U.S. patent application number 10/113246 was filed with the patent office on 2002-11-14 for chip-based isothermal amplification devices and methods.
This patent application is currently assigned to The Regents of the University of Michigan. Invention is credited to Beyer, Wayne F. JR., Burke, David T., Burns, Mark A., DeNuzzio, John D., Johnson, Brian N..
Application Number | 20020168671 10/113246 |
Document ID | / |
Family ID | 21860334 |
Filed Date | 2002-11-14 |
United States Patent
Application |
20020168671 |
Kind Code |
A1 |
Burns, Mark A. ; et
al. |
November 14, 2002 |
Chip-based isothermal amplification devices and methods
Abstract
Disclosed are methods and compositions for isothermal
amplification of nucleic acids in a microfabricated substrate.
Methods and compositions for the analysis of isothermally amplified
nucleic acids in a microfabricated substrate are disclosed as well.
The microfabricated substrates and isothermal amplification and
detection methods provided are envisioned for use in various
diagnostic methods, particularly those connected with diseases
characterized by altered gene sequences or gene expression.
Inventors: |
Burns, Mark A.; (Ann Arbor,
MI) ; Burke, David T.; (Ann Arbor, MI) ;
Johnson, Brian N.; (Ann Arbor, MI) ; DeNuzzio, John
D.; (Chapel Hill, NC) ; Beyer, Wayne F. JR.;
(Bahama, NC) |
Correspondence
Address: |
Thomas M. Boyce
FULBRIGHT & JAWORSKI L.L.P.
Suite 2400
600 Congress Avenue
Austin
TX
78701
US
|
Assignee: |
The Regents of the University of
Michigan
Ann Arbor
MI
|
Family ID: |
21860334 |
Appl. No.: |
10/113246 |
Filed: |
April 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10113246 |
Apr 1, 2002 |
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08974738 |
Nov 19, 1997 |
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6379929 |
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60031590 |
Nov 20, 1996 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/293.1 |
Current CPC
Class: |
B01J 2219/00961
20130101; B01L 3/502738 20130101; B01J 2219/00995 20130101; B01L
2400/0406 20130101; B01L 2400/0448 20130101; B01J 2219/00853
20130101; B01L 3/502707 20130101; B01J 2219/00891 20130101; C12Q
1/6844 20130101; B01L 2300/163 20130101; B01J 2219/00952 20130101;
B01L 2200/0605 20130101; B01L 2400/0688 20130101; B01L 2300/0861
20130101; B01L 7/00 20130101; B01L 2300/1805 20130101; B01L
2300/165 20130101; B01J 2219/00828 20130101; B01J 2219/00912
20130101; B01L 3/5027 20130101; B01L 2300/0887 20130101; B01L 7/54
20130101; B01J 2219/00831 20130101; B01J 2219/00783 20130101; B01J
19/0093 20130101; B01L 2400/0677 20130101; B01J 2219/00873
20130101; B01L 3/50273 20130101; B82Y 30/00 20130101; B01L 2200/10
20130101; B01L 7/52 20130101; B01L 2400/0442 20130101; B01J
2219/00826 20130101; B01J 2219/00871 20130101; B01J 2219/00986
20130101; B01L 2200/12 20130101; B01J 2219/00889 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 435/293.1 |
International
Class: |
C12Q 001/68; C12M
001/34; C12M 001/00 |
Goverment Interests
[0002] The government owns rights in the present invention pursuant
to grant number R01-HG01044 from the National Institutes of Health.
Claims
What is claimed is:
1. An apparatus for use in the isothermal amplification of a
selected nucleic acid, comprising a microfabricated substrate
defining at least a first channel, said at least a first channel
connected to a reaction chamber, and a means for isothermally
regulating the temperature of said reaction chamber.
2. The apparatus of claim 1, wherein said microfabricated substrate
further defines at least a first entry port connected to said at
least a first channel.
3. The apparatus of claim 1, wherein said microfabricated substrate
further defines at least a second channel directly or indirectly
connected to said reaction chamber.
4. The apparatus of claim 3, wherein said microfabricated substrate
further defines at least a second entry port connected to said at
least a second channel.
5. The apparatus of claim 3, wherein said at least a second channel
is connected to said at least a first channel at a point prior to
connection of said at least a first channel to said reaction
chamber.
6. The apparatus of claim 1, wherein biological reagents effective
to permit an isothermal nucleic acid amplification reaction are
disposed in said reaction chamber, or in a first or second channel
or reservoir that is directly or indirectly connected to said
reaction chamber.
7. A method for isothermal amplification of a selected nucleic
acid, comprising: a) providing a sample comprising said selected
nucleic acid, and reagents effective to permit an isothermal
amplification reaction, to a microfabricated substrate that defines
at least a first channel, said at least a first channel connected
to an isothermally regulated reaction chamber; and b) conducting an
isothermal amplification reaction to amplify said selected nucleic
acid.
8. The method of claim 7, wherein said microfabricated substrate
further comprises a flow-directing means system in operable
relation to said at least a first channel.
9. The method of claim 8, wherein said flow-directing means system
is separated from said at least a first channel by a liquid
barrier.
10. The method of claim 9, wherein said liquid barrier comprises a
first silicon oxide layer, a silicon nitride layer and a second
silicon oxide layer.
11. The method of claim 8, wherein said flow-directing means system
comprises a series of heating elements arrayed along said at least
a first channel.
12. The method of claim 11, wherein said heating elements are
comprised of aluminum.
13. The method of claim 7, wherein said sample is conveyed from
said at least a first channel to said isothermally regulated
reaction chamber by differential heating of said sample.
14. The method of claim 8, wherein said flow-directing means system
comprises a series of hydrophobic and hydrophilic surface
structures arrayed along said at least a first channel.
15. The method of claim 14, wherein said at least a first channel
is treated with a hydrophilicity-enhancing compound.
16. The method of claim 14, wherein said at least a first channel
is modified to comprise one or more hydrophobic regions.
17. The method of claim 8, wherein said flow-directing means system
comprises a gas source in fluid communication with said at least a
first channel.
18. The method of claim 7, wherein said microfabricated substrate
further defines at least a first entry port connected to said at
least a first channel.
19. The method of claim 7, wherein said microfabricated substrate
further defines at least a second channel directly or indirectly
connected to said reaction chamber.
20. The method of claim 19, wherein said microfabricated substrate
further defines at least a second entry port connected to said at
least a second channel.
21. The device of claim 19, wherein said at least a second channel
is connected to said at least a first channel at a point prior to
connection of said at least a first channel to said reaction
chamber.
22. The method of claim 7, wherein said microfabricated substrate
further defines at least a first reservoir directly or indirectly
connected to said at least a first channel or to said reaction
chamber.
23. The method of claim 7, wherein said microfabricated substrate
further defines a nucleic acid analysis component operably
connected to said isothermally regulated reaction chamber.
24. The method of claim 23, wherein said nucleic acid analysis
component is a gel electrophoresis channel.
25. The method of claim 24, wherein said nucleic acid analysis
component is a capillary gel electrophoresis channel.
26. The method of claim 23, further comprising a nucleic acid
detection means operably connected to said nucleic acid analysis
component.
27. The method of claim 26, wherein said nucleic acid detection
means is a DNA sensor means.
28. The method of claim 27, wherein said DNA sensor means detects a
radiolabel.
29. The method of claim 28, wherein said DNA sensor means is
ap-n-type diffusion diode.
30. The method of claim 27, wherein said DNA sensor means detects a
fluorescent label.
31. The method of claim 30, wherein said DNA sensor means is a
p-n-type diffusion diode combined with a wavelength filter and an
excitation source.
32. The method of claim 7, wherein said microfabricated substrate
is comprised of silicon, quartz or glass.
33. The method of claim 7, wherein said reagents comprise reagents
for conducting a Strand Displacement Amplification reaction.
34. The method of claim 7, wherein said reagents comprise reagents
for conducting a self-sustained sequence replication amplification
reaction.
35. The method of claim 7, wherein said reagents comprise reagents
for conducting a Q.beta. replicase amplification reaction.
36. The method of claim 7, wherein said reagents are prefabricated
into said reaction chamber, or into a first or second channel or
reservoir that is directly or indirectly connected to said reaction
chamber.
37. The method of claim 36, wherein said reagents are prefabricated
into said reaction chamber, first or second channel or reservoir in
a lyophilized form.
38. The method of claim 7, wherein said reagents further comprise a
DNA ligase.
39. The method of claim 7, wherein said reagents further comprise a
nuclease.
40. The method of claim 39, wherein said reagents further comprise
a restriction endonuclease.
41. The method of claim 7, wherein said sample is derived from an
animal having or suspected of having a disease.
42. The method of claim 41, wherein said sample is derived from a
human subject.
43. A method for detecting the presence of a selected nucleic acid,
comprising: a) introducing a sample suspected of containing said
selected nucleic acid, and reagents effective to permit an
isothermal amplification reaction, into a microfabricated substrate
defining at least a first channel, said at least a first channel
connected to an isothermally regulated reaction chamber; b)
conducting an isothermally regulated amplification reaction to
amplify said selected nucleic acid; and c) detecting the presence
of the amplified selected nucleic acid, wherein the presence of the
amplified selected nucleic acid confirms the presence of said
selected nucleic acid in said sample.
44. The method of claim 43, wherein said sample is derived from an
animal or human subject.
45. The method of claim 43, wherein said sample is derived from an
animal having or suspected of having a disease, and the presence of
said selected nucleic acid is indicative of the disease state.
46. The method of claim 43, wherein said sample is derived from an
animal having or suspected of having a disease, and the absence of
said selected nucleic acid is indicative of the disease state.
47. The method of claim 43, wherein said selected nucleic acid
comprises a mutation not present in the wild-type version of the
selected nucleic acid.
48. The method of claim 43, wherein said selected nucleic acid is
detected by means of a detectable label incorporated into the
amplified selected nucleic acid by said isothermal amplification
reaction.
49. The method of claim 43, wherein said selected nucleic acid is
detected by means of a labeled nucleic acid probe.
50. The method of claim 48, wherein said detectable label is a
radioisotopic, enzymatic or fluorescent label.
51. A method of making a device for use in isothermal nucleic acid
amplification, comprising preparing at least a first
microfabricated device, chip or wafer defining at least a first
channel that is operably connected to an isothermally regulated
reaction chamber.
52. A kit for conducting isothermal amplification of a selected
nucleic acid, comprising, in suitable container means: a) at least
a first microfabricated substrate defining at least a first
channel, said at least a first channel connected to an isothermally
regulated reaction chamber; and b) reagents effective to permit an
isothermal nucleic acid amplification reaction.
53. The kit of claim 52, wherein said at least a first
microfabricated substrate further defines a nucleic acid analysis
component operably connected to said isothermally regulated
reaction chamber.
54. The kit of claim 53, wherein said at least a first
microfabricated substrate further comprises a nucleic acid
detection means operably connected to said nucleic acid analysis
component.
55. The kit of claim 52, wherein said reagents effective to permit
an isothermal nucleic acid amplification reaction are disposed in
said reaction chamber, or in a first or second channel or reservoir
that is directly or indirectly connected to said reaction
chamber.
56. A diagnostic system for identifying a selected nucleic acid,
comprising at least a first microfabricated substrate defining at
least a first channel that is connected to at least a first
isothermally regulated reaction chamber; said diagnostic system
further comprising a nucleic acid analysis component and a nucleic
acid detection means in operable association with the reaction
chamber of said at least a first microfabricated substrate.
57. The diagnostic system of claim 56, further comprising, in
operable association, at least a second microfabricated substrate
defining at least a second channel that is connected to at least a
second isothermally regulated reaction chamber.
58. The diagnostic system of claim 57, wherein said at least a
first and at least a second microfabricated substrates are operably
connected in series to a single nucleic acid analysis component and
nucleic acid detection means.
59. The diagnostic system of claim 57, wherein said at least a
first and at least a second microfabricated substrates are operably
connected in parallel to at least two distinct nucleic acid
analysis components and nucleic acid detection means.
60. The diagnostic system of claim 56, further comprising reagents
effective to permit an isothermal nucleic acid amplification
reaction.
61. The diagnostic system of claim 60, wherein said reagents are
disposed in said reaction chamber, or in a first or second channel
or reservoir that is directly or indirectly connected to said
reaction chamber.
62. A method of making a nucleic acid diagnostic system, comprising
preparing at least a first microfabricated substrate defining, in a
series of operable associations, at least a first channel, an
isothermally regulated reaction chamber, a nucleic acid analysis
component and a nucleic acid analysis detection means.
Description
[0001] The present application claims the priority of co-pending
U.S. Provisional Patent Application Serial No. 60/031,590, filed
Nov. 20, 1996, the entire disclosure of which is incorporated
herein by reference without disclaimer.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the field of
molecular biology, and relates to methods for amplifying nucleic
acid target sequences in microfabricated devices. It particularly
relates to isothermal methods for amplifying nucleic acid targets
in microfabricated devices. The present invention also relates to
methods of detecting and analyzing nucleic acids in microfabricated
devices.
[0005] 2. Description of Related Art
[0006] In vitro nucleic acid amplification techniques have provided
powerful tools for detection and analysis of small amounts of
nucleic acids. The extreme sensitivity of such methods has lead to
their development in the fields of diagnosis of infectious and
genetic diseases, isolation of genes for analysis, and detection of
specific nucleic acids as in forensic medicine.
[0007] Nucleic acid amplification techniques may be grouped
according to the temperature requirements of the procedure. Certain
nucleic acid amplification methods, such as the polymerase chain
reaction (PCR.TM.--Saiki et al., 1985), ligase chain reaction
(LCR--Wu et al., 1989; Barringer et al., 1990; Barony, 1991),
transcription-based amplification (Kwoh et al., 1989) and
restriction amplification (U.S. Pat. No. 5,102,784), require
temperature cycling of the reaction between high denaturing
temperatures and somewhat lower polymerization temperatures. In
contrast, methods such as self-sustained sequence replication (3SR;
Guatelli et al., 1990), the Q.beta. replicase system (Lizardi et
al., 1988), and Strand Displacement Amplification (SDA--Walker et
al., 1992a, 1992b; U.S. Pat. No. 5,455,166) are isothermal
reactions that are conducted at a constant temperature, which is
typically much lower than the reaction temperatures of temperature
cycling amplification methods.
[0008] The SDA reaction initially developed was conducted at a
constant temperature between about 37.degree. C. and 42.degree. C.
(U.S. Pat. No. 5,455,166). This was because the exo klenow DNA
polymerase and the restriction endonuclease (e.g., HindII) are
mesophilic enzymes that are thermolabile (temperature sensitive) at
temperatures above this range. The enzymes that drive the
amplification are therefore inactivated as the reaction temperature
is increased.
[0009] Methods for isothermal Strand Displacement Amplification,
which may be performed in a higher temperature range than
conventional SDA (about 50.degree. C. to 70.degree. C.,
"thermophilic SDA"), were later developed. Thermophilic SDA is
described in European Patent Application No. 0 684 315 and employs
thermophilic restriction endonucleases that nick the hemimodified
restriction endonuclease recognition/cleavage site at high
temperature and thermophilic polymerases that extend from the nick
and displace the downstream strand in the same temperature
range.
[0010] Photolithographic micromachining of silicon has been used to
construct high-throughput integrated fluidic systems for a variety
of chemical analyses. This technology is of particular interest for
the development of devices for analysis of nucleic acids, as in
their conventional formats such analyses are typically labor- and
material-intensive. Ideally, all of the processing steps of the
amplification reaction would be conducted on the microfabricated
device to produce a completely integrated nucleic acid analysis
system for liquid transfer, mixing, reaction and detection that
requires minimal operator intervention.
[0011] Silicon and glass devices are economically attractive
because the associated micromachining methods are, essentially,
photographic reproduction techniques. Silicon structures are
processed using batch fabrication and lithographic techniques.
These processes resemble those of printing where many features may
be printed at the same time. These processes permit the
simultaneous fabrication of thousands of parts in parallel, thus
reducing system costs enormously. Today, silicon fabrication
techniques are available to simultaneously fabricate micrometer and
submicrometer structures on large-area wafers (100 cm.sup.2),
yielding millions of devices per wafer and may be used to process
either silicon or glass substrates.
[0012] These characteristics have led to the proposal of silicon
and glass as a candidate technology for the construction of
high-throughput DNA analysis devices (Woolley and Mathies, 1994;
Northrup et al., 1993; Effenhauser et al., 1994). As mechanical
materials, both silicon and glass have well-known fabrication
characteristics (Petersen, 1982). Microfabricated devices for
biochemical and fluidic manipulation are undergoing development in
many laboratories around the world (Ramsey et al., 1995; McIntyre
1996). Over the past 10 years, a number of microfluidic devices
have been developed that allow the construction of miniaturized
"chemical reactors." Individual components of the system such as
pumps (Esashi et al, 1989; Zengerle et al., 1992; Matsumoto and
Colgate, 1990; Folta et al., 1992); valves (Esashi et al., 1989,
Ohnstein et al., 1990; Smits, 1990); fluid channels (Pfahler et
al., 1990); chromatographic and liquid electrophoresis separation
systems (Terry et al., 1979; Harrison et al., 1992b-g; Manz et al.,
1991; Manz et al., 1992) are available. Although an objective of
several research groups, complete silicon-fabricated nucleic acids
analysis systems are still at the earliest stages of
development.
[0013] Other components that have been microfabricated which are
applicable to nucleic acid analysis include elements for gel
electrophoresis (Zeineh and Zeineh, 1990; Heller and Tullis, 1992;
Effenhauser et al., 1994; Woolley and Mathies, 1994, 1995; Webster
et a., 1996); capillary electrophoresis (Manz et al. 1992, 1995;
Effenhauser et al., 1993; Fan and Harrison, 1994; Jacobsen et al.,
1994a; 1994b; Jacobson and Ramsey, 1995; Ocvirk et al., 1995; von
Heeren et al., 1996); synthetic oligonucleotide arrays (Fodor et
al., 1993; Schena et al., 1995; Hacia et al., 1996); continuous
flow pumps (Lintel, 1988; Esashi et al., 1989; Matsumoto and
Colgate, 1990; Nakagawa et al., 1990; Pfahler et al, 1990; Smits,
1990; Wilding et al., 1994; Olsson et al., 1995); discrete drop
pumps (Burns et al, 1996); enzymatic reaction chambers (Northrup et
al., 1994; Wilding et al., 1994b; Cheng et al., 1996);
optical/radiation detectors (Belau et al., 1983; Wouters and van
Sprakelaar, 1993; Webster et al., 1996); and multicomponent systems
(Harrison et al., 1992, 1995; Northrup et al 1994; Jacobsob and
Ramsey 1996).
[0014] To date, a number of devices have been micromachined,
including pumps and valves (Gravensen et al., 1993; Manz et al.,
1994; Colgate and Matsumoto, 1990, Sammorco et al, 1996); reaction
chambers (Woolley and Mathies, 1994; Wilding et al., 1994); and
separation and detection systems (Weber and May, 1989, Northrup et
al., 1993, Harrison et al., 1993; Manz et al., 1992; Jacobson et
al., 1994; Schoonevald et al., 1991; Van den Berg and Bergveld,
1995; Woolley et al., 1995). Some of these have been recently
integrated together to build pharmaceutical drug closing systems
(Lammerink et al., 1993; Miyake et al., 1993) and other
microchemical systems (Nakagawa et al., 1990; Washizu, 1992; Van
den Berg and Bergveld, 1995). One device is an integrated glass
system combining DNA restriction enzyme digestion and capillary
electrophoresis (Jacobson and Ramsey 1996). An alternative format
using high-density arrays of synthesized oligodeoxynucleotides has
been demonstrated as a DNA sequence detector (Fodor et al., 1993;
Hacia et al., 1996).
[0015] Nucleic acid targets have been successfully amplified by the
PCR.TM. on such microfabricated devices, often referred to as
"chips" (U.S. Pat. No. 5,498,392; Woolley et al., 1996; Shoffner et
al., 1995; Cheng et al., 1996; Wilding et al., 1994; U.S. Pat. No.
5,589,136; U.S. Pat. No. 5, 639,423; U.S. Pat. No. 5,587,128, U.S.
Pat. No. 5,451,500) and LCR (Cheng et al., 1996; U.S. Pat. No.
5,589,136). Evaporation due to repeated exposure to high
temperatures during thermocycling is a problem. Evaporation during
PCR.TM. has been controlled by immersing the channel in oil such
that the open ends are covered, but this makes recovery of the
amplified sample difficult.
SUMMARY OF THE INVENTION
[0016] The present invention overcomes the foregoing evaporation
and recovery drawbacks, and other deficiencies inherent in the
prior art, by providing compositions and methods for use in the
isothermal amplification of nucleic acids in microfabricated
devices. In contrast to the difficulties previously perceived to
exist and the prejudices in the art, the inventors found isothermal
amplification of nucleic acids using microfabricated devices or
"chips" to be surprisingly effective. In fact, the chip-based
isothermal amplification of the present invention was discovered to
be efficient at previously untested low temperatures, despite
potentially negative effects of surface chemistry and other
proposed problems, such as stagnant temperature gradients, reduced
diffusion and mixing, and inhibition of enzyme activity.
[0017] The invention thus generally provides an apparatus, system,
device or chip, or a plurality thereof, with isothermally regulated
reaction chambers, methods of constructing single-chip and
multiple-chip analytical systems, and methods for using such
devices, chips and systems in the isothermal amplification of
nucleic acids. The invention also provides for the analysis of the
amplification products using, e.g., sequencing, gel separation,
and/or detection of the amplification products in microfabricated
devices. Further methods of the invention therefore include
laboratory methods connected with nucleic acid analysis and
clinical methods connected with the diagnosis and prognosis of
disease states.
[0018] First provided by the invention are devices, chips, wafers
or an analytical apparatus or system(s), generally of a
microfabricated or micromachined type, for use in the isothermal
amplification of selected nucleic acids. Certain preferred devices
utilize the silicon chip or silicon wafer formats. In preferred
embodiments, the devices of the present invention are
"microdevices", preferably defining micromachined structures for
use with nanoliter volumes.
[0019] The apparati, devices or chips of the invention generally
comprise a microfabricated substrate or housing defining at least a
first transport channel, or microdroplet transport channel,
operably connected to at least a first reaction chamber, and at
least a first means for isothermally regulating the temperature of
the reaction chamber.
[0020] The "means for isothermally regulating the temperature of
the reaction chamber" may be an element, such as a particular
resistor, combination of resistors, feed-back temperature detector,
and/or circuitry for temperature control, that has not been
previously used in conjunction with a microfabricated device or
chip for use in nucleic acid amplification. More preferably, the
"means" for isothermally regulating the temperature of the reaction
chamber will be a "programmable means". That is, a series of
executable and controlled steps, preferably in the form of a
computer program, the implementation of which results in the
control of the temperature of the reaction chamber within narrow
limits, such that the temperature is "substantially constant".
These computer microprocessor or programmable means, although
readily prepared by those of skill in the art, have not previously
been proposed for use in combination with a microfabricated nucleic
acid amplification device.
[0021] The microfabricated substrate of the device, chip or system
is generally constructed so that application of a fluid in one or
more transport channels will result in the fluid being conveyed at
least to the reaction chamber. Accordingly, the microfabricated
substrate inherently has a "flow-directed fabrication". The
flow-directed fabrication or construction may be based upon
gravitational attraction, thermal gradients, gas or liquid pressure
differences, differences in hydrophobic and hydrophilic surface
structures, electrowetting, and/or differences in the dielectric
constant between reagent fluids applied to the substrate and the
air or surrounding media. The manner in which a directional flow
capability is provided to the substrate is not critical to the
invention, so long as the substrate, device or system ultimately
allows for the controlled manipulation of liquids or fluids applied
thereto, and effective merging and mixing where appropriate.
[0022] In the context of this invention, a "reaction" or
"amplification" generally refers to reactions involving nucleic
acid biomolecules, such as RNA and DNA. "Nucleic acid
amplification" generally refers to the process of increasing the
concentration of nucleic acid, and in particular, the concentration
of a selected nucleic acid and/or a defined piece of a selected
nucleic acid. "Amplified or amplification products" or "amplicons"
generally define the products resulting from execution of a nucleic
acid amplification reaction.
[0023] As used herein, the term "an isothermal amplification
reaction" refers to a nucleic acid amplification reaction that is
conducted at a substantially constant temperature. It will be
understood that this definition by no means excludes certain,
preferably small, variations in temperature but is rather used to
differentiate the isothermal amplification techniques from other
amplification techniques known in the art that basically rely on
"cycling temperatures" in order to generate the amplified products.
Thus, the present invention is distinguished from PCR, which
fundamentally rests on the temperature cycling phenomenon.
[0024] It will be further understood that although the isothermal
amplification reactions of the present invention will generally be
conducted at a substantially constant temperature, the overall
execution of the amplification, diagnostic or prognostic methods of
the invention may nonetheless require certain steps to be conducted
at different temperatures. For example, moving fluids or
microdroplets through the different channels or chambers defined on
the microfabricated substrate, and/or merging and mixing samples
and reagents, may involve alterations in temperature, e.g., as may
be achieved via the use of defined heating elements.
[0025] The microfabricated substrate or housing of the invention
may be fabricated from any one of a number of suitable materials.
The materials will preferably be of the type that can be
manipulated to define the channels, reaction chambers and other
components necessary for conducting the amplification methods, and
yet will be stable enough to permit repeated use in such methods
once the defining components have been etched or otherwise imparted
onto the substrate. Certain preferred examples include, but are not
limited to, silicon, quartz and glass.
[0026] The transport channels or "microdroplet transport channels"
defined in the substrate are generally pathways, whether straight,
curved, single, multiple, in a network, etc., through which
liquids, fluids and/or gases may be passively or actively
transported. The channels are generally etched into the silicon,
quartz, glass or other supporting substrate. The present invention
requires the presence of at least a first channel that functions to
allow the transport of a fluid sample into the reaction chamber. It
will be understood that such a channel need not be of a significant
minimum length, and that the term "channel" therefore refers to a
fluid-conveying section in functional terms, rather than to
defining a structure that is necessarily long and pipe-like.
[0027] The one or more channels in the substrate connect the
various components, i.e., keep components "in communication" and
more particularly, "in fluidic communication" and still more
particularly, "in liquid communication." Such components include,
but are not limited to, gas-intake channels and gas vents. In
certain other aspects of the invention "microdroplet transport
channels" may refer to channels configured (in microns) so as to
accommodate "microdroplets."
[0028] While it is not intended that the present invention be
limited by precise dimensions of the channels or precise volumes
for microdroplets, illustrative ranges for channels may be between
0.5 and 50/.mu.m in depth (preferably between 5 and 20 .mu.m) and
between 20 and 1000/.mu.m in width (preferably 500/.mu.m), and the
volume of the microdroplets may range (calculated from their
lengths) between approximately 0.01 and 100 nanoliters (more
typically between ten and fifty).
[0029] The first microdroplet transport channel may be operably or
functionally connected to, or in liquid communication with, at
least a second microdroplet transport channel. First and second
channels may operatively interact prior to connection with at least
a first isothermally regulated reaction chamber. This would be the
first meaning of "connective channels". However, other operative
connections are envisioned, and separate transport channels that
function to deliver fluids to a common reaction chamber are still
"interactive transport channels" in the context of the present
invention in that they convey their contents to a common
destination.
[0030] The present invention is not limited to the number of
transport channels or other fluid-conveying means that may be
provided in the substrate. The number and configuration of such
channels will generally be dictated by the number of reaction
chambers and other components provided on the substrate and/or the
interaction of various individual chip elements to form a
coordinated system.
[0031] At least one isothermally regulated reaction chamber is an
important element of the present invention. As used herein, an
"isothermally regulated reaction chamber" is a chamber, preferably
one defining a microvolume receptacle, the temperature of which
chamber may be regulated in order to keep it substantially
constant. The "substantially constant" temperature may be
controlled within a few degrees, or within a single degree, or in
certain embodiments, within a few tenths of a degree.
[0032] The means for isothermally regulating the reaction chamber
may include, but are not limited to, resistors in contact with or
in proximity to the reaction chamber, temperature detectors,
resistive temperature detectors, dielectric sensors, or diodes
and/or circuitry for temperature control. As discussed, the
isothermal regulation means will preferably be a programmable
means. The actual means of conveying the heat will preferably be a
sheet resistively heated (rather than a wire), although polysilicon
and doped polysilicon and diaphragm-type heaters may also be used
in the reaction chamber.
[0033] In certain embodiments of the present invention, the
microfabricated substrate further defines at least a first entry
port operably or functionally connected to, or in liquid
communication with, at least a first microdroplet transport
channel. Any one of a variety of entry valves or ports may be used
to control application of the sample or samples.
[0034] In embodiments where the microfabricated substrate further
defines at least a second microdroplet transport channel, at least
a second entry port may be provided in operable or functional
connection, or in liquid communication with the second microdroplet
transport channel. The invention is not limited to the number of
transport channels, nor to the number of entry ports, either in
terms of ports per channel or the total number of entry ports.
[0035] "Exit ports" or "sample collection points" are also
envisioned, which are generally positioned at a downstream flow
site from the reaction chamber.
[0036] In certain aspects of the invention, the microfabricated
substrate will further comprise a flow-directing means system in
order to facilitate that directed manipulation of fluids around the
substrate. The term "flow-directing means system" is intended to
refer to one or more modifications of the substrate or other
components used in functional association with the substrate that
act to control, or further control, the transport, merging and/or
mixing of fluids or microdroplets between the components etched
onto the underlying substrate.
[0037] Certain preferred flow-directing means systems are those
that employ a surface-tension-gradient mechanism in which discrete
droplets are differentially heated and propelled through etched
channels. A series of heating elements may thus be arrayed along
the one or more microdroplet transport channels. Such resistive
heaters may be located slightly beneath the channels. In certain
aspects of the invention, the heating elements are comprised of
aluminum, although one or more or a combination of other suitable
resistive metals or materials may be employed, such as platinum,
gold, etc.
[0038] In certain aspects of the invention, "heating element" may
refer to an element that is capable of at least partially
liquefying a meltable material. A meltable material is "associated
with" a heating element when it is in proximity to the heating
element such that the heating element can at least partially melt
the meltable material. The proximity necessary will depend on the
melting characteristics of the meltable material as well as the
heating capacity of the heating element. The heating element may or
may not be encompassed within the same substrate as the meltable
material.
[0039] Other fluid-directing means systems for use in the invention
are those that comprise a gas source in fluid communication with
the one or more transport channels and other components, such that
application of differential gas pressure gradients result in the
controlled flow of gases or liquids through the micromachined
device.
[0040] Differences in hydrophobic and hydrophilic surface
structures may also be employed to control the flow or transport of
fluids through the defined channels and etched components. In such
embodiments, the transport channels and/or components may comprise
or may be manipulated to comprise one or more hydrophobic regions.
The channels and components may also be treated with a
hydrophilicity-enhancing compound or compounds prior to addition of
one or more of the biological samples or amplification reaction
reagents.
[0041] "Hydrophilicity-enhancing compounds" are generally those
compounds or preparations that enhance the hydrophilicity (water
affinity) of a component, such as a transport channel.
"Hydrophilicity-enhancing compound" is thus a functional term,
rather than a structural definition. For example, Rain-X.TM.
anti-fog is a commercially available reagent containing glycols and
siloxanes in ethyl alcohol. The fact that Rain-X.TM. anti-fog
renders a glass or silicon surface more hydrophilic is more
important than the reagent's particular formula.
[0042] In certain aspects of the invention "hydrophobic reagents"
are used to make "hydrophobic coatings" and create "hydrophobic
regions" (more water repellent) in channels. It will be understood
that the present invention is not limited to particular hydrophobic
reagents. In one embodiment, the present invention contemplates
hydrophobic polymer molecules that may be grafted chemically to the
silicon oxide surface. Such polymer molecules include, but are not
limited to, polydimethylsiloxane. In another embodiment, the
present invention contemplates the use of silanes to make
hydrophobic coatings, including but not limited to halogenated
silanes and alkylsilanes. The invention is not limited to
particular silanes; the selection of the silane is only limited in
a functional sense, ie., that it render the surface
hydrophobic.
[0043] In one embodiment, n-octadecyltrichlorosilane (OTS) is used
as a hydrophobic reagent. In another embodiment,
octadecyldimethylchlorosilane is employed. In yet another
embodiment, the invention contemplates 1H, 1H, 2H,
2H-perfluorodecyltricholorosilane (FDTS, C.sub.10H.sub.4F.sub.17S-
iCl.sub.3) as a hydrophobic reagent. In still other embodiments,
fluoroalkyl-, aminoalkyl-, phenyl-, vinyl-, bis silyl ethane- and
3-methacryloxypropyltrimethoxysilane (MAOP) are contemplated as
hydrophobic reagents. Such reagents (or mixtures thereof) are
useful for making hydrophobic coatings, and more preferably, useful
for making regions of a channel hydrophobic (as distinct from
coating the entire channel).
