U.S. patent application number 10/394484 was filed with the patent office on 2003-10-09 for method and apparatus for performing biological reactions on a substrate surface.
This patent application is currently assigned to Motorola. Invention is credited to Hawkins, George W., Johnson, W. Travis, McGarry, Mark W..
Application Number | 20030190744 10/394484 |
Document ID | / |
Family ID | 23954585 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030190744 |
Kind Code |
A1 |
McGarry, Mark W. ; et
al. |
October 9, 2003 |
Method and apparatus for performing biological reactions on a
substrate surface
Abstract
The present invention provides a method and an improved
apparatus for removing gas bubbles from a reaction chamber
comprising a flexible layer removably affixed to a substrate layer
having a multiplicity of oligonucleotide binding sites disposed
thereon, in which biological reactions are performed. The invention
specifically relates to methods and apparatus for removing gas
bubbles from a reaction chamber wherein target molecules contained
in a sample fluid are reacted with probe molecules immobilized on a
substrate having an array of oligonucleotide binding sites. The
arrays are covered with a flexible, gas permeable layer that
permits mixing of the sample fluid on the biochip and removal of
gas bubbles from the fluid by use of a means for facilitating
diffusion of gas bubbles across the flexible, gas permeable
layer.
Inventors: |
McGarry, Mark W.;
(Scottsdale, AZ) ; Johnson, W. Travis; (Chandler,
AZ) ; Hawkins, George W.; (Gilbert, AZ) |
Correspondence
Address: |
Robin M. Silva
DORSEY & WHITNEY LLP
Suite 3400
Four Embarcadero Center
San Francisco
CA
94111-4187
US
|
Assignee: |
Motorola
Amersham
|
Family ID: |
23954585 |
Appl. No.: |
10/394484 |
Filed: |
March 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10394484 |
Mar 20, 2003 |
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09492013 |
Jan 26, 2000 |
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6569674 |
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09492013 |
Jan 26, 2000 |
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09464490 |
Dec 15, 1999 |
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6589778 |
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Current U.S.
Class: |
435/287.1 |
Current CPC
Class: |
B01L 3/502707 20130101;
B01L 2400/0481 20130101; B01L 3/50273 20130101; B01L 2200/0684
20130101; B01L 2300/0887 20130101; B01L 2300/1822 20130101; G01N
2021/054 20130101; B01J 2219/00659 20130101; B01L 2400/049
20130101; G01N 2035/00158 20130101; B01L 2300/0654 20130101; C07B
2200/11 20130101; B01L 2300/123 20130101; B01L 2200/027 20130101;
C40B 40/06 20130101; B01L 2300/0877 20130101; B01L 2200/10
20130101; B01L 3/502723 20130101; B01J 2219/00702 20130101; B01L
2200/0689 20130101; G01N 2035/00267 20130101; B01L 3/50853
20130101; B01L 3/5027 20130101; B01L 2300/1827 20130101; B01L
2400/0677 20130101; B01L 2300/0636 20130101; B01L 3/502715
20130101; B01L 2300/0822 20130101; B01L 9/527 20130101; B01L
2300/1861 20130101; B01J 2219/00722 20130101; B01L 2300/1866
20130101; B01J 2219/00596 20130101; B01L 2400/0487 20130101 |
Class at
Publication: |
435/287.1 |
International
Class: |
C12M 001/34 |
Claims
What we claim is:
1. An apparatus for performing biological reactions, comprising:
(a) a substrate having a first surface and a second surface
opposite thereto, (b) an array of biomolecules positioned on the
first surface of the substrate, (c) a flexible, gas permeable layer
affixed to the first surface of the substrate by an adhesive,
wherein the adhesive is deposited on the first surface of the
substrate and encloses an area thereupon, and wherein the flexible,
gas permeable layer, the adhesive, and the first substrate surface
enclose a reaction chamber; (d) a port extending through the
substrate from the first surface to the second surface thereof and
having a first opening and a second opening, wherein the first
opening of the port is provided within the area bounded by the
adhesive and covered by the flexible, gas permeable layer and the
second opening of the port is provided on the second surface of the
substrate; (e) a removable cover positioned over the second opening
of the port; (f) a layer of a water-soluble compound that is a
solid at a first temperature and a liquid at a second, higher
temperature, the layer being positioned in the reaction chamber
between the first surface of the substrate and the flexible, gas
permeable layer; and (g) a means for facilitating diffusion of gas
bubbles across the flexible, gas permeable layer, wherein the
diffusion-facilitating means creates a pressure gradient or
concentration gradient across the flexible, gas permeable layer
thereby increasing the rate of diffusion of gas bubbles from the
reaction chamber across the flexible, gas permeable layer.
2. The apparatus of claim 1, wherein the flexible, gas permeable
layer is a porous membrane.
3. The apparatus of claim 2, wherein the porous membrane is porous
Teflon.
4. The apparatus of claim 2, wherein, when the reaction chamber
contains a fluid sample, the porous membrane prevents the sample
fluid from passing through the membrane.
5. The apparatus of claim 1, wherein the means for facilitating
diffusion of gas bubbles across the flexible, gas permeable layer
comprises a vacuum source removably affixed to the flexible, gas
permeable layer, and wherein the vacuum source is used to create a
pressure gradient across the flexible, gas permeable layer to
facilitate diffusion of gas bubbles from the reaction chamber
across the flexible, gas permeable layer.
