U.S. patent application number 11/168144 was filed with the patent office on 2007-02-22 for moving microdroplets.
This patent application is currently assigned to The Regents Of The University Of Michigan. Invention is credited to Mark A. Burns, Bishnu Gogoi, Kalyan Handique.
Application Number | 20070042503 11/168144 |
Document ID | / |
Family ID | 34682174 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070042503 |
Kind Code |
A1 |
Handique; Kalyan ; et
al. |
February 22, 2007 |
Moving microdroplets
Abstract
The movement and mixing of microdroplets through microchannels
is described employing microscale devices, comprising microdroplet
transport channels, reaction regions, electrophoresis modules, and
radiation detectors. The 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.
Inventors: |
Handique; Kalyan; (Ann
Arbor, MI) ; Gogoi; Bishnu; (Scottsdale, AZ) ;
Burns; Mark A.; (Ann Arbor, MI) |
Correspondence
Address: |
PETER G. CARROLL;MEDLEN & CARROLL, LLP
Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
The Regents Of The University Of
Michigan
|
Family ID: |
34682174 |
Appl. No.: |
11/168144 |
Filed: |
June 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09518895 |
Mar 6, 2000 |
6911183 |
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11168144 |
Jun 28, 2005 |
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08938689 |
Sep 26, 1997 |
6130098 |
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09518895 |
Mar 6, 2000 |
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08888309 |
Jul 3, 1997 |
6048734 |
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08938689 |
Sep 26, 1997 |
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08529293 |
Sep 15, 1995 |
6057149 |
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08888309 |
Jul 3, 1997 |
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Current U.S.
Class: |
436/180 |
Current CPC
Class: |
B01L 3/502715 20130101;
Y10T 436/2575 20150115; B01F 13/0084 20130101; B01L 3/502784
20130101; B01L 2400/0688 20130101; F16K 2099/008 20130101; B01L
2400/0694 20130101; B01F 13/0071 20130101; B01F 5/0085 20130101;
B01L 2400/0442 20130101; F15B 13/0807 20130101; B01J 19/0093
20130101; B01L 3/50273 20130101; F16K 99/0019 20130101; B01L
2200/0673 20130101; Y10T 436/118339 20150115; B01F 15/0232
20130101; Y10T 436/117497 20150115; B82Y 30/00 20130101; F16K
99/0061 20130101; B01F 13/0093 20130101; F16K 2099/0074 20130101;
F16K 99/0001 20130101; B01L 2300/1827 20130101; B01L 3/502738
20130101; B01L 2300/0816 20130101; B82Y 40/00 20130101; B01L
2400/0487 20130101; F16K 2099/0084 20130101; B01F 13/0072 20130101;
B01F 5/0403 20130101; B01L 2200/0605 20130101 |
Class at
Publication: |
436/180 |
International
Class: |
B01L 3/02 20070101
B01L003/02 |
Goverment Interests
[0002] This invention was made with government support awarded by
the National Institutes of Health (grant numbers NIH-R01-HG01044
and NIH-R01-HG01406). The government has certain rights in the
invention.
Claims
1. A device comprising a microdroplet transport channel etched in
substrate, said substrate selected from the group consisting of
silicon, quartz and glass, said channel comprising one or more
hydrophobic regions.
2. The device of claim 1, wherein said device further comprises a
gas-intake pathway in fluidic communication with said microdroplet
channel.
3. The device of claim 2, wherein said device further comprises a
gas vent in fluidic communication with said microdroplet
channel.
4. The device of claim 3, wherein one of said hydrophobic regions
is positioned in said channel between said gas-intake pathway and
said gas vent.
5. The device of claim 2, further comprising an air chamber in
communication with said gas-intake pathway.
6. A device comprising a microdroplet transport channel, said
channel comprising i) first and second ends, and ii) a hydrophobic
regions disposed within said channel between said first and second
ends.
7. The device of claim 6, wherein said device further comprises a
gas-intake pathway i) positioned internal to said first end of said
channel, and ii) in fluidic communication with said microdroplet
channel.
8. The device of claim 7, wherein said device further comprises a
gas vent i) positioned internal to said second end of said channel,
and ii) in fluidic communication with said microdroplet
channel.
9. The device of claim 8, wherein said hydrophobic region is
positioned in said channel between said gas-intake pathway and said
gas vent.
10. The device of claim 6, wherein said first end of said channel
comprises a inlet port for the introduction of liquid.
11. The device of claim 7, further comprising an air chamber in
communication with said gas-intake pathway.
12. The device of claim 6, wherein said device is fabricated from a
glass, quartz or silicon substrate.
13. The device of claim 12, wherein said channels are between 5 and
20 .mu.m in depth and between 20 and 1000 .mu.m in width.
Description
[0001] This application is a continuation-in-part of copending U.S.
patent application Ser. No. 08/888,309, filed Jul. 3, 1997, which
is a continuation-in-part of copending U.S. patent application Ser.
No. 08/529,293, filed Sep. 15, 1995.
FIELD OF THE INVENTION
[0003] The present invention relates to movement of microdroplets
through microchannels, and more particularly, compositions, devices
and methods to control microdroplet size and movement.
BACKGROUND
[0004] The complexity and power of biological reactions has
increased dramatically over the last thirty years. The initial
observations of the "hybridization" process, i.e., the ability of
two polymers of nucleic acid containing complementary sequences to
find each other and anneal through base pairing interaction, by
Marmur and Lane, Proc. Nat. Acad. Sci., U.S.A. 46, 453 (1960) and
Doty et al., Proc. Nat. Acad. Sci., U.S.A. 46, 461 (1960), have
been followed by the refinement of this process into an essential
tool of modern biology.
[0005] Initial hybridization studies, such as those performed by
Hayashi et al., Proc. Nat. Acad. Sci., U.S.A. 50, 664 (1963), were
formed in solution. Further development led to the immobilization
of the target DNA or RNA on solid supports. With the discovery of
specific restriction endonucleases by Smith and Wilcox, J. Mol.
Biol. 51, 379 (1970), it became possible to isolate discrete
fragments of DNA. Utilization of immobilization techniques, such as
those described by Southern, J. Mol. Biol. 98, 503 (1975), in
combination with restriction enzymes, has allowed for the
identification by hybridization of singly copy genes among a mass
of fractionated, genomic DNA.
