U.S. patent application number 12/149865 was filed with the patent office on 2012-03-22 for microarray system.
Invention is credited to Christopher G. Cooney.
Application Number | 20120071355 12/149865 |
Document ID | / |
Family ID | 41267348 |
Filed Date | 2012-03-22 |
United States Patent
Application |
20120071355 |
Kind Code |
A9 |
Cooney; Christopher G. |
March 22, 2012 |
Microarray system
Abstract
A microarray system is disclosed. The microarray system includes
a microarray formed on a planar substrate and an incubation chamber
formed around the microarray. The incubation chamber has a
plurality of interior surfaces including a bottom surface on which
the microarray is formed and a top surface that faces the bottom
surface and is generally parallel to the bottom surface. At least
one of a plurality of interior surfaces is a hydrophilic
surface.
Inventors: |
Cooney; Christopher G.;
(Severn, MD) |
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20090280997 A1 |
November 12, 2009 |
|
|
Family ID: |
41267348 |
Appl. No.: |
12/149865 |
Filed: |
May 9, 2008 |
Current U.S.
Class: |
506/16; 506/13;
506/18; 506/39 |
Current CPC
Class: |
B01J 2219/0063 20130101;
B01J 2219/00722 20130101; B01J 2219/00644 20130101; B01J 19/0046
20130101; B01J 2219/00612 20130101; B01J 2219/00637 20130101; B01J
2219/00725 20130101 |
Class at
Publication: |
506/16; 506/13;
506/18; 506/39 |
International
Class: |
C40B 40/06 20060101
C40B040/06; C40B 60/12 20060101 C40B060/12; C40B 40/10 20060101
C40B040/10; C40B 40/00 20060101 C40B040/00 |
Claims
1. A microarray system, comprising: a microarray formed on a planar
substrate; and an incubation chamber formed around said microarray,
wherein said incubation chamber comprises a plurality of interior
surfaces including a bottom surface on which said microarray is
formed and a top surface that faces said microarray, and wherein at
least one of said a plurality of interior surfaces is a hydrophilic
surface.
2. The microarray system of claim 1, wherein said hydrophilic
surface is said top surface.
3. The microarray system of claim 2, wherein said hydrophilic
surface is formed by covering said top surface with a hydrophilic
coating.
4. The microarray system of claim 2, wherein said incubation
chamber is formed by placing a gasket around said microarray and
covering said gasket with a hydrophilic tape or a hydrophilic
film.
5. The microarray system of claim 4, wherein said hydrophilic tape
or hydrophilic film is transparent.
6. The microarray system of claim 1, further comprising a cover
slip that covers said planar substrate, wherein said microarray is
formed in a recession area on said planar substrate and wherein
said incubation chamber is formed between said cover slip and said
recession area on said planar substrate.
7. The microarray system of claim 1, further comprising a cover
slip that covers said planar substrate, wherein said cover slip has
a recession area, said recession area is larger than said
microarray and is positioned on top of said microarray, and wherein
said incubation chamber is formed between said microarray and said
recession area on said cover slip.
8. The microarray system of claim 1, wherein said hydrophilic
surface comprises impregnated chemicals that lyses cell
membranes.
9. The microarray system of claim 8, wherein said hydrophilic
surface comprises a hydrophilic matrix that retains nucleic acid
from lysed cells.
10. The microarray system of claim 8, wherein said hydrophilic
surface is said top surface.
11. The microarray system of claim 8, wherein said hydrophilic
surface is said bottom surface.
12. The microarray system of claim 1, wherein said hydrophilic
surface is said bottom surface.
13. The microarray system of claim 1, further comprising a one-way
valve for loading a liquid sample into said incubation chamber.
14. The microarray system of claim 13, wherein said one-way valve
is a check valve.
15. The microarray system of claim 13, wherein said one-way valve
is a dome valve.
16. The microarray system of claim 13, wherein said one-way valve
is connected to said incubation chamber through a first
channel.
17. The microarray system of claim 1, further comprising a waste
chamber.
18. The microarray system of claim 17, wherein said waste chamber
comprises an absorbent capable of wicking liquid from said
incubation chamber.
19. The microarray system of claim 18, wherein said absorbent
comprises cellulose.
20. The microarray system of claim 17, wherein said waste chamber
has a volume that is larger than a volume of said incubation
chamber.