6. The apparatus of claim 5, wherein the vacuum source comprises:
(a) a vacuum pump, and (b) a chamber seal removably affixed to the
flexible, gas permeable layer and removably connected to the vaccum
pump, wherein the seal completely surrounds the area on the first
substrate surface containing the array of biomolecules.
7. A method for removing gas bubbles from an apparatus for
performing biological reactions having one or more reaction
chambers bounded by a substrate layer, an adhesive and a flexible,
gas permeable layer, comprising the steps of: (a) attaching to the
flexible, gas permeable layer a means for facilitating diffusion of
gas molecules across the flexible, gas permeable layer, and (b)
operating the diffusion-facilitating means until the gas bubbles
are removed from the reaction chamber.
8. A method for removing gas bubbles from an apparatus for
performing biological reactions having one or more reaction
chambers bounded by a substrate layer, an adhesive and a flexible,
gas permeable layer, comprising the steps of: (a) isolating a
reaction chamber of the apparatus by applying a reducer to the
flexible, gas permeable layer surrounding the reaction chamber,
thereby forming a seal between the reducer and the flexible, gas
permeable layer, (b) attaching a vacuum source to the reducer, and
(c) applying a vacuum to the reaction chamber until the gas bubbles
are removed from the reaction chamber.
9. In an apparatus for performing biological reactions, comprising
a substrate having a first surface and a second surface opposite
thereto, an array of biomolecules positioned on the first surface
of the substrate, a flexible layer affixed to the first surface of
the substrate by an adhesive, wherein the adhesive is deposited on
the first surface of the substrate and encloses an area thereupon;
a reaction chamber enclosed by the flexible layer, the adhesive and
the first substrate surface; a port extending through the substrate
from the first surface to the second surface thereof and having a
first opening and a second opening, wherein the first opening of
the port is provided within the area bounded by the adhesive and
covered by the flexible layer and the second opening of the port is
provided on the second surface of the substrate; a removable cover
positioned over the second opening of the port; and a layer of a
water-soluble compound that is a solid at a first temperature and a
liquid at a second, higher temperature, the layer being positioned
between the first surface of the substrate and the flexible layer,
the improvement comprising (a) a flexible layer that is a gas
permeable layer and (b) a means for facilitating diffusion of gas
bubbles across the flexible, gas permeable layer, wherein the
diffusion-facilitating means creates a pressure gradient or
concentration gradient across the flexible, gas permeable layer
such that gas bubbles diffuse from the reaction chamber to the
environment outside the flexible, gas permeable layer.
10. The improvement of claim 9, wherein the diffusion-facilitating
means comprises a vacuum source removably affixed to the flexible,
gas permeable layer and wherein the vacuum source is used to apply
a vacuum to the flexible, gas permeable layer to remove gas bubbles
from the reaction chamber.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Related Applications
[0002] This application is a continuation-in-part of U.S.
application Ser. No. 09/464,490, filed Dec. 15, 1999, which is
incorporated herein by reference.
[0003] 2. Field of the Invention
[0004] The present invention relates to an apparatus for performing
biological reactions on a substrate surface and a method for
removing gas bubbles from the apparatus. Specifically, the
invention relates to an apparatus having a flexible, gas permeable
layer affixed to a substrate layer with an adhesive, wherein the
flexible, gas permeable layer, the adhesive and the substrate layer
enclose a reaction chamber, and a means for facilitating diffusion
across the flexible, gas permeable layer. The
diffusion-facilitating means creates a pressure gradient or
concentration gradient across the flexible, gas permeable layer,
thereby increasing the rate of diffusion of gas molecules from the
reaction chamber across the flexible, gas permeable layer.
[0005] 3. Description of the Prior Art
[0006] Recent advances in molecular biology have provided the
opportunity to identify pathogens, diagnose disease states, and
perform forensic determinations using gene sequences specific for
the desired purpose. This explosion of genetic information has
created a need for high-capacity assays and equipment for
performing molecular biological assays. Most urgently, there is a
need to miniaturize, automate, standardize and simplify such
assays. While these assays were originally developed in research
laboratories working with purified products and performed by highly
skilled individuals, adapting these procedures to clinical uses,
such as diagnostics, forensics and other applications, has produced
the need for equipment and methods that allow less-skilled
operators to effectively perform the assays under higher capacity,
less stringent assay conditions.
[0007] Existing technology utilizes the binding of molecules
contained within a biologically reactive sample fluid, hereinafter
referred to as target molecules, onto molecules contained within
biologically reactive sites, hereinafter referred to as probe
molecules. The primary enabler of this technology is an apparatus
commonly referred to as a biochip, which comprises one or more
ordered microscopic arrays ("microarrays") of biologically reactive
sites immobilized on the surface of a substrate. A biologically
reactive site can be created by dispensing a small volume of a
fluid containing a biological reagent onto a discrete location on
the surface of a substrate, also commonly referred to as spotting.
To enhance immobilization of probe molecules, biochips can include
a 2-dimensional array of 3-dimensional polymeric anchoring
structures (for example, polyacrylamide gel pads) attached to the
surface of the substrate. Probe molecules such as oligonucleotides
are covalently attached to polyacrylamide-anchoring structures by
forming amide, ester or disulfide bonds between the biomolecule and
a derivatized polymer comprising the cognate chemical group.
Covalent attachment of probe molecules to such polymeric anchoring
structures is usually performed after polymerization and chemical
cross-linking of the polymer to the substrate is completed.