[0006] In 1977, two methods for DNA sequencing were reported. These
were the chemical degradation method of Maxam and Gilbert, Proc.
Nat. Acad. Sci. U.S.A. 74:560 (1977) and the enzymatic method of
Sanger et al., Proc. Nat. Acad. Sci. U.S.A. 74:5463 (1977). Both
methods generate populations of radiolabeled oligonucleotides which
begin at a fixed point and terminate randomly at a fixed residue or
type of residue. These populations are resolved on polyacrylamide
gels which allow the discrimination between oligonucleotides that
differ in length by as little as one nucleotide.
[0007] The Maxam and Gilbert method utilizes a fragment of DNA
radiolabeled at one end which is partially cleaved in five separate
chemical reactions, each of which is specific for a particular base
or type of base. The products of these chemical reactions are five
populations of labelled molecules that extend from the labeled end
to the site of chemical cleavage. This method has remained
relatively unchanged since its initial development. This method
works best for DNA sequences that lie less than 250 nucleotides
from the labeled end.
[0008] In contrast, the Sanger method is capable of sequencing
greater than 500 nucleotides in a single set of reactions. The
Sanger method is an enzymatic reaction that utilizes
chain-terminating dideoxynucleotides (ddNTPs). ddNTPs are
chain-terminating because they lack a 3'-hydroxyl residue which
prevents formation of a phosphodiester bond with the succeeding
deoxyribonucleotide (dNTP). A small amount of one ddNTP is included
with the four conventional dNTPs in a polymerization reaction.
Polymerization or DNA synthesis is catalyzed by a DNA polymerase.
There is competition between extension of the chain by
incorporation of the conventional dNTPs and termination of the
chain by incorporation of a ddNTP. A short oligonucleotide or
primer is annealed to a template containing the DNA to be
sequenced. The original protocols required single-stranded DNA
templates. The use of double-stranded templates was reported later.
The primer provides a 3' hydroxyl group which allows the
polymerization of a chain of DNA when a polymerase enzyme and dNTPs
are provided.
[0009] The original version of the Sanger method utilized the
Klenow fragment of E. coli DNA polymerase. This enzyme has the
polymerization and 3' to 5' exonuclease activity of the unmodified
polymerase but lacks 5' to 3' exonuclease activity. The Klenow
fragment has several limitations when used for enzymatic
sequencing. One limitations is the low processivity of the enzyme,
which generates a high background of fragments that terminate by
the random dissociation of the enzyme from the template rather than
by the desired termination due to incorporation of a ddNTP. The low
processivity also means that the enzyme cannot be used to sequence
nucleotides that appear more than 250 nucleotides from the 5' end
of the primer. A second limitation is that Klenow cannot
efficiently utilize templates which have homopolymer tracts or
regions of high secondary structure. The problems caused by
secondary structure in the template can be minimized by running the
polymerization reaction at 55.degree. C.
[0010] Improvements to the original Sanger method include the use
of polymerases other than the Klenow fragment. Reverse
transcriptase has been used to sequence templates that have
homopolymeric tracts. Reverse transcriptase is somewhat better than
the Klenow enzyme at utilizing templates containing homopolymer
tracts.
[0011] The use of a modified T7 DNA polymerase (Sequenase.TM.) was
the most significant improvement to the Sanger method. See
Sambrook, J. et al. Molecular Cloning, A Laboratory Manual, 2d Ed.
Cold Spring Harbor Laboratory Press, New York, 13.7-13.9 and
Hunkapiller, M. W. (1991) Curr. Op. Gen. Devl. 1:88-92.
Sequenase.TM. is a chemically-modified T7 DNA polymerase has
reduced 3' to 5' exonuclease activity. Tabor et al., Proc. Natl.
Acad. Sci. U.S.A. 84:4767 (1987). Sequenase.TM. version 2.0 is a
genetically engineered form of the T7 polymerase which completely
lacks 3' to 5' exonuclease activity. Sequenase.TM. has a very high
processivity and high rate of polymerization. It can efficiently
incorporate nucleotide analogs such as dITP and 7-deaza-dGTP which
are used to resolve regions of compression in sequencing gels. In
regions of DNA containing a high G+C content, Hoogsteen bond
formation can occur which leads to compressions in the DNA. These
compressions result in aberrant migration patterns of
oligonucleotide strands on sequencing gels. Because these base
analogs pair weakly with conventional nucleotides, intrastrand
secondary structures are alleviated. In contrast, Klenow does not
incorporate these analogs as efficiently. The main limitation to
the amount of DNA sequence that can be obtained from a single set
of chain-termination reactions using Sequenase.TM. is the resolving
power of polyacrylamide gels, not the properties of the enzyme.
[0012] The use of Taq DNA polymerase is a more recent addition to
the improvements of the Sanger method. Innis et al., Proc. Natl.
Acad. Sci. U.S.A. 85:9436 (1988). Taq polymerase is a thermostable
enzyme which works efficiently at 70-75.degree. C. The ability to
catalyze DNA synthesis at elevated temperature makes Taq polymerase
useful for sequencing templates which have extensive secondary
structures at 37.degree. C. (the standard temperature used for
Klenow and Sequenase.TM. reactions). Taq polymerase, like
Sequenase.TM., has a high degree of processivity and like Sequenase
2.0, it lacks 3' to 5' nuclease activity.
[0013] Methods were also developed for examining single base
changes without direct sequencing. These methods allow for the
"scanning" of DNA fragments for the presence of mutations or other
sequence variation. For example, if a mutation of interest happens
to fall within a restriction recognition sequence, a change in the
pattern of digestion can be used as a diagnostic tool (e.g.,
restriction fragment length polymorphism [RFLP] analysis).
[0014] With the development of these complex and powerful
biological techniques, an ambitious project has been undertaken.
This project, called the Human Genome Project (HGP), involves the
complete characterization of the archetypal human genome sequence
which comprises 3.times.10.sup.9 DNA nucleotide base pairs. An
implicit goal of the project is the recognition that all humans are
greater than 99% identical at the DNA sequence level. The
differences between people, however, provide the information most
relevant to individual health care, including potential estimates
of the risk of disease or the response to a specific medical
treatment. Upon completion of the HGP, a continuing effort of the
human genetics research community will be the examination of
differences within populations and of individual variants from the
defined archetype. While the 15-year effort of the HGP represents a
defined quantity of DNA data acquisition, the future demand for DNA
information is tied to individual genetic variation and is,
therefore, unlimited.
