applying a vacuum or pressure to a
particular reaction chamber through an opening in the chamber, or
it may be carried out using a pressure manifold employing valves as
described herein, which valves may be selectively operated to
direct pressure or vacuum to a given reaction chamber upon demand,
or according to a programmed protocol.
[0065] As referenced above, valve structures can also typically be
included in devices which incorporate the reaction chambers herein
described. Typically, these valves will include, e.g., a
deflectable diaphragm which when in a non-deflected position, rests
against a valve seat blocking fluid flow between, e.g., a reaction
chamber and a fluid channel. Deflection of the diaphragm thus
allows fluid flow between the reaction chamber and fluid
channel.
[0066] For a number of applications, it may be desirable to include
a vent within a given reaction chamber. Typically, this will be the
case where reaction conditions result in the evolution or expansion
of gas or fluid within the chamber. Such events will typically be
fitted with a poorly wetting filter plug to allow for the passage
of gas, while retaining liquid.
[0067] Control of reaction parameters within the reaction chamber
may be carried out manually, but is preferably controlled via an
appropriately programmed computer. In particular, the EMF from the
thermocouple and the input for the power source will typically be
interfaced to a computer which is programmed to receive and record
data via an analog-digital/digital-analog (AD/DA) converter. The
same computer will typically include programming for instructing
the delivery of appropriate current to allow the reaction chamber
to follow any number of predetermined time/temperature profiles,
e.g., thermal cycling for PCR, and the like. Referring now to FIG.
2, the evolution some of the objects of the present invention is
set forth. Namely, it is noted that initially, a square reactor
design was investigated (FIG. 2a), and improved upon. Reservoirs,
wells or indentations 1, 2, 3 and were each utilized for the
cycling according to the instant teachings, and are extensions of
apertures extending through a top portion of the substrate-means
(chip) as shown in prior art FIG. 1.
[0068] At an etch depth of 8 .mu.m the volume of the reactor was
approximately I .mu.L. Previously, it had been noted that while
filling the chamber, air became trapped in the reactor comers.
During thermal cycling, expansion or the trapped air forced liquid
out of the reaction chamber. To overcome this problem the reactor
was redesigned as an ellipse (FIG. 2b). The elliptical design
reduced slightly the reactor volume (0.3 .mu.L) but allowed bubble
free filling of the chip. In addition, an extra hole (numbered 5 in
FIG. 2b) was drilled close to the reaction T to allow independent
filling of the reaction chamber.
[0069] Thus, inject and separation channels could be filled with
the HEC separation buffer while the PCR reactor contained only the
amplification mixture. The majority of the work according to
preferred embodiments of the present invention utilizes elliptical
reactor patterns, etched to a depth of from about 13- to about 22
.mu.m. Deep channels permit easy replacement of the viscous sieving
matrix. Likewise, it was discovered that most preferred embodiments
were those have outside heaters and using platinum films with an
etch depth of about 20 .mu.m.
[0070] By way of further general background to the instant
teaching, planar members are also referred to herein as
"substrates," "slides" or "chips." These planar members may be made
from a variety of materials, including, e.g., plastics (press
molded, injection molded, machined, etc.), glass, silicon, quartz,
or other silica based materials, gallium arsenide, and the like.
Preferably, at least one of the planar members is glass. The cavity
or well that forms the basis of the reaction chamber is generally
disposed within the first planar member, or may be a combination of
an aperture in the first and a well region or indentation in the
second. This cavity may be machined or etched into the surface of
the first planar member, or alternative, may be prepared in the
manufacturing of the first planar member, such as where the planar
member is molded part, e.g., plastic.
[0071] Referring now once again to FIG. 2, Temperature control for
the reaction chamber is provided by a resistive heater means 4
deposited within the reaction chamber. The resistive heater means
is shown in FIG. 2 as a thin film resistive heater deposited on the
bottom surface of the reaction well. Typically, the resistive
heater means comprises a thin resistive metal film, coated with an
insulating layer (not shown) to prevent electrolysis at the surface
of the heater, and/or electrophoresis of the sample components
during operation with fluid samples.