[0044] This invention is not limited to particular dimensions for
the hydrophobic regions of the channels or components. However,
while a variety of dimensions are possible, it is generally
preferred that the regions have a width of between approximately 10
and 1000 .mu.m (or greater if desired), and more preferably between
approximately 100 and 500 .mu.m.
[0045] A surface (such as a channel surface) is "hydrophobic" when
it displays advancing contact angles for water greater than
approximately 70.degree.. In one embodiment, the treated channel
surfaces of the present invention display advancing contact angles
for water between approximately 90.degree. and approximately
130.degree.. In another embodiment, the treated microchannels have
regions displaying advancing contact angles for water greater than
approximately 130.degree..
[0046] In certain aspects of the invention, a "liquid-abutting
hydrophobic region" may refer to a hydrophobic region within a
channel which has caused liquid (e.g., aqueous liquid) to stop or
be blocked from further movement down the channel, said stopping or
blocking being due to the hydrophobicity of the region, said
stopped or blocked liquid positioned immediately adjacent to said
hydrophobic region.
[0047] Other flow-controlling or flow-directing means systems
contemplated for use in the present invention are those that rely
on the phenomenon of electrowetting, and/or differences in the
dielectric constant between the reagent fluids and air.
Electrowetting may be described as the initial intake of fluid from
a reservoir into a channel, electrowetting (or heating) may also be
used to break the channel droplet from contact with the reservoir.
Valve sealed by a movable diaphragm and/or meltable solder can also
be used to control fluid flow.
[0048] Any one of a variety of pumps, both external and internal
pumps, may be used in order to control the flow of fluids in the
context of this invention. In certain aspects of the invention a
"bubble pump" may be used as a flow-directing means. A bubble pump
operates as follows: fluid is introduced into a channel that
comprises one or more electrodes positioned such that they will be
in contact with a liquid sample placed in the channel. Two
electrodes may be employed and a potential may be applied between
the two electrodes. At both ends of the electrodes, hydrolysis
takes place and a bubble is generated. The gas bubble continues to
grow as the electrodes continue pumping electrical charges to the
fluid. The expanded bubble creates a pressure differential between
the two sides of the liquid drop which eventually is large enough
to push the liquid forward and move it through the polymer
channel.
[0049] When coupled with a capillary valve, a bubble pump can
actuate an effective quantity of fluidic samples along the channel.
The capillary valve is a narrow section of a channel. In operation,
the fluidic sample is first injected into an inlet reservoir. As
soon as the fluid is loaded, it moves in the channel by capillary
force. The fluid then passes the narrow section of the channel but
stops at the edge where the channel widens again. After the fluidic
sample is loaded, a potential is applied between two electrodes. At
both ends of the electrodes, hydrolysis occurs and bubble is
generated. The bubble keeps growing as the electrodes continue
pumping electrical charges to the fluid. The expanding bubble then
creates a pressure differential between the two sides of the liquid
drop, which eventually large enough to push the liquid forward.
[0050] The combination of bubble pump and capillary valve does not
require any moving parts and is easy to fabricate. In addition, the
device produces a well-controlled fluid motion, which depends on
the bubble pressure. The bubble pressure is controlled by the
amount of charges pumped by the electrodes. The power consumption
of the device is also minimized by this method.
[0051] In certain aspects of the invention, the flow-directing
means is separated from at least the first microdroplet transport
channel by a liquid barrier. "Liquid barrier" or "moisture barrier"
refers to any structure or treatment process on existing structures
that prevents short circuits and/or damage to electronic elements
(e.g., prevents the destruction of the aluminum heating elements).
In one embodiment, the liquid barrier may comprise a first silicon
oxide layer, a silicon nitride layer and a second silicon oxide
layer.
[0052] Further preferred aspects of the invention are those wherein
the microfabricated substrate further defines, or is operably
associated with, a nucleic acid analysis component operably
connected to or in liquid communication with the isothermally
regulated reaction chamber. The operative connection between the
nucleic acid analysis component and the reaction chamber is such
that the amplified nucleic acid products generated by the
isothermal amplification reaction can be analyzed by the nucleic
acid analysis component. The overall analytical method thus
requires that the amplified products are conveyed or otherwise
transported from the isothermally regulated reaction chamber to the
nucleic acid analysis component in a manner effective to allow
their subsequent analysis, separation, detection, or such like.
[0053] Any one of a variety of nucleic acid separation and
analytical components may be used as part of the devices or systems
of the present invention. Amplification product separation means
include those for use in separation methods based upon
chromatographic separation, including adsorption, partition,
ion-exchange and molecular sieve, and techniques using column,
paper, thin-layer and gas chromatography. Gel electrophoresis,
liquid capillary electrophoresis, e.g., in glass, fused silica,
coated and rectangular column format, polyacrylamide gel-filled
capillary columns are particularly contemplated. Gel
electrophoresis channels and/or capillary gel electrophoresis
channels may thus be etched into the substrate.
[0054] The use of a miniature electrophoresis stage for
macromolecule DNA separation is also contemplated. Using such a
system can accomplish large savings of time and funds by a
reduction in sample size, an increase in processing system speed of
the system, a increase in the number of samples handled through
massive parallelism and batch fabrication techniques.
[0055] In certain embodiments, the present invention will comprises
a nucleic acid detection means operably connected to, or in
electrical communication with, the nucleic acid analysis component.
Visualization means particularly envisioned include those using
ethidium bromide/UV and radio or fluorometrically-labeled
nucleotides, including antibody and biotin bound probes. The
nucleic acid detection means may thus include, but is not limited
to, a diode detection device with suitable filters for detection of
radioactive decay, fluorescence, visible and nonvisible light
wavelengths, and/or electromagnetic field changes.
[0056] The nucleic acid detection means may be a DNA sensor means,
e.g., one that detects a radiolabel or a fluorescent label. Such
DNA sensor means may be p-n-type diffusion diodes or p-n-type
diffusion diodes combined with a wavelength filter and an
excitation source. Silicon radiation/fluorescence detectors,
photodiodes, silicon diffused diode detectors, and other silicon
fabricated radiation detectors are also contemplated.
[0057] The control circuitry for preferable use in the device may
be "on wafer control circuitry" or "off wafer control circuitry",
the latter preferably for use in non-glass devices. In addition to
the isothermal temperature controls, the control circuitry employed
may include sample size and flow control circuits; timing circuits;
electrophoretic separation bias, data detection and transmission
control circuits; and one or more sequencer/timers to control the
overall operation.
[0058] Thus the instant devices are contemplated for use in
conducting a diagnostic test on a nucleic acid sample.
Additionally, the present devices are contemplated for use in
conducting a diagnostic or prognostic test on a biological sample
suspected of containing a selected nucleic acid. Therefore, the
present invention provides for the use of the instant devices in
the manufacture of a kit or system for the amplification of nucleic
acids. In certain aspects, the invention provides for the use of
the present devices in the manufacture of a kit or system for the
diagnosis or prognosis of a disease.
[0059] Any one or more of the isothermal amplification devices or
chips of the present invention may be formulated or packaged with
biological reagents effective to permit an isothermal nucleic acid
amplification reaction. In such aspects, the combined reagents and
devices may be considered as "isothermal nucleic acid amplification
kits". "Biological reagents effective to permit an isothermal
nucleic acid amplification reaction" are exemplified by
polymerases, nucleotides, buffers, solvents, nucleases,
endonucleases, primers, target nucleic acids including DNA and/or
RNA, salts, and other suitable chemical or biological
components.
[0060] The kits may thus be defined as comprising, in suitable
container means at least a first microfabricated substrate defining
at least a first channel, the at least a first channel connected to
an isothermally regulated reaction chamber, and reagents effective
to permit an isothermal amplification reaction.
[0061] In such kits, the first microfabricated substrate may
further define a nucleic acid analysis component operably connected
to said isothermally regulated reaction chamber and, optionally, a
nucleic acid detection means operably connected to the nucleic acid
analysis component.
[0062] The biological reagents effective for use in the
amplification reactions may be provided or packaged in any suitable
form, preferably aliquoted into suitable quantities. In certain
preferred aspects, such reagents will be provided in a dry or
lyophilized formulation. The provision of reagents, preferably in a
lyophilized form, applies to both kits, in which the reagents are
generally separately packaged, and integral devices, in which the
lyophilized reagents may be pre-fabricated into one or more etched
components on the substrate.
[0063] In certain other embodiments, an effective amount of the
amplifying reagents may be provided in a separate cartridge that is
interchangeably connected to the device, chip or system. Such
replaceable cartridges or reservoirs may be provided in the same
overall container means as the device, chip or system or may be
purchased separately as distinct items. Different replaceable
cartridges may be provided for conducting the various different
isothermal amplification reactions that are known in the art. A
number of reagent formulations may be packaged together for
alternative use according to the needs of the end user.
[0064] Diagnostic systems are also provided by the present
invention, comprising at least a first microfabricated substrate
defining at least a first channel that is connected to at least a
first isothermally regulated reaction chamber; wherein the
diagnostic system further comprises a nucleic acid analysis
component and a nucleic acid detection means in operable
association with the reaction chamber of the microfabricated
substrate.
[0065] The diagnostic systems may also comprise, in operable
association, at least a second microfabricated substrate defining
at least a second channel that is connected to at least a second
isothermally regulated reaction chamber. Third, fourth, fifth,
tenth, 20th, 50th, 100th, 500th and 1000th microfabricated
substrates may also be provided, as is the meaning of "a plurality
of microfabricated substrates".
[0066] The diagnostic systems may variously have at least a first
and at least a second microfabricated substrate, or a plurality
thereof, that are operably connected in series to a single nucleic
acid analysis component and nucleic acid detection means. The
diagnostic systems may alternatively comprise at least a first and
at least a second microfabricated substrate, or a plurality
thereof, that are operably connected in parallel to at least two
distinct nucleic acid analysis components and nucleic acid
detection means, or a plurality of such components.
[0067] In such kit and system embodiments, liquid handling,
electrophoresis, and detector components may be coupled into an
integrated format. DNA samples may move directly between sample
processing, size-separation, and product detection. The components
are controlled by electronic circuitry, fabricated on the same
silicon wafer.
[0068] Accordingly, an integrated DNA sample processing design may
be arrayed as multiple parallel units on a single silicon wafer.
The number of parallel DNA processing units per wafer may be
maximized, and circuitry used for overall control. A large number
of simultaneous isothermal amplification reactions (up to 1000 per
wafer) may be performed on such systems.
[0069] Methods of making devices for use in isothermal nucleic acid
amplification are provided by the invention, which generally
comprise preparing at least a first microfabricated device, chip or
wafer defining at least a first channel that is operably connected
to an isothermally regulated reaction chamber, preferably
isothermally regulated by a programmable means.
[0070] A method of making a nucleic acid diagnostic kit is also
provided, which generally comprises preparing at least a first
microfabricated device, chip or wafer defining at least a first
channel that is operably connected to an isothermally regulated
reaction chamber, and combining the microfabricated device with
biological reagents effective for use in an isothermal
amplification reaction. The combination may be with lyophilized
reagents, which may further be disposed in the device as an
integral component.
[0071] Methods of making a nucleic acid diagnostic system are
further provided, comprising preparing at least a first
microfabricated substrate defining, in a series of operable
associations, at least a first channel, an isothermally regulated
reaction chamber, a nucleic acid analysis component and a nucleic
acid analysis detection means.
[0072] Multi-component nucleic acid diagnostic systems may also be
manufactured by the methods of the present invention. To make a
multi-component nucleic acid diagnostic system, a plurality of
microfabricated substrates, nucleic acid analysis and detection
means are operably combined, preferably in an interactive array or
arrays. Controlling electronic circuitry and programmable
regulating means are preferably provided. Multiple parallel unit
arrays on single silicon wafers are particularly preferred.
[0073] Important aspects of the present invention are methods for
the isothermal amplification of selected nucleic acids or portions
thereof, which methods generally comprise providing or introducing
a microdroplet sample comprising or suspected of comprising the
selected nucleic acid, and reagents effective to permit an
isothermal amplification reaction, to at least a first
microfabricated substrate with an isothermally regulated reaction
chamber, as generally defined hereinabove, and conducting an
isothermally regulated amplification reaction to amplify the
selected nucleic acid or a portion thereof.
[0074] As used herein, the terms "providing" or "introducing" mean
that the sample or samples are provided or introduced into the one
or more microfabricated substrates in a manner effective to begin
their conveyance, transportation or general movement to the
isothermally regulated reaction chamber. As described hereinabove,
a number of particular flow-directing means systems may be employed
in order to convey the sample or samples to the reaction chamber.
Where differential heating is employed as the sole transport means,
or as part of the overall transport means, an important aspect of
the invention is that any samples that comprise enzymes for use in
the isothermally regulated nucleic acid amplification reaction are
"thermotransported" at a temperature below the critical temperature
of the polymerase enzyme. Preferably, all samples will be
transported at temperatures that are below the critical ranges for
substantial inactivation of the enzymes for use in the isothermal
amplification reaction. It is a surprising feature of the invention
that heat-conveying temperatures effective to transport samples
into the reaction chamber can be employed that are far enough below
the denaturation and/or inactivation temperatures of the enzymes
necessary to catalyze the isothermal nucleic acid amplifications.
The invention may thus be characterized as including a method step
of conveying said sample and/or said reagents from an initial
contact point on the microfabricated substrate to the isothermally
regulated reaction chamber at a "transportingly effective
temperature" that does not significantly denature the selected
amplification enzyme or otherwise significantly impair or reduce
its catalytic amplification activity.
[0075] The isothermal amplification reactions of the invention are
also conducted at temperatures effective and by means effective to
result in productive mixing of the one or more samples and
amplification reagents. "Effective mixing" is a functional term,
most readily characterized by the operative execution of the
amplification reaction such that amplified products may be
detected. If desired, one or more samples containing nucleic acids
and/or amplification reagents may first be "merged" prior to
mixing.
[0076] In certain definitional terms, "merging" is distinct from
"mixing." When a first and second microdroplet is merged to create
a merged microdroplet, the liquid may or may not be mixed.
[0077] In any event, irrespective of the degree of prior sample
association, the isothermal amplification reaction as a whole must
be conducted under conditions effective to adequately mix the
substrates and other components of the reaction. Prior to the
present invention, it was generally believed in the art that
effective mixing could not be achieved at the temperatures
preferred for use in the present isothermal amplification
reactions. Only the endeavors of the present inventors, conducted
despite the prejudices in the prior art, resulted in the discovery
that effective mixing could be achieved. Effective mixing is
achievable despite the viscosity of the samples and/or reagent
formulations used, and the particular biological components
employed in connection with the isothermal amplification enzyme
solutions and/or suspensions.
[0078] Those of ordinary skill in the art will be able to vary the
application of the samples and reagents and the manner of
transporting such components to the reaction chamber, in addition
to varying the particular details of the amplification reaction, in
order to ensure that a degree of mixing sufficient to result in
amplified products is achieved. Moreover, the degree of mixing in a
merged microdroplet may be enhanced by a variety of techniques
provided by the present invention, including but not limited to,
reversing the flow direction of the merged microdroplet (as
discussed herein below).
[0079] Although not in any way being limited by the following
guidance, the temperature differential believed to be effective in
conveying microdroplet samples along a microfabricated device in
accordance with the present invention should generally be a
temperature differential of at least about 10.degree. C.
Preferably, temperature differentials of at least about 11.degree.
C., 12.degree. C., 13.degree. C., 14.degree. C., 15.degree. C.,
16.degree. C., 17.degree. C., 18.degree. C., 19.degree. C.,
20.degree. C., about 25.degree. C., about 30.degree. C., about
35.degree. C. or even up to about 40.degree. C. or above may be
advantageously used in conveying microdroplet samples along a
micromachined device or substrate. It will be understood that each
of the foregoing effective conveying temperature differentials must
be analyzed in connection with the preferred operating temperatures
for any one or more particular amplifying enzyme, and that the
temperatures chosen must be below the temperature at which the
enzyme denatures or otherwise becomes significantly impaired in its
catalytic activity. In general, it is believed that temperature
differences of greater than about 30.degree. C. will be preferred
for creating microdroplet motion or movement. In certain other
embodiments, temperature differentials of about 40.degree. C. will
be effective, and these temperature gradients can be readily
generated by a number of means, particularly by the use of a series
of temperature sensors arrayed along the entire length of the one
or more conveying channels etched into the substrate.
[0080] Although an understanding of the mechanisms of action
underlying the surprising operability of the present invention is
not necessary in order to carry out the claimed amplification
methods, the inventors further point out that circulation patterns
generated in the drop during motion aid in mixing the liquid
sample. Studies using metal elements as both heaters and
temperature sensors demonstrate that a temperature differential of
20-40.degree. C. across the drop is sufficient to provide forward
motion in transport channels.
[0081] Thus for only small temperature differences across the drop
(on the order of 10.degree. C.) velocities on the order of 1 cm/s
may be obtained. This velocity is more than sufficient for
transporting liquid drops in MIDAT and other chip based
systems.
[0082] Those of ordinary skill in the art will further understand
that other physical components of the chip fabrication will impact
the temperatures effective to transport microdroplets. By way of
example only, in studies using glass capillaries, it has been found
that there is a minimum temperature difference required to move the
droplet. For instance, if the advancing angle is 36.degree. and the
receding angle is 29.degree. (with the front of the droplet being
25.degree. C.), then the back of the droplet would need to be
heated to .about.60.degree. C. for a 1 mm long droplet in a 20 mm
high channel. This is just one example situation.
[0083] The use of channel geometry and defined chip fabrications
that necessitate higher transport temperatures will naturally be
combined with the use of enzymes that are functional at higher
isothermal amplification temperatures. The choice of enzyme and
transport temperatures will be routine to those of ordinary skill
in the art, with a number of possibilities being readily available.
By way of example only, methods for isothermal SDA are available in
which temperatures of between about 50.degree. C. and about
70.degree. C. are used in conjunction with a thermophilic
amplification enzyme. Accordingly, temperatures of about 30.degree.
C., about 35.degree. C., about 40.degree. C., about 45.degree. C.,
about 50.degree. C., about 55.degree. C., about 60.degree. C.,
about 65.degree. C., about 70.degree. C., or even up to about
75.degree. C. also may be employed.
[0084] However, the calculations of the present inventors indicated
that about a 35.degree. C. difference between the front and back of
a droplet will be sufficient to initiate droplet motion in a system
with advancing angles of 36.degree. and receding angles of
29.degree. in a 20 mm high channel. Further studies of effective
transport showed that the resulting temperature difference was
40.degree. C. between the front and back of the droplet, thus
corroborating the initial determination of the requirements.
[0085] This shows that the range of transporting temperatures and
the variety of enzymes for use in the invention extends to
encompass each of the enzymes known to be suitable for use in
isothermal amplifications. For example, 3SR and Qbeta-replicase are
known to function at 37.degree. C., which can be used as part of
the effective conveying temperature. Classical SDA reactions can
also be conducted at a constant temperature between about
37.degree. C. and 42.degree. C., the preferred range identified in
U.S. Pat. No. 5,455,166 (incorporated herein by reference).
[0086] U.S. Pat. No. 5,455,166 is also incorporated herein by
reference for the purposes of exemplifying the level of skill in
the art regarding the selection of each component necessary for the
isothermal amplification reaction. For example, this patent
explains that, in addition to the DNA polymerases, the restriction
endonucleases necessary to carry out the reaction are also
mesophilic enzymes that are thermolabile at temperatures generally
above the 37-42.degree. C. advised for use in the reaction. All
such considerations will be readily employed by those of skill in
the art as they select the reagents necessary for use in the
present isothermal amplification reactions.
[0087] In terms of the isothermal amplification reaction itself,
rather than the transporting, merging and/or mixing steps, those of
ordinary skill in the art will instantly appreciate appropriate
temperatures for use in connection with the selected polymerase,
replicase or other amplification system. By way of example only,
isothermal amplification reactions involving 3SR and
Qbeta-replicase may be conducted at or about 37.degree. C. Standard
SDA isothermal amplification reactions may be conducted at a
constant temperature between about 37.degree. C. and 42.degree. C.
(including 38.degree. C., 39.degree. C., 40.degree. C. and
41.degree. C.), whereas isothermal SDA using a thermophilic enzyme
may be performed at a higher temperature range than conventional
SDA, anywhere between about 50.degree. C. and about 70.degree.
C.
[0088] Any effective temperature that will support the desired
enzymatic activity, even if sub-optimal, may be employed in the
isothermal amplification reactions of this invention. Accordingly,
the isothermal amplifications may be conducted at any substantially
constant and effective temperature, including at about 20.degree.
C., 21.degree. C., 22.degree. C., 23.degree. C., 24.degree. C.,
25.degree. C., 26.degree. C., 27.degree. C., 28.degree. C.,
29.degree. C., 30.degree. C., 31.degree. C., 32.degree. C.,
33.degree. C., 34.degree. C., 35.degree. C., 36.degree. C.,
37.degree. C., 38.degree. C., 39.degree. C., 40.degree. C.,
41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C., 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., 70.degree. C., 71.degree. C., 72.degree. C.,
73.degree. C., 74.degree. C., 75.degree. C., and the like.
[0089] It will be understood that the overall isothermal
amplification reaction is carried out in a manner effective to
result in at least detectable amounts of amplified products. "At
least detectable amounts of amplified products" refers to a yield
of amplified nucleic acid products that can be detected by
currently available nucleic acid detection means. Optical methods
using efficient fluorophores can detect atto-molar concentrations
(corresponding to .about.10.sup.5 DNA molecules) migrating in
capillary channels of 8.times.50 mm internal cross section (Woolley
and Mathies, 1994; incorporated herein by reference). Reactions for
synthesizing such DNA quantities can reasonably occur in 10 .mu.l.
An integrated system designed for picoliter volumes may require
channel dimensions on the order of 10 .mu.m.sup.2.times.100 .mu.m
(cross section.times.length).
[0090] In contrast to the negative beliefs in the prior art, the
present invention has provided methods for target amplification
efficiency surprisingly equivalent to conventional SDA reactions,
but conducted on a DNA chip. Amplifications of almost a
million-fold have already been achieved. This demonstrated that the
physical changes in the environment on the DNA chip, including
silicon contact, temperature gradients, surface interactions and
other potential inhibitors, did not adversely affect the
amplification reaction.
[0091] In certain preferred embodiments, it is believed that the
isothermal amplification reactions of the present invention will be
conducted such that the sample nucleic acids are amplified at least
about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold,
2000-fold, 5000-fold, 10,000-fold, 50,000-fold, 100,000-fold,
200,000-fold, 300,000-fold, 400,000-fold, 500,000-fold,
600,000-fold, 700,000-fold, 800,000-fold, 900,000-fold, or so, up
to and including at least about 1,000,000-fold, 2,000,000-fold or
so.
[0092] The simplicity of sample provision to microfabricated
devices is another surprising feature of the present isothermal
amplification methods. The samples may be provided in any
"silicon-compatible formulation". Prior to the present invention,
it was not known whether the various isothermal polymerases and
replicases would be operative in contact with the fabricating
structures of a microdevice, particularly the preferred silicon
formulations. The diligent studies of the present inventors have
shown that the present isothermal amplification methods function in
a "silicon-compatible manner", and the methods of the invention are
intended to be carried out in such effective manners.
[0093] The provision of the sample to the microfabricated or
micromachined devices or systems is not believed to be critical, so
long as the samples are later capable of being conveyed along the
appropriate channels. Sample sources include, but are not limited
to, continuous streams of liquid as well as static sources (such as
liquid in a reservoir). In a preferred embodiment, the source of
liquid microdroplets comprises liquid in a microchannel from which
microdroplets of a discrete size are split off. As described above,
in certain preferred embodiments, the reagents for use in the
isothermal reaction will already be comprised within a
pre-fabricated microdevice. In such embodiments, lyophilized
reagents may be rendered active by contact with the nucleic
acid-containing sample, or alternatively, they may be separately
contacted with another fluid sample, such as a buffer.
[0094] The samples comprising the nucleic acids for application in
the present isothermal amplification methods may be "laboratory
samples" for use in any one of a variety of molecular biological
embodiments. Such samples may also be "biological or clinical
samples", in which case the samples will generally be obtained from
or otherwise derived from an animal or human subject.
[0095] In any event, where the samples used are "microdroplet
samples", this term generally refers to the microdroplet themselves
and samples from which microdroplets may be made.
[0096] Whether the sample is a laboratory, biological or clinical
sample, the purity of the nucleic acids within the sample may vary
widely. The purity of the sample is controlled only by the need to
have a minimum purity necessary for successful execution of the
isothermal amplification reaction. In certain embodiments, the
sample will have been subjected to a substantial degree of
extraction or purification prior to use in the present invention,
although this is not necessary in all embodiments.
[0097] In terms of the biological samples, these may be obtained
from a variety of biological fluids, including blood, plasma,
urine, sputum, semen, and fluids obtained from homogenized tissues.
It is not believed to be necessary to limit the presence of other
biological components, such as proteins and lipids, from the
samples for use in the invention, although this may be desired in
certain embodiments and is within the level of skill of the
ordinary artisan.
[0098] In common with the sample preparation, the purity of the
reactants provided to the device and the makeup of the device
itself require some degree of biocompatability in order to achieve
the desired reaction. That is to say, that the isothermal
amplification reaction should not be substantially inhibited or
prevented by any components present within the biological sample,
contaminants within the reactants or by the characteristics or
nature of the device components, including the silicon
fabricants.
[0099] It will be understood that the particular components,
amounts of components and/or reactants and the particular
conditions of the reaction may be modified in order to optimize the
isothermal amplification reaction itself. All such variations and
modifications are routinely investigated in this field of study. By
way of example only, one may vary the concentration of any of the
components or the samples, the temperature, pH or ionic makeup of
the buffers, and generally vary any other parameter of the
amplification reaction.
[0100] It will be understood that the execution of the
amplification reaction, including the application of the samples
and the movement, mixing and distribution of the samples prior to
the actual isothermal amplification step, may also require certain
optimizations. All such variations and optimizations will be
routine to those skilled in this field of study.
[0101] All liquid distributions and manipulations may be performed
entirely within a handling system formed as channels in
micromachined silicon. Sensors may monitor the temperature and
location of liquid in the channels. The manipulation of reagents
includes the movement, merging, mixture, and temperature control of
the reagents to allow nucleic acid amplification under isothermal
reaction conditions.
[0102] In certain aspects of the present invention, the isothermal
amplification methods and the reagents provided for use in the
methods will be based upon the strand displacement amplification
reaction. Self-sustained sequence replication amplification
reactions and/or Q.beta. replicase amplification reactions may also
be used.
[0103] A preferred technique is the Strand Displacement
Amplification (SDA). The SDA reaction may be conducted at a
substantially constant temperature between about 37.degree. C. and
about 42.degree. C., or at any other effective temperature, as
exemplified herein by 52.degree. C. It was previously believed that
the low temperature requirement for SDA would prevent its use in
connection with amplification on microchip devices. However, the
inventors discovered that the potential problems of stagnant
temperature gradients and reduced diffusion and sample mixing do
not actually impact the efficiency of the SDA reaction in such
microvolume embodiments.
[0104] Thermophilic SDA may also be employed, as described in
published European Patent Application No. 0 684 315 (incorporated
herein by reference). This technique employs thermophilic
restriction endonucleases which nick the hemimodified restriction
endonuclease recognition/cleavage site at high temperature and
thermophilic polymerases which extend from the nick and displacing
the downstream strand in the same temperature range. At increased
temperature, the amplification reaction has improved specificity
and efficiency, reduced nonspecific background amplification, and
potentially improved yields of amplification products.
[0105] In terms of amplified product analysis, DNA samples may be
size-fractionated on an electrophoresis system built within or
attached or connected to the silicon substrate. Electrophoresed DNA
products may be visualized by radioactivity or fluorescence
detectors fabricated directly in the silicon wafer.
[0106] In certain aspects of the invention, the amplified nucleic
acid is detected by means of a detectable label incorporated into
the amplified selected nucleic acid by the isothermal amplification
reaction. In other aspects, it is detected by means of a labeled
probe. The label may variously be a radioisotopic, enzymatic or
fluorescent label.
[0107] The present invention further provides methods for detecting
the presence of a selected nucleic acid, comprising introducing a
sample suspected of containing the selected nucleic acid, and
reagents effective to permit an isothermal amplification reaction,
into a microfabricated substrate defining at least a first channel,
the at least a first channel connected to an isothermally regulated
reaction chamber, conducting an isothermally regulated
amplification reaction to amplify the selected nucleic acid, and
detecting the presence of the amplified selected nucleic acid,
wherein the presence of the amplified selected nucleic acid
confirms the presence of the selected nucleic acid in the
sample.
[0108] The sample may be obtained or derived from an animal or
patient having or suspected of having a disease. It will be
understood that in certain aspects of the present diagnostic and/or
prognostic methods, the presence of the ultimate amplified selected
nucleic acid will be indicative of the disease state being
analyzed. In alternative embodiments, it is the absence of
amplified nucleic acid products that is indicative of a disease
state. In either embodiment, the present invention is ideally
suited for the amplification of nucleic acids of defined sequence,
having a defined sequence element, or including a potential point
mutation, as each of the foregoing variants may be distinguished by
analyzing the amplified products resulting from execution of the
presently claimed methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0109] FIG. 1. Example of an integrated DNA analysis system,
represented schematically. The individual components of the system
are injection entry ports (A), liquid pumping channels (B),
thermally (i.e., isothermally) controlled reaction chamber (C),
electrophoresis channel (D), and DNA band migration detector (E).
Each component would have associated sensors, control circuitry,
and external connections.
[0110] FIG. 2A and FIG. 2B. A two-part approach to construction of
a silicon device of the present invention, and a silicon substrate
comprising a plurality of devices.
[0111] FIG. 2A shows one embodiment of a single microfluidic
device.
[0112] FIG. 2B shows one aspect of a silicon device comprising a
plurality of microfluidic device modules.
[0113] FIG. 3A and FIG. 3B. A schematic of one embodiment of a
device to split a nanoliter-volume liquid sample and move it using
gas from a gas source.
[0114] FIG. 3A shows the liquid sample prior to splitting.
[0115] FIG. 3B shows the liquid sample after splitting off a
microdroplet of length L. The hatched regions represent the
hydrophobic regions.
[0116] FIG. 4A and FIG. 4B. A schematic of one embodiment of a
device of the present invention to split, move and stop
microdroplets using internal gas pressure generation.
[0117] FIG. 4A shows a liquid sample prior to splitting.
[0118] FIG. 4B shows the liquid sample after splitting off a
microdroplet of length L. The hatched regions represent the
hydrophobic regions.
[0119] FIG. 5. Schematic drawing showing the principle of thermally
induced drop motion in a closed channel. The case of a single
aqueous drop in a hydrophilic channel is presented, where V is an
applied voltage, P.sub.atm is atmospheric pressure, P.sub.2 is the
receding-edge internal pressure, P.sub.2 is the advancing-edge
internal pressure, and .theta. is the contact angle of the
liquid-gas-solid interface. The contact angle will depend on the
surface characteristics of the channel and the constituents of the
drop, with a hydrophilic interaction giving .theta. between
0.degree. and 90.degree., and a hydrophobic surface giving .theta.
between 90.degree. and 180.degree.. Surface treatments can also
reduce contact angle hysteresis and, therefore, reduce the
temperature difference necessary for drop motion.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0120] I. Design of Microscale Devices for Isothermal Amplification
Reactions
[0121] The amplification of nucleic acids provides a convenient way
to diagnose a variety of disease states. However, prior to the
present invention, it was unknown whether the movement, mixing, and
merging of viscous microvolume fluids at lower temperature to
conduct isothermal amplification reactions was possible in a
microfabricated environment. Isothermal amplification reactions
employ reaction schemes and enzymes which are very different from
PCR.TM., and it is unknown whether or not the enzymology of
isothermal amplification reactions is compatible with chip hardware
and materials.
[0122] Specifically, the only enzyme necessary for PCR.TM.
amplification of DNA targets is a thermostable DNA polymerase.
Isothermal DNA amplification reactions employ additional enzymes
with different biological activities because heat is not used to
denature double-stranded nucleic acids. In addition to a DNA
polymerase, 3SR requires an enzyme with RNase H activity and an RNA
polymerase. The SDA reaction requires several very specific
enzymatic activities which are not necessary for PCR.TM. in order
to successfully amplify a target sequence. In addition to
synthesizing a new DNA strand, the DNA polymerase in SDA must lack
5'-3' exonuclease activity, either naturally or by inactivation,
incorporate the modified nucleotides required by SDA
(.alpha.thio-dNTPs or other modified dNTPs), and displace a
downstream single strand from a double stranded molecule starting
at a single stranded nick. In addition, the restriction
endonuclease in SDA must nick (i.e., cleave a single strand of) its
double stranded recognition/cleavage site when the
recognition/cleavage site is hemimodified and dissociate from its
recognition/cleavage site rapidly enough to allow the polymerase to
bind and amplify the target efficiently. The restriction
endonuclease must exhibit these activities under reaction
conditions which are compatible with the activities required of the
polymerase.