[0008] Biochips are advantageously used to perform biological
reactions on the surface thereof. Existing apparatus for performing
biological reactions on a substrate surface, however, are deficient
in that they either require unacceptably large volumes of sample
fluid to operate properly, cannot accommodate substrates as large
as or larger than a conventional microscope slide, cannot
independently accommodate a plurality of independent reactions, or
cannot accommodate a substrate containing hydrogel-based
microarrays. Most existing apparatus also do not allow introduction
of fluids in addition to the sample fluid (such as wash buffers,
fluorescent dyes, etc.) into the reaction chamber. Disposable
apparatus must be disassembled and reassembled around the biochip
every time a new fluid must be introduced. Other existing apparatus
are difficult to use in a laboratory environment because they
cannot be loaded with standard pipet tips and associated pipettor
apparatus.
[0009] Many existing apparatus also exhibit unacceptable reaction
reproducibility, efficiency, and duration. Reaction reproducibility
may be adversely affected by bubble formation in the reaction
chamber or by the use of biologically incompatible materials for
the reaction chamber. Reaction duration and efficiency may be
adversely affected by the presence of concentration gradients in
the reaction chamber.
[0010] Bubbles can form upon introduction of sample fluid to the
reaction chamber or by outgassing of the reaction chamber
materials. When gas bubbles extend over the substrate surface in an
area containing biologically reactive sites, the intended reaction
may intermittently fail or yield erroneous results because the
intended concentration of the sample fluid mixture has been
compromised by the presence of gas bubbles.
[0011] Biologically incompatible reaction chamber materials may
cause unacceptable reaction reproducibility, by interacting with
the sample fluid, thus causing the intended reaction to
intermittently fail or yield erroneous results.
[0012] Incomplete mixing of the sample fluid can introduce
concentration gradients within the sample fluid that adversely
impact reaction efficiency and duration. This effect is most
pronounced when there is a depletion of target molecules in the
local volume surrounding a biologically reactive site. During a
biological reaction, the probability that a particular target
molecule will bind to a complementary (immobilized) probe molecule
is determined by the given concentration of target molecules
present within the sample fluid volume, the diffusion rate of the
target molecule through the reaction chamber, and the statistics of
interaction between the target molecule and the complementary probe
molecule. For diagnostic assays, target DNA molecules are often
obtained in minute (<picomol) quantities. In practice, it can
take tens of hours for a hybridization reaction to be substantially
complete at the low target nucleic acid molecule levels available
for biological samples. Concentration gradients in the
hybridization chamber can further exacerbate this problem.
[0013] U.S. Pat. No. 5,948,673 to Cottingham discloses a
self-contained multi-chamber reactor for performing both DNA
amplification and DNA probe assays in a sealed unit wherein some
reactants are provided by coating the walls of the chambers and
other reactants are introduced into the chambers prior to starting
the reaction in order to eliminate flow into and out of the
chamber. No provisions are made for eliminating gas bubbles from
the chambers.
[0014] There remains a need in the art for methods and apparatus
for performing biological reactions on a substrate surface that use
a low volume of sample fluid, that accommodate substrates as large
as or larger than a conventional microscope slide, that accommodate
a plurality of independent reactions, and that accommodate a
substrate surface having one or more hydrogel-based microarrays
attached thereto. There also remains a need in the art for an
apparatus that allows introduction of fluids in addition to sample
fluid into each reaction chamber via standard pipet tips and
associated pipettor apparatus. There also remains a need in the art
for such an apparatus that increases reaction reproducibility,
increases reaction efficiency, and reduces reaction duration. There
also remains a need in this art for a simple method for removing
gas bubbles from such an apparatus. These needs are particularly
striking in view of the tremendous interest in biochip technology,
the investment and substantial financial rewards generated by
research into biochip technology, and the variety of products
generated by such research.
SUMMARY OF THE INVENTION
[0015] The invention provides an apparatus for performing
biological reactions on a substrate surface and a method for
removing gas bubbles from the apparatus to prevent interference
with biological reactions such as hybridization at reaction sites
on the substrate surface. Specifically, the method of the invention
is directed to an apparatus comprising a flexible, gas permeable
layer affixed to a biochip with an adhesive, wherein the flexible,
gas permeable layer, the adhesive, and the biochip enclose a
reaction chamber, and a means for facilitating diffusion of gas
molecules out of the reaction chamber across the flexible, gas
permeable layer. The diffusion-facilitating means creates a
pressure gradient or concentration gradient across the flexible,
gas permeable layer, thereby increasing the rate of diffusion of
gas molecules from the reaction chamber through the flexible, gas
permeable layer.
[0016] The biochip comprises a substrate having a first surface and
a second surface, wherein the first surface contains an array of
biologically reactive sites, and is preferably an oligonucleotide
array. The array is provided in an area bounded by an adhesive set
down on the first substrate surface. The flexible, gas permeable
layer, the adhesive and the first substrate surface further define
a volume comprising a reaction chamber.
[0017] The flexible, gas permeable layer preferably is deformable,
translucent, and porous. More preferably, the flexible, gas
permeable layer is selectively permeable to gas but impermeable to
liquid. Most preferably, the flexible, gas permeable layer is
selectively permeable to gases and impermeable to liquids because
the surface tension of the sample fluid prevents escape of the
liquid through the pores of the flexible membrane.
[0018] In certain embodiments of the invention, the substrate
comprises a multiplicity of oligonucleotide arrays, which are
contained in one or a plurality of areas bounded by the adhesive
and covered by the flexible, gas permeable layer.