[0072] In preferred embodiments, the thin metal film is a thin
chromium film ranging in thickness from about 200 .ANG.. Deposition
of the heater may be carried out by a variety of known methods,
e.g., vacuum evaporation, controlled vapor deposition, sputtering,
chemical decomposition methods, and the like. The protective layer
over the heater may comprise a number of nonconductive materials,
e.g., a TEFLON coating, SiO.sub.2, Si.sub.3N.sub.4, and the like.
In particularly preferred embodiments, the heater may be coated
with a SiO.sub.2 layer. The SiO.sub.2 layer may generally be
deposited over the heater film using methods well known in the art,
e.g., sputtering. Typically, this SiO.sub.2 film will be from about
1000 .ANG. to about 4000 .ANG. thick.
[0073] The resistive heater is connected to electrical leads which
allow the application of a voltage across the heater, and
subsequent heating of the reaction chamber. A variety of conducting
materials may be used as the electrical leads, however, gold leads
are preferred. In particularly preferred embodiments, the
electrical leads comprise a gold/chromium bilayer, having a gold
layer of from about 2000 .ANG. to about 3000 .ANG. and a chromium
layer of from about 250 .ANG. to about 350 .ANG.. This bilayer
structure is generally incorporated to enhance the adhesion of the
gold layer to the surface of the substrate.
[0074] The temperature sensor may also be selected from other
well-known miniature temperature sensing devices, such as
resistance thermometers which include material having an electrical
resistance proportional to the temperature of the material,
thermistors, IC temperature sensors, quartz thermometers and the
like.
[0075] The reaction chambers described herein will typically be
incorporated in devices which include additional elements for
sample manipulation, transport and analysis. In particular, the
reaction chambers described herein will typically have an opening
which is adapted for receipt of a fluid sample. Typically, these
openings will include sealable closures which prevent leakage of
the sample introduced into the chamber during operation. Sealable
openings may include e.g., a silicone septum, a sealable valve, one
way check valves such as flap valves or duck-billed check valves,
or the like. Similarly, the reaction chamber may be provided with a
means for removing the sample following the particular reaction.
This may be the same as the sample introduction means, or may
include an additional sealable opening in the reaction chamber.
[0076] In addition to openings in the chamber for sample
introduction and removal, the reaction chambers herein described
may be fluidly connected to additional reaction chambers to carry
out any number of additional reactions. For example, one reaction
chamber may be used to carry out a fragmentation reaction.
Following this fragmentation reaction, the sample may be
transported to a second reaction chamber for, e.g., PCR
amplification of desired fragments, hybridization of the fragments
to an array. Similarly, a first reaction chamber may be adapted for
performing extension reactions, whereupon their completion, the
sample may be transported to a subsequent reaction chamber for
analysis, i.e., sequencing by capillary electrophoresis.
[0077] According to a currently contemplated best mode of the
design a further modified embodiment likewise facilitates ease of
filling, in addition, allows injection of the sample from the
"heart" of the reaction chamber (FIG. 2c), by adjusting the
position of supplemental aperture 5 positionally reactive to the
rear of the disclosed design.
[0078] Several different glass types and thickness have been
evaluated for PCR-CE chip fabrication. Soda lime glass, etched to a
depth of 8 .mu.m, was used in early designs. The etched plate and
cover plate thickness was 1.1 mm and devices were patterned and
etched according to Woolley et al. To allow greater etch depth,
D263 and borofloat glasses were used in later designs and etched
according to Simpson et al. Cover plate thicknesses of these
glasses were 200 and 300 .mu.m respectively. At 200 .mu.m
thickness, substrates were difficult to handle and, for multiple
processing steps, breakage was common.
[0079] Holes were drilled in individual etched bottom plates using
a 3/4 mm diamond-tipped drill bit, using the etched pattern as a
template. Substrates were bonded to a blank glass slide using a
resin/beeswax mixture to provide support and prevent break-out
during drilling. The resin/beeswax was removed by soaking the
substrate in trichloroethylene for 1 hour and, if necessary, by
brushing the substrate gently with a nylon brush.
[0080] In the case of D263 and soda lime glass, bonding was
performed at 550.degree. C. for 3 hours. For borofloat, the
temperature was increased to 624.degree. C. Re-bonding was
frequently required, since internal metal or insulating layers
caused variations in surface topography and poor bonding was
observed in these regions.