[0123] It was not previously known if the enzymatic activities
required for such isothermal amplification reactions would be
inhibited by interaction with the surfaces of silicon
microfabricated analysis devices or by inhibitors present in the
devices (e.g., residual chemicals from microfabrication). In
addition, the change in surface-to-volume ratio which accompanies
taking an enzymatic reaction developed in a test tube to the
microchannel of a silicon microfabricated device may have
unpredictable effects, as changes in the diffusion properties of
the reactants in the channel may interfere with the amplification
reaction. In particular for SDA, the interaction of the derivatized
dNTPs with the microdevice environment, the effect of the
environment on nicking activity by the restriction endonuclease and
strand-displacing activity by the polymerase were not known. It is
known that liquid movement in a closed channel, which is a
convenient means for bringing components of the amplification
reaction into contact, is affected by the contact angle of the
liquid-gas-solid interface within the channel. Changes in the
composition of the liquid in the channel change the surface tension
and therefore the contact angle, affecting liquid movement. The
contact angle is reduced and liquid movement is facilitated by more
hydrophilic liquids such as the reaction buffers conventionally
used in PCR.TM..
[0124] Certain isothermal amplification reactions, such as SDA,
employ hydrophobic components such as glycerol and BSA, which may
unpredictably affect the surface tension properties of the liquid
and the ability to move it within the channels of microfabricated
devices, particularly when thermocapillary pumps are used. The need
to increase the amount of heat to move the liquid aliquot with a
thermocapillary pump could be incompatible with the temperature
requirements of the enzymes and the isothermal amplification
reaction.
[0125] Lowering the temperature of the amplification reaction may
also have unpredictable effects. The temperature of the reaction in
the microfabricated device is typically controlled from one side of
the chip, setting up a temperature gradient across the channel. The
temperature conditions of isothermal amplification reactions would
also be expected to alter the interactions of the reactants with
the silicon or glass surfaces of the channel. Because isothermal
amplification is conducted at constant, lower temperatures, the
temperature gradient which is produced reaches equilibrium and
becomes stagnant. In contrast, the temperature gradient in higher
temperature reactions with thermocycling is not stagnant.
Temperature fluctuations during PCR.TM. amplification serve to
minimize the gradient effect, improve diffusion of reactants and
facilitate mixing.
[0126] Mixing of reactants in the channels and chambers of the DNA
chip is of particular concern in isothermal amplification
reactions, as mixing of reactants initiates the amplification
reaction. This is not the case in PCR.TM., as all reactants
required for amplification are present together in the reaction
mix. PCR.TM. amplification of double-stranded targets does not
begin until temperature cycling is started because until that time
no single-stranded target is available to amplify. This is not the
case in isothermal amplification reactions. Because strand
separation is an enzymatic process in isothermal amplification, at
least one of the enzyme reactants (usually the polymerase) is
withheld until it is desired to begin the reaction. If the
isothermal amplification reaction starts with a heat-denaturation
step and the enzymes employed are not thermostable, all of the
enzymes for amplification are typically withheld until the
target-containing sample is cooled to the appropriate reaction
temperature. The sample containing the enzyme or enzymes must be
mixed with the remaining reagents in order for amplification to
begin.
[0127] To control initiation of the isothermal amplification
reaction and provide an integrated nucleic acid analysis system, it
is therefore highly desirable to keep the components separate on
the microfabricated device and bring them together to initiate
amplification. This requires, however, that mixing of the two
components in the channel be adequate at the lower temperatures of
isothermal amplification, and this mixing may be negatively
affected due to temperature-related decreases in diffusion and
changes in surface chemistry. The components of the amplification
reaction itself may also have negative effects on mixing within the
channel. Many amplification reactions contain reagents such as
glycerol and bovine serum albumin which increase viscosity and
could reduce mixing. The viscosity-increasing effects of these
reagents is increased at lower temperatures. It was therefore
unknown whether or not there would be adequate mixing, diffusion
and temperature regulation to produce isothermal amplification on a
silicon microfabricated device.
[0128] In certain aspects, the present invention relates to
movement of microdroplets through microchannels, and more
particularly, compositions, devices and methods to control
microdroplet size and movement. The present invention involves
microfabrication of microscale devices and reactions in microscale
devices, and in particular, movement of biological samples in
microdroplets through microchannels to, for example, initiate
biological reactions, particularly isothermal amplification of
nucleic acids.
[0129] Although there are many formats, materials, and size scales
for constructing integrated fluidic systems, the present invention
contemplates silicon microfabricated devices as a cost-effective
solution.
[0130] The present invention contemplates microscale devices,
comprising microdroplet transport channels, reaction regions (e.g,
chambers), electrophoresis modules, and radiation detectors. In a
preferred embodiment, these elements are microfabricated from
silicon and glass substrates. The various components are linked
(ie., in liquid communication) using a surface-tension-gradient
mechanism in which discrete droplets are differentially heated and
propelled through etched channels. Electronic components are
fabricated on the same substrate material, allowing sensors and
controlling circuitry to be incorporated in the same device. Since
all of the components are made using conventional photolithographic
techniques, multi-component devices can be readily assembled into
complex, integrated systems.
[0131] Continuous flow liquid transport has been described using a
microfluidic device developed with silicon (Pfahler et al., 1990).
Pumps have also been described, using external forces to create
flow, based on micromachining of silicon (Van Lintel et al, 1988).
The present invention employs discrete droplet transport in silicon
using internal forces or external forces (i.e., external forces
created by pumps).
[0132] As a mechanical building material, silicon has well-known
fabrication characteristics. The economic attraction of silicon
devices is that their associated micromachining technologies are,
essentially, photographic reproduction techniques. In these
processes, transparent templates or masks containing opaque designs
are used to photodefine objects on the surface of the silicon
substrate. The patterns on the templates are generated with
computer-aided design programs and can delineate structures with
line-widths of less than one micron. Once a template is generated,
it may be used almost indefinitely to produce identical replicate
structures. Consequently, even extremely complex micromachines may
be reproduced in mass quantities and at low incremental unit
cost--provided that all of the components are compatible with the
silicon micromachining process. While other substrates, such as
glass or quartz, can use photolithographic methods to construct
microfabricated analysis devices, only silicon gives the added
advantage of allowing a large variety of electronic components to
be fabricated within the same structure.
[0133] In one embodiment, the present invention contemplates
silicon micromachined components in an integrated analysis system,
including the elements identified schematically in FIG. 1. In this
proposed format, sample and reagent are injected into the device
through entry ports (FIG. 1-A) and they are transported as discrete
droplets through channels (FIG. 1-B) to a reaction chamber, such as
an isothermally controlled reactor where mixing and reactions, such
as isothermal nucleic acid amplification reactions (SDA,
Q.beta.-replicase, etc), restriction enzyme digestion, ligation,
phosphorylation, dephosphorylation, sequencing or other enzymatic
or chemical reaction known to those of skill in the art occur (FIG.
1-C). The biochemical products are then moved by the same method to
an electrophoresis module (FIG. 1-D) where migration data is
collected by a detector (FIG. 1-E) and transmitted to a recording
instrument. Importantly, the fluidic and electronic components are
designed to be fully compatible in function and construction with
the biological reactions and reagents.
[0134] A. Two-Part Approach to Construction
[0135] Most of the devices of the invention are hybrid
micromechanical devices (two substrates bonded together). The
purpose of using this method is to allow the fabrication of
micromechanical devices out of a variety of materials (silicon,
glass, fused silica, quartz, etc.). The devices have chamber
volumes that are easily handled (sample loading, component
analysis, etc.) and chamber walls that are transparent (sample
loading, fluorescent detection, etc.). The hybrid system also gives
flexibility in choosing materials in one section of the unit
without affecting other pans of that same unit.
[0136] The invention may comprise two separate wafers of either the
same or different materials, including but, not limited to,
silicon, glass, or quartz are micromachined independently. The
pieces are then bonded together using a variety-of techniques
(polyimide, UV-curing cements, anodic bonding, etc.). For
transparency, a glass or quartz wafer is usually used on one side
of the hybrid. In general, the sensors, heaters, and other
electronic components may be patterned onto one wafer and etch
channels into the other. The electronic components may use 5 .mu.m
wire width over channel regions so that, if the glass wafer has the
electronic components patterned on it, the contents of the channels
may be seen.
[0137] FIG. 2A shows a two-part approach to construction.
Microchannels (100) are made in the silicon substrate (200) and the
structure is bonded to a glass substrate (300). The two-part
channel construction technique requires alignment and bonding
processes but is amenable to a variety of substrates and channel
profiles. In other words, for manufacturing purposes, the two-part
approach allows for customizing one piece (i.e., the silicon with
channels and reaction formats) and bonding with a standardized
(non-customized) second piece, e.g., containing standard electrical
pads (400).
[0138] Hundreds or thousands of copies of a particular component
can be made simultaneously across the entire silicon wafer surface
(FIG. 2B; for example, but not limited to, a wafer that is 0.5 mm
thick and 100 mm in diameter). The components are made by
sequential deposition, ion implantation, or etching of thin layer
materials in defined patterns. Materials that are commonly used
include silicon oxide, silicon nitride, and various metals and
alloys.
[0139] The technology of silicon fabrication is essentially a
photolithographic method for making machines. Once a "template" or
"stencil" pattern has been prepared, additional copies of the
machines are replicated at minimal cost and effort. The density of
components is limited by line-width considerations and the
designing abilities of the engineers. Complete devices are made in
batches and can often exceed thousands of replicates per
fabrication run. Additionally, silicon fabrication has benefited
from massive industrial commitment over the past 20 years. The
characteristics of the fabrication steps are known and have been
incorporated into intelligent design software or computer-aided
design and manufacturing packages (CAD/CAM).
[0140] B. Channel Design and Construction
[0141] In silicon micromachining, a technique to form closed
channels involves etching an open trough on the surface of a
substrate and then bonding a second, unetched substrate over the
open channel. There are a wide variety of isotropic and anisotropic
etch reagents, either liquid or gaseous, that can produce channels
with well-defined side walls and uniform etch depths. Since the
paths of the channels are defined by the photo-process mask, the
complexity of channel patterns on the device is virtually
unlimited. Controlled etching can also produce sample entry holes
that pass completely through the substrate, resulting in entry
ports on the outside surface of the device connected to channel
structures.
[0142] The present invention contemplates a variety of
silicon-based, microdroplet transport channel-containing devices.
In one embodiment, the device comprises: a housing comprised of
silicon, a microdroplet transport channel etched in the silicon, a
microdroplet receiving means in liquid communication with a
reaction region via said transport channels, and a liquid barrier
disposed between the transport channels and a microdroplet
flow-directing means. In one embodiment, the device is assembled in
two parts. First, the channels are etched in any number of
configurations. Second, this piece is bonded with a silicon-based
chip containing the electronics. This allows for both customization
(in the first piece) and standardization (in the second piece).
[0143] In certain aspects of the invention "conveying" may refer to
causing to be moved through, as in the case where a microdroplet is
conveyed through a transport channel to a particular point, such as
a reaction region. Conveying may be accomplished via a
flow-directing means.
[0144] The present invention also contemplates devices and methods
for the sealing of channels with meltable material. In one
embodiment, the device comprises a meltable material disposed
within a substrate and associated with a heating element.
[0145] In one embodiment, the present invention contemplates a
method comprising providing a device having a meltable material
disposed within a substrate and associated with a heating element,
and heating the meltable material with the heating element such
that the meltable material at least partially liquefies and such
that the substrate is not damaged. The method may further comprise
allowing the liquefied meltable material to cool. While the present
invention is not limited by the size of the channel, in one
embodiment the substrate further comprises a microdroplet channel
disposed in the substrate, the meltable material is disposed within
the microdroplet channel.
[0146] In another embodiment, the present invention contemplates a
method for restricting fluid flow in a channel comprising providing
a device comprising a meltable material disposed within a
substrate, the meltable material associated with a heating element;
and a diaphragm positioned such that, when extended, it touches the
meltable material, extending the diaphragm such that it touches the
meltable material, and heating the meltable material with the
heating element such that the meltable material at least partially
liquefies and such that the substrate is not damaged. In one
embodiment the method further comprises allowing the meltable
material to cool. While the present invention is not limited by the
size of the channel, in one embodiment, the substrate further
comprises a microdroplet channel disposed in the substrate, the
meltable material disposed within the microdroplet channel.
[0147] In certain aspects of the invention "meltable material" may
refer to a material that is at least semi-solid (and preferably
completely solid) at ambient temperature, will liquefy when heated
to temperatures above ambient temperature, and will at least
partially resolidify when cooled. Preferably, meltable material at
least partially liquefies at a temperature such that the substrate
is undamaged. That is to say, at the temperature the meltable
material liquefies, the substrate and other metals in the substrate
does not liquefy (readily tested as set forth in Example 6) and
does not change its properties. By "changing properties" it is
meant that the substrate or metal maintains it structural
integrity, does not change its conductivity and does not liquefy.
Thus, the characteristic of being meltable is not necessarily
associated with a particular melting point. Examples include, but
are not limited to, solder, wax, polymer and plastic.
[0148] In certain aspects of the invention "solder" may refer to a
metal or alloy that is a meltable material. Preferably, the solder
is a lower temperature solder, such as set forth in U.S. Pat. No.
4,967,950, herein incorporated by reference. "Lower temperature
solder" means a eutectic alloy. While the present invention is not
limited to a specific solder, one preferred solder composition for
the paste is a 63:37 eutectic alloy of tin:lead. Another compatible
solder is a 90% metal composition having a 63:35:2 eutectic alloy
of tin:lead:silver. Other desired solder compositions such as
eutectic Pb:Sn, Pb:In, Pb:In:Sn, etc.
[0149] The present invention also contemplates a method for
restricting fluid flow in a channel, comprising providing a main
channel connected to a side channel and disposed within a
substrate, meltable material disposed within the side channel and
associated with a heating element, and a movement means connected
to the side channel such that application of the movement means
induces the meltable material to flow from the side channel into
the main channel, heating the meltable material such that the
meltable material at least partially liquefies, and applying the
movement means such that the liquefied meltable material flows from
the side channel into the main channel. While the present invention
is not limited by the movement means, in one embodiment the
movement means is forced air. In one embodiment the method further
comprises allowing the meltable material to cool. While the present
invention is not limited by the size of the channel, in one
embodiment, the main channel and the side channel are microdroplet
channels.
[0150] While the present invention is not limited by the nature of
the substrate, in one embodiment the substrate comprises silicon or
glass. Likewise, the present invention is not limited by the
composition of the meltable material. In one embodiment, the
meltable material comprises solder. In a preferred embodiment, the
solder comprises 40:60 Sn:Pb. In other embodiments, the meltable
material is selected from a group consisting of plastic, polymer
and wax. Likewise, the present invention is not limited by the
placement of the meltable material in the substrate. In another
embodiment, the meltable material is placed adjacent to a channel,
while in another embodiment it is placed near the junction of more
than one channel.
[0151] II. Microfabrication of Silicon-Based Devices
[0152] As noted previously, silicon has well-known fabrication
characteristics and associated photographic reproduction
techniques. The principal modem method for fabricating
semiconductor integrated circuits is the so-called planar process.
The planar process relies on the unique characteristics of silicon
and comprises a complex sequence of manufacturing steps involving
deposition, oxidation, photolithography, diffusion and/or ion
implantation, and metallization, to fabricate a "layered"
integrated circuit device in a silicon substrate. See e.g., Miller,
U.S. Pat. No. 5,091,328, hereby incorporated by reference.
[0153] For example, oxidation of a crystalline silicon substrate
results in the formation of a layer of silicon dioxide on the
substrate surface. Photolithography can then be used to selectively
pattern and etch the silicon dioxide layer to expose a portion of
the underlying substrate. These openings in the silicon dioxide
layer allow for the introduction ("doping") of ions ("dopant") into
defined areas of the underlying silicon. The silicon dioxide acts
as a mask; that is, doping only occurs where there are openings.
Careful control of the doping process and of the type of dopant
allows for the creation of localized areas of different electrical
resistivity in the silicon. The particular placement of acceptor
ion-doped (positive free hole, "p") regions and donor ion-doped
(negative free electron, "n") regions in large part defines the
interrelated design of the transistors, resistors, capacitors and
other circuit elements on the silicon wafer. Electrical
interconnection and contact to the various p or n regions that make
up the integrated circuit is made by a deposition of a thin film of
conductive material, usually aluminum or polysilicon, thereby
finalizing the design of the integrated circuit.
[0154] Of course, the particular fabrication process and sequence
used will depend on the desired characteristics of the device.
Today, one can choose from among a wide variety of devices and
circuits to implement a desired digital or analog logic
feature.
[0155] In a preferred embodiment, channels were prepared on 500
.mu.m thick glass wafers (Dow Corning 7740) using standard
aqueous-based etch procedures. The initial glass surface was
cleaned and received two layers of electron beam evaporated metal
(20 nm chromium followed by 50 nm gold). Photoresist Microposit
1813 (Shipley Co.) was applied 4000 rpm, 30 sec; patterned using
glass mask 1 and developed. The metal layers were etched in
chromium etchant (Cr-14, Cyantek Inc.,) and gold etchant (Gold
Etchant TFA, Transene Co.,) until the pattern was clearly visible
on the glass surface. The accessible glass was then etched in a
solution of hydrofluoric acid and water (1:1, v/v). Etch rates were
estimated using test wafers, with the final etch typically giving
channel depths of 20 to 30 .mu.m. For each wafer, the depth of the
finished channel was determined using a surface profilometer. The
final stripping (PRS-2000, J. T. Baker) removed both the remaining
photoresist material and the overlying metal.
[0156] In one embodiment, channels etched on glass in the
above-described manner, were bonded to the heater-element wafer in
a two-part construction approach using optical adhesive (SK-9 Lens
Bond, Sumers Laboratories, Fort Washington, Pa.). The bond was
cured under an ultraviolet light source (365 nm) for 12 to 24
h.
[0157] Initial device design involved single layers of silicon.
However, experience showed these to be inadequate to prevent short
circuiting due to (necessary) liquid microdroplets within the
channels (see studies described below). The preferred design
involves a triple layer of oxides. Such a preferred device capable
of moving and mixing nanoliter droplets was constructed by bonding
a planar silicon substrate to channels etched in a glass cover. A
series of metal heaters was inlaid on the silicon substrate as two
parallel lanes merging into a single lane (a "Y"-shape). The
heating elements were formed by first coating the wafer with a 1.0
.mu.m layer of thermal silicon dioxide. Next, 0.35 .mu.m deep, 5
.mu.m wide grooves were reactive-ion etched (RIE) into the silicon
dioxide following the pattern set in an overlying photoresist.
Aluminum was deposited (0.35 .mu.m) across the entire wafer using
electron beam evaporation and the metal layer was "lifted-off" from
all surfaces having intact photoresist using a stripping solution.
The metal inlay process gives a relatively planar surface and
provides a uniform base for deposition of a solution-impermeable
barrier layer. The barrier layer is made by a sequence of three
plasma-enhanced chemical vapor depositions (PECVD): 1.0 .mu.m
silicon oxide (SiO,), 0.25 .mu.m silicon nitride (Si.sub.xN.sub.y)
and 1.0 .mu.m silicon oxide (SiO,). Some heating elements were also
used as resistive temperature sensors.
[0158] Heater elements were fabricated as follows. Silicon wafer
(p-type, 18-22 1/2-cm, boron concentration .ANG. 10.sup.15
cm.sup.-3) was used as a substrate for growth of SiO.sub.2 thermal
oxide (1 .mu.m); photoresist (AZ-5214-E, Hoescht-Celanese) was
applied and spun at 3000 rpm, 30 sec. The resist was patterned
(metal 1) and developed. Reactive ion etch (RIE, PlasmaTherm, Inc.)
was performed to 0.35 .mu.m depth into the SiO.sub.2 layer at the
following conditions: CHF.sub.3, 15 sccm (standard cubic
centimeters per min); CF.sub.4, 15 sccm; 4 mTorr; DC bias voltage
of 200V, 100 W, 20 min. The etch depth was measured by profilometer
and 0.35 .mu.m metallic aluminum was electron beam deposited. The
resist and overlying metal was lifted off by development using
Microposit 11 12A remover in solution (Shipley Co.,). The barrier
layers consist of sequentially deposited 1 .mu.m SiOX, 0.25 .mu.m
Si.sub.xN.sub.y, and 1 .mu.m SiOX using plasma-enhanced chemical
vapor deposition (PECVD). RIE was used to etch contact holes to the
metal layer using a second mask (CHF.sub.3, 15 sccm; CF.sub.4, 15
seem; 4 mTorr; and DC bias voltage of 200V, 100 W, 120 min).
[0159] The elements are arrayed as two parallel lanes, each 500
.mu.m wide, merging into one lane. The individual heaters consist
of paired aluminum wires (5 .mu.m) winding across the 500 .mu.m
wide region. The broad metal areas on either side of the elements
are bonding locations for connection to external circuitry. The
width of the aluminum element is 5 .mu.m. The channel is uniformly
etched 500 .mu.m wide and approximately 20 .mu.m deep.
[0160] The heating-element wafer was bonded to a glass wafer
containing etched channels with the same "Y" format. An aqueous
chemical etch of concentrated hydrofluoric acid was used to produce
channels with defined side walls and uniform depth. The etched
channels are defined by a chromium/gold mask and are 500 .mu.m wide
and approximately 20 .mu.m deep. The complementary silicon heater
and glass channel wafers were aligned and then bonded with adhesive
to form the finished device.
[0161] Each heating element used as a temperature sensor is
preferably first calibrated by measurement of electrical resistance
at 22.degree. C. and 65.degree. C. under constant voltage;
intermediate temperatures are estimated by linear
interpolation.
[0162] A. Microchannel Construction
[0163] There are two basic techniques that may be used for
construction of channel structures. The first technique uses a
chemical or a reactive ion etch to form open channels on selected
areas of a substrate. These channels can range in width from 10
.mu.m to the full thickness of the wafer (500 .mu.m). The open
channels are sealed by bonding of a second substrate as a cap on
top of the first one. Common bonding techniques include anodic
bonding, fusion bonding, melting, and epoxy bonding. Holes at
specific locations for injection of the sample are then etched from
the backside of the cap wafer. Bonded structures have been
successfully applied in the implementation of capillary liquid
electrophoresis systems etched on glass substrates (Burggraf et
al., 1993, ; Harrison et al., 1993). Most bonded structures are
simple discrete channel devices with a limited number of electrical
components and interconnects. However, the bonded nature of the
device means that the substrate material containing the electrical
components may be different than the cap material, adding great
flexibility to device design.
[0164] The second technique for the fabrication of channels relies
on the sacrificial etch technique (Mastrangelo and Muller 1989). In
this technique, the channel is formed from a patterned thin film
that determines the channel height. The film is covered by the
deposition of a thick cap material and access holes are opened
through it. The sacrificial material defining the channel is next
removed by chemical etching through the access holes, and finally
the channel is sealed by plugging the access holes. The main
advantage of this fabrication approach is that the channel
fabrication takes place entirely on one side of the substrate;
hence this technique is referred as surface micromachining. The
ability to pattern channels on the surface of the substrate brings
a great deal of flexibility. Surface micromachined channels may be
fabricated on substrates with complex topographies of
interconnects, sensors, and control electronics. In surface
micromachined devices, the analytical instrumentation is built
along with the channel on the same physical substrate.
[0165] The devices by both the hybrid (bonded) and monolithic
(surface machining) designs have been constructed. For bonded
structures, both glass and silicon substrates to form channels (500
.mu.m wide), and t-circuits (aluminum heater circuitry, each wire
filament is 5 .mu.m wide) have been used. The monolithic device is
compatible with conventional NMOS device fabrication.
[0166] B. Channel Fabrication
[0167] The channels are made of diffused silicon on the bottom and
a thin film cap on the top. This type of channel may be routed
through low-mass diaphragm-type heaters needed for the reaction. On
the top layer, a set of thin film electrodes and heaters is
constructed. Both the channels and entry port components can be
formed by etching of silicon. The depth of etching can be
controlled by prior doping of the silicon material with an etch
stop (boron).
[0168] The surface treatment of the channels may be done by
immersing the open channel in organosilane or a self-assembled
monolayer coating, with oxygen reactive ion etching removing the
surface from unwanted areas. Heating elements, dielectric sensors,
and connecting wires may be made from sputtered aluminum metal and
conventional masking. The sequential activation of heating elements
can be computer controlled through external circuitry, and a
printed circuit board connector.
[0169] C. Channel Treatment
[0170] Prior to performing microdroplet motion and biological
reactions, the channels are preferably treated by washing with
base, acid, buffer, water and a hydrophilicity-enhancing compound,
followed by a relatively high concentration solution of
non-specific protein. "Hydrophilicity-enhancing compounds" are
those compounds or preparations that enhance the hydrophilicity of
a component, such as the hydrophilicity of a transport channel. The
definition is functional, rather than structural. For example,
Rain-X.TM. anti-fog is a commercially available reagent containing
glycols and siloxanes in ethyl alcohol. However, the fact that it
renders a glass or silicon surface more hydrophilic is more
important than the reagent's particular formula.
[0171] "Hydrophobic reagents" are used to make "hydrophobic
coatings" in channels. It is not intended that the present
invention be limited to particular hydrophobic reagents. In one
embodiment, the present invention contemplates hydrophobic polymer
molecules that can be grafted chemically to the silicon oxide
surface. Such polymer molecules include, but are not limited to,
polydimethylsiloxane. In another embodiment, the present invention
contemplates the use of silanes to make hydrophobic coatings,
including but not limited to halogenated silanes and alkylsilanes.
In this regard, it is not intended that the present invention be
limited to particular silanes; the selection of the silane is only
limited in a functional sense, ie., that it render the surface
hydrophobic.
[0172] In various aspects of the invention,
n-octadecyltrichlorosilane (OTS), octadecyldimethylchlorosilane,
1H, 1H, 2H, 2H-perfluorodecyltricho- lorosilane (FDTS,
C.sub.10H.sub.4F.sub.17SiCl.sub.3), fluoroalkyl-, aminoalkyl-,
phenyl-, vinyl-, bis silyl ethane- or
3-methacryloxypropyltrimethoxysilane (MAOP) are contemplated as
hydrophobic reagents. Such reagents (or mixtures thereof) are
useful for making hydrophobic coatings, and more preferably, useful
for making regions of a channel hydrophobic (as distinct from
coating the entire channel).
[0173] In a preferred embodiment, the channels are washed with
approximately 100 .mu.l each of the following solutions in series:
0.1N NaOH; 0.1N HCl; 10 mM Tris-HCl (pH 8.0), deionized, H.sub.2O,
Rain-X Anti-Fog (a hydrophilicity-enhancing compound commercially
available from Unelko Corp., Scottsdale, Ariz), and 500 .mu.g/.mu.l
bovine serum albumin (non-specific protein commercially available
in restriction enzyme grade from GIBCO-BRL). The wafer was placed
on a stereoscope stage (Olympus SZ1145), and the contact pads for
the heating elements were connected to a regulated power supply.
Heating occurred by passing approximately 30 volts through the
element in short pulses and observing the movement rate of the
droplets. A detectable reduction in droplet volume from evaporation
was noted in each study, usually of less than 30%. Droplet movement
was recorded with a Hamamatsu video camera on videotape.
[0174] It is not intended that the present invention be limited to
particular dimensions for the hydrophobic regions of the present
invention. While a variety of dimensions are possible, it is
generally preferred that the regions have a width of between
approximately 10 and 1000 .mu.m (or greater if desired), and more
preferably between approximately 100 and 500 .mu.m.
[0175] A surface (such as a channel surface) is "hydrophobic" when
it displays advancing contact angles for water greater than
approximately seventy degrees. In one embodiment, the treated
channel surfaces of the present invention display advancing contact
angles for water between approximately ninety (90) and
approximately one hundred and thirty (130) degrees. In another
embodiment, the treated microchannels have regions displaying
advancing contact angles for water greater than approximately one
hundred and thirty (130) degrees.
[0176] D. Glass Channel and Chamber Fabrication
[0177] The channel and the chamber fabrication begins by depositing
0.4 .mu.m metallic layer of Gold (Electron beam deposition) on the
surface of 500 .mu.m thick glass water (Dow Coming 7740). A 0.06
.mu.m layer of chromium is used as the adhesion layer. Photoresist
is applied and patterned using glass mask 1 and developed. The
metal layers are etched in gold etchant (Gold Etchant TFA, Transene
Co.) and Chromium etchant (CR-14, Cyantec Inc.). The accessible
glass is then etched in a solution of freshly prepared hydrofluoric
and nitric acid (7:3, v/v). The etch rate is approximately 5
.mu.m/min and the etch depth is conveniently measured using a
surface profilometer. The metal layers are removed and the wafer
rinsed in DI water, air dried and oven dried at 100.degree. C. for
20 min. The following processing steps are done for patterning
hydrophobic regions onto the glass surface.
[0178] 1. Hydrophobic Patterning of Glass Substrate
[0179] A 1.5 .mu.m thick aluminum layer was electron beam
deposited, covering the etched channels and chamber. A thick
photoresist (AZ 4620) is applied and spun at 750 rpm for 50 sec .
The resist is patterned (SAM Mask) and developed. The exposed
aluminum is etched in aluminum etchant. The photoresist is stripped
off in hot PRS 2000 (J. T. Baker). The samples are then cleaned in
acetone, isopropyl alcohol and DI water for 5 min each and the
water dried off in a 100.degree. C. oven of 10-15 min. The samples
are then dipped in a 1% OTS solution in toluene for 10-15 min. The
SAM deposition was carried out in a chemical hood. The samples were
then rinsed in toluene, isopropyl alcohol and DI water for 5 min
each. Next, they were put in aluminum etchant until all metallic
aluminum was removed. The samples were then rinsed in DI water and
air dried. For the devices with the inlet from the top, holes were
drilled by electrochemical discharge drilling.
[0180] The glass side was then aligned on top of the silicon side
and then bonded together using optical adhesive (SK-9 Lens Bond,
Sumers Laboratories, Fort Washington, PA). The bond was cured under
an ultraviolet light source (365 nm) for 24 h.
[0181] E. Heaters and Resistive Temperature Detectors
[0182] The fabrication process for the heater and temperature
detector begins by using Silicon water (p-type, 18-22 alun-cm,
boron concentration .about.10.sup.15 cm.sup.3) as a substrate for
growth of S102 thermal oxide (1 .mu.m). A 0.5 .mu.m metallic
Aluminum film is electron beam deposited. Photoresist PR 1827 is
applied and spun at 4000 rpm for 30 sec, patterned (metal 1) and
developed. The exposed aluminum is etched in aluminum etchant and
the photoresist stripped to define the metal heater.
[0183] Photoresist is spun again and a second lithography is done
(metal 2). A 0.15 .mu.m layer of platinum ("Pt") is electron beam
deposited. A 0.03 .mu.m thick titanium metal layer (electron beam
deposited) is used as the adhesion layer. The resist and the
overlying metal is lifted off by development using Microposit 1112A
remover in solution (Shipley Co.). This platinum metal will be used
as the resistive thermal detector. Next, 0.7 .mu.m of low
temperature oxide (LTO) of silicon is deposited to act as the
barrier layer and the hydrophilic substrate. A third lithography is
done and the LTO is etched in buffered hydrofluoric acid to open
contacts to the metal contact pads. The further processing steps
are done to pattern hydrophobic regions onto the hydrophilic
silicon oxide surface.
[0184] 1. Hydrophobic Patterning of Silicon Oxide Substrate
[0185] A 0.1 .mu.m layer of chromium metal is electronbeam
deposited on the processed water. Photoresist PR 1827 is applied
and spun at 2000 rpm for 30 sec. The resist is patterned (SAM mask)
and developed. The exposed chromium metal is etched in chromium
etchant to expose the silicon oxide and the photoresist is then
stripped off. The samples are then cleaned in acetone, isopropyl
alcohol and DI water for 10 min each, air dried and oven dried at
100.degree. C. for 5 min. The samples are then put in 1 wt %
octadecyltrichlorosilane (OTS) solution in toluene for 15-30 min.
OTS deposits on the samples as a self assembled monolayer (SAM).
The samples are then rinsed in toluene, isopropyl alcohol and DI
water for 5 min each, and then oven dried (100.degree. C., 5 min).
Next, they are put in chromium etchant to remove the chromium layer
below. The SAM on the chromium metal gets lifted off as a result of
this. The samples were then rinsed in DI water and air dried,
resulting in regions of intact hydrophobic regions on a hydrophilic
oxide substrate. Heater elements and RTDs have also been fabricated
on a quartz substrate. The fabrication steps are similar to that of
the silicon processing steps.