21. The microarray system of claim 17, wherein said waste chamber
is connected to said incubation chamber through a second
channel.
22. The microarray system of claim 21, wherein said waste chamber
comprises an absorbent placed at a distance from said second
channel to control wicking rate.
23. The microarray system of claim 21, wherein said second channel
comprises an inlet section, a funnel shape connecting section, and
an outlet section, wherein said inlet section has a diameter that
is larger than a diameter of said outlet section.
24. The microarray system of claim 17, wherein said waste chamber
is vented to atmosphere through a venting channel.
25. The microarray system of claim 1, wherein said substrate is
glass.
26. The microarray system of claim 1, wherein said substrate is
plastic.
27. The microarray system of claim 1, wherein said microarray is an
oligonucleotide array.
28. The microarray system of claim 1, wherein said microarray is a
protein array.
29. The microarray system of claim 28, wherein said protein array
is an antibody array.
30. The microarray system of claim 1, wherein said microarray is
formed by a gel spot printing method.
31. A microarray system, comprising: a microarray formed on a
planar substrate; an incubation chamber, wherein said incubation
chamber surrounds said micro array; a dome valve for loading a
liquid sample into said incubation chamber; and a channel
connecting said one-way valve to said incubation chamber.
32. A microarray system, comprising: a microarray formed on a
planar substrate; an incubation chamber, wherein said incubation
chamber surrounds said micro array; a waste chamber containing an
absorbent; and a channel connecting said waste chamber to said
incubation chamber.
33. The microarray system of claim 32, wherein said incubation
chamber comprises an hydrophilic interior surface.
Description
TECHNICAL FIELD
[0001] The technical field is microarray systems and, in
particular, microarray systems having an incubation chamber coupled
with a one-way valve and/or a waste chamber.
BACKGROUND
[0002] Microarrays offer great potential for performing complex
analyses of samples by carrying out multiple detection reactions
simultaneously. Typically, a microarray of multiple spots of
reactant molecules is formed on a planar substrate such as a glass
microscope slide, usually in a two-dimensional grid pattern. Liquid
sample and reagents are then applied to the slide to contact
multiple spots simultaneously. Various reaction steps may be
performed with the bound molecules in the microarray, including
exposure of bound reactant molecules to the liquid sample and
reagents and washing steps. The progress or outcome of the reaction
may be monitored at each spot in the microarray in order to
characterize either material(s) immobilized on the slide or
material(s) in a liquid sample.
[0003] Microarray analysis usually requires an incubation period
that ranges from minutes to hours. The duration of the incubation
period is assay dependent and is determined by a variety of
factors, such as the type of reactant, degree of mixing, sample
volume, target copy number, and density of the array. During the
incubation period, target molecules in the liquid sample must be in
intimate contact with the microarray probes. The incubation is
usually performed in an incubation chamber. The incubation chamber
is typically formed by forming a gasket around the microarray. The
gasket is covered with a cover slip to form an enclosed chamber.
The cover slip can be made of a transparent material, such as
glass, to allow optical interrogation of the microarray after the
incubation.
[0004] If the cover slip does not have an entry port and a vent,
the liquid sample and other reagents need to be added to the
incubation chamber before the cover slip is placed on top of the
gasket. If the reaction mixture is filled to the rim of the gasket,
the reaction mixture may leak out of the side of the gasket,
compromising the gasket/cover seal and increasing the risk of
contaminating the environment. Cover slips with holes for filling
and venting circumvent these two problems. However, filling the
incubation chamber through holes on the cover slip often risks the
introduction of air bubbles or air pockets into the incubation
chamber. Moreover, surface tension of a liquid sample or a reaction
mixture may also prevent the liquid sample or reaction mixture from
completely filling the incubation chamber. A partially filled
chamber may result in a false negative if an air pocket covers an
array spot and prevents contact between the array spot and the
liquid sample or reaction mixture.
SUMMARY
[0005] A microarray system is disclosed. The microarray system
includes a microarray formed on a planar substrate and an
incubation chamber formed around the microarray. The incubation
chamber has a plurality of interior surfaces including a bottom
surface on which the microarray is formed and a top surface that
faces the bottom surface and is generally parallel to the bottom
surface. At least one of the plurality of interior surfaces is a
hydrophilic surface.