[0081] In addition to glass, plastic was also investigated as a
PCR-CE chip material. PCR reaction wells were formed by drilling an
appropriately sized hole in polymethylmethacrylate (PMMA). PMMA
plates were bonded together by heating at 135.degree. C. for 1 hour
and cooling slowly. The compatibility of PMMA with PCR is discussed
later.
[0082] Resistive heaters were deposited on the PCR-CE chip by thin
film deposition (.about.2000 .ANG.) of a metal on either the
internal or external surface of the PCR reaction chamber. Chrome or
platinum/titanium metal films were used. Titanium (.about.200
.ANG.) acts as an adhesion layer for platinum (.about.1500 .ANG.)
and the combination was found to adhere very well to glass. By
comparison chrome adhesion was worse. Chrome layers were deposited
by evaporation, while platinum/titanium were plasma sputtered. Both
techniques yielded films of comparable quality. Evaporation of
platinum/titanium films proved more difficult.
[0083] Chrome heaters suffered significantly from oxidation both
during the bonding process and subsequently during repeated thermal
cycling experiments. After bonding, a chrome heater had an
approximate resistance of 20.OMEGA.. This rose to above 100.OMEGA.
after several weeks of experimental usage. At high resistance
heating becomes increasingly more difficult (V=IR, P=I.sup.2R).
Platinum/titanium heaters were not found to suffer from this
problem.
[0084] Initial experiments were performed with heaters deposited on
the internal, etched surface of the chip. This approach, however,
was improved upon since currents required to heat the chip to
95.degree. C. caused degradation in the heater side-wall coating
and eventual loss in electrical continuity. The situation can be
likened to a fuse-blowing mechanism, whereby the metal melts when
the current exceeds a particular amperage. Many attempts were made
to thicken the metal side-wall coatings, including angling the
substrate relative to the source during chrome evaporation and
continually rotating the substrate during plasma deposition of
platinum/titanium.
[0085] As an alternative, heaters were deposited on the external
flat surface of the chip. Aside from overcoming problems with
side-wall coatings, this solution had a number of other advantages.
Firstly, since heaters were deposited after bonding, metal films
were not subjected to high temperatures which resulted in
oxidation. Secondly, heaters could be removed and redeposited on a
bonded chip. Thirdly, since the heater was not in direct contact
with the PCR reaction mixture, no insulating coating (such as
SiO.sub.2) was required to provide a PCR compatible surface.
[0086] On the downside, the 200 .mu.m separation of the heat source
from the reactor caused temperature lag. This however, proved only
a minor problem because equilibration times were fast and the
reactor temperature was still sensed directly by an internal
thermocouple.
[0087] Platinum/titanium films had a resistance in the range of
from about 2-5.OMEGA.. It was necessary to impedance match the ac
amplifier output with the heater resistance to prevent distortion
of the sine wave at higher voltage outputs. Electrical connection
to heaters was made using a conductive cement which could be
removed and reapplied at any time.
[0088] Temperature of the PCR reaction mixture was monitored during
thermal cycling by a thin film chrome/gold thermocouple. Sensing
and reference junctions of the thermocouple were formed by overlay
of a 1500 .ANG. chrome layer with a 1500 .ANG. gold layer. It was
necessary to use 100 .ANG. of chrome as an adhesion layer for the
gold. During chip bonding at >500.degree. C. these two layers
would diffuse into one another to form an alloy producing a
thermocouple with a response not predicted by consideration of the
difference in the Seebek coefficient for each metal. Thus, the
thermocouple on each chip required calibration prior to use,
although in general the response was in the range 55,000.degree.
C./V-65,000.degree. C./V. The measured resistance of each
thermocouple was around 100.OMEGA..
[0089] In common with heaters, thermocouple deposition on the
etched surface of the chip suffered from poor side-wall coating and
it was therefore necessary to deposit thermocouples on the flat
surface of the 300 .mu.m top plate, on the flip-side to the
heaters.
[0090] To gain electrical access to the thermocouples, two 1.4 mm
holes were drilled in the etched bottom plate prior to bonding.
After bonding, conductive cement was used to extend the connections
to the chip edge, where flat bladed copper clips could be
attached.