[0186] Once the appropriate chemicals are added to the DNA sample,
the solution may be passed through several different temperatures.
The mixed solution may be transported to a uniformly heated
reaction chamber of the unit. Once in the chamber, the temperature
of the solution may be increased using local heaters and
temperature sensors. The temperature of the ends of the drops may
be monitored and maintained at the same temperature to prevent the
drop from leaving the reaction zone. If the drop does begin to
move, local temperature gradients could quickly stabilize the drop.
The cooling of the drop may be accomplished by simple conduction of
the heat through the walls of the channel to ambient
temperature.
[0187] F. Fluid Mixing Chamber
[0188] The mixing chamber consists of an enlarged portion of the
microchannel structure, with one or more microchannels connected to
the chamber. The mixing chamber is suspended on a thin silicon
nitride diaphragm. This construction allows for excellent thermal
isolation, as needed for low power heat cycling of the mixture.
Construction of membrane suspended structures has been demonstrated
(Mastrangelo et al., 1991). The heating is effected with a set of
concentric resistors (heaters) that are placed on the periphery of
the mixing chamber. This design, along with the high thermal
conductivity of the liquid sample, makes the chamber temperature
quite uniform. Along with the heaters, temperature sensors (diodes)
are constructed on the diaphragm to monitor the temperature of the
mixture. The low mass construction of the chamber allows for rapid
heating cycles. Temperature control may handle samples of variable
volume and heat capacity. The chamber also contains a set of
electrodes and heating elements to drive the mixture out of the
chamber at the completion of the reaction.
[0189] G. Electrophoresis and Detector Component Design
[0190] The present invention contemplates one or more gel
electrophoresis modules as a component of the microscale device.
Reducing the thickness of the electrophoresis channel may improve
resolution. Thinner gels dissipate heat more readily and allow
higher voltages to be used, with concomitant improvements in
separation. The position and width of the electrophoresis detector
are also critical to the ultimate resolution of the electrophoresis
system. A micromachined electronic detector, such as a photodiode,
placed in the underlying silicon substrate may be less than one
micron from the gel matrix and can have a width of 5 microns or
less. Since the gel length required for the resolution of two
migrating bands is proportional to the resolution of the detector,
the incorporation of micron-width electronic detectors can reduce
the total gel length required for standard genotyping by at least
an order of magnitude.
[0191] To demonstrate that standard gel electrophoresis can operate
in micron-diameter channels, modules were fabricated using etched
glass channels and fluorescent-labeled DNA (YOYO intercalating
dye). Polyacrylamide gel electrophoresis of a complex DNA mixture
was performed in a channel 500 .mu.m wide and 20 .mu.m deep. The
electrophoresis was performed with the positive electrode to the
right and the DNA sample applied at the left. The DNA sample
(Bluescript KS digested with MspI) is labeled with intercalating
UV-fluorescent dye (YOYO-1) and is visualized under incandescent
light. Separation of the component bands is clearly visible less
than 300/.mu.m from the buffer reservoir-to-gel interface. The high
resolution of the detector (in this case, a microscope) allowed the
use of an unusually short gel, resolving several closely eluting
bands.
[0192] H. Miniature Electrophoresis Chamber
[0193] A 20 .mu.m.times.500 .mu.m.times.4 cm channel etched into a
glass wafer was used as an electrophoresis chamber. The channels
may be made by three different processes: a glass channel
wet-etched, a silicon channel dry-etched (RIE), or a silicon
channel wet-etched. Although the edges of the channel are rough and
the walls of the channels are not vertical, the floor of the
channel is quite smooth. Better channels may also be constructed
with silicon as the base material using dry or wet etching. The
glass channel was then bonded to a quartz slide using UV-cure
cement and loaded with a 15% polyacrylamide gel and 1.times. TBE
running buffer. The gel was loaded with DNA ladder (BSKSIMSPI
50-500 bp), stained with TOTO fluorescent dye, and placed in a
.about.3 volt/cm field for 30 minutes. At these short times and low
voltages, separation into visibly resolved bands is obtained.
[0194] I. Integrated Electrophoresis/Detection Device
[0195] Monolithic devices created from silicon have the advantage
that no bonding is necessary and that electronic components may be
integrated with the mechanical system in any location. A silicon
micromachined gel electrophoresis channel integrated with a silicon
radiation/fluorescence detector underneath it was fabricated. The
"die" measures 1.25.times.1.25 cm and contains about 20 different
types of gel devices. Among these devices, the channel width varies
from 20 to 150 .mu.m, and the channel height is approximately 3
.mu.m. There are different channel formats including straight,
folded, and looped channels, each of which has at least one DNA
detector. The longest channel on this wafer is a 9.5 cm long folded
channel. For folded channels, as long as the channel bends are
paired curves, it may be shown that the electric field is uniform
around the bend and the solute bands start and end as uniform
bands.
[0196] The structure primarily comprising a silicon diffused diode
detector (Kemmer, 1980; Wouters and van Sprakelaar 1993) fabricated
underneath a gel channel. The diode is fabricated on a high purity
p-type float zone substrate to assure a good carrier lifetime. A
layer of silicon dioxide is used as a passivation layer below a
silicon nitride blocking layer. The electrodes for the
electrophoresis stage are formed by deposition and patterning of
n+polycrystalline silicon. The channel for the microgel is built
with two layers of phosphoslicate glass as described in Mastrangelo
and Muller 1989; Mastrangelo and Muller 1989. The cap of the
channel is deposited using a thin silicon nitride dielectric and a
2 m-thick undoped polysilicon shell. A series of etching holes are
patterned on the side or top of the shell down to the
phosphosilicate glass and used to sacrificially etch the
phosphosilicate glass (Mastrangelo and Muller, 1989) thus forming
the channel cavity. The cavity can then be refilled with
polyacrylamide gel.
[0197] The invention has tested the radiation/fluorescence
detectors and performed simple DNA separations with them. The
experiments were performed using a P.sup.32 labeled DNA source
placed on top of the detector. Note that the chip used for this
test did not have the channels formed on the surface and contained
as an insulating layer. Pulse-shaped (Knoll, 1989) scope traces or
the measured signals from the diode detector were detected from
sample DNA. Not only is the response rapid (.apprxeq.1 .mu.s), but
a single decay event (each trace is from only one particle) may be
detected.
[0198] Fluorescent DNA may also be detected with the same detector.
A detector was mounted in a 24 pin IC package and covered with a
SYBR green gel filter (the filter was .about.1 cm from the detector
surface). A glass slide was placed over the filter and .about.40
.mu.l of 0.03 .mu.g/.mu.g of SYBR green labeled DNA solution was
placed on the slide (contained by silicon grease wells). The sample
was illuminated using a Ziess Axioskop UV source with a .about.490
nm filter. The reverse current was measured with an HP 4145B
semiconductor parameter analyzer as a function of the bias voltage.
The signal from the SYBR sample is approximately twice the control
signal (DI water). Although this experiment was not performed under
optimum conditions, it clearly demonstrates that the detector is
capable of detecting fluorescent DNA.
[0199] Sample separation experiments have also been performed using
this detector. A 100 .mu.m ID capillary tube filled with 10%
polyacrylamide was glued on top of a detector (same as that
described above) approximately 1.5 cm from the sample injection
end. A 100 bp and a 300 bp PCR.TM. product (50/50 mixture) was
electrokinetically injected into the channel for approximately 5
min using a field of 25 V/cm after which the sample well was
flushed and refilled with running buffer. The results of the 125
minute run show the detection of the radioactive primers and the
two PCR.TM. products. Note that, although the radiation detection
scheme may not be used in the final sequencing system, it is very
useful to evaluate the electrophoresis chambers until the
necessary, fluorescent filters are constructed and tested.
[0200] The present invention contemplates an electrophoresis unit
that integrates a micromachined channel and an electronic DNA
detector. The channel is constructed using a sacrificial etch
process on a single silicon wafer rather than the bonded
surface-etch method described earlier. In the sacrificial etch
technique, the channel configuration is patterned by depositing on
the wafer surface an etch-sensitive material (phosphosilicate
glass, SiO.sub.2.P.sub.x) with a thickness equivalent to the
desired channel height. A triple-layer overlay of plasma-enhanced
chemical vapor deposited silicon nitride, undoped polycrystalline
silicon, and silicon nitride
(Si.sub.xN.sub.y/polySi/Si.sub.xN.sub.y) completely covers the
sacrificial material with the exception of small access holes on
the top or sides. A selective liquid etch removes the sacrificial
layer material, but not the overlay or the underlying substrate.
The sacrificial etch technique results in a complete channel being
formed directly on the substrate containing the electronic
components. The 3 .mu.m deep channel has two buffer reservoirs on
either end with integral phosphorus-doped polycrystalline silicon
electrodes. The channel height formed by this technique
(.about.3/.mu.m) is considerably smaller than the height of the
bonded structures due to the limitations of the sacrificial layer
deposition and the strength of the overlying layer. Note that, for
these channel dimensions, liquid drops would have volumes on the
order of picoliters.
[0201] The diffusion regions of the doped-diffusion diode radiation
detector elements fabricated on a silicon wafer are approximately
300 .mu.m long and 4 .mu.m wide, and are flanked by the guard ring
shielding electrodes.
[0202] A radiation detector, consisting of a 10 .mu.m wide
"p-n"-type diode with a 5 .mu.m wide guard ring around the outer
edge, is fashioned directly into the silicon substrate underneath
the channel. In this implementation, an integral radiation detector
was chosen because of high sensitivity (a single decay event),
small aperture dimensions, and well-know fabrication and response
characteristics. On this electrophoresis system, a 1 cm long, 3
.mu.m thick gel is able to perform as separation on a 80 and a 300
base-pair fragment of DNA. It should be noted that this diode,
although currently configured for high-energy beta particle
detection, can also operate as a photon detector. With proper
wavelength filters and light sources, detection of fluorescence
emission may be accommodated with a similar device.
[0203] Radiation detectors were prepared as follows. A 200 1/2-cm,
float zone, boron-doped, p-type silicon wafer was used as a
substrate. Diffused layers of phosphorus (5.times.10.sup.4
cm.sup.-2) and boron (1.times.10.sup.15 cm.sup.-2) were
ion-implanted onto the sample in lithographically-defined regions;
thermal silicon oxide was grown (0.2 .mu.m at 900.degree. C.) over
the wafer; and contact holes were etched to the diffusion layer
using buffered hydrofluoric acid solution (5:1). A 3.3 .mu.m layer
of Microposit 1400-37 photoresist was patterned to define the metal
pads; 50 nm chromium followed by 400 nm gold was evaporated over
the resist; and the metallization lifted off the regions retaining
the resist. A layer of Microposit 1813 photoresist was applied
across the wafer and baked for 110.degree. C. for 30 min to form an
aqueous solution barrier. Radioactive phosphorus (.sup.32P) decay
events could be detected using a sample of labeled DNA in PCR.TM.
reaction buffer placed on the photoresist layer. The detector was
connected to a charge-sensitive preamplifier (EV-Products 550A),
followed by a linear shaping amplifier and a standard
oscilloscope.
[0204] An oscilloscope trace of output from the radiation detector
showing individual decay events from .sup.32P-labeled DNA was
generated after the aqueous DNA sample was placed directly on the
detector and sampled for 30 sec. The screen is displaying a
vertical scale of 0.5V/division and horizontal scale of 20
.mu.sec/division.
[0205] J. Gel Voltage and Temperature Control Circuits
[0206] The control circuitry and software for the integrated DNA
sample processing and sequencing devices are a further aspect of
the invention. In particular, the devices will require circuitry
for signal buffering and for the multiplexing of control signals. A
microprocessor, either external or on-wafer, determines the
synchronization of events on the device and store the output
information.
[0207] Temperature control of gel occurs by heating with
polysilicon or thin metal resistors imbedded in the surface of the
wafer immediately beneath the channel. The precise temperature
control of the gel is required as minute fluctuations contribute to
the dispersion of the migrating sample and non-uniform bands. The
power distribution and optimal heater placement is determined for
each electrophoresis design by solving the relevant heat transfer
equations. As long as the walls of the electrophoresis channel are
maintained at the appropriate temperature and the height of the
channel is constructed uniformly, the internal temperature of the
across the gel should not vary by more than 1.0.degree. C. and be
maintained at any arbitrary temperature.
[0208] Although the electrophoresis voltages may be low, the
potential use of high voltages in the gel electrophoresis channels
will necessitate care in fabricating the silicon oxide/silicon
nitride/silicon oxide insulating layer. Silicon nitride and silicon
oxide have a breakdown field voltage of about 200-1000 V/.mu.m
(Sze, 1967; Harari, 1977; Sze, 1981). Consequently, the layers
between the silicon circuitry (including the diode detectors) and
the electrically active gel are approximately 2 to 4 microns thick.
The possible presence of minute "pinholes" in the LPCVD deposited
layers must also be carefully monitored, since such holes can
provide local weak points in the insulation of the silicon
circuitry. However, the routine use of silicon nitride as a mask
for wet etch processes in solid-state fabrication indicate that
pinholes are insignificant.
[0209] Glass may be used as their substrate material. In a
glass-based device, any associated on-wafer circuitry must be
constructed on polysilicon thin films adjacent to the
electrophoresis channels (Tickle, 1969). As an alternative are
designs that energize small fractions of the channel at a time,
thereby decreasing the voltage required without sacrificing
resolution. Cyclical or "loop" 9.5 cm channels were constructed to
test this (Sun and Hartwick, 1994). However, since active
electrodes are in immediate contact with the get matrix care must
be exercised so as not to irreversibly adsorb the DNA samples on
the electrodes. Alternative gel channel designs are possible.
[0210] In one embodiment of the device of the present invention,
the device comprises a glass top bonded to a silicon substrate
containing the heater, the contact pad and the resistive
temperature detector. The glass side has channels and chambers
etched into it. Inlet and overflow ports, a gas vent and a air
chamber are also part of this embodiment.
[0211] III. Fluid Movement
[0212] The present invention contemplates a method for moving
microdroplets, comprising providing a liquid microdroplet disposed
within a microdroplet transport channel etched in silicon, the
channel in liquid communication with a reaction region via the
transport channel and separated from a microdroplet flow-directing
means by a liquid barrier, and conveying the microdroplet in the
transport channel to the reaction region via the microdroplet
flow-directing means. It is intended that the present invention be
limited by the particular nature of the microdroplet flow-directing
means. In one embodiment, it comprises a series of aluminum heating
elements arrayed along the transport channel and the microdroplets
are conveyed by differential heating of the microdroplet by the
heating elements.
[0213] It has been found empirically that the methods and devices
of the present invention may be used with success when, prior to
the conveying described above the transport channel (or channels)
is treated with a hydrophilicity-enhancing compound. It is not
intended that the invention be limited by exactly when the
treatment takes place. Indeed, there is some flexibility because of
the long-life characteristics of some enhancing compounds.
[0214] It has also been found empirically that the methods and
devices of the present invention may be used with success when
regions of the microchannel are treated with hydrophobic reagents
to create hydrophobic regions. By using defined, hydrophobic
regions at definite locations in microchannels and using a pressure
source, one can split off precise nanoliter volume liquid drops
(i.e., microdroplets) and control the motion of those drops though
the microchannels.
[0215] In one embodiment employing such hydrophobic regions (or
"hydrophobic patches"), the present invention contemplates a method
for moving microdroplets, comprising providing microdroplet
transport channel (or a device comprising a microdroplet transport
channel), the channel having one or more hydrophobic regions and in
communication with a gas source; introducing liquid into the
channel under conditions such that the liquid stops at one of the
hydrophobic regions so as to define a source of liquid
microdroplets disposed within the channel and a liquid-abutting
hydrophobic region, and separating a discrete amount of liquid from
the source of liquid microdroplets using gas from the gas source
under conditions such that a microdroplet of defined size comes in
contact with, and moves over, the liquid-abutting hydrophobic
region.
[0216] In one embodiment, the gas from the gas source enters the
channel from a gas-intake pathway in communication with the
microdroplet transport channel and exits the channel from a gas
vent that is also in communication with the microdroplet transport
channel. It is preferred, in this embodiment, that the introduction
of liquid into the channel (as set forth in the above-described
method) is such that the liquid passes over the gas-intake pathway
and the desired size of the microdroplet is defined by the distance
between the gas-intake pathway and the liquid-abutting hydrophobic
region. In this embodiment, introduction of the gas (as set forth
in the above-described method) forces the microdroplet to pass over
the liquid-abutting hydrophobic region and pass by (but not enter)
the gas vent.
[0217] In another embodiment employing such hydrophobic regions (or
"hydrophobic patches"), the present invention contemplates a method
for moving microdroplets, comprising: providing a device comprising
a microdroplet transport channel etched in silicon, the channel
having one or more hydrophobic regions and in communication with a
gas source; introducing liquid into the channel under conditions
such that the liquid stops at one of the hydrophobic regions so as
to define a source of liquid microdroplets disposed within the
channel and a liquid abutting hydrophobic region, and separating a
discrete amount of liquid from the source of liquid microdroplets
using gas from the gas source under conditions such that a
microdroplet of defined size comes in contact with, and moves over,
the liquid-abutting hydrophobic region.
[0218] Again, it has been found empirically that there is a need
for a liquid barrier between the liquid in the channels and the
electronics of the silicon chip. A preferred barrier comprises a
first silicon oxide layer, a silicon nitride layer, and a second
silicon oxide layer.
[0219] The present invention further contemplates a method for
merging microdroplets comprising providing first and second liquid
microdroplets, a liquid microdroplet delivering means, and a
device, said device comprising a housing comprised of silicon,
first and second microdroplet transport channels etched in the
silicon and connecting to form a third transport channel containing
a reaction region, a microdroplet receiving means in liquid
communication with the reaction region via the transport channels,
and microdroplet flow-directing means arrayed along the first,
second and third transport channels delivering the first liquid
microdroplet via the microdroplet delivering means to the first
transport channel, delivering the second liquid microdroplet via
the microdroplet delivering means to the second transport channel,
and conveying the microdroplets in the transport channels to the
reaction region in the third transport channel via the microdroplet
flow-directing means, thereby merging the first and second
microdroplets to create a merged microdroplet.
[0220] In one embodiment, said first microdroplet comprises nucleic
acid and the second microdroplet comprises a nuclease capable of
acting on the nucleic acid. In this embodiment, it is desirable to
enhance the mixing within the merged microdroplet. This may be
achieved a number of ways. In one embodiment for mixing, after the
conveying of step, the flow direction is reversed. It is not
intended that the present invention be limited by the nature or
number of reversals. If the flow direction of the merged
microdroplet is reversed even a single time, this process increases
the mixing of the reactants.
[0221] The present invention contemplates methods, compositions and
devices for the creation of microdroplets of discrete (i.e.,
controlled and predetermined) size. The present invention
contemplates the use of selective hydrophobic coatings to develop a
liquid-sample injection and motion system that does not require the
use of valves. In one embodiment, the present invention
contemplates a method of lift-off to pattern hydrophobic and
hydrophilic regions on glass, quartz and silicon substrates,
involving the deposition of a hydrophobic reagent (such as a
self-assembled monolayer film of OTS) on a silicon oxide surface
pattered by a metal layer and subsequent removal of the metal to
give hydrophobic patterns. Other substrates such as plastics may
also be used after depositing a think film of silicon oxide or
spin-on-glass.
[0222] Previous work in patterning hydrophobic surfaces have been
done by photocleaving of such monolayer films. The photocleaving
procedure uses Deep-UV exposure to make the molecules of the
monolayer hydrophilic. By contrast, the present invention
contemplates a method which eliminates the use of high-power UV
source; rather the preferred method of the present invention uses
microfabrication procedures.
[0223] Following the proper hydrophobic patterning of the surface
(e.g., the surface of a microdroplet transport channel), the
present invention contemplates the placement of a patterned etched
glass cap over the pattern on a flat surface. The
hydrophobic/hydrophilic channels thus formed can then be used to
move precise nanoliter-volume liquid samples.
[0224] FIG. 3A and FIG. 3B show a schematic of one embodiment of a
device (10) to split a nanoliter-volume liquid sample and move it
using external air, said device having a plurality of hydrophobic
regions (hatched regions). Looking at FIG. 3A, liquid (shown as
solid black) placed at the inlet (20) is drawn in by surface forces
and stops in the channel at the liquid-abutting hydrophobic region
(40), with overflow handled by an overflow channel and overflow
outlet (30). In the embodiment shown in FIG. 3A, the from of the
liquid moves by (but does not enter) a gas-intake pathway (50) that
is in fluidic communication with the channel; the liquid-abutting
hydrophobic region (40) causes the liquid to move to a definite
location. Gas from a gas source (e.g., air from an external air
source and/or pump) can then be injected (FIG. 3B, lower arrow) to
split a microdroplet of length "L". The volume of the microdroplet
split-off (60) is pre-determined and depends on the length "L" and
the channel cross-section. To prevent the pressure of the gas
(e.g., air) from acting towards the inlet side, the inlet (20) and
overflow ports (30) may be blocked or may be loaded with excess
water to increase the resistance to flow.
[0225] The patterned surfaces may also be used to control the
motion of the drop. By placing a hydrophobic gas vent (70) further
down the channel, one can stop the liquid microdroplet (60) after
moving beyond the vent (70). As the drop (60) passes the vent (70),
the air will go out through the vent (70) and will not push the
drop further.
[0226] One can start moving the drop (60) again by blocking the
vent (70). By using a combination of hydrophobic air pressure
lines, hydrophobic vents and strategic opening and/or closing of
vents, one can move the liquid drop back and forth for mixing or
move it to precise locations in a channel network to perform
operations such as heating, reaction and/or separations.
[0227] In addition to using external air, one can also use
internally generated air pressure to split and move drops. FIG. 4A
and FIG. 4B show a schematic of one embodiment of a device (110) of
the present invention to split (e.g., define), move and stop
microdroplets using internal gas (e.g., air) pressure generation,
said device having a plurality of hydrophobic regions (hatched
regions). Looking at FIG. 4A, liquid (shown as solid black) placed
at the inlet (120) is drawn in by surface forces and stops in the
channel at the liquid-abutting hydrophobic region (140), with
overflow handled by an overflow channel and overflow outlet (130).
In the embodiment shown in FIG. 4A, the front of the liquid moves
by (but does not enter) a gas-intake pathway (150) that is in
fluidic communication with the channel. By heating air trapped
inside chambers (180) that are in fluidic communication with the
microdroplet transport channel via the gas-intake pathway (150), an
increased pressure may be generated. The magnitude of the pressure
increase inside a chamber of volume V is related to the increase in
temperature and may be estimated by the Ideal Gas relation.
[0228] Increasing the temperature of the gas (e.g., air) will cause
the pressure inside the chamber to rise until the pressure is high
enough to split off a drop (160) and move it beyond the
liquid-abutting hydrophobic region (140). In order to avoid the
problem of the expanded air heating up the liquid, the chamber may
be placed at a distance from the transport channel. Moreover,
having the heaters suspended inside the air chamber or placing them
on a thin insulation membrane will not only avoid cross-talk, but
will involve a minimal power consumption.
[0229] The compositions and methods are suitable for devices having
a variety of designs and dimensions, including, but not limited to,
devices with chamber volumes from 0.24 mm.sup.3to 0.8 mm.sup.3 for
channel dimensions of 40 .mu.m by 500 .mu.m. Drop splitting and
motion is seen with 1-3 sec using voltages between 4.5 volts to 7.5
volts (the resistance of the heaters varied between 9.5 ohms to 11
ohms). The size of the drop split is between approximately 25 and
approximately 50 nanoliters, depending on the value "L" used for
the channel design. Keeping the heaters actuated keeps the
microdroplet moving almost to the end of the channel (a distance of
around 125 mm); the time taken depends on the voltage applied to
the heater and the volume of the chamber. Initiation of drop motion
is seen sooner for the operation of devices with smaller chambers.
While an understanding of precise mechanisms is not needed for the
successful practice of the present invention, it is believed that
with smaller chamber, the volume is smaller and higher values of
pressure are achieved more quickly. The maximum temperatures
reached near the heater are approximately 70.degree. C. measured by
the RTD.
[0230] A. Movement of Discrete MicroDroplets
[0231] The present invention contemplates microscale devices,
comprising microdroplet transport channels having hydrophilic and
hydrophobic regions, reaction chambers, gas-intake pathways and
vents, electrophoresis modules, and detectors, including but not
limited to radiation detectors. In some embodiments, the devices
further comprise air chambers to internally generate air pressure
to split and move microdroplets (i.e., "on-chip" pressure
generation).
[0232] The present invention describes the controlled movement of
liquid samples in discrete droplets in silicon. Discrete droplet
transport involves a system using enclosed channels or tubes to
transport the liquid to the desired locations (FIG. 1-B). Within
the channels, discrete liquid reagent microdroplets may be
injected, measured, and moved between the biochemical analysis
components. Discrete droplet movement has three advantages. First,
each sample droplet is separated from all others so that the risk
of contamination is reduced. Second, in a uniform channel, the
volume of each sample may be determined by merely measuring the
droplet length. Third, the motion of these droplets may be
accomplished with heating (i.e., using internal forces and no
moving parts). Movement is performed using thermal gradients to
change the interfacial tension at the front or back of the droplets
and, thus, generate pressure differences across the droplet. For
example, a droplet in a hydrophilic channel may be propelled
forward by heating the back interface. The local increase in
temperature reduces the surface tension on the back surface of the
droplet and, therefore, decreases the interfacial pressure
difference. The decreased pressure difference corresponds to an
increase in the local internal pressure on that end of the droplet
(PI increases). The two droplet interfaces are no longer in
equilibrium, with P.sub.1 greater than P.sub.2, and the pressure
difference propels the droplet forward.
[0233] That is to say, forward motion may be maintained by
continuing to heat the droplet at the rear surface with successive
heaters along the channel, while heating the front surface may be
used to reverse the motion of the droplet. Applying a voltage to
the wire beneath the channel generates heat under the edge of the
droplet. Heating the left interface increases the internal pressure
on that end of the droplet and forces the entire droplet to the
right. The pressure on the interior of the droplet may be
calculated knowing the atmospheric pressure, P.sub.atm, surface
tension, .sigma., and the dimensions of the channel. For a circular
cross-section, the interior pressure, P.sub.i, is given by
P.sub.i=P.sub.atm (4.sigma. cos .theta.)/d where d is the diameter
of the channel and .theta. is the contact angle. Since .sigma. is a
function of temperature (.sigma.=.sigma..sub.o(1-bT) where
.sigma..sub.o and b are positive constants and T is the
temperature), increasing the temperature on the left end of the
droplet decreases the surface tension and, therefore, increases the
internal pressure on that end. The pressure difference between the
two ends then pushes the droplet towards the direction of lower
pressure (i.e., towards the right). The aqueous droplet shown is in
a hydrophilic channel (0<.theta.<90); for a hydrophobic
channel (90<.theta.<180), heating the right edge would make
the droplet move to the right.
[0234] Contact angle hysteresis (the contact angle on the advancing
edge of the droplet is larger than the contact angle on the
retreating edge) requires a minimum temperature difference before
movement will occur. The velocity of the droplet after motion
begins may be approximated using the equation .nu.=AEPd.sup.2/32
.mu.L where AEP is the pressure difference, .mu. is the viscosity
of the solution, and L is the length of the droplet. The present
invention contemplates temperature differences of greater than
30.degree. C. to create movement. Studies using temperature sensors
arrayed along the entire channel indicate that a differential of
approximately 40.degree. C. across the droplet is sufficient to
provide motion. In these studies, the channel cross-section was
20/.mu.m.times.500/.mu.m, and the volume of each of these droplets
may be calculated from their lengths and is approximately 100
nanoliters for a 1 cm long droplet.
[0235] B. Integrated Fluid Handling System
[0236] Although there are many designs currently available for
liquid handling in micromachined devices, a preferred method uses
individual drops propelled by induced gradients in surface tension.
The principle behind the technique is to inject the samples into
the device as discrete drops. These drops, once inside the
channels, may be propelled by changing the forces on the two drop
surfaces. For instance, if the drops are in a hydrophilic channel
(glass), the interfacial tension acts outward from both ends. Since
the surface tension of water decreases with increasing temperature,
heating the left side of the drop causes the drop to be propelled
towards the right. Splitting, merging, and mixing of such drops may
also be accomplished by careful control of drop location in
micromachined channels.
[0237] In certain embodinments, the channels contain approximately
30 heaters and 10 temperature sensors along the length of the
channels. Location sensors can sense the location, length, and,
therefore, the volume of the drop. The base material of the chip is
silicon with silicon oxide and nitride layers used for insulation.
The resistive heaters in the channel may be made from a variety of
materials including platinum, aluminum, and doped polysilicon; in
one aspect the chip has gold heaters. These resistive heaters are
inlaid into the insulating oxide to provide a smooth (<1 .mu.m)
surface for the upper insulating layer: failure to make the upper
surface smooth can result in layer instabilities and device failure
during heating in an aqueous environment. Silicon or glass channels
may be attached to the substrate with a variety of adhesive
techniques. Anodic, UV cure cement, and polyimide bonding have been
used in the invention, though other methods may be used, and are
known to those of skill in the art.
[0238] Drop motion was induced by changes in the surface tension in
a glass channel glued to a silicon substrate using UV-cure cement.
Note that the surface conditions, solution conditions, and channel
geometry, all affect the motion of the drop. Careful attention must
be paid to both the construction procedure and the surface
preparation procedure or drop motion will not occur. By being able
to move drops in this manner, the mixing of two drops (for sample
injection) or the splitting of one drop into two (for post reaction
treatment) may be accomplished.
[0239] C. Characteristics of Micro-Scale Fluids
[0240] In miniaturization of a DNA processing system, most
components may be designed similar to their benchtop equivalents.
Fabrication at the micron level may then be accomplished using
known silicon characteristics. Some specific components are easily
miniaturized. For instance, a heating element on a silicon wafer
will, for most applications, be able to heat a sample much more
rapidly than a larger scale heater. This increase in efficiency is
due to a decrease in the distance over which thermal energy must
travel and to the reduced mass of the sample being heated.
[0241] In contrast to the heating of samples, the movement of
samples is more complicated. The diameter of the "tubing" through
which samples will flow in the proposed system may be reduced to a
channel width as small as 10 .mu.m. This extremely small diameter
will change the typical characteristics of the fluid flow. Methods
to move liquids become much more difficult in the nanoliter volume
range. The Reynold's number (Re) of a liquid system is an
indication of the ease with which a liquid will move, and is
defined as Equation 1:
Re=(.nu.)(d)(r/.mu.) (1)
[0242] where .nu. is the velocity, d is the diameter of the tubing,
r is the density of the solution, and .mu. is its viscosity. Using
the Reynold's number, comparing a 1 cm diameter tube to a 10 .mu.m
diameter channel would result in a Re decrease from about 100 to
10.sup.-2 (for water moving with a velocity of 1 cm/s).
[0243] One method of coping with this new flow regime (very low Re)
would be to use higher pressure pumps. High pressure liquid
chromatography (HPLC) system (d.about.10 .mu.m) typically run at
thousands of pounds per square inch (PSI) pressure, while standard
liquid chromatography systems (d.about.100 .mu.m) can operate with
only several PSI. In the silicon wafer system, a pump-based
propulsion mechanism may be fabricated by designing an "in-chip"
peristaltic pump (Folta et al., 1992). This micropump design
consists of a heating element within a thermopneumatic chamber. The
thernopneumatic chamber, when heated, causes a membrane along the
flow channel to "bulge". Peristaltic pumping occurs by "bulging" of
a set of thermopneumatic actuators in series (Van Lintel, 1988;
Pohl, 1978). Unfortunately, these pumps must generate a relatively
high pressure to move the liquids through micrometer-sized
tubing.
[0244] Another method for moving small volumes of liquid is to use
gradients in surface tension (Edwards et al., 1991). If a thin
capillary, tube is inserted into water, the liquid in the
capillary, will rise a centimeter or so above the surface of the
surrounding water. This rise is due to the force of surface tension
acting on the meniscus as defined by Equation 2:
F=(.pi.)(d)(.sigma.)(cos .theta.) (2)
[0245] where d is the inside diameter of the tube, .sigma. is the
surface tension (force/length) and .theta. is the contact angle
(Osipow, 1962). If a smaller diameter capillary is used, the
decrease in force is proportional to d but the decrease in weight
of water per unit height in the capillary decreases by d.sup.2.
Therefore, for very large diameter tubes, the forces of surface
tension can usually be neglected due to the large mass of fluid.
However, for small tubes, pores, or channels, the force of surface
tension may be great compared to the mass of liquid being moved.
This "wicking" of liquid is a common occurrence and may be observed
when a porous solid is brought in contact with a solution (i.e.,
paper towel and water).