[0015] Current DNA genotyping technologies are adequate for the
detailed analysis of samples that range in number from hundreds to
thousands per year. Genotyping projects on the order of millions of
assays, however, are beyond the capabilities of today's
laboratories because of the current inefficiencies in (i) liquid
handling of reagent and DNA template solutions, (ii) measurement of
solution volumes, (iii) mixing of reagent and template, (iv)
controlled thermal reaction of the mixed solutions, (v) sample
loading onto an electrophoresis gel, and (vi) DNA product detection
on size-separating gels. What is needed is methodology that allows
for a high-volume of biological reactions without these existing
inefficiencies.
SUMMARY OF THE INVENTION
[0016] 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.
[0017] 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).
[0018] In a preferred embodiment, these elements are
microfabricated from silicon and glass substrates. The various
components are linked (i.e., in liquid communication) using
flow-directing means, including but not limited to, a flow
directing means comprising 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.
[0019] 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.
[0020] The present invention contemplates a method for moving
microdroplets, comprising: (a) providing a liquid microdroplet
disposed within a microdroplet transport channel etched in silicon,
said channel in liquid communication with a reaction region via
said transport channel and separated from a microdroplet
flow-directing means by a liquid barrier; and (b) conveying said
microdroplet in said transport channel to said reaction region via
said 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 said transport
channel and the microdroplets are conveyed by differential heating
of the microdroplet by the heating elements.
[0021] It has been found empirically that the methods and devices
of the present invention can 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.
[0022] It has also been found empirically that the methods and
devices of the present invention can 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.
[0023] In one embodiment employing such hydrophobic regions (or
"hydrophobic patches"), the present invention contemplates a method
for moving microdroplets, comprising: (a) providing microdroplet
transport channel (or a device comprising a microdroplet transport
channel), said channel i) having one or more hydrophobic regions
and ii) in communication with a gas source; (b) introducing liquid
into said channel under conditions such that said liquid stops at
one of said hydrophobic regions so as to define i) a source of
liquid microdroplets disposed within said channel and ii) a
liquid-abutting hydrophobic region; and (c) separating a discrete
amount of liquid from said source of liquid microdroplets using gas
from said gas source under conditions such that a microdroplet of
defined size i) comes in contact with, and ii) moves over, said
liquid-abutting hydrophobic region.
[0024] In one embodiment, said gas from said gas source enters said
channel from a gas-intake pathway in communication with said
microdroplet transport channel and exits said channel from a gas
vent that is also in communication with said microdroplet transport
channel. It is preferred, in this embodiment, that the introduction
of liquid into the channel (as set forth in part b of the
above-described method) is such that i) the liquid passes over the
gas-intake pathway and ii) 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 part c of the
above-described method) forces the microdroplet to i) pass over the
liquid-abutting hydrophobic region and ii) pass by (but not enter)
the gas vent.
[0025] In another embodiment employing such hydrophobic regions (or
"hydrophobic patches"), the present invention contemplates a method
for moving microdroplets, comprising: (a) providing a device
comprising a microdroplet transport channel etched in silicon, said
channel i) having one or more hydrophobic regions and ii) in
communication with a gas source; (b) introducing liquid into said
channel under conditions such that said liquid stops at one of said
hydrophobic regions so as to define i) a source of liquid
microdroplets disposed within said channel and ii) a liquid
abutting hydrophobic region; and (c) separating a discrete amount
of liquid from said source of liquid microdroplets using gas from
said gas source under conditions such that a microdroplet of
defined size i) comes in contact with, and ii) moves over, said
liquid-abutting hydrophobic region.
[0026] 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.
[0027] The present invention further contemplates a method for
merging microdroplets comprising: (a) providing first and second
liquid microdroplets, a liquid microdroplet delivering means, and a
device, said device comprising: i) a housing comprised of silicon,
ii) first and second microdroplet transport channels etched in said
silicon and connecting to form a third transport channel containing
a reaction region, iii) a microdroplet receiving means in liquid
communication with said reaction region via said transport
channels, and iv) microdroplet flow-directing means arrayed along
said first, second and third transport channels; (b) delivering
said first liquid microdroplet via said microdroplet delivering
means to said first transport channel; (c) delivering said second
liquid microdroplet via said microdroplet delivering means to said
second transport channel; and (d) conveying said microdroplets in
said transport channels to said reaction region in said third
transport channel via said microdroplet flow-directing means,
thereby merging said first and second microdroplets to create a
merged microdroplet.
[0028] In one embodiment, said first microdroplet comprises nucleic
acid and said second microdroplet comprises a nuclease capable of
acting on said nucleic acid. In this embodiment, it is desirable to
enhance the mixing within the merged microdroplet. This can be
achieved a number of ways. In one embodiment for mixing, after the
conveying of step (d), 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 said merged
microdroplet is reversed even a single time, this process increases
the mixing of the reactants.
[0029] The present invention contemplates a variety of
silicon-based, microdroplet transport channel-containing devices.
In one embodiment, the device comprises: i) a housing comprised of
silicon, ii) a microdroplet transport channel etched in said
silicon, iii) a microdroplet receiving means in liquid
communication with a reaction region via said transport channels,
and iv) a liquid barrier disposed between said 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. Secondly, 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).
[0030] 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.
[0031] In one embodiment, the present invention contemplates a
method comprising: a) providing a device having a meltable material
disposed within a substrate and associated with a heating element;
and b) heating said meltable material with said heating element
such that said meltable material at least partially liquifies and
such that said substrate is not damaged. The method may further
comprise c) allowing said liquified meltable material to cool.
While the present invention is not limited by the size of the
channel, in one embodiment said substrate further comprises a
microdroplet channel disposed in said substrate, said meltable
material is disposed within said microdroplet channel.