[0019] Each of the reaction chambers also preferably include a
first port, and certain embodiments further include a second port,
that transverses the substrate and comprises a first opening on the
first substrate surface and a second opening on the second
substrate surface. The openings of these ports on the second
substrate surface are covered by a removable cover, most preferably
a foil tape. The openings of these ports on the first substrate
surface are provided within the area bounded by the adhesive.
[0020] The adhesive, the flexible, gas permeable layer and the
substrate also enclose a reaction chamber that is filled prior to
use with a water-soluble compound. The water-soluble compound is
preferably a solid at a temperature most preferably at or below
room temperature, and a liquid at higher temperatures, most
preferably below about 100.degree. C.
[0021] In preferred embodiments, the diffusion-facilitating means
creates a pressure differential across the flexible, gas permeable
layer. In more preferred embodiments, the diffusion-facilitating
means comprises a vacuum source removably affixed to the flexible,
gas permeable layer, wherein the vacuum source is used to apply a
vacuum to the flexible, gas permeable layer. Most preferably, the
vacuum source comprises a vacuum pump connected by a length of
plastic tubing to a reducer that completely encloses the area
defined by the adhesive and is removably sealed to the flexible,
gas permeable layer.
[0022] The chamber is also optionally supplied with a roller, most
preferably a patterned roller, positioned in contact with the
flexible, gas permeable layer and movable longitudinally across the
surface of the chamber for mixing sample fluid and wash solutions
as required.
[0023] Specific preferred embodiments of the present invention will
become evident from the following more detailed description of
certain preferred embodiments and the claims.
DESCRIPTION OF THE DRAWINGS
[0024] Presently preferred embodiments of the invention are
described with reference to the following drawings.
[0025] FIGS. 1A-1D are views of an illustrative embodiment of the
present invention illustrating the preparation of a chamber of the
invention for reaction. FIG. 1A is a cross-sectional view of the
apparatus illustrating a reaction chamber prefilled with a
water-soluble compound. FIG. 1B is a cross-sectional view of the
apparatus illustrating the mixing of the water-soluble compound and
the biological sample fluid. FIG. 1C is a cross-sectional view of
the apparatus illustrating a chamber filled with the sample
fluid/water-soluble compound mixture, wherein the first and second
ports are covered with a seal. FIG. 1D is a top plan view of the
apparatus illustrating the pattern of adhesive defining the
individual areas containing the arrays of oligonucleotide
probes.
[0026] FIG. 2 is an exploded perspective view of the array of
biomolecular probes showing the positioning of the gel pads on the
substrate of a preferred embodiment of the invention.
[0027] FIG. 3 is an exploded cross-sectional view of a chamber
showing the array of gel pads of a preferred embodiment of the
invention.
[0028] FIG. 4 is an exploded cross-sectional view of a port
illustrating the conical shape of the port of a preferred
embodiment of the invention.
[0029] FIG. 5 is a perspective view of the label layer, the
flexible, gas permeable layer and the adhesive of a preferred
embodiment of the invention.
[0030] FIG. 6 is a cross-sectional view of a preferred embodiment
of the present invention illustrating the application of vacuum to
a reaction chamber.
[0031] FIG. 7 illustrates the assembly and use of a preferred
embodiment of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] The present invention provides an apparatus for performing
high-capacity biological reactions on a biochip comprising a
substrate having an array of biological binding sites. The
invention also provides a method for removing gas bubbles from the
apparatus. The invention is specifically directed to methods for
removing gas bubbles from an apparatus comprising a reaction
chamber having one or more arrays, preferably comprising arrays
consisting of gel pads, and most preferably comprising arrays
consisting of 3-dimensional polyacrylamide gel pads, wherein
biological reactions are performed by reacting a biological sample
containing a target molecule of interest with a complementary probe
molecule immobilized on the biochip.
[0033] As used herein, the term "array" refers to an ordered
spatial arrangement, particularly an arrangement of immobilized
biomolecules or polymeric anchoring structures.
[0034] As used herein, the term "addressable array" refers to an
array wherein the individual elements have precisely defined x and
y coordinates, so that a given element at a particular position in
the array can be identified.
[0035] As used herein, the terms "probe" and "biomolecular probe"
refer to a biomolecule used to detect a complementary biomolecule.
Examples include antigens that detect antibodies, oligonucleotides
that detect complimentary oligonucleotides, and ligands that detect
receptors. Such probes are preferably immobilized on a
substrate.
[0036] As used herein, the terms "bioarray," "biochip" and "biochip
array" refer to an ordered spatial arrangement of immobilized
biomolecules or polymeric anchoring structures on a solid
supporting substrate. Preferred probe molecules include nucleic
acids, oligonucleotides, peptides, ligands, antibodies and
antigens; oligonucleotides are the most preferred probe
species.
[0037] The invention is advantageously used for performing
biological reactions using biochips 18, preferably at room
temperature or at slightly elevated temperatures. Biochips, as used
in the art, encompass substrates containing arrays or microarrays,
preferably ordered arrays and most preferably ordered, addressable
arrays, of biological molecules that comprise one member of a
biological binding pair. Typically, such arrays are oligonucleotide
arrays comprising a nucleotide sequence that is complementary to at
least one sequence that may be or is expected to be present in a
biological sample. Alternatively, peptides or other small molecules
can be arrayed in such biochips for performing, inter alia,
immunological analyses (wherein the arrayed molecules are antigens)
or assaying biological receptors (wherein the arrayed molecules are
ligands, agonists or antagonists of said receptors).