[0091] The oxidation of chrome regions of the thermocouple during
the bonding resulted in approximately 25% off all thermocouples
failing (R=.infin..OMEGA.). Alternative combinations of metals
(platinum/titanium and gold and platinum/titanium and silver) were
investigated to overcome this problem, but in these cases the emf
generated per .degree..degree.C., was less than for chrome/gold and
difficult to measure accurately. Metal/alloy combinations such as
copper/constantan were not available for deposition in the
microlab.
[0092] Since metal surfaces were found to inhibit PCR
amplification, an insulating layer was coated over the temperature
sensing junction of the thermocouple. In addition, the thermocouple
was electrically isolated from the solution, since application of
an injection voltage across the reactor caused electrolysis of
water and bubble formation. For D263 and sodalime glass, silicon
dioxide was used as an insulating layer, while a borofloat coating
was used if the chip was fabricated from borofloat. In both cases
the film was plasma sputtered to a depth of .about.2000 .ANG.. Over
gold regions of the thermocouple these coatings were poor,
suffering from pinholes and crazing. Coatings over chrome regions,
by comparison were considerably improved. A moderately good coating
of SiO.sub.2, or borofloat over the entire junction was achieved by
applying a final capping chrome layer over gold coated regions of
the thermocouple. This additional layer did not affect the
performance of the thermocouple.
[0093] Despite its distance from the heated region of the chip (4-5
cm), the thermocouple reference junction experienced a small
temperature rise during thermal cycling, especially at longer
denaturation hold times. The maximum increase was around 1.degree.
C. per 10 cycles. The increase was compensated for by periodic
adjustment of the reference set point in the PCR program. In
general, however, the temperature rise was <1.degree. C. over 30
cycles.
[0094] Each thermocouple was individually calibrated using a series
of temperature indicating fluids supplied by Omega. A thin smear
applied to the top surface of the chip dried almost instantly to
mark. Heating caused the mark to liquefy at a stated temperature.
Accuracy of melting was .+-.1% and the time response was on the
order of milliseconds. The temperature gradient across the chip was
assumed linear and calculated by simultaneously monitoring the
temperature on both sides of the chip during heating. Further
confirmation of the temperature accuracy was obtained by monitoring
the boiling point of ethanol and/or water.
[0095] Dependent on the chip design, a number of different
procedures were used to fill PCR-CE chips with the HEC separation
matrix and the PCR reaction mixture. The primary objective was to
fill the reactor with PCR mix and the remainder of the chip with
HEC. In this way the HEC acts as an electrophoretic valve,
restricting amplification to the heated reactor region and
preventing contamination of injection and separation channels with
PCR mix.
[0096] To fill reaction chambers bubble free, it was necessary to
first flush the chamber with ethanol to wet the reactor surfaces.
Ethanol has no inhibitory effect on Taq polymerase activity at the
10% level. For PCR-CE devices of the design shown in FIG. 2b, HEC
was flushed from well 3 of the separation channel as far as well 5
of the PCR chamber, thus filling inject and separation channels
with the sieving matrix. The PCR chamber was next flushed with
ethanol from well 4, followed by PCR reaction mixture. Due to the
viscosity of the HEC buffer and the resistance to flow in the
inject channel, this procedure fills only the reactor and no
reaction mix is observed to flow into the inject channel. HEC also
prevents flow of PCR reaction mixture during thermal cycling.
[0097] A PCR chip was filled with a solution of Linearized pUC19
and electropherograms in FIG. 3 represent analysis of the injection
cross plug before thermal cycling (a), after thermal cycling (b)
and after thermal cycling following application of an injection
voltage (c). Results show that the reaction mix remains confined to
the reaction chamber during thermal cycling and is only transported
to the injection cross after application of the appropriate
voltages.
[0098] Devices of the design shown in FIG. 2c were filled in a
different manner. Initially, HEC was flushed from reservoir 3 to a
point just below the cross channel. Ethanol, followed by PCR
reaction mix, was then flushed from reservoir 4, filling the
reactor and injection region. A small air bubble was trapped
between the amplification solution and the HEC. Next, HEC was
flushed from reservoir 4 again until the air bubble migrated to
reservoir 1. At this point the bubble was removed from the
reservoir and replaced by fresh HEC. By monitoring movement of the
bubble it was possible to ensure only the inject and separation
channels were filled with HEC.