[0032] In another embodiment, the present invention contemplates a
method for restricting fluid flow in a channel comprising a)
providing a device comprising: i) a meltable material disposed
within a substrate, said meltable material associated with a
heating element; and ii) a diaphragm positioned such that, when
extended, it touches said meltable material; b) extending said
diaphragm such that it touches said meltable material; and c)
heating said meltable material with said heating element such that
said meltable material at least partially liquifies and such that
said substrate is not damaged. In one embodiment the method further
comprises d) allowing said 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 said substrate, said meltable material disposed within
said microdroplet channel.
[0033] The present invention also contemplates a method for
restricting fluid flow in a channel, comprising: a) providing: i) a
main channel connected to a side channel and disposed within a
substrate, ii) meltable material disposed within said side channel
and associated with a heating element, and iii) a movement means
connected to said side channel such that application of said
movement means induces said meltable material to flow from said
side channel into said main channel; b) heating said meltable
material such that said meltable material at least partially
liquifies; and c) applying said movement means such that said
liquified meltable material flows from said side channel into said
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 d) allowing said
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.
[0034] 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.
DEFINITIONS
[0035] The following definitions are provided for the terms used
herein:
[0036] "Biological reactions" means reactions involving
biomolecules such as enzymes (e.g., polymerases, nucleases, etc.)
and nucleic acids (both RNA and DNA). Biological samples are those
containing biomolecules, such proteins, lipids, nucleic acids. The
sample may be from a microorganism (e.g., bacterial culture) or
from an animal, including humans (e.g. blood, urine, etc.).
Alternatively, the sample may have been subject to purification
(e.g. extraction) or other treatment. Biological reactions require
some degree of biocompatability with the device. That is to say,
the reactions ideally should not be substantially inhibited by the
characteristics or nature of the device components.
[0037] "Chemical reactions" means reactions involving chemical
reactants, such as inorganic compounds.
[0038] "Channels" are pathways (whether straight, curved, single,
multiple, in a network, etc.) through a medium (e.g., silicon) that
allow for movement of liquids and gasses. Channels thus can connect
other 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.
[0039] "Microdroplet transport channels" are channels configured
(in microns) so as to accommodate "microdroplets." 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 and microdroplets are as follows:
the channels can 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 can
range (calculated from their lengths) between approximately 0.01
and 100 nanoliters (more typically between ten and fifty).
[0040] "Conveying" means "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 can be
accomplished via flow-directing means.
[0041] "Flow-directing means" is any means by which movement of
liquid (e.g. a microdroplet) in a particular direction is achieved.
A variety of flow-directing means are contemplated, including but
not limited to pumps such as a "bubble pump" described below. A
preferred directing means employs a surface-tension-gradient
mechanism in which discrete droplets are differentially heated and
propelled through etched channels. For continuous flow of liquids,
pumps (both external and internal) are contemplated.
[0042] A "bubble pump" is one embodiment of a flow-directing means,
liquid is introduced into a channel, said channel comprising one or
more electrodes positioned such that they will be in contact with a
liquid sample placed in said channel. Two electrodes can be
employed and a potential can 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.
[0043] When coupled with the capillary valve, a bubble pump can
actuate a certain quantity of fluidic samples along the channel.
The capillary valve is essentially a narrow section of a channel.
In operation, the fluidic sample is first injected in the 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 on 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.
[0044] 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. Furthermore, the power
consumption of the device is minimal.
[0045] "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.
[0046] "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 chmically 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, i.e. that it render the surface
hydrophobic.
[0047] In one embodiment, n-octadecyltrichlorosilane (OTS) is used
as a hydrophobic reagent. In another embodiment,
octadecyldimethylchlorosilane is employed. In yet another
embodiment, the present invention contemplates
1H,1H,2H,2H-perfluorodecyltricholorosilane (FDTS,
C.sub.10H.sub.4F.sub.17SiCl.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).
[0048] 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.
[0049] 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.
[0050] A "liquid-abutting hydrophobic region" is 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.
[0051] "Initiating a reaction" means causing a reaction to take
place. Reactions can be initiated by any means (e.g., heat,
wavelengths of light, addition of a catalyst, etc.)
[0052] "Liquid barrier" or "moisture barrier" is 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 of
the present invention, the liquid barrier comprises a first silicon
oxide layer, a silicon nitride layer, and a second silicon oxide
layer.
[0053] "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. Moreover, the degree of mixing in a merged
microdroplet can be enhanced by a variety of techniques
contemplated by the present invention, including by not limited to
reversing the flow direction of the merged microdroplet.
[0054] "Nucleic Acid Amplification" involves increasing the
concentration of nucleic acid, and in particular, the concentration
of a particular piece of nucleic acid. A preferred technique is
known as the "polymerase chain reaction." Mullis et al., U.S. Pat.
Nos. 4,683,195 and 4,683,202, hereby incorporated by reference,
describe a method for increasing the concentration of a segment of
target sequence in a mixture of genomic DNA without cloning or
purification. This process for amplifying the target sequence
consists of introducing a molar excess of two oligonucleotide
primers to the DNA mixture containing the desired target sequence.
The two primers are complementary to their respective strands of
the double-stranded sequence. The mixture is denatured and then
allowed to hybridize. Following hybridization, the primers are
extended with polymerase so as to form complementary strands. The
steps of denaturation, hybridization, and polymerase extension can
be repeated as often as needed to obtain are relatively high
concentration of a segment of the desired target sequence. The
length of the segment of the desired target sequence is determined
by the relative positions of the primers with respect to each
other, and therefore, this length is a controllable parameter. By
virtue of the repeating aspect of the process, the method is
referred to by the inventors as the "Polymerase Chain Reaction"
(hereinafter PCR). Because the desired segment of the target
sequence become the dominant sequences (in terms of concentration)
in the mixture, they are said to be "PCR-amplified."
[0055] "Substrate" as used herein refers to a material capable of
containing channels and microdroplet transport channels. Examples
include, but are not limited to, silicon and glass.
[0056] "Meltable material" as used herein refers to a material that
is at least semi-solid (and preferably completely solid) at ambient
temperature, will liquify when heated to temperatures above ambient
temperature, and will at least partially resolidify when cooled.
Preferably, meltable material at least partially liquifies at a
temperature such that the substrate is undamaged. That is to say,
at the temperature the meltable material liquifies, the substrate
and other metals in the substrate does not liquify (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 liquify. 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.