[0038] One characteristic of biochips is the manner in which the
arrayed biomolecules are attached to the surface of the biochip.
Conventionally such procedures involve multiple reaction steps,
often requiring chemical modification of the solid support itself.
Even in embodiments comprising absorption matrices, such as
hydrogels, present on a solid support, chemical modification of the
gel polymer is necessary to provide a chemical functionality
capable forming a covalent bond with the biomolecule. The
efficiency of the attachment chemistry and strength of the chemical
bonds formed are critical to the fabrication and ultimate
performance of the microarray.
[0039] Preferably, polyacrylamide hydrogels and gel pads are used
as binding layers to adhere biological molecules to surfaces,
wherein said biological molecules include but are not limited to
small molecule ligands, hormomes, nutrients, metabolites, proteins,
peptides, oligonucleotides, polynucleotides, and larger nucleic
acid fragments. The gel pads for use with the apparatus of the
present invention are conveniently produced as thin sheets or
slabs, typically by depositing a solution in between two glass
surfaces (such as glass plates or microscope slides) using a spacer
to obtain the desired thickness of the polyacrylamide gel, wherein
the solution comprises a monomer, most preferably an acrylamide
monomer, a crosslinker such methylene bisacrylamide, a catalyst
such as N,N,N',N'-tetramethylethylendiamine (TEMED) and an
initiator such as ammonium persulfate for chemical polymerization,
or 2,2-dimethoxy-2-phenyl-acetophone (DMPAP) for
photopolymerization. Generally, the acrylamide monomer and
crosslinker are prepared in a solution of about 4-5% acrylamide
(having an acrylamide/bisacrylamide ratio of 19:1) in
water/glycerol, with a small amount of initiator added. The
solution is polymerized and crosslinked either by ultraviolet (UV)
radiation (e.g., 254 nm for at least about 15 minutes, or other
appropriate UV conditions, collectively termed
"photopolymerization"), or by thermal initiation at elevated
temperature (e.g., typically at about 40.degree. C.). Following
polymerization and crosslinking, the top glass slide is removed
from the surface to uncover the gel. The pore size (and hence the
"sieving properties") of the gel can be controlled by changing the
amount of crosslinker and the percent of the monomer in the
polymerization solution. The pore size also can be controlled by
changing the polymerization temperature.
[0040] In the fabrication of polyacrylamide hydrogel arrays (i.e.,
patterned gels) used as binding layers for biological molecules,
the acrylamide solution typically is imaged through a mask during
the UV polymerization/crosslinking step. The top glass slide is
removed after polymerization, and the unpolymerized monomer is
washed away (developed) with water, leaving a fine feature pattern
of polyacrylamide hydrogel, which is used to produce the
crosslinked polyacrylamide hydrogel pads. Further, in an
application of lithographic techniques known in the semiconductor
industry, light can be applied to discrete locations on the surface
of a polyacrylamide hydrogel to activate these specified regions
for the attachment of an oligonucleotide, an antibody, an antigen,
a hormone, hormone receptor, a ligand or a polysaccharide on the
surface (e.g., a polyacrylamide hydrogel surface) of a solid
support (see, for example, International Application, Publication
No. WO 91/07087, incorporated by reference).
[0041] For hydrogel-based arrays using polyacrylamide, biomolecules
(such as oligonucleotides) are covalently attached by forming an
amide, ester or disulfide bond between the biomolecule and a
derivatized polymer comprising the appropriate cognate chemical
group. Covalent attachment of the biomolecule to the polymer is
usually performed after polymerization and chemical cross-linking
of the polymer is completed
[0042] Alternatively, oligonucleotides bearing 5'-terminal
acrylamide modifications can be used that efficiently copolymerize
with acrylamide monomers to form DNA-containing polyacrylamide
copolymers (Rehman et al., 1999, Nucleic Acids Research 27:
649-655). Using this approach, stable probe-containing layers can
be fabricated on supports (e.g., microtiter plates and silanized
glass) having exposed acrylic groups. This approach has been
commercially marketed as "Acrydite.TM." capture probes (available
from Mosaic Technologies, Boston, Mass.). The Acrydite moiety is a
phosporamidite that contains an ethylene group capable of
free-radical copolymerization with acrylamide, and which can be
used in standard DNA synthesizers to introduce copolymerizable
groups at the 5' terminus of any oligonucleotide probe.
[0043] With reference to the illustration provided in FIG. 1, the
invention provides an apparatus 10 comprising a biochip, which
itself comprises a substrate 11 having a first surface 12 and a
second surface 13 opposite thereto, and a flexible, gas permeable
layer 16 affixed to the first substrate surface 12 by an adhesive
15. On the first surface 12 is an area 14 bounded by adhesive 15
and completely covered by flexible, gas permeable layer 16.
Flexible, gas permeable layer 16, adhesive 15, and first substrate
surface 12 enclose a volume or reaction chamber 25. The ratio of
volume 25 to area 14 is preferably from about 0.025 .mu.L/mm.sup.2
to about 0.25 .mu.L/mm.sup.2, more preferably from about 0.1
.mu.L/mm.sup.2 to about 0.25 .mu.L/mm.sup.2, and most preferably
from about 0.1 .mu.L/mm.sup.2 to about 0.2 .mu.L/mm.sup.2.
[0044] As shown in FIG. 2, an array 17 of biomolecules, which is
preferably affixed to first substrate surface 12, is positioned
between flexible, gas permeable layer 16 and first substrate
surface 12 in area 14. In preferred embodiments, the array
comprises at least about 400, more preferably at least about 1000,
and most preferably at least about 10,000 biomolecular probes.