[0246] By controlling the magnitude and direction of the surface
forces, the movement of small sample volumes in the interior of
capillary tubes may be controlled. Several researchers have
described moving small drops through silicon channels using this
principle (Beni and Tenan, 1981; Matsumoto and Colgate, 1990; Fuhr
et al., 1992). The technique may be best understood by examining
the liquid drop contained in a glass capillary. For glass,
.theta.=0, consequently, the force due to surface tension is
pulling the drop both to the right and to the left, and is
perfectly balanced. If the surface tension on the left side of the
drop is decreased or the surface tension on the left side is
increased, the drop will be pulled to the right. Movement to the
left may be accomplished in a similar fashion (decreasing the
surface tension on the left or increasing the surface tension on
the right). Most previous work has changed the surface tension
force by altering the channel wall hydrophobicity, and
consequently, the contact angle .theta..
[0247] Alternatively, the surface force may be altered through
changes in the liquid surface. It is known that the surface tension
of liquids is a strong function of both temperature and surface
electrical charge (Osipow, 1962). Matsumoto and colleagues used
electrostatic control to develop a surface tension driven micropump
(Beni and Tenan, 1981; Matsumoto and Colgate, 1990). Because of the
possibility of charge attraction with the DNA molecules in
solution, temperature control may be a preferred choice for
changing liquid surface tension in the invention. For most liquids,
surface tension decreases nearly linearly with increased
temperature.
[0248] Modeling this dependence may be accomplished using the
linear empirical expression. Equation 3:
.sigma.=.sigma..sub.o(1-bT) (3)
[0249] where .sigma..sub.o and b are constants (Beni and Tenan,
1981; Matsumoto and Colgate, 1990). This expression means that
increases in temperature result in linear decreases in surface
tension. An empirical model was obtained by a linear fit of
.sigma..sub.o versus temperature for pure water. The change in
surface tension with temperature for pure water is approximately
-0.16 dyne/cm (Probstein, 1989) and remains nearly constant over
all temperatures for liquid water. It is the magnitude of this
change that can serve as the driving force for fluid movement;
therefore, knowledge of this parameter's magnitude is necessary for
predictions of liquid velocities in a capillary system.
[0250] D. Micro-Scale Fluid/Solute Parameters
[0251] As demonstrated in Equation 4, the velocity profile for
fluid motion in a capillary tube depends upon several
characteristics of the liquid and its interface with the flow
chamber. These include: .DELTA..sigma., the surface tension
difference between the drop ends, d, the capillary diameter, .mu.,
the liquid viscosity, and L the drop length.
.nu..sub.ave(.DELTA..sigma.)(d)/(8)(.mu.)(L) (4)
[0252] For example, the liquid viscosity, the surface tension
difference between the ends of the drop, and the contact angle
between the liquid and the flow chamber all influence the magnitude
of fluid motion. However, the flow chamber dimensions and geometry
affect the shape of the velocity profile of the liquid.
[0253] E. Fluid Viscosity
[0254] Fluid rheology is the study of how a fluid reacts to a
stress (force/area). For instance, a common class of fluids, termed
Newtonian fluids, exhibit a regular response to a fluid stress. The
behavior of a Newtonian fluid may be expressed in terms of its
constitutive equation, which states that the shear stress
(force/area) is proportional to the local velocity gradient (Bird
et al., 1960):
Shear Stress=(viscosity)(velocity gradient) (5)
[0255] where the constant of proportionality, the viscosity of the
fluid, is an indication of the resistance to flow.
[0256] The dilute aqueous DNA solutions are stored in a Tris-HCl
(10 mM), EDTA (1 mM) buffer solution. Although the DNA
concentration is presumably too low to affect the physical behavior
of the macroscopic fluid, the Newtonian behavior of the water-based
solvent was checked. Viscosity measurements, as a function of shear
rate, were taken for pure Tris-HCl/EDTA buffer in addition to 1,
50, and 105 microgram/ml samples of DNA solution. The results
indicate Newtonian behavior for all three DNA concentrations and
for the Tris-HCl/EDTA buffer. As expected, the viscosities of four
samples tested were Newtonian, and had viscosities very close to
that for water at 25.degree. C. (ie., lcP).
[0257] F. Surface Characteristics
[0258] The contact angle (force/contact length) between a fluid and
its solid surface is an extremely important parameter in surface
tension driven flow. The magnitude of this force is directly
related to the cosine of the contact angle between the liquid and
the solid flow chamber. To maximize this force, a perfectly
hydrophilic (contact angle of 0.degree., cos 0.degree.=13 or
perfectly hydrophobic (contact angle of 180.degree., cos
180.degree.=-1) surface is preferred. For example, clean glass
surfaces are extremely hydrophilic and form a 0.degree. contact
angle with pure water producing the maximum surface tension.
Surface treatments of glass can produce hydrophobic surfaces. Two
hydrophobic glass surface treatment processes have been examined.
First, a silane treatment was followed by addition of a long chain
aldehyde (decyl aldehyde). In the second treatment, a commercial
brand Rain-X used. Interestingly, the Rain-X treatment was the
easiest to apply and produced a more hydrophobic surface than the
silane treatment. However, the Rain-X contact angle was still much
less than optimal 180.degree. making it a less than ideal surface
for surface tension driven flow.
[0259] G. Surface Tension
[0260] From equation 4, the change in surface tension can serve as
the driving force for fluid motion. One embodiment of the invention
is described as a micromechanical integrated DNA analysis
technology, or MIDAT. In the MIDAT system a temperature difference
between the ends of the drop will be used to produce a surface
tension difference. For pure water, the change in surface tension
with temperature is -0.15 dyn/cm-.degree. C. (Probstein, 1989) and
is constant over the entire liquid range of water (Osipow, 1962).
Because the DNA solutions being used are very dilute, the surface
tension values are expected to be identical to water.
[0261] Using the Krus Interfacial Tensiometer K8, the surface
tension of both pure water and buffer solution was measured at
several temperatures between 15.degree. C. and 55.degree. C. As
expected, the buffer and the water solutions exhibit nearly
identical slopes. Also, the surface tensions of DNA solutions at
several concentrations between 0 and 120 ug/ml were measured. The
DNA concentrations were chosen to reflect a range relevant to
standard PCR.TM. reaction conditions (1 .mu.g/ml, 50 .mu.g/ml and
105 .mu.g/ml). There is little to no change in surface tension with
varying DNA concentrations at 25.degree. C. These values, ranging
from 70-71 dyn/cm, are very close to those described for pure water
at 25.degree. C. (72 dyn/cm).
[0262] H. Capillary Drop Movement
[0263] As an initial demonstration of surface tension driven flow,
a small volume of water was moved in a 0.5 mm inside diameter glass
capillary. This was accomplished by heating one of the liquid to
air interfaces on the drop, thereby imbalancing the surface tension
present in the two surfaces of the drop. The 1.5 centimeter long
drop was moved approximately 3 cm forward and back using a hot
water spray (80.degree. C.) as a heating system. The spray was
washed over the glass capillary near the back end of the drop, and
followed the drop as it moved. A rough estimate of the velocity of
the drop may be calculated from the timed video images. The drop is
moving at approximately 0.5 cm/sec, which is of the same order of
magnitude as the theoretical velocity prediction, as calculated
from Equation 4.
[0264] I. Silicon Microfabrication and Integrated Systems.
[0265] FIG. 2B shows a physical layout of a constructed chip. It
consists of a two wafer bonded structure. One of the wafers is made
of silicon and the other is glass. In the glass wafer, two levels
of thin-film aluminum are patterned to make electrodes,
interconnects, and heaters for the driven mechanisms. On the
silicon wafer, the chip is patterned with microchannels and sample
inlets and outlets. The two wafers are bonded together to complete
the system. The sample is moved inside the channel using a linear
array of electrical devices.
[0266] Three propulsion mechanisms are contemplated other than the
thermal surface tension method for fluid propulsion chips.
Microchannels with electrowetting (Beni and Tenan, 1981; Matsumoto
and Colgate, 1990; Washizu, 1992), dielectrophoretic, and thermal
gradient (Van Lintel, 1988; Pohl, 1978) drives have been
fabricated. Briefly, electrowetting propulsion relies on
charge-induced change in the hydrophobicity (wetting)
characteristics of the channel wall. Induction of a current along
the channel makes the wall more hydrophilic, drawing the liquid
drop toward the activated electrode. Dielectrophoresis utilizes the
difference in dielectric constant between water and air. A liquid
drop will be preferentially draw in between the plates of a charged
capacitor. Each chip is designed to move samples in the 5-50 nL
range.
[0267] Twenty-seven devices were fabricated from a single 100 mm
diameter wafer. The chips are cut out of the wafer and bonded to a
printed circuit (PC) board. The setup is constructed to control the
signals to each microchannel heater or electrode using a computer
for sample droplet formation, separation, and movement control. The
complete fabrication process requires 5 lithographic steps and was
completed in one week. Microchannels with thicknesses of 20, 50,
100, and 200 .mu.m deep and 500-1000 .mu.m width were patterned.
Each of these was fabricated with 20, 50, or 150 electrodes along
the microchannel length.
[0268] In determining whether a drop will move due to surface
tension gradients, the two key parameters are the magnitude of the
surface tension (.sigma.) and the contact angle (.theta.). For
channels in silicon wafers, the surface is easily oxidized,
producing a glass-like surface whose contact angle is approximately
zero. This implies that the surface is hydrophilic and the liquid
will "wet" the walls of the channel. For a drop in this channel, a
force balance on a horizontally oriented drop gives
(.pi.)(d)(.sigma..sub.left)=(.pi.)(d)(.sigma.).sub.right (6)
[0269] from equation 2. Since the surface tension is constant for a
liquid at constant temperature, the forces on each side of the drop
are identical, thus the drop remains motionless.
[0270] Knowing that surface tension is a function of temperature,
the surface tension on one side of the drop may be selectively
changed. The surface tension of water decreases as the temperature
at the liquid-solid interface increases. Therefore, using a
microheater located slightly beneath the surface of the channel,
the surface tension on one side of the drop may be reduced while
keeping the other side constant. Using the heater in combination
with a thermocouple (or other thermosensor), the temperature, and
therefore the surface tension, at that edge may be accurately
controlled. The unequal heating will accelerate the drop away from
the heat source. Sensors fabricated beneath the channel may be used
to locate the edge of the drop. (Dielectric sensors may be used for
this application, as the dielectric constant of water is different
from that of air.) By sensing this movement and turning on
sequential heating elements at the rear edge of the moving drop,
the drop may be propelled down the flow channel in a "bucket
brigade" fashion. Sequence control of the heater activation may be
performed by quadrature electrical signals.
[0271] The velocity at which the drop will move may be determined
by balancing the force generated by the surface tension gradient
with the drag caused by the fluid flowing through the channel. The
average steady-state velocity for pressure-driven flow in a
capillary tube, termed Poiseuille flow, is given as equation 7:
.nu..sub.ave=[(.DELTA.P)(d.sup.2)]/[(32)(.mu.)(L)] (7)
[0272] where .DELTA.P is the pressure difference between the drop
ends, d is the capillary diameter, .mu. is the liquid viscosity and
L is the drop length (Bird et al., 1960). The pressure difference
is a result of the curvature at the interface. Use of Young-Laplace
equation relating the pressure difference across a curved interface
(Probstein, 1989), such as in a hydrophilic capillary system,
results in Equation 4 for calculating the average steady-state
velocity for surface tension driven flow:
.nu..sub.ave=[(.DELTA..sigma.)(d)]/[(8)(.mu.)(L)] (4)
[0273] where .DELTA..sigma. is the difference in surface tension
between the ends of the drop. Thus for only small temperature
differences across the drop (on the order of 10.degree. C.)
velocities on the order of 1 cm/s may be obtained. This velocity is
more than sufficient for transporting liquid drops in MIDAT and
other chip based systems.
[0274] It should be noted that other methods also exist for moving
a drop by changes in surface tension. The drop may be moved by
changing the hydrophobicity of the channel surface
(electrowetting). The surface may be made hydrophobic by a variety
of chemical surface treatments. Imparting an electrostatic charge
to the channel wall surface at the right edge of the drop, and
thereby decreasing the contact angle will have the same effect as
heating it: the drop will move to the right. These methods are
particularly attractive as they are not greatly effected by the low
Reynold's numbers associated with moving small liquid volumes.
[0275] J. MIDAT Channel Injection
[0276] Accurate and reproducible injection of a small liquid volume
into micromachined channels may be accomplished using these
principles. At the sample injection port, the channel immediately
adjacent to the input reservoir contains a section that is
hydrophobic. This portion of the channel contains a series of
electrodes that can change the channel hydrophobicity
(electrowetting).
[0277] A drop of solution is placed on the hydrophilic input
reservoir to form a sessile drop. The reservoir is connected to the
entrance of a microchannel that delivers the sample into the MIDAT
device. A portion of the channel is then made hydrophilic by
charging a set of electrodes. Once the required amount of liquid is
drawn into the device, the region near the junction of the
reservoir and the channel is made locally hydrophobic by turning
off the most proximal electrode. Alternatively, a brief burst of
heating at the junction could vaporize a small quantity of the
sample and break the continuity of the drop. In either
configuration, no further in-flow of liquid occurs. The drop is
then moved forward by electrowetting or thermal surface tension
effects, as discussed previously. Replacement air is drawn in
through a small hydrophobic-coated channel. The volume of the drop
is fixed by the cross-sectional area of the channel and the
distance between cleavage point and the leading drop edge. Sample
volumes as low as 10.sup.-12 liters may be manipulated by this
system.
[0278] During the movement of solutions, the biological activity of
the samples must be preserved. Since the channel may be designed to
almost any dimension, the surface area/volume ratio may be kept low
to avoid surface denaturation of reaction mixture protein
components (i.e., DNA polymerase). Adsorption of solutes onto the
surface of the channels must be minimized and may be controlled by
proper treatment of the channel with various dopants. Conversely,
the desorption of silicon dopants into the reaction solutions must
be carefully monitored, as these may affect biochemical
reactions.
[0279] When it is necessary to move the fluid within the channels
or chambers of the device, pressure (e.g., air pressure) may be
applied to an opening in the channel or chamber (e.g., the inlet
port). When pressure is used to move the liquid, there is
preferably a second opening or exit port which may be used to apply
pressure in the opposite direction or to remove the liquid from the
device. Alternatively, the fluid may be moved within the channel
using a thermocapillary pump as described by Burns, et al. (1996).
The thermocapillary pump has the advantage of providing a
self-contained miniaturized device in which movement of discrete
aliquots within the channels requires no moving parts or
valves.
[0280] IV. Flow Control with Sealed Valves
[0281] The present invention contemplates the use of sealed valves
to control fluid flow. While the present invention is not limited
to a particular sealing method, in one embodiment, an actuating
force pushes a diaphragm against a valve seat to restrict fluid
flow and the diaphragm is then sealed to the valve seat. In such an
embodiment, the solder pads are associated with a heating element
that can melt the solder. This liquefied solder flows over areas of
the valve seat and diaphragm to cover contamination, cracks and
crooks between the diaphragm and valve seat. With the actuating
force still holding the diaphragm and valve-seat together, the
heating element is turned off to allow the solder to cool and
re-solidify. Upon solidification, the actuating force may be
released and the valve is sealed. To open the valve again, the
solder may be liquefied without applying an actuation force.
[0282] In certain aspects of the invention "diaphragm" may refer to
an element capable of being manipulated such that it can at least
partially block the passage of fluid in a channel in one position
(extended) and permit the flow of fluid in a channel in another
position. An "actuating force" is a force that is capable of
extending a diaphragm. A "valve seat" is an element designed to
accept a portion of the diaphragm when extended. A "movement means"
is a means capable of moving liquefied meltable material (e.g.,
force air, magnetic field, etc.).
[0283] In a preferred embodiment, the valve is designed such that
solder pads are placed on the diaphragm or valve seat. While the
present invention is not limited to a precise method of placing
these solder pads, it is specifically contemplated that they may be
electroplated.
[0284] V. Mixing Biological Samples in Reactions
[0285] Droplet motion (described generally above) is contemplated
as one step in a pathway. The other steps typically involve sample
mixing and a controlled reaction. For example, the integral heaters
arrayed along the entire surface of the channel used for droplet
motion also allow for a region of a channel to be used as a thermal
reaction chamber. For sample mixing prior to the reaction, a
Y-channel device is one embodiment of the invention. In such a
device, a first droplet containing a first sample (e.g., nucleic
acid) is moved along one channel of the Y-channel device, and a
second droplet containing a second sample (e.g., a restriction
digest enzyme in digestion buffer) is moved along the other channel
of the Y-channel device.
[0286] Following sample merging there is the concern that the
combined samples have not been properly mixed. That is to say, if
two similar microdroplets enter the single channel in laminar flow
at the same flow rate, they will form an axially uniform droplet
but will not be mixed width-wise. Width-mixing may be accomplished
in a number of ways.
[0287] First, there is diffusion, although, for large DNA
molecules, the characteristic time for this mixing could be on the
order of several hours or more. Circulation patterns generated
inside the droplets during movement and heating significantly
reduce this time. In this regard, the present invention
contemplates maintaining the mixture as a heated mixture (e.g.,
maintaining the temperature at 65.degree. C. for 10 min) using the
integral heaters and temperature sensors.
[0288] Second, the present invention contemplates mixing by
reversing the flow direction of the mixture over a relatively short
distance in the channel. While a variety of reverse flow approaches
are possible, one or two direction changes over a distance
comprising approximately two droplet lengths has been found to be
adequate.
[0289] Finally, there is the mixing approach wherein the mixture is
moved against or over physical obstacles. For example, the mixture
may be either "crashed" back against merge point of the Y-channel
or simply moved over deliberate imperfections in the channel (i.e.,
"roller coaster" mixing).
[0290] Successful mixing, of course, may be confirmed by
characterization of the product(s) from the reaction. Where product
is detected, mixing has been at least partially successful. The
present invention contemplates, in one embodiment, using
electrophoresis to confirm product formation.
[0291] A restriction digest was performed by mixing a DNA sample
with an enzyme solution and heating the resulting mixture. The
solutions were injected into the ends of the y-channel (simple
capillary action drew the samples into the channels). The drops
were then moved using the embedded heaters and, once the combined
drop was in the single channel, it was heated to a constant
temperature. Comparison of the results of this digestion with that
performed on commercial thermocyclers indicated little
difference.
[0292] A. Biocompatibility
[0293] A 5 mm.times.5 mm heater surface for is used as a polymerase
chain reaction thermocycler. The cross-shaped loop that divides the
region into four heating zones is an RTD (resistive temperature
detector). The construction of this chamber is identical to the
y-channel heaters described earlier. In fact, the chips are
processed on the same wafers. Reactions may be carried out on this
surface using either walled polymer vessels for large-volume
reactions or etched caps for small-volume (.about.0.5 .mu.l)
reactions.
[0294] PCR.TM. was run on this chip. The reaction was carried out
on the surface of this chip using a polypropylene ring cemented to
the chip as the vessel walls. 20 gm of reaction mix was covered
with oil to prevent evaporation and the solution was cycled through
94.degree. C., 55.degree. C., and 72.degree. C. using a digital
controller (National Instruments LabView, programmed VI, Macintosh
Quadra 950 computer). Using such a controller based in LabView
allows change in the function and design of the controller without
the expense of circuit construction. As the electrophoresis gel
indicates, the oxide surface of the chip and the heaters did not
damage the enzyme or inhibit the reaction; the chip results appear
identical to the control run on a commercial thermocycler.
Extensive biocompatibility tests indicate that the results of the
reaction are very sensitive to controller settings and to the
materials used for construction (Burns, 1994).
[0295] B. Reaction Parameters
[0296] Solutions containing the DNA samples and solutions
containing the reagents for the reaction must both be added to the
MIDAT unit, thoroughly mixed, and reacted at the proper
temperature. The mixing of solutes at very small length scales is
both simple and complex. The simplicity arises because the radial
distance that the solutes must diffuse is relatively small and,
therefore, any radial mixing will occur quite rapidly. For
instance, in a 1 .mu.m channel, the characteristic time for
diffusion a typical solute (D=10.sup.-5 cm.sup.2/s) is
approximately (Probstein, 1989):
t=L.sup.2/D=(10.sup.-4 cm).sup.2/(10.sup.-5 cm.sup.2/s).apprxeq.1
ms (8)
[0297] Even for larger solutes with diffusion coefficients of
10.sup.-6 to 10.sup.-8 cm.sup.2/s, the mixing time is under 1
s.
[0298] The complexity arises because the mixing lengthwise in a
channel of length 1 cm or longer is not rapid (t.apprxeq.several
days). Care must be taken to assure that uniform mixing occurs
along the length of a drop of solution. This uniform concentration
may be assured in several ways. First as two drops are mixed, they
will join at the front ends. This joining may be accurately
controlled using dielectric sensors and heaters, as discussed
before. If the drops are in a hydrophilic channel, each meniscus
will naturally join. By controlling the force driving each drop,
the liquids may be added at precisely the rate to yield a uniform
axial concentration.
[0299] In addition to this precise control of the liquid motion,
uniform axial concentrations may be assured due to the flow pattern
generated within the drop moving in the channel. The liquid near
the surface of the channel, due to intermolecular forces, remains
motionless while liquid in the center of the drop is moving forward
at twice the average velocity of the drop. At the back edge of the
drop, this stagnant liquid is "picked off" the walls of the channel
by surface tension while at the front of the drop, the liquid is
deposited by surface wetting phenomena. In this way, the liquid is
constantly circulating from the front of the drop, down the side of
the drop, and returning through the center. During this travel, the
solutes are rapidly mixing radially with the different velocity
streams. The net result is that, as long as the drop travels
approximately one or two drop lengths, complete mixing of the drop
should occur.
[0300] VI. Isothermal Amplification Reactions
[0301] A. Enzymatic Reactions
[0302] The channels of the DNA chip may be constructed in any
configuration appropriate for the selected reaction protocol. The
complete amplification reaction, including the target and the other
components for the amplification reaction, may prepared and mixed
outside of the DNA chip. The complete amplification reaction is
then placed into the channel of the DNA chip and, if necessary,
moved to a region of the channel in contact with a heater element
which maintains the desired reaction temperature. Alternatively,
the reaction may be performed in a device in which the sample
containing the target and the sample containing the enzymes and
other components of the amplification reaction are maintained as
separate liquid aliquots until the reaction is to be initiated. At
that time, the two liquid aliquots may be brought into contact by
means of pressure, a thermocapillary pump, other equivalent means,
such that they mix and react in a region of the channel which is
maintained at the desired reaction temperature by a heater element.
In an alternative embodiment, the channels of the DNA chip may be
in the form of a "Y" such that a liquid aliquot containing the
target placed in one arm of the "Y" is kept separate from the
enzymes and amplification reagents in the other arm of the "Y".
Using pressure applied to the inlet port of each arm of the "Y" or
thermocapillary pumps in contact with each arm, the two liquid
aliquots are moved into contact at the junction of the two arms and
allowed to mix and react at a selected reaction temperature
maintained by the heater element in the region of the channel which
forms the stem of the "Y". Other configurations for the channels
and device designs which also employ reaction chambers and/or
detection areas will be apparent to those skilled in the art and
are intended to be included within the scope of the invention. If
desired, mixing may be enhanced by moving the liquid aliquot back
and forth by alternately applying pressure on each side or
alternately heating each side of the aliquot, though other
equivalent means may be substituted.
[0303] In certain preferred embodiments of the invention, the
device for use in the isothermal amplification of a selected
nucleic acid further comprises one or more of the reagents for an
isothermal nucleic acid amplification reaction. Such reagents may
include polymerases, nucleotides, buffers, solvents, nucleases,
endonucleases, primers, target nucleic acids including DNA and/or
RNA, salts, and other suitable chemical or biological components.
In certain preferred aspects, these reagents may be provided in dry
or lyophilized form. In other embodiments the reagents may be
dissolved in a suitable solvent.
[0304] In certain embodiments one or more of the reagents,
including nucleotides, buffers, salts, chemicals, solvents,
primers, target nucleic acids including DNA and/or RNA,
polymerases, endonucleases, nucleases, and chemical or biological
components suitable for the isothermal reaction mixture are added
to the at least first and/or second microdroplet transport channels
separately or in various combinations. In other preferred
embodiments of the invention one or more of the nucleotides,
buffers, salts, chemicals, solvents, primers, target nucleic acids
including DNA and/or RNA, polymerases, endonucleases, nucleases,
and chemical or biological components suitable for an isothermal
amplification reaction are contained in, in liquid communication
with, operably or functionally connected to, and/or provided with
the microfabricated substrate. In certain other embodiments one or
more or the reagents for an isothermal reaction may be contained in
a detachable reservoir that may be contained in or attached to an
inlet port, channel, or reservoir so to be in liquid communication
with, and/or operably or functionally connected to the
microfabricated substrate. In certain preferred embodiments the
reagents may be in dry or lyophilized form. In other embodiments
the reagents may be dissolved in a suitable solvent.
[0305] Any isothermal nucleic acid amplification method may be
performed on the DNA chips essentially as described in the art. The
lower, constant temperature and complex enzymology of isothermal
amplification does not inhibit the reaction in the DNA chip format.
That is, it has unexpectedly been found that movement and mixing of
the liquid aliquots is not significantly compromised, that the
enzymes involved in isothermal amplification are not significantly
inhibited, and that the predicted stagnant temperature gradient
does not prevent efficient amplification. Thermophilic SDA (tSDA)
is a preferred amplification method for application to DNA chips
because of its high amplification factors and rapid results.
Amplification reactions are generally performed in the
microfabricated device in a volume of about 0.6 .mu.L-3 .mu.L, but
the dimensions of the channels and/or reaction chambers may be
altered to accommodate larger or smaller reaction volumes.
[0306] Nucleic acid used as a template for amplification is
isolated from cells contained in the biological sample, according
to standard methodologies (Sambrook et al., 1989). The nucleic acid
may be genomic DNA or fractionated or whole cell RNA. Where RNA is
used, it may be desired to convert the RNA to a complementary DNA.
In one embodiment, the RNA is whole cell RNA and is used directly
as the template for amplification.
[0307] Pairs of primers that selectively hybridize to nucleic acids
corresponding to an isolated target sequence for amplification are
contacted with the isolated nucleic acid under conditions that
permit selective hybridization. The term "primer", as defined
herein, is meant to encompass any nucleic acid that is capable of
priming the synthesis of a nascent nucleic acid in a
template-dependent process. Typically, primers are oligonucleotides
from ten to twenty base pairs in length, but longer sequences can
be employed. Primers may be provided in double-stranded or
single-stranded form, although the single-stranded form is
preferred.
[0308] Once hybridized, the nucleic acid:primer complex is
contacted with one or more enzymes that facilitate
template-dependent nucleic acid synthesis. Multiple rounds of
amplification, also referred to as "cycles," are conducted until a
sufficient amount of amplification product is produced.
[0309] Next, the amplification product is detected. In certain
applications, the detection may be performed by visual means.
Alternatively, the detection may involve indirect identification of
the product via chemiluminescence, radioactive scintigraphy of
incorporated radiolabel or fluorescent label or even via a system
using electrical or thermal impulse signals (Affymax
technology).
[0310] B. Types of Nucleic Acid Amplification
[0311] A number of template dependent processes are available to
amplify nucleotide sequences present in a given template sample. It
is not intended that the present invention be limited by the nature
of the reactions carried out in the microscale device. Reactions
include, but are not limited to, chemical and biological reactions.
Biological reactions include, but are not limited to sequencing,
restriction enzyme digests, RFLP, nucleic acid amplification, and
gel electrophoresis. It is also not intended that the invention be
limited by the particular purpose for carrying out the biological
reactions. In one medical diagnostic application, it may be
desirable to differentiate between a heterozygotic and homozygotic
target and, in the latter case, specifying which homozygote is
present. Where a given genetic locus might code for allele A or
allele a, the assay allows for the differentiation of an AA from an
Aa from an aa pair of alleles. In another medical diagnostic
application, it may be desirable to simply detect the presence or
absence of specific allelic variants of pathogens in a clinical
sample. For example, different species or subspecies of bacteria
may have different susceptibilities to antibiotics; rapid
identification of the specific species or subspecies present aids
diagnosis and allows initiation of appropriate treatment.
[0312] Preferred methods are Strand Displacement Amplification
(SDA) and thermophilic SDA for carrying out isothermal
amplification of nucleic acids which involves multiple rounds of
strand displacement and synthesis, i.e., nick translation.
[0313] In this method, either before or after the template nucleic
acids are denatured, a mixture comprising an excess of all four
deoxynucleosidetriphosphates, wherein at least one of which is
substituted, a polymerase and an endonuclease are added. (If high
temperature is used to denature the nucleic acids, unless
thermophilic enzymes are used, it is preferable to add the enzymes
after denaturation.) The substituted deoxynucleosidetriphosphate
should be modified such that it will inhibit cleavage in the strand
containing the substituted deoxynucleotides but will not inhibit
cleavage on the other strand. Examples of such substituted
deoxynucleosidetriphosphates include 2'deoxyadenosine
5'-O-(1-thiotriphosphate), 5-methyldeoxycytidine 5'-triphosphate,
2'-deoxyuridine 5'-triphosphate and 7-deaza-2'-deoxyguanosine
5'-triphosphate.
[0314] The mixture comprising the reaction components for target
generation and SDA can optionally include NMP (1-methyl 2
pyrrolidinone), glycerol, polyp(ethylene glycol), dimethyl
sulfoxide and/or formamide. The inclusion of such organic solvents
is believed to help alleviate background hybridization
reactions.
[0315] It should be appreciated that the substitution of the
deoxynucleotides may be accomplished after incorporation into a
strand. For example, a methylase, such as M. Taq I, could be used
to add methyl groups to the synthesized strand. The methyl groups
when added to the nucleotides are thus substituted and will
function in similar manner to the thiosubstituted nucleotides.
[0316] It further should be appreciated that if all the nucleotides
are substituted, then the polymerase need not lack the 5' forward
arrow 3' exonuclease activity. The presence of the substituents
throughout the synthesized strand will function to prevent such
activity without inactivating the system.
[0317] The selection of the endonuclease used in this method should
be such that it will cleave a strand at or 3' (or 5') to the
recognition sequence. The endonuclease further should be selected
so as not to cleave the complementary recognition sequence that
will be generated in the target strand by the presence of the
polymerase, and further should be selected so as to dissociate from
the nicked recognition sequence at a reasonable rate. It need not
be thermophilic. Endonucleases, such as HincII, HindII, AvaI,
Fnu4HI, Tth111I, and NciI are preferred.
[0318] One can envision several alternative nicking enzyme systems
in addition to those detailed in this application. For example, it
is generally regarded that class IIS restriction endonucleases
(e.g., Fokl) contain two DNA cleavage centers within a single
polypeptide unit. If one of the cleavage centers was inactivated,
such as through site directed mutagenesis, the resultant nicking
enzyme could be used in an amplification system not requiring
modified deoxynucleosidetriphosphates. As an additional example,
the restriction enzyme EcoRi has been shown to preferentially
cleave one strand in noncanonical recognition sites or when its
canonical recognition site is flanked by an oligopurine tract
(Thielking et al., 1990; Lesser et al., 1990; Venditti & Wells,
1991). As another example, the restriction enzyme DpnI (available
from New England Biolabs, Beverly Mass.) cleaves a recognition site
containing me.sup.6 dA on both strands. DpnI or an analogous
restriction enzyme may be able to nick the methyl containing strand
of a hemimethylated recognition site. Such a system would employ
SDA primers (P.sub.1 and P.sub.2) with methylated recognition
sequences along with unmodified deoxynucleosidetriphosphates.
Alternatively, certain restriction enzymes are known to cleave the
nonmethylated strand of a hemimethylated recognition site (e.g.,
MspI and me.sup.5 dC). Such a system would use a methylated
deoxynucleosidetriphosphate. Finally, one could use origin of
replication proteins to nick one strand of a recognition
sequence.
[0319] Polymerases useful in this method include those that will
initiate 5'-3' polymerization at a nick site. The polymerase should
also displace the polymerized strand downstream from the nick, and,
importantly, should also lack any 5' forward arrow 3' exonuclease
activity. It should be appreciated that a polymerase ordinarily
having such exonuclease activity may be deemed to "lack" such
activity if that activity is blocked by the addition of a blocking
agent.
[0320] An additional feature of this method is that it does not
require temperature cycling. Many amplification methods require
temperature cycling in order to dissociate the target from the
synthesized strand. In this method, a single temperature may be
employed after denaturation has occurred. The temperature of the
reaction should be high enough to set a level of stringency that
minimizes non-specific binding but low enough to allow specific
hybridization to the target strand. In addition proper temperature
should support efficient enzyme activity. From about 37.degree. C.
to about 42.degree. C. has been found to be a preferred temperature
range.
[0321] The SDA reaction initially developed was conducted at a
constant temperature between about 37.degree. C. and 42.degree. C.