[0057] "Solder" as used herein refers 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.
[0058] "Heating element" as used herein refers to an element that
is capable of at least partially liquify 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.
[0059] "Diaphragm" as used herein refers 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 liquified meltable material (e.g., force air, magnetic
field, etc.).
[0060] A "source of liquid microdroplets" is a liquid source from
which microdroplets can be made. Such 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.
DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1 is a schematic of an integrated analysis system of
the present invention.
[0062] FIG. 2 shows a two-part approach to construction of a
silicon device of the present invention.
[0063] FIG. 3 shows a schematic of one embodiment of a device to
split a nonoliter-volume liquid sample and move it using gas from a
gas source.
[0064] FIG. 4 shows a schematic of one embodiment of a device of
the present invention to split, move and stop microdroplets using
internal gas pressure generation.
[0065] FIG. 5 is a schematic showing the principle of
thermally-induced liquid microdroplet motion in a closed
channel.
[0066] FIG. 6A shows a selected frame of a videotape wherein two
microdroplets are at their starting locations in the branches of
the Y-channel.
[0067] FIG. 6B shows movement by heating the left interface of both
microdroplets.
[0068] FIG. 6C shows the microdroplets at the intersection.
[0069] FIG. 6D shows the merging of the microdroplets to form the
combined microdroplet. The open arrowheads in the figure indicate
the rear meniscus and the filled arrowheads the leading meniscus
for each microdroplet.
[0070] FIG. 7A is a photomicrograph of inlay-process heater
elements on the surface of a silicon wafer.
[0071] FIG. 7B is a scanning electron micrograph (SEM) of an
inlay-process heater wire in cross section (the arrows indicate the
deposited aluminum, silicon dioxide, and silicon nitride
layers).
[0072] FIG. 7C is a SEM of a channel formed on glass using a
wet-etch process, shown in cross section with the etched face of
the wafer immediately adjacent to the intersection of two
channels.
[0073] FIG. 8A is a schematic of one embodiment of the device of
the present invention wherein the device comprises a glass top
bonded to a silicon substrate.
[0074] FIG. 8B is a schematic showing one embodiment of the
fabrication steps for fabrication of components in silicon.
[0075] FIG. 8C is a schematic showing one embodiment of the
fabrication steps for fabrication of components in glass.
[0076] FIG. 9A is a photomicrograph of polyacrylamide gel
electrophoresis in a wide etched-glass channel.
[0077] FIG. 9B is a photomicrograph of a set of four
doped-diffusion diode radiation detector elements fabricated on a
silicon wafer.
[0078] FIG. 9C is an oscilloscope trace of output from the
radiation detector showing individual decay events from
.sup.32P-labeled DNA.
[0079] FIG. 10 is a photo of gel electrophoresis of PCR reactions
wherein potentially inhibiting components were added directly to
the PCR.
[0080] FIG. 11 is one embodiment of a test device for the present
invention.
[0081] FIG. 12 is a schematic of one embodiment for manufacturing a
sealable valve of the present invention.
[0082] FIG. 13 is a schematic of one embodiment for the layout of
the movable sealing means of the present invention.
[0083] FIG. 14 is a photograph showing water droplets separated by
lines of hydrophobic and hydrophilic regions patterned according to
the methods of the present invention.
[0084] FIG. 15 is photograph showing simply paterning according to
the methods of the present invention to create multiple
droplets.
[0085] FIG. 16 are schematics and photographs of one embodiment of
the device of the present invention utilizing a heater.
DESCRIPTION OF THE INVENTION
[0086] The present invention relates to microfabrication and
biological reactions in microfabricated devices, and in particular,
movement and mixing of biological samples in microdroplets through
microchannels. The description of the invention involves I) the
design of microscale devices (comprising microdroplet transport
channels, reaction chambers, electrophoresis ports, and radiation
detectors) using silicon and glass substrates, II) the creation (or
definition) of microdroplets having a discrete size, III) movement
of discrete microdroplets using a surface-tension-gradient
mechanism in which discrete microdroplets are differentially heated
and propelled through etched channels, IV) flow control with sealed
valves, and V) mixing of biological samples for reactions.
[0087] I. Design of MicroScale Devices
[0088] 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. Silicon is the material used for the construction of
computing microprocessors and its fabrication technologies have
developed at an unprecedented pace over the past 30 years. While
this technology was initially applied to making microelectronic
devices, the same techniques are currently being used for
micromechanical systems.
[0089] Continuous flow liquid transport has been described using a
microfluidic device developed with silicon. See J. Pfahler et al.,
Sensors and Actuators, A21-A23 (1990), pp. 431-434. Pumps have also
been described, using external forces to create flow, based on
micromachining of silicon. See H. T. G. Van Lintel et al., Sensors
and Actuators 15:153-167 (1988). By contrast, the present invention
employs discrete droplet transport in silicon (i.e., in contrast to
continuous flow) using internal forces (i.e., in contrast to the
use of external forces created by pumps).
[0090] 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 can be used almost indefinitely to produce identical replicate
structures. Consequently, even extremely complex micromachines can
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.
[0091] 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 (A) and they are transported as discrete
droplets through channels (B) to a reaction chamber, such as a
thermally controlled reactor where mixing and reactions (e.g.,
restriction enzyme digestion or nucleic acid amplification) occur
(C). The biochemical products are then moved by the same method to
an electrophoresis module (D) where migration data is collected by
a detector (E) and transmitted to a recording instrument (not
shown). Importantly, the fluidic and electronic components are
designed to be fully compatible in function and construction with
the biological reactions and reagents.
[0092] In silicon micromachining, a simple 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.
[0093] FIG. 2 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).
[0094] II. The Creation of MicroDroplets
[0095] 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 i) 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 ii) subsequent removal of the metal
to give hydrophobic patterns. Other substrates such as plastics can
also be used after depositing a think film of silicon oxide or
spin-on-glass.
[0096] 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
simple microfabrication procedures.
[0097] 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.
[0098] FIG. 3 shows a schematic of one embodiment of a device (10)
to split a nonoliter-volume liquid sample and move it using
external air, said device having a plurality of hydrophobic
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 front 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 the pressure of the gas
(e.g. air) from acting towards the inlet side, the inlet (20) and
overflow ports (30) can be blocked or may be loaded with excess
water to increase the resistance to flow.