Array 17 most preferably further comprises gel pads 22. FIG. 3
provides an exploded cross-sectional view of a portion of array 17
illustrating the gel pads 22. Each gel structure 22 is preferably
cylindrical, most preferably having about a diameter of about 100
microns and a thickness of about 25 microns. The distance between
each site within each array 17 is most preferably about 300
microns.
[0045] A layer of a water-soluble compound 28 having a melting
point of about 30.degree. C. to about 60.degree. C., more
preferably of about 35.degree. C. to about 50.degree. C., and most
preferably of about 35.degree. C. to about 45.degree. C. is
deposited in volume 25 bounded by first substrate surface 12,
flexible, gas permeable layer 16, and adhesive 15. Preferably, the
water-soluble compound is biocompatible, does not stick to or clog
the pores of flexible, gas permeable layer 16, and serves to
prevent mechanical damage to gel pads 22. In a preferred
embodiment, the compound is polyethylene glycol, most preferably
polyethylene glycol 600. The compound 28 is deposited so that the
entire volume 25, with the exception of that portion of volume 25
occupied by array 17, comprises compound 28.
[0046] Array 17 can be positioned on surface 12 by providing
markings, most preferably holes or pits in surface 12, that act as
fiducials or reference points on surface 12 for accurate placement
of array 17. The presence of said fiducials is particularly
advantageous in embodiments comprising a multiplicity of arrays 17
in one or a multiplicity of areas 14 on surface 12, wherein
accurate placement of said arrays is required for proper spacing
and orientation of the arrays on the reaction chamber.
[0047] Substrate 11 further comprises a first port 19 that
transverses the substrate from the first surface 12 to the second
surface 13 and forms first and second openings 29 and 30 on said
first and second surfaces, respectively. The first port 19 serves
as an input port and is positioned in substrate 11. so that the
first opening 29 is provided within the area 14 bounded by adhesive
15 on first surface 12. In further preferred embodiments, substrate
11 further comprises a second port 20 that transverses the
substrate from first surface 12 to second surface 13 and forms
first and second openings 31 and 32 on said first and second
surfaces, respectively. Second port 20 serves as an outlet port and
is positioned in substrate 11 so that the first opening 31 opens
within area 14 bounded by the adhesive 15 on the first surface 12.
The second openings of ports 19 and 20 are covered with a removable
and replaceable cover 21. In preferred embodiments, replaceable
cover 21 is a stopper, a gasket, or tape, most preferably foil
tape.
[0048] Input and output ports 19 and 20 are preferably shaped to
accept a plastic pipette tip, most preferably a 10 .mu.L pipette
tip or a 200 .mu.L pipette tip. In preferred embodiments, input and
output ports 19 and 20 are generally in the shape of a truncated
cone, as shown in FIG. 4, wherein the end of the cone having the
smaller diameter forms the first opening of each port 29 and 31,
respectively, and the end of the cone having the larger diameter
forms the second opening of each port 30 and 32, respectively. This
shape creates a seal between the pipette tip and the port, enhances
visibility of the port for operator accuracy and prevents
protrusion of the pipette tip into volume 25, thereby preventing
potential damage to components therein, particularly the flexible,
gas permeable layer 16. In these embodiments, each port preferably
has a diameter on second substrate surface 13 of from about 1.0 mm
to about 2.0 mm, and a diameter on first substrate surface 12 of
from about 0.3 mm to about 0.6 mm. The conical walls of ports 19
and 20 form an angle 54 with the second substrate surface 13, which
is preferably less than 90.degree.. Most preferably, angle 54 is
less than or equal to the contact angle 55 of the biological sample
fluid 26. Most preferably, angle 54 is equal to contact angle 55
such that the surface of the fluid in the port is flat. For aqueous
solutions, this angle is about 60.degree.. This geometric
arrangement provides a port that tends not to leak, but instead
wicks fluid into volume 25 so that the second substrate surface 13
is dry when replaceable cover 21 is applied.
[0049] Substrate 11 is fabricated from any solid supporting
substance, including but not limited to plastics, metals, ceramics,
and glasses. Most preferably, substrate 11 is made from silicon or
glass (for accuracy and stiffness), molded plastics (which reduce
cost of manufacture and thermal inertia), or ceramics (for the
incorporation of microfluidic elements including integrated heating
elements). Most preferably, substrate 11 is glass.
[0050] Adhesive 15 is prepared using an adhesive suitable for
forming a water-tight bond between substrate 11 and flexible, gas
permeable layer 16, including, but not limited to, high temperature
acrylics, rubber-based adhesives, and silicone-based adhesives. The
shape of adhesive 15 is configured to contain array 17. Adhesive 15
can be deposited on first substrate surface 12 in a pattern to
produce area 14 in any desired shape, and is most preferably
deposited to define an ellipsoidal area 14. Adhesive 15 can be
deposited using inkjet printing or offset printing methods, or by
die cutting the desired shapes from a sheet of adhesive material.
In addition, a substantial portion of first surface 12 can be
covered with adhesive and portions of the substrate which are not
desired to retain adhesive properties can be hardened
preferentially, for example, by ultraviolet curing. In these
embodiments, portions retaining adhesive properties can be defined
using a mask and thereby retain adhesive properties necessary to
affix flexible, gas permeable layer 16 to surface 12. In
embodiments using the die cut adhesive material, the adhesive
material is preferably a double sided adhesive tape, and more
preferably a double sided adhesive tape having no carrier. Adhesive
15 is most preferably set down in a layer between 1 and 100 .mu.m
thick, more preferably between 25 and 75 .mu.m thick, and most
preferably about 50 .mu.m thick.