(U.S. Pat. No. 5,455,166, incorporated herein by reference,). This
was because the exo.sup.- Klenow DNA polymerase and the restriction
endo nuclease (e.g., Hindu) are mesophilic enzymes which are
thermolabile (temperature sensitive) at temperatures above this
range. The enzymes which drive the amplification are therefore
inactivated as the reaction temperature is increased.
[0322] Methods for isothermal Strand Displacement Amplification
which may be performed in a higher temperature range than
conventional SDA (about 50.degree. C. to 70.degree. C.,
"thermophilic SDA") were later developed. Thermophilic SDA is
described in published European Patent Application No. 0 684 315
and employs thermophilic restriction endonucleases which nick the
hemimodified restriction endonuclease recognition/cleavage site at
high temperature and thermophilic polymerases which extend from the
nick and displacing the downstream strand in the same temperature
range. At increased temperature, the amplification reaction has
improved specificity and efficiency, reduced nonspecific background
amplification, and potentially improved yields of amplification
products. In addition, the need to add the enzymes in a separate
step after the initial heat denaturation of double stranded targets
may be eliminated when enzymes which are stable at the denaturation
temperature are used. UDG decontamination of target-specific
amplicons in the SDA reaction is also more efficient when the
amount of nonspecific background amplicons is reduced.
[0323] Another method, called Repair Chain Reaction (RCR), involves
annealing several probes throughout a region targeted for
amplification, followed by a repair reaction in which only two of
the four bases are present. The other two bases can be added as
biotinylated derivatives for easy detection. Target specific
sequences can also be detected using a cyclic probe reaction (CPR).
In CPR, a probe having 3' and 5' sequences of non-specific DNA and
a middle sequence of specific RNA is hybridized to DNA that is
present in a sample. Upon hybridization, the reaction is treated
with RNase H, and the products of the probe identified as
distinctive products that are released after digestion. The
original template is annealed to another cycling probe and the
reaction is repeated.
[0324] Qbeta Replicase, described in PCT Application No.
PCT/US87/00880, incorporated herein by reference, may also be used
as still another amplification method in the present invention. In
this method, a replicative sequence of RNA that has a region
complementary to that of a target is added to a sample in the
presence of an RNA polymerase. The polymerase will copy the
replicative sequence that can then be detected.
[0325] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[alpha-thio]-triphosphates in one strand of a restriction site
may also be useful in the amplification of nucleic acids in the
present invention.
[0326] Yet another amplification method is described in PCT
Application No. PCT/US93/07138, which is incorporated herein by
reference, may be used in accordance with the present invention.
This method of amplification features treating a target sequence
with a first oligonucleotide (that has a complexing sequence
sufficiently complementary to a 3'-end portion of the target
sequence to hybridize therewith (this alone is termed a primer),
and that has a sequence 5' to the complexing sequence that includes
a sequence which, in double-stranded form, acts as a promoter for
an RNA polymerase (this arrangement is termed a promoter-primer),
and a second oligonucleotide (which is a primer or promoter-primer
that has a complexing sequence sufficiently complementary to the
complement of the target sequence to hybridize therewith), under
conditions in which an oligonucleotide/target sequence complex may
be formed and DNA and RNA synthesis may occur. In this invention,
one or both of the first and second oligonucleotides is a mixture
of a blocked and an unblocked oligonucleotide sequence (blocked
oligonucleotides have a modified 3' end to prevent or reduce the
rate and/or extent of primer extension by a DNA polymerase), or a
mixture of oligonucleotides with different 3' modifications. Such a
mixture significantly enhances the efficiency of the specific
amplification reaction compared to use of only blocked or only
unblocked oligonucleotides.
[0327] The amplification method synthesizes RNA copies of a target
sequence by use of a mixture of blocked and unblocked
promoter-primers, or promoter-primers with different 3'
modifications, consisting essentially of the same nucleic acid
sequence in a ratio that provides for lessened non-specific
byproducts. The amplification process occurs spontaneously and
isothermally under a broad range of conditions.
[0328] Still another amplification methods described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025, each of which is incorporated herein by reference
in its entirety, may be used in accordance with the present
invention. In the former application, "modified" primers are used
in a PCR-like, template- and enzyme-dependent synthesis. The
primers may be modified by labeling with a capture moiety (e.g.,
biotin) and/or a detector moiety (e.g., enzyme). In the latter
application, an excess of labeled probes are added to a sample. In
the presence of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the target sequence is released
intact to be bound by excess probe. Cleavage of the labeled probe
signals the presence of the target sequence.
[0329] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR Gingeras et al.,
PCT Application WO 88/10315, incorporated herein by reference. In
NASBA, the nucleic acids can be prepared for amplification by
standard phenol/chloroform extraction, heat denaturation of a
clinical sample, treatment with lysis buffer and minispin columns
for isolation of DNA and RNA or guanidinium chloride extraction of
RNA. These amplification techniques involve annealing a primer
which has target specific sequences. Following polymerization,
DNA/RNA hybrids are digested with RNase H while double stranded DNA
molecules are heat denatured again. In either case the single
stranded DNA is made fully double stranded by addition of second
target specific primer, followed by polymerization. The
double-stranded DNA molecules are then multiply transcribed by an
RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction,
the RNA's are reverse transcribed into single stranded DNA, which
is then converted to double stranded DNA, and then transcribed once
again with an RNA polymerase such as T7 or SP6. The resulting
products, whether truncated or complete, indicate target specific
sequences.
[0330] Davey et al., EPA No. 329 822 (incorporated herein by
reference in its entirety) disclose a nucleic acid amplification
process involving cyclically synthesizing single-stranded RNA
("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be
used in accordance with the present invention. The ssRNA is a
template for a first primer oligonucleotide, which is elongated by
reverse transcriptase (RNA-dependent DNA polymerase). The RNA is
then removed from the resulting DNA:RNA duplex by the action of
ribonuclease H (RNase H, an RNase specific for RNA in duplex with
either DNA or RNA). The resultant ssDNA is a template for a second
primer, which also includes the sequences of an RNA polymerase
promoter (exemplified by T7 RNA polymerase) 5' to its homology to
the template. This primer is then extended by DNA polymerase
(exemplified by the large "Klenow" fragment of E. coli DNA
polymerase I), resulting in a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence can be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies can
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification can be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence can be
chosen to be in the form of either DNA or RNA.
[0331] Miller et al., PCT Application WO 89/06700 (incorporated
herein by reference in its entirety) disclose a nucleic acid
sequence amplification scheme based on the hybridization of a
promoter/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. This
scheme is not cyclic, ie., new templates are not produced from the
resultant RNA transcripts. Other amplification methods include
"RACE" and "one-sided PCR" (Frohman, 1990 incorporated by
reference).
[0332] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide, may also be used in the amplification step of
the present invention.
[0333] One of the best known amplification methods is the
polymerase chain reaction (referred to as PCR.TM.) which is
described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and
4,800,159, and each incorporated herein by reference in
entirety.
[0334] Briefly, in PCR.TM., two primer sequences are prepared that
are complementary to regions on opposite complementary strands of
the marker sequence. An excess of deoxynucleoside triphosphates are
added to a reaction mixture along with a DNA polymerase, e.g., Taq
polymerase. If the marker sequence is present in a sample, the
primers will bind to the marker and the polymerase will cause the
primers to be extended along the marker sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
marker to form reaction products, excess primers will bind to the
marker and to the reaction products and the process is
repeated.
[0335] A reverse transcriptase PCR amplification procedure may be
performed in order to quantify the amount of mRNA amplified.
Methods of reverse transcribing RNA into cDNA are well known and
described in Sambrook et al., 1989. Alternative methods for reverse
transcription utilize thermostable, RNA-dependent DNA polymerases.
These methods are described in WO 90/07641, filed Dec. 21, 1990,
incorporated herein by reference. Polymerase chain reaction
methodologies are well known in the art.
[0336] Another method for amplification is the ligase chain
reaction ("LCR"), disclosed in EPA No. 320 308, incorporated herein
by reference in its entirety. In LCR, two complementary probe pairs
are prepared, and in the presence of the target sequence, each pair
will bind to opposite complementary strands of the target such that
they abut. In the presence of a ligase, the two probe pairs will
link to form a single unit. By temperature cycling, as in PCR,
bound ligated units dissociate from the target and then serve as
"target sequences" for ligation of excess probe pairs. U.S. Pat.
No. 4,883,750 describes a method similar to LCR for binding probe
pairs to a target sequence.
[0337] Following any amplification, it may be desirable to separate
the amplification product from the template and the excess primer
for the purpose of determining whether specific amplification has
occurred. In one embodiment, amplification products are separated
by agarose, agarose-acrylamide or polyacrylamide gel
electrophoresis using standard methods (Sambrook et al., 1989).
[0338] Alternatively, chromatographic techniques may be employed to
effect separation. There are many kinds of chromatography which may
be used in the present invention: adsorption, partition,
ion-exchange and molecular sieve, and many specialized techniques
for using them including column, paper, thin-layer and gas
chromatography.
[0339] Amplification products must be visualized in order to
confirm amplification of the marker sequences. One typical
visualization method involves staining of a gel with ethidium
bromide and visualization under UV light. Alternatively, if the
amplification products are integrally labeled with radio- or
fluorometrically-labeled nucleotides, the amplification products
can then be exposed to x-ray film or visualized under the
appropriate stimulating spectra, following separation.
[0340] In one embodiment, visualization is achieved indirectly,
Following separation of amplification products, a labeled, nucleic
acid probe is brought into contact with the amplified marker
sequence. The probe preferably is conjugated to a chromophore but
may be radiolabeled. In another embodiment, the probe is conjugated
to a binding partner, such as an antibody or biotin, and the other
member of the binding pair carries a detectable moiety.
[0341] In one embodiment, detection is by Southern blotting and
hybridization with a labeled probe. The techniques involved in
Southern blotting are well known to those of skill in the art and
can be found in many standard books on molecular protocols. See
Sambrook et al, 1989. Briefly, amplification products are separated
by gel electrophoresis. The gel is then contacted with a membrane,
such as nitrocellulose, permitting transfer of the nucleic acid and
non-covalent binding. Subsequently, the membrane is incubated with
a chromophore-conjugated probe that is capable of hybridizing with
a target amplification product. Detection is by exposure of the
membrane to x-ray film or ion-emitting detection devices.
[0342] One example of the foregoing is described in U.S. Pat. No.
5,279,72 1, incorporated by reference herein, which discloses an
apparatus and method for the automated electrophoresis and transfer
of nucleic acids. The apparatus permits electrophoresis and
blotting without external manipulation of the gel and is ideally
suited to carrying out methods according to the present
invention.
[0343] VII. Analysis and Manipulation of Amplification Products
[0344] A. Electrophoresis
[0345] The biochemical and electrophoretic manipulations for
successful DNA sequencing are well characterized, but have not been
assembled into a simple automated processing system. The use of
silicon photolithographic fabrication techniques allows components
to be compatible, readily assembled as a single device, and
inexpensive to mass produce.
[0346] One type of device contains several components: liquid
injection ports, self-pumping channels based upon surface-force
gradient phenomena, a thermally isolated amplification chamber, a
decision split point, and gel electrophoresis channels. Next to,
and underneath, these components are the system detectors and the
controlling circuitry. Within this system a sample is injected,
moved to a specific location, and the enzymatic sequencing
reactions are performed. A portion of the sequencing product is
isolated and sent to a preliminary electrophoresis gel for
screening. Using the preliminary information, sequence data
acquisition may be optimized by dividing the remaining product
between electrophoresis gels having different resolution
characteristics.
[0347] 1. Construction of a Miniature Electrophoresis System.
[0348] Existing electrophoresis technology is able to size
fractionate sequencing reactions 800 basepairs and greater using
gels 50-100 .mu.m thick and 50 cm long. These results are
duplicated using micromachined 5-100 .mu.m channels. Short channels
of about 1 cm length will be linear, longer channels of about 5 cm
will use folded columns.
[0349] The invention will use technology for DNA electrophoresis
and construct the system using microfabrication techniques. The
existing technology for DNA sequencing has shown that a
polyacrylamide gel 400 microns thick and 55 cm long can easily
provide single-base resolution of DNA fragments 100-400 base pairs
in length when operated in snapshot mode. The invention may
duplicate this separation on a micromachined substrate by using a
serpentine channel and an etched glass or silicon substrate.
However, as has been reported in the literature, separation should
be possible on a much smaller gel.
[0350] The first step in miniaturizing this technology is to
analyze the resolution obtained in a typical gel and determine how
the dimensions and operating conditions of a smaller unit would
affect the resolution of the migrating bands. A radiograph of a 55
cm long, 400 .mu.m thick polyacrylamide sequencing gel that was run
for 2 h at 2000 V (58 W) using radioactively tagged DNA was used to
estimate .mu., .DELTA..mu., and D.sub.eff. While the smaller DNA
fragments have moved to the end of the gel, the 400 bp fragments
are only 10 cm down from the sample wells. To the left of this run
is a "G` reaction run for 5, 10, 15, 30, 45, and 60 min at the same
voltage. The fragments passing the 10 cm point have been resolved.
Thus, if a detector had been placed at 10 cm and monitored
continuously (finishline mode), all bands would have been resolved.
By using a single detector at the end of the gel and monitoring
that detector continuously, the invention is able to use a shorter
overall gel length. The net voltage that needs to be applied to the
gel would be less to give the same electric field strength (i.e.,
for a 10 cm gel, only 360 V need to be applied to get the same
field).
[0351] While this qualitative experiment indicates that the peaks
should be resolved in only 10 cm for finishline operation, the
invention may use the definition of resolution to quantitatively
describe the operation for a variety of operating voltages and
channel dimensions.
[0352] For a finishline run, the time of the run is just the length
of the channel divided by the velocity of the slowest DNA fragment:
the time, therefore, is
t=L.sup.2/(.mu..sub.slowV) (9)
[0353] where L is the channel length, .mu..sub.slow is the mobility
of the slowest band, and V is the applied voltage. The resolution
between bands may be defined as 1 R = 2 ( z 2 - z 1 ) ( w 1 + w 2 )
( 10 )
[0354] where z.sub.i is the location of the center of each band and
w.sub.i is the width of the band (measured at baseline). The width
of the peaks may be approximated by
w.sub.i=(32D.sub.eff,avet).sup.1/2 (11)
[0355] where D.sub.eff,ave is the average dispersion coefficient
between the two bands. Knowing that the difference in spatial
locations (z.sub.2-z.sub.1) is easily calculated from the
electrophoretic mobilities, R may be rewritten as
R=1/4.DELTA..mu.V/L(t/2D.sub.eff,avet) (12)
[0356] Plugging Equation (9) into Equation (12) obtains
R=1/4(V/(2D.sub.eff,ave)).sup.1/2.DELTA..mu..sub.slow/.mu..sub.slow.sup.1/-
2 (13)
[0357] where D.sub.eff,ave is the average dispersion coefficient
between the two bands and .DELTA..sub.slow is the difference in
mobility between the slowest and next slowest fragments.
[0358] Using a radiograph of a 55 cm long, 400 .mu.m thick
polyacrylamide sequencing gel that was run for 2 h at 2000 V (58 W)
gel, these equations may calculate whether the 360 volts in the 10
cm gel would be able to obtain 400 bp resolution. The mobility,
.mu..sub.400, is given by (dL)/(Vt)=3.1.times.10.sup.-5
cm.sup.2/Vs. Since the 399 and the 400 bp-length fragments are 0.03
cm apart (band 399 and 397 are 0.06 cm apart, therefore 399 and 400
are .apprxeq.0.06/2 cm apart), then .DELTA..mu., is
((.DELTA.d)L)/(Vt)=9.2.times.10.sup.-8 cm.sup.2/Vs. D.sub.deff is
more difficult to obtain. If the plot of the width of a bands vs,
t.sup.1/2, D.sub.eff,ave may be found, using equation 11, from the
slope of the straight line through the points. This was difficult
to do for the 400 bp band but the plot of this information for the
133 bp long fragment may obtain an estimate of
D.sub.eff,ave=7.times.10.sup.-8 cm.sup.2/s. This value is
significantly above the molecular diffusion coefficient of DNA in
free solution (.about.10.sup.-9 cm 2/s) and probably represents
both the dispersion caused by the gel matrix and the low resolution
of the x-ray film (Lang and Coates, 1968). Note that this
calculation assumes that the sample was applied in an
infinitesimally small sample volume; the zero intercept on the plot
indicates that this is essentially true. The microfluidic devices
in the MIDAT system may be used to introduce the sample in a
similar fashion as what is done on the large scale.
[0359] The invention may calculate the resolution that should be
obtain based on the above approximate and the 360 V in the 10 cm
finishline gel. Using .DELTA..mu.=10.sup.-7 cm.sup.2/Vs,
.mu.=10.sup.-5 cm.sup.2/Vs, D.sub.eff,ave 10.sup.-8 cm.sup.2/s
(this value was decreased to compensate for the band broadening
caused by the film), and Equation 13, R.apprxeq.1.0. Although an R
value of 1 usually defines a adequate separation, peaks can still
be resolved at significantly lower R values. This implies that
voltages as low as 200 V (R=0.75) may be used and still achieve
adequate resolution. These voltages pose little problems in either
the glass or silicon devices. Note that, D.sub.eff in a variety of
different media; D.sub.eff should be a function not only of the
matrix used but also of the distance traveled.
[0360] The length of the channel governs the minimum spatial
resolution the detector must possess; this spatial distance would
be equal to the separation distance between the two most difficult
peaks. Knowing that .DELTA.z=.DELTA..mu.V/L t and using Equations 9
gets
.DELTA.z=L.DELTA..mu..sub.slow/.mu..sub.slow (14)
[0361] Using the parameters obtained above, .DELTA.z=0.003 L. Thus
if the electrophoresis channel was 10 cm long, the spatial
resolution of the detector must be 0.03 cm (the distance actually
obtained between the last two peaks).
[0362] The thickness of the gel governs both the quantity of DNA in
a migrating band and the heat dissipation of the channel. The
quantity of the DNA in the band must be matched with the
sensitivity of the detector. Based on the fluorescent probes in
current sequencing gels, electrophoresis channels 20-100 .mu.m high
should not have a problem with sensitivity of the fluorescent
detector.
[0363] The temperature profile in the gel must remain constant to
avoid viscosity/mobility gradients that could distort the bands.
For symmetric gels, the temperature rise in the center of the gel
may be calculated by solving the heat conduction equation with
constant-temperature boundary conditions and a heat generation
term. Published solutions are available and are of the form
(Geankoplis, 1993)
.DELTA.T=(SH.sup.2)/(2k) (15)
[0364] where .DELTA.T is the difference between the surface
temperature and the center of the gel, S is the heat generation per
volume in the gel, H is the thickness of the gel, and k is the
thermal conductivity of the solution. Knowing the resistivity of
the solution in the gel (.rho.), one can obtain an equation for
.DELTA.T in terms of operating variables: 2 T = ( H V / L ) 2 8 k (
16 )
[0365] Assuming a solution resistivity of 500 .OMEGA.cm and thermal
conductivity of 0.006 W/cm K, .DELTA.T for a typical sequencing gel
(0.4 mm thick, 55 cm long run at 2000 volts (.about.50 watts)) is
less than 0.1.degree. C. Note that this analysis assumes that the
wails of the gels were kept at constant, equal temperatures;
microfabricated heaters and temperature sensors can easily
accomplish this. Equation 8 can be rearranged to solve for the
height of the channel:
H.ltoreq.1.5L/V (17)
[0366] where V is in volts. Note that this equation was derived for
the specific test gel that was used and for a temperature
difference of 0.1.degree. C.; the equation would need to be derived
for other gel/polymer systems. Note also that, for their test gel,
the equation correctly calculates that H.ltoreq.0.4 mm.
[0367] There are a variety of other considerations for scale-down
of DNA sequencing systems. For instance, the mobilities and
dispersion coefficients may be functions of field intensities or
channel wall materials. However, the analysis presented here
provides the framework with which to design micromachined
systems.
[0368] 2. Summary of Channel Specifications
[0369] The channel specification will use three basic criteria:
First, the resolution will be measured and designed to be above
0.8:
R=1/4(V/(.sub.2D.sub.di
eff,ave)).sup.1/2.DELTA..mu..sub.slow/.mu..sub.slo- w (13)
[0370] Second, the spatial resolution of the detector will be
designed to give adequate spatial resolution:
.DELTA.z=L.DELTA..mu..sub.slow/.mu..sub.slow (14)
[0371] Third, the height of the channel will be small enough to
prevent temperature gradients:
H.ltoreq.1.5L/V (17)
[0372] Thus, to sequence a 400 base-pair fragment by standard
techniques, a 55 cm channel that was 400 .mu.m high is used. The
invention may be able to run the same sample in a 10 run channel
that is 100 .mu.m high. Both of these designs may be constructed
using silicon microfabrication.
[0373] The use of very small diameter capillary systems for
electrophoretic separations has been well established since the
early 1980's (Jorgenson and Lukacs, 1981; Kuhr, 1990). Capillary
electrophoresis has an enormous theoretical resolving power and has
been commercially applied to a number of analytical systems (Datta,
1990; Gordon et al., 1988). A variety of liquid capillary formats
are available, having glass, fused silica, coated, and rectangular
columns. Automated injectors and high sensitivity detectors are
also being actively developed. More recently, polyacrylamide
gel-filled capillary columns have become available for use with DNA
fragment separation (Mathes and Huang, 1992; Swerdlow et al., 1992;
Drossman et al, 1990).
[0374] Standard liquid capillary electrophoresis has been reported
as an integrated silicon chip technology (Manz et al., 1991; Manz
et al., 1992; Lammcrink et al., 1993). The channel dimensions of
the chip-integrated liquid capillary electrophoresis system were
approximately 30 .mu.m wide by 10 .mu.m deep. These columns had
very long effective lengths (several cm), and were constructed as
spirals on the surface of the silicon chip. Very high voltages were
used in the separation, on the order of several kilovolts. As a
consequence, breakdown of the silicon substrate was seen as a major
obstacle to routine use. As gel columns of the MIDAT may be
micromachined channels etched on silicon chips, however, much lower
voltages are required for gel electrophoresis (1 to 10 volts/cm).
Glass capillary gel electrophoresis has been described with 50
.mu.m to 75 .mu.m inner diameter tubing, and effective column
lengths of from 50 cm to a few millimeters (Pentoncy et al., 1992;
Heller and Tullis, 1992). Theoretical models of DNA electrophoresis
have been useful for estimating the matrix pore structure needed
for a particular separation application (Datta, 1990; Gordon et
al., 1988).
[0375] 3. Electrophoretic Separation
[0376] Following the amplification reaction, the replicated DNA
fragments may be transported to the separation system, again by
means of heating-induced surface tension propulsion. In the MIDAT
system, the separation may be performed using a chip-integrated
capillary gel electrophoresis system. Additionally, the switch to a
square channeled capillary obtained from the micromachining process
may benefit the separation process. Turner (1993) has cited many
reports that square capillaries are advantageous to circular ones
due to their higher surface-volume ratio in providing for more
effective heat dissipation.
[0377] The gel is polymerized inside a channel that is made
entirely using thin films. The unpolymerized acrylamide enters the
channel from a sessile drop using an injection scheme similar to
that described previously. After filling, the acrylamide monomers
are polymerized in situ by the addition of catalyst or by
photoinduction. Channel walls may need to be chemically treated to
alter the wetting properties or surface charge (Kolb and Cerro,
1991).
[0378] The sample may be directly moved to the anode chamber. The
complex mixture of reagents in the isothermal amplification
reaction, including unincorporated labeled nucleotide monomers, may
necessitate a pre-electrophoresis separation step. This step may be
accomplished by low-molecular weight dialysis. Microporous
membranes may be fabricated in silicon (Petersen, 1982), and may
provide an on-chip dialysis mechanism.
[0379] A low voltage field (1-10 V/cm) is applied to the gel column
to induce electrophoretic motion. This field strength will allow
fractionation of DNA fragments of the PCR.TM. size-range in about
10 min on a 1 cm-long column (Pentoncy et al., 1992; Heller and
Tullis, 1992). Switched-field electrophoresis schemes may also be
used for more rapid DNA fragment discrimination. In order to reduce
the area of the separation stage, the column may be folded. Such
arrangement is more compact and can increase DNA fragment
resolution by several fold.
[0380] B. Detection
[0381] 1. Detector Specifications and Construction
[0382] The detection scheme uses previously tested high-sensitivity
semiconductor diode detectors placed about 0.5-1 .mu.m beneath the
electrophoresis stage. A preferred detection structure has a
lightly-doped diffusion diode (Kemmer, 1980; Wouters and van
Sprakelaar 1993) suitable for measurements of both .beta. radiation
decay from .sup.32P labeling isotopes and visible-light wavelength
photons from fluorescent labels. The detector consists of an n+
diffusion onto a lightly-doped 100 p-type float zone silicon
substrate with resistivity of 100 .OMEGA.-cm. The n+ diffusion is
buried under the electrophoresis stage and separated from it by a
thin dielectric layer or layers. Fluorescence-based detection may
be performed using these detectors as photodiodes and adding a thin
film optical filter layer(s) placed between the gel and
detector.
[0383] The operation of the diffusion detector is as follows. First
a reverse bias is applied between the n+ and the substrate,
creating a depletion region. Due to the low doping of the
substrate, the depletion region is approximately 8 .mu.m deep for a
10 V bias. When a .beta. particle or photon traverses the depleted
area, electron hole pairs are generated. Carriers generated in the
depletion region are readily swept to the electrodes generating a
short current burst. When the mean free path of the impacting
particle is greater than the depletion region, additional carriers
diffuse through the substrate until they reach the edge of the
depletion region where they are collected back over time. Therefore
the detector current consists of a sharp peak (corresponding to the
charge generated in the depletion region) followed by a long tail
caused by the diffusing carriers. Diffusion detectors are highly
sensitive to small charge packets, responsive to the initial
position of the ionization charge, and easily fabricated in
silicon. Approximately 50% of the charge is collected within 1 ns
from the decay.
[0384] Because of the close proximity to the gel, these detectors
can pinpoint the position of a radioactive decay or light emission
event within 0.5 .mu.m inside the gel. In simple radiation
detection mode, each detector is capable of sensing a single decay
of a .beta. particle from a .sup.32P DNA label yielding an average
of 15,000 electrons per event and a charge of 2.5 fC. However, this
extremely small charge packet demands the use of low-noise and
low-parasitic capacitance instrumentation amplifiers. Therefore,
the detectors will have on-wafer low-capacitance buffer amplifiers
implemented in NMOS technology. Effective charge gains of 5 V/fC
and noise level of 50 electrons are prepared. In existing CCD
charge amplifiers, smaller charge packets have been sensed on
silicon fabricated devices at noise levels of 10-18 electrons
(Hynecek, 1992).
[0385] The primary improvements to the existing diode detector
involve conversion to a narrow wavelength visible light detector.
Since the detector can function as a fluorescence detector as long
as the appropriate filters are used, the invention may include the
design and construction of optical filter materials directly on the
silicon substrate and diffusion diode. The electrophoresis channels
are separated from the underlying silicon wafer and electronic
components by layers of silicon oxide and silicon nitride (made by
low pressure chemical vapor deposition, LPCVD). The same
fabrication method may be used for production of optical filters.
First, the spectral absorbance characteristics of silicon nitride
are well known and vary depending on the stoichiometric ratios of
silicon to nitrogen (Philipp, 1993; Macleod, 1986). The LPCVD
method allows control of Si:N ratios, and for most ratios, some
range of the visible spectrum is completely transmitted. In
addition, stoichiometric SiO.sub.2 is transparent to visible light
and much of the UV-range, while pure crystalline silicon (Si) is
opaque. Second, the sequential layering of silicon oxide and
silicon nitride layers of approximately one-quarter the passed
wavelength (0.25.times..lambda.) can produce narrow wavelength
interference filters. The invention uses the known optical
properties of silicon-based thin film materials to design and
construct interference/absorbance filters, including primarily
LPCVD deposition of silicon nitride and silicon oxide over the
detector. The final filters may require less than 10 .mu.m of
material to achieve complete UV blocking.
[0386] Spatial resolution is an essential requirement in detectors
used for separations. The spatial resolution determines the
accuracy in the localization of an emission event within the
sieving material. Detector structure design may assist in
determining the position of an emission source. Three distinct
phenomena affect the detector resolution. First, in order for the
position of the impact to faithfully reflect the location of the
DNA fragment, the distance between the gel and detector must be
small. In the integrated structure, the detector is in direct
contact with the gel channel, hence resolution loss through
dispersion is minimal. Second, the detector capture width must be
small. The diffusion diode detectors have a capture width of 2
.mu.m and are easily formed with lithographic techniques.
[0387] Thirdly, to prevent sensing of emissions from adjacent DNA
fragments, the detector response must be insensitive to events
originating outside its capture width. The use of a guard ring
around the sensing electrode (Belcarz et al., 1970) eliminates
spurious signals. The ring collects charges generated outside the
capture range of interest, preventing them from interacting with
the central detector. The resulting structure is a low-noise,
low-leakage detector. Further improvements on the localization are
accomplished using charge division techniques (Knoll, 1989; Alberi
and Radeka, 1976; Gerber et al.; 1977; Belau et al., 1983;). The
position of the source of the emission event is calculated from the
difference in the two outputs V1 and V2. The respective charges
collected on a set of electrodes are used to estimate the centroid
of the decay through the resistive network. Localization of the
decay event within 0.5 .mu.m may be possible. Other detector
structures are based on MOSFET structures since these devices are
directly compatible with NMOS process.
[0388] 2. Implementation of Detection Circuits
[0389] The charge collected by the diffusion detector from a .beta.
particle or photon yields approximately 10.sup.4 electrons per
event. Semiconductor radiation detectors of this type are typically
(Knoll, 1989) connected to electronic amplifiers. The detector is
essentially a diode in reverse bias subject to a transient pulse of
charge lasting a few nanoseconds. The output of the detector is fed
directly to a low noise op-amp (typically a JFET buried channel
input device) which integrates the pulse of current and generates a
step in the potential at its output. The virtual ground of the
op-amp maintains a constant potential difference across the
detector; therefore its output is independent of parasitics
connected to this node. The parasitic cancellation allows the
implementation of most of the circuits off of the device. Any
leakage through the diode will induce an offset at the op-amp
output. Since the charge packet is very small, the corresponding
integrator capacitor should also be small. A 250 fF capacitor
yields a change of 10 mV at the integrator output. Hence, it will
be desirable to make the resistor as high as possible to minimize
the noise of the circuit and to retain the capacitor charge as long
as possible (a 10 .OMEGA. resistor yields a retention time of 2.5
sec). Any drifts in the leakage current, such as response to a
temperature change, will lead to large drifts in the output voltage
of the op-amp and may drive the op-amp outside its linear operation
regime.
[0390] For the diffusion diode detector circuit, the invention uses
an on-wafer circuit that eliminates most of these parasitic
effects. The circuit consist of a current source depletion load, an
enhancement discharge transistor, inverters linked to a
non-inverting amplification stage, and a low-pass filter. The
circuits are fabricated using a 3 .mu.m NMOS process.
[0391] 3. DNA Detection
[0392] An embodiment of the invention is the DNA sample detector.
Two primary detection schemes are contemplated. First, fluorescent
DNA labels are commercially available and may be detected using
optical p-n photodiodes constructed below the electrophoresis
column. The optical transducers are very small with areas on the
order of 5 pm. Many detectors may be constructed in a small area,
permitting multiple detector sampling of each electrophoresis
column, if desired. The signal of each detector may be multiplexed
with an on-chip circuit.
[0393] The fluorescent tags may be excited by an external laser
scanning system or, more simply, a uniform source of light. Special
attention is required for wavelength discrimination and detection
of the faint fluorescent signals. For ethidium bromide-stained DNA,
the excitation wavelength is 302 nm and fluorescent emission is 590
nm. The silicon nitride base of the electrophoresis channel absorbs
all wavelengths less than -500 nm; thus blocking the UV radiation
and transmitting the fluorescent signal. Alternatively, fluorescent
DNA labels are becoming available with a variety of excitation and
emission spectra (Middendorf et al., 1992).
[0394] A method for detection uses radioactively labeled DNA
products. Silicon fabricated radiation detectors have been used
since the earlier 1960's (Bertolini, 1968; Deme, 1971; Knoll,
1979), and are extremely sensitive. The basic structure is similar
to that of the p-n photodetector. The incoming radiation ionizes
the silicon creating free carriers that are collected by the
reversed bias diode. The energy needed to create an electron hole
pair is about 3 eV. Typical decay energies of .beta.-emitting DNA
labeling isotopes (.sup.32P, .sup.33P, .sup.35S) are in the 50 to
500 keV region. These energies can create a collected charge of
10-13 coulombs per event and an easily detectable current of a few
microamps. To prevent the collection of radiation-generated
carriers from adjacent regions of the chip, a shielding ring is
constructed around the n+detector. The radiation detector may prove
less expensive than the optical scheme, as radioactive DNA tracers
are less expensive than fluorescent labels.