[0099] The patterened surfaces can 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.
[0100] One can start moving the drop (60) again by blocking the
vent (70). By using a combination of hydrophobic air pressure lines
(not shown), 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.
[0101] In addition to using external air, one can also use
internally generated air pressure to split and move drops. FIG. 4
shows 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. 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 can
be generated. The magnitude of the pressure increase inside a
chamber of volume V is related to the increase in temperature and
can be estimated by the Ideal Gas relation:
[0102] 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 consuption.
[0103] 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.1 to 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 seconds 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 12.5 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.
[0104] III. Movement of Discrete MicroDroplets
[0105] 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 can 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 can be determined by merely measuring the
droplet length. Third, the motion of these droplets can be
accomplished with simple 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 (FIG.
5). For example, a droplet in a hydrophilic channel can 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
(P.sub.1 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.
[0106] That is to say, forward motion can be maintained by
continuing to heat the droplet at the rear surface with successive
heaters along the channel, while heating the front surface can 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 can be
calculated knowing the atmospheric pressure, P.sub.atm' the 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.
[0107] 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 can be approximated using the equation .nu.=Pd.sup.2/32
.mu.L where P 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
thirty (30) degrees Centigrade to create movement. Experiments
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 experiments, the
channel cross-section was 20 .mu.m.times.500 .mu.m, and the volume
of each of these droplets can be calculated from their lengths and
is approximately 100 nanoliters for a 1 cm long droplet.
[0108] IV. Flow Control with Sealed Valves
[0109] 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 liquified 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 can be
released and the valve is sealed. To open the valve again, the
solder can be liquified without applying an actuation force.
[0110] 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 can be
electroplated.
[0111] V. Mixing Biological Samples in Reactions
[0112] 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 contemplated (FIG. 6A). 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 (FIGS. 6B and 6C).
[0113] Following sample merging (FIG. 6D), 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 can
be accomplished in a number of ways.
[0114] First, there is simple 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 minutes) using
the integral heaters and temperature sensors.
[0115] 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.
[0116] Finally, there is the mixing approach wherein the mixture is
moved against or over physical obstacles. For example, the mixture
can 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).
[0117] Successful mixing, of course, can 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.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0118] The description of the preferred embodiments involves: I)
microfabrication techniques for manufacture of silicon-based
devices; II) channel treatment for optimum flow and
reproducibility; III) channel treatment for fabricating hydrophobic
regions, and IV) component design (particularly the electrophoresis
module and the radiation detectors).
[0119] 1. Microfabrication of Silicon-Based Devices
[0120] As noted previously, silicon has well-known fabrication
characteristics and associated photographic reproduction
techniques. The principal modern 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., W.
Miller, U.S. Pat. No. 5,091,328, hereby incorporated by
reference.
[0121] 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.
[0122] 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.
[0123] 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 seconds; 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.
[0124] 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
hours.
[0125] Initial device design by the present inventors 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 experiments 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) (FIG. 7A). 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.sub.x), 0.25 .mu.m silicon nitride
(Si.sub.xN.sub.y), and 1.0 .mu.m silicon oxide (SiO.sub.x) (FIG.
7B). Some heating elements were also used as resistive temperature
sensors.
[0126] Heater elements were fabricated as follows. Silicon wafer
(p-type, 18-221/2-cm, 100, 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 seconds. 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 minute); CF.sub.4, 15 sccm; 4 mTorr; DC bias
voltage of 200V, 100 W, 20 minutes. 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 1112A remover in solution (Shipley
Co.). The barrier layers 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 (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 sccm; 4 mTorr; and DC bias
voltage of 200V, 100 W, 120 minutes).
[0127] As shown in FIG. 7, 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 in FIG. 7C has identical width and design configurations as
the heating element lanes in FIG. 7A, and is uniformly etched 500
.mu.m wide and approximately 20 .mu.m deep.
[0128] 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.
[0129] 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.
[0130] II. Channel Treatment
[0131] 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. 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 experiment, usually of
less than 30%. Droplet movement was recorded with a Hamamatsu video
camera on videotape.
[0132] III. Channel Treatment for Hydrophobic Regions
[0133] In one embodiment of the device of the present invention
(FIG. 8A), the device comprises a glass top (810A) bonded to a
silicon substrate (810B) containing the heater (891), the contact
pad (892) and the resisitive temperature detector (893). The glass
side has channels and chambers etched into it. FIG. 8A shows the
inlet (820) and overflow (830) ports, a gas vent (870) and a air
chamber (880).
Heaters and Resisitive Temperature Detectors
[0134] The fabrication process for the heater and temperature
detector (FIG. 8B) 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 SlO.sub.2 thermal oxide (1 .mu.m) (STEP 1).
A 0.5 .mu.m metallic Aluminum film is electron beam deposited.
Photoresis PR 1827 is applied and spun at 4000 rpm for 30 seconds,
patterned (metal 1) and developed. The exposed aluminum is etched
in Aluminum etchant and the photoresist stripped to define the
metal heater (STEP 2).
[0135] Photoresist is spun again and a second lithography is done
(metal 2). A 0.15 .mu.m layer of platimun ("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 dydrophilic substrate (STEP 4). 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.
Hydrophobic Patterning of Silicon Oxide Substrate
[0136] 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 seconds. 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 (STEP 5). The samples are then cleaned in
Acetone, Isopropyl Alcohol and DI water for 10 minutes each, air
dried and oven dried at 100.degree. C. for 5 minutes. The samples
are then put in 1 wt % octadecyltrichlorosilane (OTS) solution in
Toluene for 15-30 minutes. OTS deposits on the samples as a Self
Assembled Monolayer (SAM) (STEP 6). The samples are then rinsed in
Toluene, Isopropyl alcohol and DI water for 5 minutes each, and
then oven dried (100.degree. C., 5 minutes). 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 (STEP
7). Heater elements and RTDs have also been fabricated on a quartz
substrate. The fabrication steps are similar to that of the silicon
processing steps.
Glass Channel and Chamber Fabrication
[0137] The channel and the chamber fabrication (FIG. 8C) 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
Corning 7740) (STEP 1). 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 (STEP 2). 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 (STEP 3) and the wafer rinsed in DI water, air
dried and oven dried at 100.degree. C. for 20 minutes. The
following processing steps are done for patterning hydrophobic
regions onto the glass surface.