[0006] Also disclosed is a microarray system having a microarray
formed on a planar substrate, an incubation chamber formed around
the microarray; a dome valve for loading a liquid sample into the
incubation chamber; and a channel connecting the one-way valve to
the incubation chamber.
DESCRIPTION OF THE DRAWINGS
[0007] The detailed description will refer to the following
drawings, wherein like numerals refer to like elements, and
wherein:
[0008] FIG. 1 is a schematic of an embodiment of an incubation
chamber of a microarray system.
[0009] FIG. 2 is a schematic of a dome valve in a support housing
with a penetrating pipette tip.
[0010] FIG. 3 is a schematic of an embodiment of a microarray
system with a waste chamber.
[0011] FIG. 4 is a schematic of another embodiment of a microarray
system with a waste chamber.
[0012] FIG. 5 is a schematic of an embodiment of an integrated
microarray system.
[0013] FIG. 6 is a schematic showing the dimensions of an
embodiment of a microarray system.
[0014] FIG. 7 is a composite of pictures showing four microarray
incubation chamber assemblies (panel A) used to evaluate wicking of
the liquid into the waste chamber and the hybridization results
(panel B).
[0015] FIG. 8 is a composite of pictures showing an embodiment of
an integrated microarray system (panel A) and the hybridization
results from the microarray system (panel B).
[0016] FIG. 9 is a composite showing a schematic of an embodiment
of a microarray system (panel A), an array map (panel B), and the
hybridization result (panel C).
DETAILED DESCRIPTION
[0017] This description is intended to be read in connection with
the accompanying drawings, which are to be considered part of the
entire written description of this invention. The drawing figures
are not necessarily to scale and certain features of the invention
may be shown exaggerated in scale or in somewhat schematic form in
the interest of clarity and conciseness. In the description,
relative terms such as "front," "back" "up," "down," "top" and
"bottom," as well as derivatives thereof, should be construed to
refer to the orientation as then described or as shown in the
drawing figure under discussion. These relative terms are for
convenience of description and normally are not intended to require
a particular orientation. Terms concerning attachments, coupling
and the like, such as "connected" and "attached," refer to a
relationship wherein structures are secured or attached to one
another either directly or indirectly through intervening
structures, as well as both movable or rigid attachments or
relationships, unless expressly described otherwise.
[0018] The term "microarray," as used herein, refers to an ordered
array of spots presented for binding to ligands of interest. A
microarray consists of at least two spots. The ligands of interest
includes, but are not limited to, nucleic acids, proteins,
peptides, polysaccharides, antibodies, antigens, viruses, and
bacteria.
[0019] The term "hydrophilic surface" as used herein, refers to a
surface that would form a contact angle of 60.degree. or smaller
with a drop of pure water resting on such a surface. The term
"hydrophobic surface" as used herein, refers to a surface that
would form a contact angle greater than 60.degree. with a drop of
pure water resting on such a surface. Contact angles can be
measured using a contact angle goniometer.
[0020] The term "incubation chamber," as used herein, refers to an
enclosed space around a microarray. The incubation chamber, when
filled with a liquid sample, allows the microarray to be submerged
in the liquid sample so that target molecules in the liquid sample
can maintain intimate contact with the microarray probes.
[0021] Described herein is a microarray system having an incubation
chamber with a hydrophilic surface. The use of a hydrophilic
surface that contacts the liquid as it enters the chamber allows
complete filling of the incubation chamber.
[0022] As noted above, surface tension of a liquid sample or a
reaction mixture often prevent the liquid sample or reaction
mixture from completely filling a small space, such as the
incubation chamber of a microarray system. Surface tension is the
result of the attraction between the molecules of the liquid by
various intermolecular forces. In the bulk of the liquid, each
molecule is pulled equally in all directions by neighboring liquid
molecules, resulting in a net force of zero. At the surface of the
liquid, the molecules are pulled inwards by other molecules deeper
inside the liquid and are not attracted as intensely by the
molecules in the neighboring medium (be it vacuum, air or another
fluid). Therefore all of the molecules at the surface are subject
to an inward force of molecular attraction which can be balanced
only by the resistance of the liquid to compression. This inward
pull tends to diminish the surface area, and in this respect a
liquid surface resembles a stretched elastic membrane. Accordingly,
the liquid squeezes itself together until it has the locally lowest
surface area possible. The net result is that the liquid may
maintain a near-spherical shape inside the small space and does not
fill the corners, especially square corners of the small space. The
typical small gap that separates the cover from the microarray
surface often compresses the liquid into a cylindrical shape.