[0099] During thermal cycling, reactor wells were filled with
mineral oil to prevent evaporation. In the absence of the oil,
liquid evaporated from the wells at different rates causing
siphoning. In addition, it was found that the lower surface tension
of the mineral oil (and thus better wetting capabilities) prevented
bubble formation. Ordinarily, rough drilled surfaces were poorly
wet with the PCR amplification mixture and during thermal cycling
trapped air would expand and force liquid out of the wells. This
was prevented when wells were filled with mineral oil.
[0100] One major advantage of a microfabricated PCR device is the
ability to do fast thermal cycling. A portion of a typical
temperature profile recorded for a glass PCR-CE device is shown in
FIG. 4. Heating rates in this case are 20.degree. C./sec whilst
cooling rates are 2.degree. C./sec. Cooling rates may be
significantly increased using forced air convection. The
temperature of the reactor was controlled using proportional band
control. Using this type of control the actual temperature never
reaches the set temperature, always a slightly lower value. The
temperature band in which the proportional control is active is set
by the gain parameter (set in the software).
[0101] The higher the gain value the narrower the proportional band
and the closer the actual temperature is to the set point. At lower
gain values the proportional band is wider and the temperature
difference between the actual and set temperatures is larger. Lower
gain, values are advantageous for precise temperature control. For
the majority of experiments a gain value of 20 was used. This gave
a temperature offset of 3.degree. C. at a set temperature of
95.degree. C. Offsets were accounted for by means of a blank
calibration cycle. The accuracy of temperature control at this gain
value was .+-.1.degree. C. determined at the denaturation
temperature.
[0102] Early versions of the thermal cycling control software
included heating and cooling times as part of the user-set hold
time. This was problematic on the first PCR cycle as heating
occurred from room temperature and the time taken to reach the set
temperature was longer than for subsequent cycles. Recently, the
program has been modified such that the hold time at each
temperature does not begin until the actual temperature is within a
user-defined percentage of the set point.
[0103] PCR amplification was performed using plasmids with M13
forward and reverse priming sites. Plasmids used were pBluescript
SK+, pUC19 (with an insert) and M13mp18. The use of plasmids as
templates is advantageous because of the high concentration of DNA
available. For example, the Perkin-Elmer HIV-1 PCR kit is supplied
with an HIV positive control at a concentration of 10.sup.3 copies
per .mu.L. By contrast dilution of plasmid targets permit starting
concentrations of 10.sup.6 copies and higher. While reamplification
or a reaction is possible to generate higher starting copy number.
Often this was undesirable due to the formation of non-specific
product. Table I shows starting template and amplicon size,
2 Template Amplicon size/bp pBluescript SK+ 260 pUC19 (with insert)
487 M13mp18 136
[0104] Table 1: PCR product size versus starting template. Primers
used for amplification were: 5'-CCCAG TCACG ACGTT GTAAA ACG-3'
(forward primer) and 5'-AGCGG ATAAC AATTT CACAC AGG-3' (reverse
primer). Standard cycling conditions on the PE480 were 95 (60
sees), 55 (60 sees), 72.degree. C. (90 secs) for 30 cycles.
[0105] CE separations of the amplified products are shown in FIG.
5. Referring to the figure, capillary electrophoresis separation of
M13 mp 18 (sized independently against Phix 174 at 120 bp) are
shown in FIG. 5a. FIG. 5b shows chip separation of pBluescript (260
bp) sized against pBr322-BBstN, while FIG. 5c is chip separation of
pUC19 (with insert 487 bp) sized against PBr322-BstN1. Analysis of
the M13 amplicon was performed on a capillary system, while
pBluescript and pUC19 amplifications were analyzed in a
microchannel.
[0106] The effect of the reactor surface on the efficiency of PCR
amplification was studied. A series of HIV PCR mixtures were flowed
through a chip reactor, containing an SiO.sub.2 coated
thermocouple, and then amplified under standard conditions in the
PE480. Amplification efficiencies of the flow-through reactions
were compared to positive control reactions (no chip contact) using
agarose gel electrophoresis. To obtain equivalent amplification it
was necessary to coat the surface of the glass reactor with linear
polyacrylamide (LPA) and add bovine serum albumin (BSA) to the PCR
buffer at concentration of 50 .mu.g/mL (or higher). An LPA coated
surface alone resulted in reduced amplification, while a naked
glass surface (no coating) was found to completely inhibit the PCR
reaction (no PCR band observed on a gel).