Hydrophobic Patterning of Glass Substrate
[0138] A 1.5 .mu.m thick Aluminum layer was electron beam
deposited, covering the etched channels and chamber (STEP 4). A
thick photoresist (AZ 4620) is applied and spun at 750 rpm for 50
seconds (STEP 5). The resist is patterned (SAM Mask) and developed.
The exposed Aluminum is etched in aluminum etchant. The photoresist
is stripped off (STEP 6) in hot PRS 2000 (J. T. Baker). The samples
are then cleaned in Acetone, Isopropyl alcohol and DI water for 5
minutes each and the water dried off in a 100.degree. C. oven of
10-15 minutes. The samples are then dipped in a 1% OTS solution in
Toluene for 10-15 minutes (STEP 7). The SAM deposition was carried
out in a Chemical hood. The samples were then rinsed in Toluene,
Isopropyl Alcohol and DI water for 5 minutes each. Next, they were
put in Aluminum etchant until all metallic aluminum was removed
(STEP 8). 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.
[0139] 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 hours.
[0140] IV. Component Design
[0141] The present invention contemplates one or more gel
electrophoresis modules as a component of the microscale device.
Theoretical and empirical research has indicated that reducing the
thickness of the electrophoresis channel leads to improved
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 can 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.
[0142] To demonstrate that standard gel electrophoresis can operate
in micron-diameter channels, modules were fabricated using etched
glass channels identical to FIG. 6B and fluorescent-labeled DNA
(YOYO intercalating dye). Polyacrylamide gel electrophoresis of a
complex DNA mixture is shown in FIG. 9A 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 white vertical line is the gel-to-buffer interface. The
DNA sample (BluescriptKS 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.
[0143] 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 (FIGS. 6C and 6D). 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.
[0144] FIG. 9B is photomicrograph of a set of four doped-diffusion
diode radiation detector elements fabricated on a silicon wafer.
For each element, the three parallel dark lines define the
diffusion regions of the central the detector flanked by the guard
ring shielding electrodes. The diffusion regions are approximately
300 .mu.m long and 4 .mu.m wide.
[0145] 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 (i) high sensitivity (a single decay event),
(ii) small aperture dimensions, and (iii) 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.
[0146] Radiation detectors were prepared as follows. A 2001/2-cm,
100, float zone, boron-doped, p-type silicon wafer was used as a
substrate. Diffused layers of phosphorus (5.times.10.sup.14
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 minutes to
form an aqueous solution barrier. Radioactive phosphorus (.sup.32P)
decay events could be detected using a sample of labeled DNA in PCR
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.
[0147] FIG. 9C shows an oscilloscope trace of output from the
radiation detector showing individual decay events from
.sup.32P-labeled DNA. The aqueous DNA sample was placed directly on
the detector and sampled for 30 seconds. The screen is displaying a
vertical scale of 0.5V/division and horizontal scale of 20
.mu.sec/division.
EXPERIMENTAL
[0148] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
[0149] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); .mu.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 minute).
Example 1
[0150] 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.
[0151] 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 was 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.
[0152] As a follow-up approach, a thinner layer (500 angstroms) of
aluminum was tried. This gave 1/10th 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.
[0153] 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 2
[0154] 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.
[0155] 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 hrs. 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.
[0156] 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
experiments 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.
[0157] To obtain hydrophobic surfaces, the present inventors tried
coating capillaries 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 .about.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 3
[0158] 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.
[0159] Average Flow Through a Circular Channel:
<v>=-.DELTA.P*[R.sup.2/(8 .mu.L] where:
[0160] .DELTA.=value at back-value at front end of droplet
[0161] .DELTA.P=(1/R)*(.DELTA.G)=pressure difference between
droplet ends
[0162] .DELTA.G=change in surface tension between the ends of the
droplet.
[0163] R=channel radius
[0164] L=droplet length .mu.=viscosity
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).
[0165] Therefore: <v>=-(.DELTA.G)*(1/R)*[R.sup.2/(8
.mu.L)]=[-0.16*.DELTA.T*R/(8 .mu.L)] where:
.DELTA.T=T.sub.back-T.sub.front giving: <v>=[0.16*R/(8
.mu.L)]*(T.sub.back-T.sub.front). 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 (1D) 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.
[0166] 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:
G.sub.front/G.sub.back<(R.sub.front/R.sub.back)*(cos
.beta..sub.back/cos .beta..sub.front) where: .beta.=contact
angle.
[0167] 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 us 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 experiments 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.
[0168] Discrete droplet motion in a micromachined channel structure
using thermal gradients is demonstrated in the videorecorded images
of FIG. 6. The device consists of a series of aluminum heaters
inlaid on a planar silicon dioxide substrate (similar to the
structure shown in FIG. 2) 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 can be calculated from their
lengths and is approximately 50 nanoliters.
[0169] The heaters along the entire surface of the channel shown in
FIG. 6 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 minutes 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 (not shown).
Example 4
[0170] 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.
[0171] 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.
[0172] Several different epoxies and glues were investigated, as
listed below. TABLE-US-00001 Adhesive Form Dries Texture Comments
1. Dymax UV Glue Gel Clear Rubbery Cures on UV exposure. 2.
Carter's Rubber Goo Yellow/ Rubbery Dries quickly Cement Clear and
stringy when thinned. 3. Borden's Krazy Liquid Clear Hard Thin,
dries on Glue first contact. 4. UHU Bond-All Gel/ Clear Hard Dries
quickly Goo and stringy when thin. 5. Dennison Paste Clear Hard
Will not flow Permanent Glue on applying. Stick 6. Elmer's Glue-All
Thick White Hard Slow drying. (Borden) Liquid 7. Liquid Nails Thin
Wood- Hard Thick, dries Paste like quickly when thinned. 8. Devcon
5-Minute Gel Yellow/ Hard Thick, cures Epoxy Clear on about 5 min.
9. Scotch Double- Tape Clear Rubbery Tape. Stick Tape 10. Dow
Corning Thick Frosty Soft Seals but does High Vacuum Gel not bond.