[0023] In the case of microarray systems, the liquid that fills the
incubation chamber is most likely a water-based liquid, such as a
hybridization buffer or washing buffer. The surface tension of the
water-based liquid may be overcome by coating at least a portion of
the interior surface of the incubation chamber with a hydrophilic
material.
[0024] FIG. 1 shows an embodiment of an incubation chamber. In this
embodiment, the incubation chamber 10 is formed around a microarray
20, which consists of a plurality of array spots 22 printed or
formed on the top surface 32 of a planar substrate 30. The surface
32 also forms the bottom surface of the incubation chamber 10. The
top of the chamber 10 is covered with a cover slip 40. The
incubation chamber 10 can be of any size or shape that matches the
dimension of the planar substrate 30, which is typically a glass or
plastic slide.
[0025] In this embodiment, the incubation chamber 10 is formed by
placing a gasket 34 on top of the planar substrate 30 and covering
the gasket 34 with the cover slip 40. In another embodiment, the
incubation chamber 10 is formed by creating a pocket or recession
area in the planar substrate 30 (by molding or etching, for
example), printing the microarray 20 at the bottom of the pocket or
recession area, and covering the pocket or recession area with the
cover slip 40. In yet another embodiment, the pocket or recession
area is formed on the cover slip 40, which is then placed directly
on top of the planar substrate 30.
[0026] The incubation chamber 10 is usually formed around the
microarray 20 so as to reduce the liquid volume needed for a
hybridization or any other reactions in the incubation chamber 10.
In one embodiment, the incubation chamber has a foot print of about
0.1-10 cm.sup.2, preferably about 0.5-5 cm.sup.2, and a height of
about 0.05-5 mm, preferably about 0.1-1 mm. In one embodiment, the
total volume of the incubation chamber is in the range of 1-250
.mu.l.
[0027] Depending on its shape, the incubation chamber 10 may have
several interior surfaces, including a bottom surface on which the
microarray 20 is formed, a top surface that faces downward to the
bottom surface and is generally parallel to the bottom surface, and
one or more side surfaces. For the purpose of ensuring uniform
filling of the incubation chamber 10, not all interior surfaces
need to be hydrophilic. In one embodiment, only the top surface of
the incubation chamber 10 is hydrophilic. In another embodiment,
only the bottom surface of the incubation chamber 10 is
hydrophilic. In another embodiment, both the top and bottom
surfaces are hydrophilic. In yet another embodiment, all interior
surfaces of the incubation chamber are hydrophilic.
[0028] A hydrophilic surface is a surface that attracts water.
Hydrophilic surfaces typically contain molecules that are
charge-polarized and capable of hydrogen bonding. In one
embodiment, the planar substrate 30 or the cover slip 40 is made of
a hydrophilic material and hence provide a hydrophilic bottom
surface or hydrophilic top surface, respectively. In another
embodiment, the top surface or the bottom surface of the incubation
chamber 10 is coated with an insoluble hydrophilic material.
Examples of the hydrophilic material include, but are not limited
to, hydrophilic polymers such as poly(N-vinyl lactams),
poly(vinylpyrrolidone), poly(ethylene oxide), poly(propylene
oxide), polyacrylamides, cellulosics, methyl cellulose,
polyanhydrides, polyacrylic acids, polyvinyl alcohols, polyvinyl
ethers, alkylphenol ethoxylates, complex polyol monoesters,
polyoxyethylene esters of oleic acid, polyoxyethylene sorbitan
esters of oleic acid, and sorbitan esters of fatty acids; inorganic
hydrophilic materials such as inorganic oxide, gold, zeolite, and
diamond-like carbon; and surfactants such as Triton X-100, Tween,
Sodium dodecyl sulfate (SDS), a.mmonium lauryl sulfate, alkyl
sulfate salts, sodium lauryl ether sulfate (SLES), alkyl benzene
sulfonate, soaps, fatty acid salts, cetyl trimethylammonium bromide
(CTAB) a.k.a. hexadecyl trimethyl animonium bromide,
alkyltrimethylanimonium salts, cetylpyridinium chloride (CPC),
polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC),
benzethonium chloride (BZT), dodecyl betaine, dodecyl dimethylamine
oxide, cocamidopropyl betaine, coco ampho glycinate alkyl
poly(ethylene oxide), copolymers of poly(ethylene oxide) and
poly(propylene oxide) (commercially called Poloxamers or
Poloxamines), alkyl polyglucosides, fatty alcohols, cocamide MEA,
cocamide DEA, cocamide TEA. Surfactants can be mixed with reaction
polymers such as polyurethanes and epoxies to serve as a
hydrophilic coating. In another embodiment, the top surface or the
bottom surface of the incubation chamber 10 is made hydrophilic by
atmospheric plasma treatment.