[0395] Amplification may be detected either in the DNA chip or
after removal of the amplified sample. If the amplified sample is
removed for post-amplification detection (either through the inlet
port or through a second outlet port), the channels and/or chambers
are preferably washed with additional liquid and the wash liquid
added to the amplified sample for detection. If amplification is to
be detected within the microfabricated device, the liquid may be
moved through the channels to a separate area containing reagents
for a detection reaction and means for detecting the amplification
products (e.g., labeled probes for hybridization detection and
means for detecting the hybridized label or microelectrophoresis
channels and means for detecting the amplification products by
electrophoresis). Alternatively, amplification products may be
detected in the same area where the amplification reaction takes
place when the detection system is compatible with or a component
of the amplification reaction, as discussed below. Amplification
products may be detected by hybridization to an assay probe which
is typically tagged with a detectable label. The detectable label
may be conjugated to the probe after chemical synthesis or it may
be incorporated into the probe during chemical synthesis, for
example in the form of a label-derivatized nucleotide. Such labels
are known in the art and include directly and indirectly detectable
labels. Directly detectable labels produce a signal without further
chemical reaction and include such labels as fluorochromes,
radioisotopes and dyes. Indirectly detectable labels require
further chemical reaction or addition of reagents to produce the
detectable signal. These include, for example, enzymes such as
horseradish peroxidase and alkaline phosphatase, ligands such as
biotin which are detected by binding to label-conjugated avidin,
and chemiluminescent molecules. The probes may be hybridized to
their respective amplification products in solution, on gels, or on
solid supports. Following hybridization, the signals from the
associated labels are developed, detected and optionally
quantitated using methods appropriate for the selected label and
hybridization protocol. The amount of signal detected for each
amplification product may be used to indicate the relative amount
of amplification product present. Ligand labels may also be used on
assay probes to facilitate capture of the hybrid on a solid phase
(capture probe).
[0396] An alternative method for detecting amplification products
is by polymerase extension of a primer specifically hybridized to
the target sequence. The primer is labeled as described above, for
example with a radioisotope, so that the label of the primer is
incorporated into the extended reaction product. This method is
described by Walker, et al. (1992b) and Walker, et al. (1992a).
Another method for detecting amplified target and control sequences
is a chemiluminescent method in which amplified products are
detected using a biotinylated capture probe and an
enzyme-conjugated detector probe as described in U.S. Pat. No.
5,470,723. After hybridization of these two assay probes to
different sites in the assay region of the target sequence, the
complex is captured on a streptavidin-coated microtiter plate, and
the chemiluminescent signal is developed and read in a
luminometer.
[0397] The foregoing detection methods are generally used for
post-amplification detection, either after removing the sample from
the microfabricated device or in a separate detection area of the
chip containing reagents and detecting means. As another
alternative for detection of amplification products, a signal
primer (essentially a detector probe which is extended by
polymerase, displaced and rendered double-stranded in a target
amplification-dependent manner) as described in EP 0 678 582 may be
included in the amplification reaction. In this embodiment, labeled
secondary amplification products are generated in a target
amplification-dependent manner and may be detected as an indication
of target amplification in a homogeneous assay format either
post-amplification or in real-time (i.e., during amplification).
The DNA chip assay formats of the invention are particularly
well-suited to real-time homogeneous amplification detection, as
the label of the detection system (e.g., a signal primer) may be
detected through the glass or silica walls of the channel or
reaction chamber as amplification is occurring in the liquid
aliquot. When the signal primer is labeled with a fluorescent
label, the increase in fluorescence polarization as the signal
primer becomes double-stranded may be monitored in this manner,
either in real-time or at a selected endpoint in the amplification
reaction.
[0398] C. Fluidic and Electronic Integration of the Sequencing
System
[0399] Using the invention's micromachined fluid-handling
capabilities, they integrate the template preparation, biochemical
reactions, and electrophoresis systems on a single device.
Concurrent integration of electronic components (detector, heaters,
liquid detectors, and temperature sensors) allows the construction
of a self-contained sequencing system. The invention will use
sequencing technology in their microfabricated electrophoresis
devices. The widths of the channels are (20-100 .mu.m) are on the
same order of those currently being produced commercially and the
interior material of the channels is silicon oxide (glass). While
most substrates are only 10 cm in diameter (i.e., the largest
linear dimension constructed is 10 cm), longer channels may be
constructed by using a serpentine channel (Jacobson et al., 1994).
Both radiation and fluorescence detectors may be constructed
beneath these channels to provide either a snapshot (many detectors
beneath the channel or finishline (one detector at the end) mode
for running separations. The temperature of the gel may also be
measured and controlled to insure that no gradients exist across
the gel. These separation systems may be microfabricated in either
silicon or glass.
[0400] 1. Elimination of Sequencing Bottlenecks Using Intelligent
Systems
[0401] The system of integrated fluid-handling, electrophoresis,
detector, and circuitry components allows feedback and
decision-making directly within the device. In one embodiment
information-based processing is used to reduce both the systemic
and random errors for each sequencing sample, and to improve
reproducibility, error-detection, length of readable data, and
compatibility with existing sequencing protocols.
[0402] The invention assembles individual components for DNA sample
handling and DNA sequencing into increasingly complex, integrated
systems. The incorporation of steps that normally occur in large
volumes "on the bench" will reduce the bottlenecks associated with
current large-scale DNA sequencing efforts. Since each individual
sequencing preparation, reaction, and electrophoresis run has its
own set of devices, bottlenecks cannot occur within the integrated
system.
[0403] D. Chip Multitasking
[0404] It is contemplated that using micromachining techniques,
reaction and separation units that are impossible or impractical to
build by any other techniques are constructed. For instance, an
electrophoresis chamber with hundreds of DNA detectors along its
length may be constructed for the same cost as constructing a
chamber with only one sensor. Also, several hundred of these
chambers may be processed on a single wafer with no additional cost
(aside from dicing and other post-wafer processing costs). The same
technology that makes transistors in integrated circuits so cheap
may allow these complicated, integrated systems to be produced for
a fraction of what their larger-scale equivalents cost.
[0405] 1. Fluidic Control and Integration
[0406] The controlled movement and mixing of nanoliter drops in
micron-scale channels has been demonstrated using a
differential-heating propulsion mechanism Control circuitry may
maintain uniform biochemical reaction conditions and to
reproducibly measure and detect the location of individual drops.
The individual micro fluidic components for DNA sequencing may
maintain compatibility among the devices. A variety of
photofabricated, integrated DNA analysis systems is
contemplated.
[0407] 2. Photolithographic Components as Design Tools
[0408] Once a device component has been developed on a computer
aided design program, it is replicated across the surface of the
wafer as many times as desired. Each additional reiteration of the
component or group of components does not cost appreciably more,
since the entire wafer is processed uniformly. The machines are
reproduced photographically. DNA analysis is a highly repetitive
task, requiring many identical devices generating data with very
uniform characteristics. Silicon photolithographic fabrication
provides multiple identical devices cheaply.
[0409] 3. Modules for Specific Multi-Step Tasks
[0410] To perform a DNA analysis task, the individual components is
linked and function as an integrated device. A set of tasks which
are often found together in a molecular biology protocol are
designed as a functional group or module. The module may be
replicated at multiple locations in the larger device, wherever the
specific tasks are required. Since each sample has its own set of
devices at each step, no time or effort is lost waiting for batch
processes to occur, and there are no points where process
bottlenecks occur.
[0411] 4. Incorporation of Earlier Sample Processing Steps in the
System
[0412] Increasingly complex devices may be assembled from the
individual components and basic functional modules. The modules do
not perform any template handling, and consequently require
well-characterized template as starting material. The entire
processing stream may be incorporated onto the device, with all
steps included within the silicon-fabricated environment. This
embodiment will eliminate process bottlenecks since each sample
will have its own dedicated series of instruments.
[0413] As an example of an "intelligent" system, the modules take
advantage of the ability to hold a sample in reserve, while portion
of the sample is being examined. The determination of a DNA
template size and quantity prior to more extensive processing is an
use of this capability. Size information, for example, can inform
the temperature, number of cycles, and electrophoresis conditions
of a cycle sequencing run.
[0414] A single source DNA is used to supply three sequencing
reactions. Template DNA is amplified from the source independently
for each sequencing reaction. The template is then divided into two
samples and one is assayed for quality and size by gel
electrophoresis. The remaining template is then treated to remove
unincorporated primers and dNTPs prior to cycle sequencing. The
information obtained by analysis of half of the sample is used to
determine the reaction parameters of later steps. This figure is
presented only as an example: alternative template preparation
strategies are contemplated.
[0415] 5. Reduction of Systemic and Random Error
[0416] Once a fundamental design is established in the
microfabricated format, it is a minor additional expense to prepare
and run additional gels for each synthesis reaction. Rerun gels
are, in fact, one of the major custom-handling difficulties of
current large sequencing groups. As an embodiment, one design would
generate two gel reads for each Sanger reaction. Double gel runs,
under different conditions, may be able to resolve bands that
rnigrate anomalously under a single condition. If two parallel gels
are run, the output data must then be merged and compared to
resolve the differences between gel reads.
[0417] A second method to reduce error involves duplicate synthesis
reaction conditions. For example, longer gel reads are possible (up
to 1000 bp) using a combination of modified dideoxy:deoxy ratios
and extended gel electrophoresis lengths. The original template is
divided in to two samples, one half receiving standard Sanger
reaction mix, the other a modified mix which emphasizes longer read
length. Both reactions are then run on sequencing gels, and their
output merged. This example describes one possible system developed
from individual components: a large number of alternative
strategies are contemplated.
[0418] The MIDAT system is constructed from a conventional silicon
wafer using advanced micromechanical fabrication techniques. In
certain embodiments, it is contemplated that silicon wafers are
constructed with 100 to 1000 parallel MIDAT processing units. The
multiplex wafer may be capable of simultaneous genotyping an
equivalent number of DNA samples, and may provide computer-readable
data in less than 3 hours. Miniaturization of DNA analysis results
in significant savings in reagents, enzyme, sample handling, and
sample processing time.
[0419] As a result of the enormously flexible design
characteristics of silicon, improved versions may be developed
rapidly as the basic biochemical methods advance. Consequently,
other methods of nucleic acid amplification and analysis should be
compatible with the MIDAT system.
[0420] 6. Integration of Micromachined Components on a Single
Substrate
[0421] It is contemplated the invention will comprise hundreds of
control and detector connections. In practice, the number of
external connections may be limited by the chip size. By
integrating the system with on chip electronics, it may be
controlled using as little as 5 external leads. One embodiment of
the invention is on-chip circuitry to control the operation of the
MIDAT system. These circuits may be implemented on the same
substrate as the fluidic parts. On-chip integrated control
circuitry may result in a highly compact and efficient design
capable of making real-time control decisions. The system may
comprise a sample size and flow control circuit, temperature
cycling and timing circuit, electrophoretic separation bias, data
detection and transmission, and a sequencer/timer to control the
overall operation. All the data will be transmitted in serial form
between an external computer and the MIDAT chip.
[0422] A multicomponent, integrated device includes the elements in
FIG. 1. The sections in the diagram represent fundamental process
components fabricated on silicon. Sample and reagent are injected
into the device through entry ports or reservoirs (A), and
individual liquid drops are pumped through channels (B) to a
thermally controlled reactor, where mixing and restriction enzyme
digestion or DNA amplification occurs (C). The drop movement is
controlled by simple heating, as differential heating of the two
ends of a drop in a capillary tube produces motion (i.e., a
thermocapillary pump; Bums et al., 1996). After reaction, the
biochemical products are moved by the same pumping method to an
electrophoresis channel (D), where DNA migration data are collected
by an integral photodiode (E). The output data are sent off the
integrated device for signal processing and DNA band
identification.
[0423] Additional components may be added to the system, provided
the channel connection format remains consistent. Such components
may comprise low temperature polymer-based channels. A silicon
wafer with two liquid reservoirs, 1000.times.1000.times.25 .mu.m),
each connected to a 200.times.25-.mu.m channel. The channel and
reservoir structures are made of a low-temperature polymer
(p-xylylene) using a sacrificial etch procedure. Platinum
electrophoresis electrodes are visible within each reservoir.
Additional platinum surface electrodes and photodiode detectors
have been placed beneath the channels. The interior channel opening
is .about.100.times.25 .mu.m. Peltier cooling surfaces, optical
sensors, and ultraviolet filters for continuous spectrophotometric
analysis may be present.
[0424] Using photolithographic fabrication, a silicon wafer having
>30 different electrophoresis channels and integral detectors
within a 1.25.times.1.25-cm unit area has been developed. The
devices provide a reproducible test platform for understanding gel
electrophoresis at a micron-size scale (Webster et al., 1996). The
silicon components have provided considerable preliminary
information on channel and detector formats. The overall
arrangement of components across several 1.25.times.1.25-cm DNA
processing unit repetitions on a single wafer. Within each unit are
.about.30 different electrophoresis channel and photodiode
configurations. The components include an opening to one
electrophoresis channel, an associated photodiode, buffer
reservoir, external contact points for electronic control, and
connections for the integral electrodes and photodiodes. The
channels are made using a silicon nitride sacrificial etch process
and have an interior cross section of 40.times.5 .mu.m.
[0425] VIII. Kits
[0426] All the essential materials and reagents required for the
various aspects of the present invention may be assembled together
in a kit. The kit generally will comprise reagents to provide the
necessary reaction mixture for nucleic acid amplification,
including polymerases, nucleotides, buffers, solvents, nucleases,
endonucleases, primers, target nucleic acids including DNA and/or
RNA, salts, and other suitable chemical or biological components,
and a microfabricated substrate defining at least a first channel
connected to an isothermally regulated reaction chamber. One or
more of the reagents for the reaction mixture may be contained in
the microfabricated device and/or in a separate reservoir. When the
components of the kit are provided in one or more liquid solutions,
the liquid solution is preferably an aqueous solution, with a
sterile aqueous solution being preferred.
[0427] In a particularly preferred embodiment, the components of
the kit may also be provided in dried or lyophilized forms. When
reagents or components are provided as a dried form, reconstitution
generally is by the addition of a suitable solvent. It is
envisioned that the solvent also may be provided in another
container means. The kits of the invention may also include an
instruction sheet defining the use of the microfabricated substrate
to amplify nucleic acids.
[0428] The kits of the present invention also will typically
include a means for containing the reagent vials and
microfabricated substrate in close confinement for commercial sale
such as, e.g., injection or blow-molded plastic containers into
which the desired vials are retained.
[0429] IX. Diagnostics
[0430] The diagnostic system of the present invention generally
involves determining either the type or the amount of a wild-type
or mutant nucleic acid segment amplified from a biological sample
using the chip-based devices of the invention. The biological
sample may be from a patient suspected of having a variety of
diseases including cancer. Irrespective of the disease, it will be
understood that the detection of a mutant is likely to be
diagnostic of a disease, and that the detection of altered amounts
of the target nucleic acid segment is also likely to have
diagnostic implications, particularly where there is a reasonably
significant difference in amounts between the patient and samples
from a normal subject.
[0431] The type or amount of the target nucleic acid present within
a biological sample, such as a tissue sample, may be determined or
identified by means of a molecular biological assay, particularly
an isothermal nucleic amplification reaction in a microfabricated
substrate defining at least a first channel connected to an
isothermally regulated reaction chamber connected to a nucleic
analysis component and a detector for the amplified product.
Additionally, any of the foregoing microfabricated substrate
nucleic acid amplification, processing, and detection systems may
be employed as a diagnostic system in the context of the present
invention.
[0432] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered to function well in
the practice of the invention, and thus may be considered to
constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes may be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
[0433] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); M (micromolar); N
(Normal); mol (moles); mmol (millimoles); .mu.mol (micromoles);
nmol (nanomoles); gm (grams); mg (milligrams); .mu.g (micrograms);
L (liters); ml (milliliters); .mu.l (microliters); cm
(centimeters); mm (millimeters); .mu.m (micrometers); nm
(nanometers); .degree. C. (degrees Centigrade); Ci (Curies); MW
(molecular weight); OD (optical density); EDTA
(ethylenediamine-tetracetic acid); PAGE (polyacrylamide gel
electrophoresis); UV (ultraviolet); V (volts); W (watts); mA
(milliamps); bp (base pair); CPM (counts per min).
EXAMPLE 1
[0434] This example is a minimal fully integrated device and would
include the elements identified in FIG. 1. In the chip format,
sample and reagent are injected into the device through entry ports
(FIG. 1A) and the solutions pumped through channels (FIG. 1B) to a
thermally controlled reactor where mixing and isothermal nucleic
acid amplification reactions (SDA, Q.beta.-replicase, etc.),
restriction enzyme digestion, ligation, phosphorylation,
dephosphorylation, sequencing, other nucleic acid amplification
reactions (e.g. PCR ), or other enzymatic or chemical reaction
known to those of skill in the art occurs (FIG. 1C). The
biochemical products may then moved by the same or a different
pumping method to an electrophoresis channel (FIG. 1D), where
nucleic acid migration data are collected by a detector (FIG. 1E)
and exported as electronic information. A component of the system
is a thermocapillary pump capable of connecting diverse individual
elements.
[0435] The microfabricated elements in this example are capable or
performing several processing steps in conventional DNA analysis.
The individual elements have the potential for combination into a
complete DNA genotype analysis processing path. Each component was
developed using only silicon or glass photolithographic production
methods. As a consequence, all components retain the ability to be
fabricated concurrently on the same substrate wafers. The use of
common fabrication methods allows the assembly of increasingly
complex, multicomponent, integrated systems from a small, defined
set of standardized elements. Fine control of discrete drop
location is only dependent on the density of individual heating
elements or other fluid movement devices along the channel.
Detection of the drop location within the channel may be performed
by using capacitors or conductive wires as sensors. Because the
thermocapillary pump mechanism requires no external force (other
than application of heat), it should remain scaleable within a wide
range of integrated device sizes. Finally, because each droplet is
moved uniquely, devices that incorporate branching pathways or
parallel sample analysis present no inherent obstacle, other than
requiring more complex electronic control circuitry.
[0436] Thermocapillary Pump
[0437] The thermocapillary pump provides movement of discrete drops
in micron-sized channels with no moving parts or valves. A pumping
system based on individual drop movement has three advantages for
DNA and/or nucleic acid analysis: samples may be readily divided
and mixed, the sample volume may be determined by measuring the
drop length, and each sample is kept separate, reducing the risk of
cross-contamination.
[0438] Motion of discrete liquid samples in micron-sized channels
may be accomplished by differentially heating the drop interfaces
(FIG. 5). In channels, a pressure difference occurs across the
liquid-air interface (i.e., capillary pressure). The pressure
difference, .DELTA.P.sub.c, is a function of the surface tension
and, for rectangular channels, is given by
.DELTA.P.sub.c=P.sub.atm-P.sub.liquid=(2 cos .theta.)(1/h+1/w),
(18)
[0439] where .theta. is the contact angle, h is the channel height,
w is the channel width, and .sigma. is the liquid-vapor interfacial
tension given by
.sigma.=.sigma..sub.o(1-bT), (19)
[0440] where .sigma..sub.o and b are positive constants and T is
the temperature (Probstein, 1989). Increasing the temperature on
one end of the drop decreases the surface tension and, therefore,
increases the internal pressure on that end. The pressure
difference between the two ends pushes the drop towards the
direction of lower pressure at a rate given by (ignoring edge
effects, h<<w)
<.nu.<=(h/6 .mu.L)[(.sigma. cos .theta.).sub.a-(.sigma. cos
.theta.).sub.r], (20)
[0441] where .mu. is the viscosity, L is the length of the drop,
and the subscripts a and r refer to the advancing and retreating
interfaces, respectively. Note that contact angle hysteresis
(.theta..sub.a.noteq..th- eta..sub.r) requires a threshold pressure
difference for positive motion (Tenan et al., 1982, Dussan,
1979).
[0442] A device capable of moving and mixing nanoliter drops using
differential heating was constructed by bonding a surface-etched
glass wafer to a silicon substrate. A standard aqueous acid
wet-etch is used to produce channels on the 0.5-min glass wafer
having two parallel lanes merging into a single lane of the same
cross-sectional dimensions (a Y shape) with dimensions 500 .mu.m
wide and 25 .mu.m deep. Metal heaters are patterned on the silicon
substrate having the same Y format and are protected from liquid by
a thin-film barrier. The elements are designed to match the channel
layout and are arrayed as two parallel lanes, each 500 .mu.m wide,
merging into one lane. The individual heaters consist of paired
aluminum wires winding across a 500.times.500 .mu.m region. Broad
metal areas on either side of the elements are bonding locations
for connection to external circuitry. The heaters are formed by
using an inlay process to prevent defects in the barrier layer. A
scanning electron micrograph of a heater wire in cross section
showed the deposited aluminum, silicon oxide, and silicon nitride
layers. The plasma-enhanced chemical vapor deposition process for
forming the silicon oxide and silicon nitride layers results in an
undefined stoichiometry; therefore, the layers are designated
SiO.sub.x or Si.sub.xN.sub.y. The width of the aluminum element is
5 .mu.m. The complementary heater and channel wafers are aligned
and bonded with an adhesive to form the finished device
[0443] Heater Element Wafer Fabrication
[0444] Heater elements were made with a silicon wafer (p-type,
18-22 fl-cm, boron.apprxeq.concentration.apprxeq.10.sup.15
cm.sup.-3) as a substrate for growth of SiO.sub.2 thermal oxide (1
.mu.m). A photoresist (AZ-5214-E; Hoescht-Celanese) was applied to
the wafer and spun at 3000 rpm for 30 sec. The resist was patterned
using a mask (M1) and developed. Reactive ion etching (PlasmaTherm,
St. Petersburg, Fla.) was performed to 0.35-.mu.m depth into the
SiO.sub.2 layer at the following conditions: CHF.sub.3, 15 standard
cubic centimeters per minute (sccm); CF.sub.4, 15 sccm; 4 mTorr; dc
bias voltage of 200 V, 100 W, 20 min. The etch depth was measured
by profilometer, and 0.35-.mu.m metallic aluminum was electron beam
deposited. The resist and overlying metal were lifted off by
development using Microposit 1112A remover in solution (Shipley,
Marlboro, Mass.). The barrier layers covering the aluminum elements
consist of sequentially deposited 1 .mu.m SiO.sub.x, 0.25 .mu.m
Si.sub.xN.sub.y, and 1 .mu.m SiO.sub.x using plasma-enhanced
chemical vapor deposition. Reactive ion etch was used to etch
contact holes to the metal layer using a second mask (M2) with
conditions: CHF.sub.3, 15 sccm; CF.sub.4, 15 sccm; 4 M Torr; and dc
bias, voltage of 200 V, 100 W, 120 min. Each heating element used
as a temperature sensor was calibrated by measurement of electrical
5 resistance at 22.degree. C. and 65.degree. C. under constant
voltage; intermediate temperatures were estimated by linear
interpolation.
[0445] Channel Wafer Fabrication
[0446] Channels were prepared on 500-.mu.m-thick glass wafers (Dow
Corning 7740) using standard aqueous-based etch procedures. The
initial glass surface was cleaned and received two layers of
electron beam-evaporated metal (20 nm chromium, followed by 50 nm
gold). Photoresist (Microposit 1813) was spun at 4000 rpm for 30
sec, patterned-using a mask (GI), and developed. The metal layers
were etched in chromium etchant (Cr-14; Cyantek, Newark, Calif.)
and gold etchant (Gold Etchant TFA; Transene, Rowley, Mass.) until
the pattern was clearly visible on the glass surface. The
accessible glass was then etched in a solution of hydrofluoric acid
and water (1:1). Etch rates were estimated using test wafers, with
the final etch giving channel depths of 20-30 .mu.m. For each
wafer, the depth of the finished channel was determined using a
surface profilometer. The final stripping steps removed the
remaining photoresist material (PRS-2000; J. T. Baker) and metal
layers (Cr-14 and Gold Echant TFA).
[0447] Glass-to-Silicon Wafer Bonding and Channel Pretreatment
[0448] Channels etched on glass were bonded to the heater element
wafer using a thin film of applied optical adhesive (SK-9 Lens
Bond; Sumers Laboratories, Fort Washington, PA). The bond was cured
under a UV light source (365 nm) for 12-24 h. Tests of cured
adhesive samples indicated little or no inhibition of restriction
endonuclease or thermostable DNA polymerase. Prior to each
drop-motion study, the bonded channels were prepared by washing
with .apprxeq.100 .mu.l each of the following solutions in series:
0.1 M NaOH, 0.1 M HCl, 10 mM Tris. HCl (pH 8.0), deionized
H.sub.2O, Rain-X Anti-Fog (Unelko, Scottsdale, Ariz., and bovine
serum albumin at 500 .mu.g/ml (restriction enzyme grade;
GIBCO/BRL).
[0449] Movement and Mixing of Liquid Sample
[0450] Two 80-nl drops at their starting locations in the branches
of the Y-channel; the hydrophilic surface of the channel allows the
process to occur spontaneously. The drop volumes are 60 nl and are
calculated from the drop length and the known channel cross
section. Activating the heaters under the left interfaces propels
the drops forward to the channel intersection where they meet and
join to form a single larger drop. The combined drop is stopped by
turning off all heating elements and may be reversed by heating the
right interface. Additionally, circulation patterns generated in
the drop during motion aid in mixing the liquid sample studies
using the metal elements as both heaters and temperature sensors
demonstrate that a temperature differential of 20-40.degree. C.
across the drop is sufficient to provide forward motion in this
particular channel.
[0451] Other sample-handling operations may be performed with this
device. For example, drop splitting may be accomplished in two
ways. First, a drop may be moved from the single channel, past the
Y-channel intersection, and into the two separate channels. While
the motion of the drop is accomplished by heating the retreating
interface, the amount of liquid that enters each of the two
channels may be controlled, by selectively heating one of the
advancing interfaces. The drop will preferentially move into the
less-heated branch channel. Alternatively, splitting may be
performed on a drop held in a single channel by localized heating
at the drop's center until a bubble of water vapor forms. Continued
heating of the expanding water-vapor bubble propels the two
drop-halves in opposite directions. Although an increased
gas-phase, pressure is responsible for this latter motion, properly
placed, air vents in the channel may allow the split drops to be
moved independently using thermocapillary pumping.
[0452] To confirm compatibility of the propulsion system with DNA
samples and enzymes, an integrated system was tested combining drop
motion, sample mixing, and controlled thermal reaction (FIGS.
1A-C). A sample containing plasmid DNA (supercoiled BluescriptSK;
Stratagene) was loaded into one branch of the Y channel, and a
second sample containing Taq I restriction enzyme and digestion
buffer was loaded into the other. After sample merging by
thermocapillary pumping, the combined drop was maintained at
65.degree. C. for 10 min using the integral heaters and temperature
sensors. Capillary gel electrophoresis of the reaction products
confirmed that DNA digestion on the silicon device was similar to
reactions performed in a standard polypropylene vessel. The
enzymatic reaction occurred by moving two drops (Taq I restriction
enzyme and supercoiled plasmid) down separate channels using the
thermocapillary technique, combining the drops, and heating the
merged sample to 65.degree. C. in the channel. After the reaction,
the sample was expressed from the microfabricated device and
analyzed by conventional capillary get electrophoresis. The
electrophoretic chromatogram shows complete digestion products
(elution time, 18-22 min) and minor residual undigested DNA
(elution time, 34 min).
[0453] Drop Motion and Restriction Enzyme Digestion
[0454] The bonded channel device was placed on a stereoscope stage
(Olympus SZ1145), and the contact pads for the heating elements
were connected to a regulated power supply. Aqueous samples were
applied to each of the Y-channel branches by gently touching a
suspended drop to channel opening and allowing capillary action to
draw the sample into the device. Measurements of drop length in the
channel provided a visual check of the loaded volumes. Heating of
the drops occurred by passing ,.apprxeq.30 V dc through the element
in short pulses and observing the movement of the drops. A small
detectable reduction in drop volume from evaporation was noted in
each study, usually <30% of the initial drop length. Drop
movement was recorded with a Hamamatsu (Middlesex, N.J.) video
camera on videotape, and still images were obtained from the
videotape without modification.
[0455] For the restriction enzyme digestion of DNA, a drop
containing 0.2 unit of Taq I restriction enzyme in reaction buffer
(100 mM NaCl/10 mM MgCl.sub.2/10 mM Tris-HCl, pH 8.0), 150 nl total
volume was introduced into one branch of a Y-channel while a drop
containing 150 nl of 0.1 .mu.g of supercoiled plasmid per .mu.l
(Bluescript SK; Stratagene) was introduced into the other.
Following drop motion, digestion occurred by holding the drop at a
previously calibrated 65.degree. C. for 10 min using .apprxeq.4 V
de. The single channel portion of the device was uniformly heated
by using seven contiguous heater elements, and the temperature was
monitored by measuring electrical resistance. The electronic
control system consisted of a National Instruments (Austin, Tex.)
LabView controller and virtual instrument software operating on an
Apple Macintosh 950.
[0456] PCR.TM. on Silicon Wafer Surfaces
[0457] PCR.TM. was performed using standard buffer and primer
concentration conditions for Thermus aquaticus DNA polymerase
enzyme (Mullis and Faloona, 1987, Amheim and Erlich, 1992). PCR.TM.
temperature profiles were as follows: 94.degree. C. for 4 min,
preincubation; 94.degree. C. for 1 min, 62.degree. C. for 1 min,
72.degree. C. for 1 min, 35 cycles; 72.degree. C. for 10 min, final
extension. The primer set is specific for a portion of the mouse
Tfe3 locus and produces a 460-bp-amplified product (primer A,
5'-TAAGGTATGCCCCTGGCCAC-3' (SEQ ID NO:1); primer B,
5'-AAGGTCAGCACAGAGTCCTCA-3') (SEQ ID NO:2 (Roman et al., 1992). For
each experimental run a complete 75-.mu.l reaction mixture was
prepared using 100 ng of purified genomic mouse DNA as template and
divided into three reactions of 25 .mu.l each. The first reaction
was maintained at room temperature for 2 h; the second was reacted
in a thin-wall polypropylene tube under mineral oil and cycled in a
standard thermal cycler; and the third was placed on the surface of
the described heater wafer within a small polypropylene ring (4 mm
diameter, 1.5 mm height) and covered with light mineral oil. Wafer
temperatures were determined by measuring changes in heater element
resistance and were controlled by a National Instruments LabView
controller and software operating on an Apple Macintosh 950. On
completion of the reactions, the three samples were examined for
efficiency of amplification by agarose gel electrophoresis and
ethidium bromide staining.
[0458] Capillary Gel Electrophoresis.
[0459] Following PCR.TM. amplification or restriction enzyme
digestion, DNA genotyping reactions are typically analyzed by gel
electrophoresis. To demonstrate that standard DNA gel
electrophoresis can operate in micron-sized channels identical to
those used for drop motion, studies were performed using etched
glass channels bonded to planar quartz. Channels etched on glass
were bonded to a quartz microscope slide using SK-9 optical
adhesive and 24-h UV-illuminated curing. A 10% acrylamide
electrophoresis mix (10% acrylamidel0.3% bis-acrylamide/89 mM Tris.
HCl/89 mM sodium borate/10 mM EDTA/0.001%
N,N,N',N"-tetramethylethylenedi- amine/0.01% ammonium persulfate)
was injected into the channel and allowed to polymerize. Following
polymerization, the slide was immersed in a horizontal
electrophoresis apparatus containing gel running buffer (89 mM
Tris-HCl/89 mM sodium borate/10 mM EDTA). A 50-.mu.l sample of 100
ng of DNA per .mu.1 (Bluescript SK plasmid digested with Msp I)
containing 0.01% YOYO-1 dye (Molecular Probes) was placed at the
negative electrode opening of the channel, and current was applied
until a green fluorescing band appeared at the buffer-to-gel
interface (12 V/cm, .apprxeq.2 min). The remaining DNA solution was
rinsed away, replaced by running buffer, and electrophoresis was
continued by applying current at 12 V/cm for 120 min. The gel was
photographed under an incandescent light source and viewed using an
Olympus stereo microscope and Nikon 35 mm camera with no filters.
Separation of the component bands in a range of 100-1000 bp is
clearly visible <1 mm from the buffer reservoir-to-gel
interface. The high resolution of the detector (in this case, a
conventional stereo microscope at x 10 magnification) allowed the
use of an unusually short gel, and resolved several migrating
bands.
[0460] Capillary gel electrophoresis of DNA samples was performed
using a Beckman P/ACE instrument with a laser-induced fluorescence
detector and 37 cm length, 100 .mu.m diameter, linear
polyacrylamide gel capillary according to manufacturer's
recommendation. Samples were stained, then injected
electrokinetically using a water-stacking procedure and run at 7400
V dc for 45 min.