Grease 11. Nujol Mineral Liquid Clear Runny Neither seals Oil
(Perkin (doesn't spread Elmer) on glass) nor bonds. 12. Household
Goop Gel/ Clear Rubbery Contact cement Goo which dries stringy. 13.
Permatex Gel/ Yellow/ Rubbery Dries quickly Weather Strip Goo Clear
and stringy when Cement thinned. 14. Thick Gel Super Gel Clear Hard
Does not cure on Glue contact but does quickly. 15. DAP Weldwood
Goo Orange/ Rubbery Contact cement Contact Cement Clear which gets
stringy when thinned. 16. Scotch (3M) Thin Yellow/ Rubbery Spray.
`Gooey` Photo Mount Goo Clear but not stringy. Spray Adhesive 17.
Silicone Resin Liquid Clear Smooth Spray. Dries to (spray) Lacquer
thin, clear, and (GC Electronics) sealed coating.
[0173] 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.
[0174] 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 5
[0175] This example describes a nucleic acid amplification reaction
on a silicon-based substrate. The established DNA biochemistry
steps for PCR 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.
[0176] To assess biocompatability, components were added to a
standard PCR reaction. The results (see FIG. 10) indicated that
crystalline silicon 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.
[0177] To form a biologically compatible heating element, the
present inventors began by coating a standard silicon wafer 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 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.
[0178] A device fabricated with metal resistive heaters and
oxide/nitride/oxide coating was tested for biological compatibility
and temperature control by using PCR amplification of a known DNA
template sample. The reaction was carried out on the planar device
using twenty microliters of PCR reaction mix covered with mineral
oil to prevent evaporation. The reaction mixture was cycled through
a standard 35-cycle PCR 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 experiments
performed on a commercial PCR thermocycler gave similar results. A
series of PCR 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 (not shown).
[0179] From the above it should be evident that the present
invention can 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 6
[0180] In this example, a test structure is fabricated (FIG. 11).
The test structure is very simple (FIG. 11). 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.sub.2 serves as an insulator between the
Si substrate and the other metal layers, which function as solder
pads and heating elements. The Ti layer (250 A) 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.
[0181] A thermo-pneumatic microvalve is utilized in the test
structure. The schematic and process flow of the microvalve is
shown in FIG. 12. A corrugated diaphragm is chosen for its larger
deflection and higher sensitivity. The diaphragm (side length=1000
um, thickness=3 um, boss size length=500 um boss thickness=10 um)
has a deflection of 27 um 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. As set
forth in FIG. 12, ten masks are anticipated to fabricate the
microvalve.
[0182] FIG. 9a shows a portion of a silicon substrate 10, which 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 12
of the silicon wafer containing substrate 10 is lapped, polished
and cleaned in the normal and accepted manner. Isotropic etching
using reactive ion etching (RIE) forms the diaphragm corrugations
14 with photoresist as the masking material.
[0183] FIG. 12B shows the definition of deep boron diffusion areas
16 to form the rims, center bosses, inlet and outlet holes of the
finished device. FIG. 12C shows the deposition of shallow boron
diffusion areas 18 to form a diaphragm. The various metal layers,
including solder 20, 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.
[0184] Following this, FIG. 12D shows the definition of oxide layer
22 to serve as insulator of the solder of the finished device. Ti
adhesion/Ni/Au barrier and wettable pads 24 are then deposited as
shown in FIG. 12E. The solder mold 26 of Ni and photoresist is then
defined as shown in FIG. 12F) and the first Ni channel 28 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 30 (FIG. 12G).
[0185] A second Ni channel 32 is defined by Ni and photoresist as
set forth in FIG. 12H, and inlet 34 and outlet 36 holes are defined
using EDP to remove the sacrificial layers (FIG. 121).
[0186] Lastly, a Ti/Pt heater in glass 38 is anodically bonded to
the silicon substrate (FIG. 12J). Freon-11 fills the cavity through
a hole (not shown) in the glass substrate. This hole is created
from a diamond drill bit and sealed with epoxy.
Example 7
[0187] 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 8
[0188] In light of the results of the experiment 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 9
[0189] In this example, a device and method for blocking fluid flow
in a channel is described. FIG. 13 sets forth a test schematic for
these embodiments. 60:40 Sn:Pb solder 40, associated with a heating
element 42, is placed within a side channel 44. The heating element
42 at least partially liquifies the solder 40 and air flow 46 moves
the liquified solder from the side channel into a main channel 48
and cooled, blocking the main channel.
Example 10
[0190] 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.
[0191] Line widths as narrow as 10 .mu.m were patterned on silicon
substrates using the methods of the present invention. FIG. 14
shows water drops separated by lines of hydrophobic (A) and
hydrophilic regions (B) 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..
[0192] One can also define hydrophobic regions in etched channels
in glass by performing the lithography using a thick resist. It was
found emprically 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 11
[0193] 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. FIG. 15
shows a simple use of this patterning technique to split a liquid
droplet into multiple liquid droplets. A concentric pattern of
alternating hydrophobic (dark) and hydrophilic (white) sectors was
imparted to a silicon substrate (FIG. 15A; the diameter of the
circle is 1 cm) using the methods of the present invention as
described above. A water drop was placed on the pattern (FIG. 15B)
and the excess water pulled away using a pipet, resulting in
multiple drops separated from each other (FIG. 15C).
Example 12
[0194] In this example, experiments are describe 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.l) 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.
[0195] 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 13
[0196] FIGS. 16A-E are schematics and photographs of one embodiment
of the device (910) of the present invention (in operation)
utilizing a heater. FIG. 16A shows that liquid placed at the inlet
(920) stops at the hydrophobic interfaces, and more specifically,
stops at the liquid-abutting hydrophobic region (940). The inlet
(920) and overflow (930) 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 (991)
was actuated by an applied voltage. The flow of current caused
resistive heating and subsequently increases the air temperature in
the chamber (980) and, therefore, the pressure. After the pressure
builds up to a particular value, a microdrop splits and moves
beyond the hydrophobic patch (FIG. 16B). 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.
[0197] To stop or block the moving drop at a location, two
strategies can 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 (FIG. 16C). 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 (FIG. 16D),
the drop stops moving farther (FIG. 16E). 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.
[0198] 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).
* * * * *