[0029] Alternatively, the bottom surface or top surface of the
incubation chamber may be covered with a commercially available
hydrophilic tape or film. Examples of hydrophilic tape include, but
are not limited to, Adhesives Research (AR) tape 90128, AR tape
90469, AR tape 90368, AR tape 90119, AR tape 92276, and AR tape
90741 (Adhesives Research, Inc., Glen Rock, Pa.). Examples of
hydrophilic film include, but are not limited to, Vistex.RTM. and
Visguard.RTM. films from (Film Specialties Inc., Hillsborough,
N.J.), and Lexan HPFAF (GE Plastics, Pittsfield, Mass.). Other
hydrophilic surfaces are available from Surmodics, Inc. (Eden
Prairie, Minn.), Biocoat Inc. (Horsham, Pa.), Advanced Surface
Technology (Billerica, Mass.), and Hydromer, Inc. (Branchburg,
N.J.).
[0030] In one embodiment, the hydrophilic tape or film has
sufficient transparency to allow optical interrogation of the
microarray from the top of the incubation chamber. In another
embodiment, the hydrophilic surface is created by coating the top
surface of the incubation chamber with a hydrophilic coating. In
another embodiment, the hydrophilic surface is created by simply
replacing the cover slip 40 with a hydrophilic tape or hydrophilic
film.
[0031] In yet another embodiment, the hydrophilic surface is a
hydrophilic matrix with impregnated chemicals that lyses cell
membranes, denaturing proteins, and traps nucleic acids. The
hydrophilic matrix would perform two functions, purification of the
sample and uniformly wicking of the sample into the incubation
chamber. In one embodiment, the hydrophilic matrix is FTA
paper.RTM. (Whatman, Florham Park, N.J.). Biological samples are
applied to the FTA.RTM. paper and cells contained in the sample are
lysed on the paper. The paper is washed to remove any non-DNA
material (the DNA remains entangled within the paper). The DNA is
then eluted for subsequent microarray analysis. Alternatively, the
bound DNA may be amplified in situ for microarray detection without
an elution step.
[0032] The FTA paper.RTM. can be used as an opposing surface to the
array (i.e., the top surface of the incubation chamber).
Alternatively, the microarray may be printed on the FTA paper.RTM.
and a transparent cover slide on top of the incubation chamber
would allow visualization of the microarray. In another embodiment,
PCR reagents may be introduced into the incubation chamber for
amplification of a nucleic acid sample on the FTA paper.RTM.. In
this embodiment, the amplification will be performed inside the
incubation chamber 10.
[0033] The microarray 20 can be any type of microarray, including
but not limited to, nucleotide microarrays and protein microarrays.
In one embodiment, the microarray 20 is formed using the printing
gel spots method described in e.g., U.S. Pat. Nos. 5,741,700,
5,770,721, 5,981,734, 6,656,725 and U.S. patent application Ser.
Nos. 10/068,474, 11/425,667 and 60/793,176, all of which are hereby
incorporated by reference in their entirety.
[0034] In another embodiment, the microarray system further
contains a one-way valve for introducing a liquid (e.g., a sample,
a hybridization buffer, or a washing buffer) into the incubation
chamber 10. The sample is introduced into the incubation chamber 10
through the one-way valve to prevent environmental contamination,
which is an important concern in certain applications such as the
detection of biological warfare agents. The one-way valve can be a
check valve or a dome valve that is placed at an inlet port of the
incubation chamber 10. Dome valves of various sizes are
commercially available (e.g., from Minivalve International, Yellow
Springs, Ohio). In an embodiment shown in FIG. 2, the dome valve 50
contains two components: a dome-shaped valve body 52 and a back
seal 54. The back seal has a hole (not shown) that allows an
introducer 56 to penetrate the back seal 54. The introducer 56 may
be any liquid delivering device having a pointed tip to penetrate
the back seal 54. In this embodiment, the introducer 56 is a
pipette tip. In another embodiment, the introducer 56 is a syringe
needle.