[0461] Additional DNA Analysis System Components
[0462] Using microfabrication processes compatible with the
construction of the thermocapillary pump channels, a thermal
cycling plat-form, a gel electrophoresis chamber, and a DNA
detector were fabricated and tested. PCR.TM. thermal cycling was
performed on a silicon substrate using heaters and temperature
sensors from the same processed wafer as the thermocapillary pump.
In this thermal reaction chamber device, a group of four closely
spaced heater elements were tested to ensure compatibility with the
standard PCR.TM. biochemical reactions. The device successfully
amplified a single-copy sequence from total genomic mouse DNA in
small aqueous drops (10-25 .mu.l) placed on the processed silicon
surface and covered with mineral oil to prevent evaporation.
However, variations in PCR.TM. amplification efficiency as large as
4-fold were observed between repetitions of the study.
[0463] Diffusion Diode Wafer Fabrication
[0464] Integral DNA sensor elements were fabricated on the surface
of silicon wafers to electronically detect migrating DNA bands. A
sensor capable of detecting decay events from radioactively labeled
DNA may be fabricated on the surface of silicon wafers as p-n-type
diffusion diode. Radiation detection was chosen for the initial
device since such diodes have a high sensitivity, small aperture
dimensions, and well-known fabrication and response
characteristics. Testing of the device with .sup.32P-labeled DNA
demonstrates that it readily functions as a sensor capable of
detecting single impacting events. For each diode element, the
diffusion regions of the central detector are .apprxeq.300 gm long
and 4 .mu.m wide and guard ring shield the electrodes. This diode,
although currently configured for high-energy .beta. particle
detection, can also operate as a fluorescent light detector when
combined with a matched fluorophore, wavelength filter, and
excitation source.
[0465] Diode detectors were prepared on 200 .OMEGA.-cm, (100),
boron-doped, p-type silicon wafer substrates. Diffused layers of
phosphorus (5.times.10.sup.14 cm.sup.-2) and boron (1.times.10
cm.sup.-2) were ion-implanted onto the sample in lithographically
defined regions (mask D1); thermal silicon oxide was grown (0.2
.mu.m at 900.degree. C.) over the wafer; and contact holes were
etched to the diffusion layer with buffered hydrofluoric acid
solution. A 3.3-.mu.m layer of photoresist (Microposit 1400-37) was
patterned to define the metal pads (mask D2); 50-nm chromium
followed by 400-nm gold was evaporated over the resist; and the
metallization lifted off the regions retaining the resist. In some
initial radiation sensitivity tests, a layer of photoresist
(Microposit 1813) was applied across the wafer and baked for
110.degree. C. for 30 min to form an aqueous solution barrier.
Additional studies used a double layer of plasma-enhanced chemical
vapor deposition silicon oxide and silicon nitride as a barrier,
similar to the layers described for the heater-element wafer.
Radioactive phosphorus (.sup.32P) decay events were detected using
a sample of labeled DNA in PCR.TM. buffer placed on the barrier
layer. To test sensitivity, the detector was connected to a
charge-sensitive preamplifier (model 550A, EV-Products, Saxonburg,
PA), followed by a linear shaping amplifier and a standard
oscilloscope, and events were computer recorded.
[0466] The resolving ability of DNA gel electrophoresis systems may
be improved by the proximity and narrow width of silicon-based
detectors placed immediately beneath the gel channel.
Microfabricated diodes may be placed within 1 micron of the gel
matrix and can have an aperture of 5 microns or less. Since the gel
length required for the resolution of two migrating bands is
proportional to the resolution of the detector, the incorporation
of micron-width electronic detectors may significantly reduce the
total gel length required for DNA analysis without sacrificing
band-reading accuracy.
[0467] Currently, optical methods using efficient fluorophores can
detect atto-molar concentrations (corresponding to
.apprxeq.10.sup.5 DNA molecules) migrating in capillary channels of
8.times.50 .mu.m internal cross section (Woolley and Mathies,
1994). Reactions for synthesizing such DNA quantities can
reasonably occur in 10 .mu.l. An integrated system designed for
picoliter volumes may require channel dimensions on the order of 10
.mu.m.sup.2.times.100 .mu.m (cross section.times.length). At this
size, thousands of individual devices would occupy a single
100-mm-diameter wafer.
EXAMPLE 2
[0468] Isothermal Amplification in a Silicon Chip
[0469] The compatibility of the isothermal amplification reagents
(available from Becton Dickinson), particularly enzymes, with the
silicon DNA chip assay format was investigated. The components of
an SDA reaction for amplification of the IS6110 element of
Mycobacterium tuberculosis, except for the enzymes, were assembled
externally to the chip, denatured in a boiling water bath for 2 min
and cooled to 52.degree. C. for 2 min. The enzymes were added to
bring the total volume to 50 .mu.L containing 35 mM
K.sub.2HPO.sub.4 pH 7.6, 50 mM NaCl; 10 mM TRIS pH 7.6, 9 mM
MgOAc.sub.2, 1.4 mM dCTP.alpha. S, 0.2 mM TrP, 0.2 mM dGTP, 0.2 mM
dATP, 18.5% (v/v) glycerol, 1 mM DTr, 500 ng human DNA, 500 nM SDA
primers (SI and S2), 2.5 mM SDA bumpers (B.sub.1 and B.sub.2),
10.sup.6 M. tuberculosis genomes containing the IS6110 target, 160
units BsoBI and 13 units exo.sup.- Bst polymerase. The
amplification and bumper primers were as follows, with the BsoBI
recognition sequence shown in bold and the IS6110 target binding
sequence underlined:
1 5'-CGATTCCGCTCCAGACTTCTCGGGTCTACTGAGATCCCCT-3' (S1) (SEQ ID NO:3)
5'-ACCGCATCGAATGCATCTCTCGGGTAAGGCGTACTCGACC-3' (S2) (SEQ ID NO:4)
5'-CGCTGAACCGGAT-3' (B1) (SEQ ID NO:5) 5'-TCCACCCGCCAAC-3' (B2)
(SEQ ID NO:6)
[0470] A 4 .mu.L sample of the amplification reaction was
immediately placed in a 60 .mu.m deep, 5.1 cm long glass channel
etched in 7740 PYREX (Dow Coming) and adhered to a silicon chip,
filling the entire channel. The channel was open at both ends. The
channel chip was placed on a heater element wafer in contact with
about one third of the sample, and the temperature was held at
52.degree. C. for up to 30 min to allow the amplification reaction
to proceed. To remove the sample, about 5 .mu.L of amplification
reaction buffer without the enzymes was placed at one end of the
channel and the sample was withdrawn from the other end using a
sequencing pipette tip. This process was repeated four times to
wash the channel. The total volume recovered was about 20 .mu.L.
The amplification reaction was then stopped by boiling in a water
bath and amplification was detected in a chemiluminescent assay as
described in U.S. Pat. No. 5,470,723. The biotinylated capture
probe and the alkaline phosphatase labeled detector probe used in
the assay are described in Spargo, et al. (1993). As a control, the
same SDA reaction was performed in a test tube in the conventional
manner. Target amplification efficiency was equivalent in the
conventional SDA reaction and on the DNA chip, with amplification
of almost a million-fold. This demonstrated that the physical
changes in the environment on the DNA chip, including temperature
gradients, inhibitors and surface interactions, did not adversely
affect the amplification reaction.
[0471] The ability of the separate components of the amplification
reaction to adequately mix within the channels of the DNA chip was
then investigated. In one study, 160 nL of enzyme mix was placed in
a channel prepared as described above. The target was denatured at
95.degree. C. in amplification buffer (3.84 .mu.L) and cooled to
52.degree. C. prior to loading into the channel with the enzyme.
The total volume of the reaction mix filled the entire channel.
Amplification was allowed to proceed at 52.degree. C. for 16 min
and assayed as before. In a second study, the target in
amplification buffer (1.5 .mu.L) was loaded into the channel and
moved over the heating element using air pressure. Using the heater
element the temperature was raised to 80.degree. C. for about 10
sec (temperature spiked to about 95.degree. C.) to denature the
target, then cooled to 52.degree. C. The enzyme mix (1.5 .mu.L) was
added to the channel with the denatured target to fill the channel.
The amplification reaction was performed (15 min reaction time) and
assayed as before. In an additional study, 1 .mu.L of enzyme mix
was loaded into one end of the channel and 1 .mu.L of target in
amplification buffer was loaded into the other end. The portion of
the channel containing the target was heated to 80.degree. C. for
about 15 sec to denature the nucleic acids (temperature spiked to
about 90.degree. C.) and cooled to 52.degree. C. The two samples
were brought into contact by applying air pressure to the open ends
of the channel until the target and enzyme aliquots moved into
contact with each other. The reaction was held at 52.degree. C. on
the heater element for 15 min, then removed with washing as
described above. Chemiluminescent detection of amplification
products in all of these studies revealed efficient amplification
of the target, indicating adequate mixing and diffusion of the
reactants in all channel configurations and protocols tested.
EXAMPLE 3
[0472] This example describes approaches to the problem of forming
a moisture barrier over electrical elements of the microscale
device. Initial prototypes employed 5000 angstroms of aluminum and
covered it with PECVD SiO.sub.x. Upon testing, it was determined
that the liquids were penetrating this later and destroying the
aluminum heating elements.
[0473] Without clear evidence what was causing this problem, it was
hypothesized that the step height of the aluminum was causing
cracks in the passivation layer (the oxide). In order to alleviate
the cracking problem, a layer of Si.sub.xN.sub.y tried between two
layers of SiO.sub.x, with the thought that the additional thickness
would overcome the cracking caused by the step height. It did
not.
[0474] As a follow-up approach, a thinner layer (500 angstroms) of
aluminum was tried. This gave {fraction (1/10)}th the step height
of the original prototype devices. On top of this aluminum, a
triple layer of SiO.sub.x, Si.sub.xN.sub.y, and SiO.sub.x was
employed. Moreover, the process for making the Si.sub.xN.sub.y
layer was changed to one which would give a more dense layer. This
appeared to solve the problem. However, the thinner layer of
aluminum created a higher resistance which was not acceptable. It
was determined that one needed a way to generate thicker layers of
aluminum for lower resistance, yet keep the surface relatively
smooth (planar). An etch back process was used (now called "the
inlay process") to accomplish the task. By etching back into a
layer of SiO.sub.x depositing aluminum in the resulting cavity,
then stripping the resist mask, a surface was obtained with a step
height low enough to prevent cracking of the passivation
layers.
[0475] It was also discovered that the metal bonding pads were not
adhering well to the initial PECVD SiO.sub.x layer. To overcome the
problem, the process was modified by using a wet thermal SiO.sub.2
layer.
EXAMPLE 4
[0476] This example describes approaches to enhancing droplet
motion by surface treatment. In this regard, the principle of using
surface tension to cause droplets to move may be applied to either
hydrophilic or hydrophobic surfaces. Glass, for instance, is
naturally hydrophilic with a near zero contact angle with water.
Because the oxide coating of the present invention is made
principally of the same material as glass, it was expected that the
devices would also exhibit near zero angles. It was discovered,
however, that the actual construction materials had contact angles
far from zero, thus enhancing the effects of contact angle
hysteresis (discussed in greater detail in Example 3). For
instance, water gave a contact angle (static) of .about.42.degree.
on polyamide, .about.41.degree. on SiO.sub.2 (major component of
most glasses), .about.62.degree. on silicone spray. To enhance the
surface effectiveness, several treatment processes for both
hydrophilic and hydrophobic surfaces were tried, as described
below.
[0477] To improve the hydrophilicity of a surface, several cleaning
procedures were tried. It has been reported that surface
contamination and/or roughness can reduce the hydrophilicity of
surfaces. Therefore, a high concentration chromic acid cleaning, a
high concentration sulfuric acid cleaning a baking procedure (to
600.degree. C. for 8 h to burn off contaminates), and surface
coatings were tried. The acid cleaning procedures were not as
effective as the baking procedure; however, neither proved to be
compatible with the devices since the concentrated acids would
attack the aluminum pads and the high temperature could peal the
aluminum (melting pt. .about.660.degree. C.) or break the adhesive
bond between the heater chip and the channel.
[0478] Rain-X antifog (commercially available) as a treatment was
observed to work. This is a surface treatment which makes surfaces
hydrophilic. Although, the resulting surfaces may not be 0.degree.,
by using this coating the entire surface gets treated giving a
uniform surface for the droplet. Experimentally, it was found that
Rain-X antifog treatments greatly enhanced droplet motion studies
using heat. Another such treatment which was tested but which did
not work was a material called SilWet. This material is used in the
agriculture industry for enhancing the wetting of plants with
agricultural sprays.
[0479] To obtain hydrophobic surfaces, capillaries were coated with
Rain-X and silane treatments. Neither of these gave angles much
greater than 90.degree., therefore, would not work with this
mechanism. These treatments would have to have given angles
180.degree. to be useful for hydrophobic studies of motion.
Eventually, it was discovered that one could apply a teflon coating
that was sufficiently hydrophobic to possibly warrant future
tests.
EXAMPLE 5
[0480] This example describes approaches to droplet motion by heat
treatment. As noted previously (above), the contact angle on the
advancing end of a liquid droplet in motion (known as the advancing
contact angle) is greater that the that on the receding end
(receding contact angle). In the case of a hydrophilic surface -
such as water on glass - this tends to create a back pressure
countering attempts at forward motion by heating the back side of a
droplet. This is best shown by a simple model describing laminar
flow through a channel.
[0481] Average Flow Through a Circular Channel:
[0482] <v>=-.DELTA.P*[R.sup.2/(8 .mu.L]
[0483] where:
[0484] .DELTA.=value at back-value at front end of droplet
[0485] .DELTA.P=(1/R)*(.DELTA.G)=pressure difference between
droplet ends
[0486] .DELTA.G=change in surface tension between the ends of the
droplet.
[0487] R=channel radius
[0488] L=droplet length
[0489] .mu.=viscosity
[0490] Also, for water, .DELTA.G=-constant*.DELTA.T, where
temperature increases lower the surface tension of most liquids
(constant=0.16 dyn/cm for water).
[0491] Therefore:
[0492] <v>=-(.DELTA.G)*(1/R)*[R.sup.2/(8
.mu.L)]=[-0.16*.DELTA.T*R/(- 8 .mu.)]
[0493] where:
[0494] .DELTA.T=T.sub.back-T.sub.front
[0495] giving: <v>=[0.16*R/(8
.mu.L)]*(T.sub.back-T.sub.front)
[0496] This expression indicates that any heating on the back end
of the droplet (if the front remains at a lower temperature) will
cause the liquid droplet to move. This was not the case
experimentally, however. By way of studies using glass capillaries,
it was found that there was a minimum temperature difference
required to move the droplet. This effect is believed to be the
result of contact angle hysteresis (CAH). In CAH, the advancing
contact angle is greater than the receding contact angle resulting
in a sort of back pressure which must be overcome to achieve
droplet movement. CAH occurs when the interface is placed in motion
(dynamic angles). To account for this effect, it was included in a
steady-state (ID) model for flow. For instance, if the advancing
angle is 36.degree. and the receding angle is 29.degree. (with the
front of the droplet being 25.degree. C.), then the back of the
droplet would need to be heated to .about.60.degree. C. for a 1 mm
long droplet in a 20 .mu.m high channel. This is just one example
situation.
[0497] It was discovered experimentally, however, that the channel
dimension and fluid parameters (other than surface tension) do not
affect whether or not the droplet will move. They do determine the
magnitude of motion (if it occurs). What does determine whether
motion will occur or not is the following inequality: 3 G front / G
back > ( R front / R back ) * ( cos back / front ) where : =
contact angle .
[0498] The present calculations suggest that a .about.35.degree. C.
difference between the front and back of a droplet should be
sufficient to initiate droplet motion in a system with advancing
angles of 36.degree. and receding angles of 29.degree. in a 20
.mu.m high channel. Experimental testing of actual devices however,
showed that the front of the droplet heats relatively quickly thus
reducing the temperature difference needed for movement between the
front and the back of the droplet. This effect required the
invention to use higher voltages to obtain droplet motion. Voltages
typically in the range of .about.30.degree. Volts were found to be
required to obtain motion. Further studies showed that the
resulting temperature difference was .about.40.degree. C. between
the front and back of the droplet thus corroborating the initial
determination of the requirements.
[0499] Discrete droplet motion in a micromachined channel structure
using thermal gradients was demonstrated. The device consists of a
series of aluminum heaters inlaid on a planar silicon dioxide
substrate and bonded by glue to a wet-etched glass channel (20
.mu.m depth, 500 .mu.m width). Liquid samples were manually loaded
into the two channels on the left using a micropipette. Heating the
left interface of each droplet propels it toward the intersection
of the channels. At the intersection, the droplets meet and join to
form a single larger droplet. Note that, since the channel
cross-section is 20 .mu.m.times.500 .mu.m, the volume of each of
these droplets may be calculated from their lengths and is
approximately 50 nanoliters.
[0500] The heaters along the entire surface of the channel allow it
to be used as a thermal reaction chamber in addition to a
droplet-motion device. The upper droplet in the figure contains a
DNA sample, while the lower contains a restriction digest enzyme
(TaqI) and digestion buffer. Following sample merging, the combined
droplet was maintained at 65.degree. C. for 30 min using the
integral heaters and temperature sensors. The completed enzymatic
reaction was confirmed by expressing the droplet from the right end
of the channel and loading it onto a capillary gel electrophoresis
system with a laser-induced fluorescence detector. The chromatogram
produced by the silicon-device sample was similar to chromatograms
generated from DNA digests runs in a standard polypropylene
microreaction vessel.
EXAMPLE 6
[0501] This example describes various approaches for bonding
channels to the substrate which contains circuitry for heating and
temperature sensing of the device of the present invention (see
discussion of two-part construction, above). First attempts
involved Polyamide; regular polyamide was unsatisfactory in that it
was found the two pieces would not stick together.
[0502] Follow-up attempts involved a photo-definable Polyamide.
This produced a sticky surface, but would not give a perfect seal
along the channel. It was discovered that the solvents released
during the final baking process were causing pockets in the
polyamide layer. An adhesion layer was needed which would seal by
`curing` and not release solvents.
[0503] Several different epoxies and glues were investigated, as
listed in Table 1 below.
2 TABLE 1 Adhesive Form Dries Texture Comments 1. Dymax UV Glue Gel
Clear Rubbery Cures on UV exposure. 2. Carter's Rubber Goo
Yellow/Clear Rubbery Dries quickly and Cement stringy when thinned.
3. Borden's Krazy Liquid Clear Hard Thin, dries on first Glue
contact. 4. UHU Bond-All Gel/Goo Clear Hard Dries quickly and
stringy when thin. 5. Dennison Paste Clear Hard Will not flow on
Permanent Glue applying. Stick 6. Elmer's Glue-All Thick White Hard
Slow drying. (Borden) Liquid 7. Liquid Nails Thin Paste Wood-like
Hard Thick, dries quickly when thinned. 8. Devcon 5-Minute Gel
Yellow/Clear Hard Thick, cures on Epoxy about 5 min. 9. Scotch
Double- Tape Clear Rubbery Tape. Stick Tape 10. Dow Corning Thick
Gel Frosty Soft Seals but does not High Vacuum bond. Grease 11.
Nujol Mineral Oil Liquid Clear Runny Neither seals (Perkin Elmer)
(doesn't spread on glass) nor bonds. 12. Household Goop Gel/Goo
Clear Rubbery Contact cement which dries stringy. 13. Permatex
Weather Gel/Goo Yellow/Clear Rubbery Dries quickly on Strip Cement
stringy when thinned. 14. Thick Gel Super Gel Clear Hard Does not
cure on Glue contact but does quickly. 15. DAP Weldwood Goo
Orange/Clear Rubbery Contact cement Contact Cement which gets
stringy when thinned. 16. Scotch (3M) Thin Goo Yellow/Clear Rubbery
Spray. "Gooey" Photo Mount but not stringy. Spray Adhesive 17.
Silicone Resin Liquid Clear Smooth Spray. Dries to (spray) Lacquer
thin, clear, and (GC Electronics) sealed coating.
[0504] A preferred glue was a UV cured glue, although the process
of applying the UV glue is tedious and requires some practice to
avoid putting the glue in places where it does not belong, e.g., in
the channels.
[0505] Hydroxide bonding and screen printing of bonding substances
was also attempted. Another option was glass tape, but the high
temperatures required to melt the tape appeared to be too high for
the present devices.
EXAMPLE 7
[0506] This example describes a nucleic acid amplification reaction
on a silicon-based substrate. The established DNA biochemistry
steps for PCR.TM. occur within physiological conditions of ionic
strength, temperature, and pH. Thus, the reaction chamber
components have design limitations in that there must be
compatibility with the DNA, enzymes and other reagents in
solution.
[0507] To assess biocompatability, components were added to a
standard PCR.TM. reaction. The results indicate PCR.TM. works well
with bond-all glue, goop glue, rubber cement, vacuum grease,
silicone spray, reaction vial plastic, stainless steel, wire
thermocouple, crushed glass, and glass capillary, but indicated
that crystalline silicon, crushed silicon, rubber gasket,
polyamide, UV glue, cured silicone sealer, and liquid nails glue
may not be the ideal material for biological compatibility. Given
these results, it may be desirable to modify the surface of the
micromachined silicon substrate with adsorbed surface agents,
covalently bonded polymers, or a deposited silicon oxide layer.
[0508] To form a biologically compatible heating element, a
standard silicon wafer was coated with a 0.5 .mu.m layer of silicon
dioxide. Next, a 0.3 .mu.m deep, 500 .mu.m wide channel was etched
into the silicon oxide and gold or aluminum was deposited (0.3
.mu.m thick). This inlay process results in a relatively planar
surface and provides a base for deposition of a water-impermeable
layer. The impermeable layer is made by a sequence of three plasma
enhanced vapor depositions: silicon oxide (SiO.sub.x), silicon
nitride (Si.sub.xN.sub.y) and silicon oxide (SiO.sub.x). Since the
materials are deposited from the vapor phase the precise
stoichiometries are not known. A thin metal heater design was used
for this device rather than the doped-silicon resistive heaters
previously demonstrated for micromachined PCR.TM. reaction
chambers, since the narrow metal inlay allows viewing of the liquid
sample through a transparent underlying substrate, such as glass or
quartz. Also, the use of several independent heating elements
permits a small number to operate as highly accurate resistive
temperature sensors, while the majority of elements are functioning
as heaters.
[0509] A device fabricated with metal resistive heaters and
oxide/nitride/oxide coating was tested for biological compatibility
and temperature control by using PCR.TM. amplification of a known
DNA template sample. The reaction was carried out on the planar
device using twenty microliters of PCR.TM. reaction mix covered
with mineral oil to prevent evaporation. The reaction mixture was
cycled through a standard 35-cycle PCR.TM. temperature cycling
regime using the integral temperature sensors linked to a
programmable controller. Since the reaction volume was
significantly larger than intended for the original heater design,
a polypropylene ring was cemented to the heater surface to serve as
a sample containment chamber. In all test cases, the presence of
amplified reaction products indicated that the silicon dioxide
surface and the heater design did not inhibit the reaction.
Parallel amplification studies performed on a commercial PCR.TM.
thermocycler gave similar results. A series of PCR.TM.
compatibility tests indicated that the reaction on the device is
very sensitive to controller settings and to the final surface
material in contact with the sample.
[0510] From the above it should be evident that the present
invention may be adapted for high-volume projects, such as
genotyping. The microdroplet transport avoids the current
inefficiencies in liquid handling and mixing of reagents. Moreover,
the devices are not limited by the nature of the reactions,
including biological reactions.
EXAMPLE 8
[0511] In this example, a test structure is fabricated. The main
part is constructed from a two mask process with five layers of
materials on top of the Si substrate. Proceeding from the lowest to
the uppermost layer, the SiO, serves as an insulator between the Si
substrate and the other metal layers, which function as solder pads
and heating elements. The Ti layer (250A) is for adhesion of Ni.
The layers of Ni (1000 A) and Au (1000 A) act as a diffusion
barrier for the solder. The Au layer also serves as a wettable pad.
Finally, the layer of solder is for bonding two substrates
together. The solder will melt by heating the metal layers. Another
substrate that will be bonded has the same construction except for
the solder.
[0512] A thermo-pneumatic microvalve is utilized in the test
structure. A corrugated diaphragm is chosen for its larger
deflection and higher sensitivity. The diaphragm (side length=1000
.mu.m, thickness=3 .mu.m, boss size length=500 .mu.m boss
thickness=10 .mu.m) has a deflection of 27 .mu.M at an applied
pressure of 1 atm. This applied pressure is generated by a
thermo-pneumatic mechanism, which provides a greater actuation
force. A pressure of 1 atm is generated in the cavity between the
diaphragm and glass by Freon-11 when it is heated 11.degree. C.
above room temperature. Ten masks are expected to fabricate the
microvalve.
[0513] A portion of a silicon substrate is a p-type (100)-oriented
Si wafer of normal thickness and moderate doping (>1 cm). The
preferred wafer thickness, however, is ordinarily a function of the
wafer diameter. The upper surface of the silicon wafer containing
substrate is lapped, polished and cleaned in the normal and
accepted manner. Isotropic etching using reactive ion etching (RIE)
forms the diaphragm corrugations with photoresist as the masking
material.
[0514] Deep boron diffusion areas form the rims, center bosses,
inlet and outlet holes of the finished device. The deposition of
shallow boron diffusion areas to form a diaphragm. The various
metal layers, including solder, are then deposited. The deep and
shallow boron diffusion processes define the shape of the diaphragm
and the etch-stop for the dissolved wafer process.
[0515] Following this, the definition of oxide layer to serve as
insulator of the solder of the finished device. Ti adhesion/Ni/Au
barrier and wettable pads are then deposited. The solder mold of Ni
and photoresist is then defined and the first Ni channel is created
by surface-micromachined using photoresist as sacrificial layers.
The Ni channel hole is defined using EDP to remove the sacrificial
layers, and define an channel hole.
[0516] A second Ni channel is defined by Ni and photoresist, and
inlet and outlet holes are defined using EDP to remove the
sacrificial layers.
[0517] Lastly, a Ti/Pt heater in glass is anodically bonded to the
silicon substrate. Freon fills the cavity through a hole in the
glass substrate. This hole is created from a diamond drill bit and
sealed with epoxy.
EXAMPLE 9
[0518] In this example, a low melting point solder was intended to
be utilized in the test structure. Because a universally useful
solder-sealed microvalve will be used in a gas phase
microanalytical system, it is not desirable to use a high melting
point (m.p.) solder (>200.degree. C.), which might affect the
gas properties. In addition, a high m.p. solder may affect other
components on the device, such as integrated circuits, and increase
power consumption. As a result, low melting point solder is
required. Bismuth-bearing solders have the lowest m.p.'s of
47-138.degree. C. However, when a test structure was dipped into a
pool of solder belonging to this group, all the metal layers
dissolved into the solution of solder. Moreover, this solder was
not selective in wetting the surface of the test structure.
EXAMPLE 10
[0519] In light of the results of the study set forth in Example 7,
an attempt was made with commonly available 60:40 Sn:Pb solder
(m.p. 183.degree. C.). When the test structure was dipped into a
solution of this solder, the metal layers remained intact.
Furthermore, these layers demonstrated excellent wettability for
the solder, i e., the solder was confined only to the areas of
metals.
EXAMPLE 11
[0520] In this example, a device and method for blocking fluid flow
in a channel is described. 60:40 Sn:Pb solder, associated with a
heating element, is placed within a side channel. The heating
element at least partially liquefies the solder and air flow moves
the liquefied solder from the side channel into a main channel and
cooled, blocking the main channel.
EXAMPLE 12
[0521] In this example, a device, which was fabricated using
lift-off method described above to pattern hydrophobic regions on
glass and silicon substrates, was testing for the separation of
water droplets. For the device, a patterned metallic thin film was
used to expose regions that were chosen to be made hydrophobic on a
hydrophilic substrate. Chromium, Gold or Aluminum was used as the
metal layer; the choice of the metal being based on process
compatibility with other processing steps and step height coverage
of the etched channels.
[0522] Line widths as narrow as 10 .mu.m were patterned on silicon
substrates using the methods of the present invention. Water drops
separated by lines of hydrophobic and hydrophilic regions patterned
by this new technique (the width of the hydrophilic line in the
middle is 1 mm). The contact angle of water on the OTS (SAM) coated
silicon oxide surface was measured to be approximately
110.degree..
[0523] One can also define hydrophobic regions in etched channels
in glass by performing the lithography using a thick resist. It was
found empirically that cleaning of the substrates prior to
immersion in the OTS (SAM) solution is important; improper cleaning
results in films that partially covers the surface.
EXAMPLE 13
[0524] The results of Example 10, above, demonstrate that
hydrophobic and hydrophilic patterns enable one to define and
control the placement of aqueous liquids, and more specifically
microdroplets of such liquids, on a substrate surface. Use of this
patterning technique to split a liquid droplet into multiple liquid
droplets. A concentric pattern of alternating hydrophobic and
hydrophilic sectors was imparted to a silicon substrate (the
diameter of the circular substrate was 1 cm) using the methods of
the present invention as described above. A water drop was placed
on the pattern and the excess water pulled away using a pipet,
resulting in multiple drops separated from each other.
EXAMPLE 14
[0525] In this example, studies to position a water front inside a
channel using straight channels (depth ranging from 20-40 .mu.m and
width between 100-500 .mu.m) with a 500 .mu.m wide hydrophobic
region (or patch) patterned a few millimeters away from the side
inlet. Water was placed at the inlet using a sequencing pipette
(Sigma, least count 0.5 .mu.1) and was drawn into the channel by
surface forces. The water front stopped at the hydrophobic patch if
a controlled amount of liquid was placed at the inlet. However, if
the channels were overloaded, the liquid would tend to overrun the
hydrophobic patch. This behavior was prominent in the channels with
smaller cross-section.
[0526] To eliminate the over-running of the liquid over the
patches, an overflow channel was introduced in the design to stop
the water running over the hydrophobic patch (such as that shown
FIG. 3). The dimensions of the channels varied in depth and width
as before. Water placed at the inlet is drawn in and splits into
two streams at the intersection point. The two fronts move with
almost equal velocity until the front in the main channel reaches
the hydrophobic patch. The front in the main channel stopped at the
hydrophobic patch; however, the other front continued to move to
accommodate the excess injected water. Using this overflow channel
design, one can successfully stop aqueous liquids for the full
range of variation in channel dimensions.
EXAMPLE 15
[0527] One embodiment of the device of the present invention (in
operation) utilized a heater. Liquid placed at the inlet stops at
the hydrophobic interfaces, and more specifically, stops at the
liquid-abutting hydrophobic region. The inlet and overflow ports
were blocked or heavily loaded with excess liquid to ensure that
the pressure generated acts only in the direction away from the
inlet holes. The heater resistor was actuated by an applied
voltage. The flow of current caused resistive heating and
subsequently increases the air temperature in the chamber and,
therefore, the pressure. After the pressure builds up to a
particular value, a microdrop splits and moves beyond the
hydrophobic patch. The drop keeps moving as long as the heater is
kept on; the drop velocity decreases as it moves further away.
While it is not intended that the present invention be limited by
the mechanism by which this takes place, it is believed that the
added volume (the volume by which the drop has moved) brings about
a decrease in the pressure.
[0528] To stop or block the moving drop at a location, two
strategies may be employed. In the first method, the inlet and
overflow ports were opened to the atmosphere and the heater was
slowly turned off. The temperature inside the chamber falls quickly
to around room temperature, thereby reducing the pressure inside
the chamber. The water from the inlet flows into the chamber to
relieve the pressure. In the second method, a hydrophobic vent was
placed away from the chamber to the right. As soon as the moving
drop goes past the hydrophobic vent, the drop stops moving farther.
Cooling the chamber to room temperature at this instant will cause
air to flow back through the vent to relieve the low pressure in
the chamber.
[0529] From the above, it should be clear that the compositions,
devices and methods of the present invention permit on-chip
actuation using etched chambers, channels and heaters. There is no
requirement for mechanical moving parts and the patterns are
readily fabricated. While the operations described above have been
for simple designs, the present invention contemplates more
complicated devices involving the introduction of multiple samples
and the movement of multiple microdroplets (including simultaneous
movement of separate and discrete droplets).
[0530] All of the compositions and/or methods and/or apparatus
disclosed and claimed herein may be made and executed without undue
experimentation in light of the present disclosure. While the
compositions and methods of this invention have been described in
terms of preferred embodiments, it will be apparent to those of
skill in the art that variations may be applied to the compositions
and/or methods and/or apparatus and in the steps or in the sequence
of steps of the method described herein without departing from the
concept, spirit and scope of the invention. More specifically, it
will be apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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