[0051] Flexible, gas permeable layer 16 preferably covers an area
of from about 5 mm.sup.2 to about 1400 mm.sup.2, more preferably
from about 5 mm.sup.2 to about 600 mm.sup.2, and most preferably
from about 100 mm.sup.2 to about 600 mm.sup.2. Flexible, gas
permeable layer 16 is made of any flexible solid substance,
including but not limited to plastics, including polypropylene,
polyethylene, and polyvinylidene chloride (sold commercially as
Saran.RTM. wrap) plastics, rubbers, including silicone rubbers,
high temperature polyesters, and porous Teflon.RTM.. Flexible, gas
permeable layer 16 is preferably deformable, porous, and
biocompatible. Flexible, gas permeable layer 16 also preferably
impermeable to liquids in order to prevent evaporation of water
from the volume contained between the flexible, gas permeable layer
and the substrate. Most preferably, flexible, gas permeable layer
16 is selected to have physical, chemical and mechanical properties
such that the surface tension of sample fluid 26 prevents passage
of the sample fluid through the pores of the membrane, while
allowing passage of gas molecules across the flexible, gas
permeable layer. Preferably, the pore size of flexible, gas
permeable layer 16 is between 0.2 and 3.0 .mu.m, more preferably
between 0.2 and 1 .mu.m, and most preferably about 0.2 .mu.m.
Flexible, gas permeable layer 16 also preferably is translucent and
should be able to withstand temperatures of between 50.degree. C.
and 95.degree. C. for a period of between 8 and 12 hours without
shrinkage. In a preferred embodiment, the flexible, gas permeable
layer is porous Teflon.RTM.. Membranes having these characteristics
are available from Pall Specialty Materials.
[0052] In preferred embodiments, as shown in FIG. 5, the invention
further comprises a label layer 57 which is die cut in the same
manner as the adhesive to form windows 58 that correspond in
location to areas 14 on first substrate surface 12. The label layer
is preferably a thick film having a layer of adhesive, and most
preferably is an Avery laser label. The label layer is applied to
the outer surface of the flexible, gas permeable layer, preferably
by vacuum lamination. Areas 14 are visible through windows 58 in
label layer 57.
[0053] A means for facilitating diffusion across the flexible, gas
permeable layer is removably affixed to the flexible, gas permeable
layer, or the label layer when present, in order to remove gas
bubbles from the reaction chambers. The diffusion-facilitating
means creates a pressure gradient or concentration gradient across
flexible, gas permeable layer 16, thereby increasing the rate of
diffusion of gas molecules from the sample fluid 26 contained in
volume 25 across flexible, gas permeable layer 26. A preferred
embodiment of the diffusion-facilitating means, wherein the
diffusion-facilitating means creates a pressure gradient across
flexible, gas permeable layer 16, is shown in FIG. 6. In this
embodiment, a vacuum source 70 is removably affixed to flexible,
gas permeable layer 16. In preferred embodiments, vacuum source 70
comprises a vacuum pump 71, a chamber seal 72 that completely
surrounds area 14 and is removably affixed to flexible, gas
permeable layer 16, and a length of plastic tubing 73 connecting
vacuum pump 71 to reducer 72. The chamber seal may be a suction
cup, a reducer, or any other structure having similar chemical and
mechanical properties. Most preferably, the plastic tubing is
polyurethane tubing. Most preferably the chamber seal is made of
polyvinylidene fluoride (sold under the name Kynar.RTM. by Elf
Atochem North America).
[0054] Diffusion-facilitating means that create a concentration
gradient across the membrane are also preferred. Concentration
gradients are created, for example, by providing a flow of inert
gas across flexible, gas permeable layer 16, wherein the molecules
of the inert gas are too large to pass through flexible, gas
permeable layer 16, while the gas contained in volume 25 passes
freely through flexible, gas permeable layer 16. Those skilled in
the art will be able to select the characteristics of flexible, gas
permeable layer 16 and diffusion-facilitating means that are
appropriate for their selected sample fluid 26.
[0055] Array 17 contained in area 14 on first substrate surface 12
is covered with a water-soluble compound 28, which protects and
seals the biochip prior to use and prevents degradation or other
damage to the array. Any water-soluble compound 28 having a melting
point of about 30.degree. C. to about 60.degree. C., more
preferably of about 35.degree. C. to about 50.degree. C., and most
preferably of about 35.degree. C. to about 45.degree. C. is
advantageously used in filling volume 25 between array 17 and
flexible, gas permeable layer 16. Preferably, the compound is
polyethylene glycol, most preferably polyethylene glycol 600. It is
a particularly preferred feature of reaction apparatus 10 of the
invention that water-soluble compound 28 fills the entirety of the
volume 25 and more preferably also fills at least a portion of
input port 19. This inhibits formation of air bubbles in volume 25
because compound 28 is first melted, then carefully mixed with
sample fluid 26 within volume 25 using a roller 40 without
producing air bubbles in sample fluid 26. The lack of air bubbles
in volume 25 enhances efficiency of the biological reaction by
ensuring that interactions, such as hybridization, between the
target molecules in the sample and probe molecules comprising the
array 17 or gel pads of biochip 18 are capable of proceeding
without interference from such air bubbles.