[0107] Absorption of Taq polymerase to the reactor walls was
identified as a prime investigatory concern. A 0.5 .mu.L spike of
Taq into an unsuccessful amplification followed by reamplification
yielded a positive result. However, it was not possible to simply
increase the starting Taq concentration. Even at 4.times. Taq no
band was observed after amplification of a reaction flowed through
a naked reactor. Thus, all on-chip PCR reactions were carried out
in an LPA coated reactor with 50 .mu.g/mL of BSA added to the
amplification buffer.
[0108] A PMMA surface was not found to significantly inhibit the
PCR reaction at all. Amplification of a pBluescript target was
carried out in PCR tubes coated with a thin layer of PMMA.
Amplification efficiencies compared to a positive control reaction
amplified in a polypropylene tube, were comparable. High resolution
separations of ds DNA (271/281 bp of Phi X-174 almost baseline)
were also possible in PMMA chips without coating of the channel
walls.
[0109] The efficiency of amplification in glass PCR-CE chips was
further evaluated by cycling PCR reactions in a chip containing no
heater or thermocouple components. Temperature cycling was
accomplished using the heating block of the PE480. Temperature was
monitored using the internal thermocouple of the instrument and an
externally chip-mounted copper-constantan thermocouple. Cycling
temperatures were adjusted to compensate for temperature gradients
across the chip. Amplified pUC19 (with insert) reactions were
flushed from the chip, collected into 5 .mu.L of water and analyzed
on a separate CE chip. Under standard cycling conditions
(95.degree. C. (60 s), 55.degree. C. (60 s), 72.degree. C. (90 s);
30 cycles) no product peak was observed in the separation.
[0110] A reduction in the hold time at the denaturation
temperature, however, yielded a successful amplification of the 487
bp fragment, with an amplification efficiency comparable to that of
the positive control reaction (FIG. 5). Since chip PCR reaction
volumes were sub-microliter and thermal equilibration was fast, it
is likely that 60 second hold times at 95.degree. C. were
sufficient to denature Taq polymerase early on in the reaction.
Clearly reduced hold times at set cycling temperatures is desirable
to speed chip amplification times. This data, however, demonstrates
that it is also critical to preserve enzyme activity.
[0111] Using the above experimental set-up, on-line amplification
and separation of reaction products was performed. Prior to thermal
cycling, the PCR reactor was filled with reaction mix and the
injection and separation channels with 0.75% HEC solution
containing 0.2 .mu.M thiazole orange. Access holes were again
filled with mineral oil to prevent evaporation. After thermal
cycling was complete, the mineral oil was removed and replaced with
the separation buffer. FIG. 6 shows successful amplification of the
PCR fragment.
[0112] A successful chip amplification of an M13mp 18 target
(on-chip heating and temperature control), with off-line capillary
electrophoresis separation of the amplified product, was also
demonstrated. On-chip analysis of the PCR reaction was not possible
because of metal deposition in the separation channel during the
fabrication process. Instead, the PCR sample was flushed from the
chip and collected into 5 .mu.L of water.
[0113] FIG. 8 shows a comparison of the chip amplified reaction and
positive and negative control amplifications performed in the
Perkin-Elmer instrument. Results indicate a comparable
amplification efficiency for the chip reaction compared with the
control. Despite the 10 fold dilution of the chip reaction, peak
heights are observed to be similar since samples were
electrokinetically injected onto the capillary (proportional
increase in stacking). Negative control reactions were identical to
positive controls, except that target DNA was not added to the
amplification reaction. As a further control, the unamplified
positive control reactions were injected onto the capillary. No
peak was observed for these reactions either (data not shown).
[0114] Having described preferred embodiments of the invention with
reference to the accompanying drawings, it is to be understood that
the invention is not limited to those precise embodiments, and that
various changes and modifications mat be effected therein by one of
skill in the art without departing from the scope or spirit of the
invention as defined in the appended claims.
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