[0056] Alternative embodiments of the reaction apparatus 10 of the
invention encompass a multiplicity of arrays 17 confined in a
multiplicity of areas 14 underneath flexible, gas permeable layer
16, each area comprising an array 17 and being supplied with first
port 19 and, optionally, second port 20. In these embodiments,
adhesive 15 is deposited on first substrate surface 12 in a pattern
that defines each of areas 14, and flexible, gas permeable layer 16
is applied to adhesive 15 to encompass areas 14 on said
surface.
[0057] In certain embodiments of the invention, reaction apparatus
10 is produced containing array 17 or a multiplicity of arrays 17
as disclosed herein, wherein the chamber is provided ready-to-use
by the addition of sample fluid 26 comprising one or a multiplicity
of target molecules. In alternative embodiments, reaction apparatus
10 is provided without array 17, and permits insertion thereof by a
user. In these embodiments, at least one edge of flexible, gas
permeable layer 16 is not adhered to first substrate surface 12
until array 17 is inserted.
[0058] In the use of the reaction apparatus 10 of the invention, an
amount of a sample fluid 26, most preferably comprising a
biological sample containing a target molecule, is added to the
reaction chamber through first port 19. Before application of the
sample fluid 26 to the chamber, volume 25 is most preferably heated
to a temperature greater than or equal to the melting temperature
of water-soluble compound 28. When melted, sample fluid 26 can be
added to the chamber and mixed with the water-soluble compound, as
shown in FIG. 1B. Preferably, water-soluble compound 28 does not
adversely affect the reaction taking place in the chamber. More
preferably, the amount of compound 28 is chosen such that the
efficiency of the biological reaction is improved when compound 28
is mixed with sample fluid 26.
[0059] In embodiments of the chamber comprising first port 19 but
not second port 20, the sample fluid is preferably introduced into
the chamber after compound 28 is melted, and then the fluid is
cycled into and out of the chamber, most preferably using a
pipette, until fluid 26 and compound 28 are fully mixed, and the
sample fluid evenly distributed over the surface of array 17, or
mixed into gel pads 22 comprising certain embodiments of said
arrays. Alternatively, sample fluid 26 is evenly distributed over
the surface of array 17, or mixed into gel pads 22 by physically
manipulating flexible, gas permeable layer 16, as more fully
described below. In these embodiments, sample fluid 26 is removed
after the reaction is completed, and array 17 is washed by the
cycling a sufficient volume of a wash solution 27 into and out of
the chamber, most preferably using a pipette.
[0060] In embodiments of the chamber comprising both first port 19
and second port 20, the sample fluid is preferably introduced into
the chamber after compound 28 is melted, and then the fluid is
cycled into and out of the chamber, most preferably using at least
one pipette, until fluid 26 and compound 28 are fully mixed, and
the sample fluid evenly distributed over the surface of array 17,
or mixed into gel pads 22 comprising certain embodiments of said
biochips. Sample fluid 26 is removed after the reaction has been
completed using outlet port 20, and the biochip washed by the
addition and cycling of a sufficient volume of a wash solution 27
into and out of the chamber, most preferably using a pipette. In
these embodiments, the wash solution can also be continuously
provided by addition through the input port and removal through the
output port. In certain embodiments, biochip 18 containing array 17
is removed from the chamber for development or further
manipulations as required.
[0061] Prior to commencing the reaction, the reaction apparatus 10
is degassed using vacuum source 70. Preferably a vacuum of between
13 and 27 kPa (100 to 200 torr), more preferably a vacuum of
between 13 and 20 kPa (100 to 150 torr), and most preferably a
vacuum of about 13 kPa (100 torr) is applied. Preferably the vacuum
is applied for between 10 seconds and 2 minutes, more preferably
between 10 seconds and 1 minute, most preferably between 10 seconds
and 30 seconds. Vacuum source 70 is then detached from flexible,
gas permeable layer 16, and volume 25 is visually inspected for the
presence of gas bubbles.
[0062] The Examples that follow are illustrative of specific
embodiments of the invention and various uses thereof. They are set
forth for explanatory purposes only, and are not to be taken as
limiting the invention.
EXAMPLE 1
[0063] Removing Gas Bubbles from of a Reaction Chamber
[0064] The process of assembling a chamber according to the present
invention is illustrated in FIG. 7.
[0065] A four reaction-chamber apparatus is manufactured using a
layer of 0.2 .mu.m porous Teflon unsupported membrane as the
flexible, gas permeable layer, following the procedure provided in
U.S. application Ser. No. 09/464,490, incorporated by reference
herein. Each reaction chamber is filled with 75 .mu.L of a sample
fluid containing biological target molecules by injection through a
300 .mu.L pipette tip (VWR Part No. 53510-084) using a 200 .mu.L
pipettor (Rainin P-200). Bubbles are visually detectable in the
chambers after injection.
[0066] A reaction chamber is isolated by applying a Cole-Parmer
Kynar 1/4".times.5/8" barbed reducer (Part No. 31513-31) directly
to the frame layer and forming a seal around the chamber. A "house"
vacuum source is connected to the reducer by a length of
polyurethane tubing. A vacuum of 200 torr is applied for two
minutes. Visual inspection of the chamber following application of
the vacuum shows no gas bubbles remaining in the chamber.
[0067] The reaction apparatus is maintained at 25.degree. C. and
atmospheric pressure for 8 hours until the reaction proceeds to
completion. No appreciable evaporation of water from the chamber is
observed.
[0068] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
* * * * *