[0035] The dome valve 50 allows easy access with the introducer 56
and conforms to the tip of the introducer 56 as the tip enters the
dome valve 50 through the back seal 54. After the introducer 56 is
withdrawn, the opening on the back seal 54 is spontaneously closed
to prevent the sample from leaking out of the incubation chamber 10
from the dome valve 50. Therefore, the dome valve 50 acts as both a
pierceable septum and a check valve. The dome valve may be
installed on a microarray assembly through the supporting structure
58. Tn one embodiment, the dome valve is connected to the
incubation chamber 10 through an inlet port 11 and inlet channel 14
(FIG. 3).
[0036] In yet another embodiment, the microarray system further
includes a waste chamber. Many optical readers, such as the Aurora
Photonics Port Array 5000.TM. microarray reader, give improved
signal-to-noise ratios when reading dry images. Therefore, it is
advantageous to incorporate a waste chamber into the microarray
system to remove liquid from the incubation chamber before placing
the microarray in a microarray reader. Referring now to FIG. 3, the
incubation chamber 10 is connected to a waste chamber 60 formed on
the same microarray slide.
[0037] The waste chamber 60 can be of any shape and typically has a
volume that is greater than the volume of the incubation chamber
10. Tn one embodiment, the waste chamber is formed in a gasket tape
which is then attached to the substrate 30 (See FIG. 1) on which
the microarray 20 is printed. In yet another embodiment, the
substrate 30 has a cut-out on its top surface. The cut-out has a
size and position that match the size and position of the waste
chamber 60 in the gasket 34 so that the waste chamber 60, once
formed between the substrate 30 and the gasket 34, would have a
depth that is greater than the depth of the incubation chamber 10.
In another embodiment, the substrate 30 is made of a plastic
material so that a cut-out may be easily made on the substrate 30.
In yet another embodiment, both the incubation chamber 10 and the
waste chamber 60 are formed in the substrate 30 without using the
gasket 34. The waste chamber 60, however, may have a depth that is
greater than the depth of the incubation chamber 10.
[0038] In one embodiment, the waste chamber 60 contains an
absorbent 62 that, once in contact with the liquid in the
incubation chamber 10, wicks the liquid from the incubation chamber
10, therefore allowing the microarray 20 to be read in a dry
state.
[0039] The absorbent 62 can be any material capable of retention of
a relative large quantity of liquid. In one embodiment, the
absorbent 62 is made of an aggregate of fibers. In another
embodiment, the absorbent 62 is a nonwoven fabric produced in a
through-air bonding process. The constituent fibers of the nonwoven
fabric can be hydrophilic synthetic fibers, natural cellulose
fibers of pulp or the like, or regenerated cellulose fibers. The
fibers may be coated or infiltrated with a surfactant or a
hydrophilic oil to improve liquid absorbance. Not limited to the
through-air bonding process, the nonwoven fabric for use herein may
be produced in any other process such as a spun-bonding process, an
air laying process, a spun-lacing process, etc. In one embodiment,
the absorbent 62 is a cellulose paper (C048) from Millipore
(Billerica, Mass.)
[0040] Referring again to FIG. 3, the waste chamber 60 is connected
to the incubation chamber 10 through a channel 12. The channel 12
serves dual purposes. When filled with the liquid, the channel 12
provides a liquid passage way between the incubation chamber 10 and
the waste chamber 60. When filled with air, the channel 12
separates the incubation chamber 10 from the waste chamber 60 and
prevents premature wicking by the absorbent 62 in the waste chamber
60.
[0041] The liquid inside the incubation chamber 10 is removed by
forcing the liquid inside the incubation chamber 10 into the
channel 12 and establishing a contact between the liquid in the
channel 12 and the absorbent 62 in the waste chamber 60. The
contact may be established by applying a pressure to the liquid in
the incubation chamber 10 to push the liquid out of the channel 12
or by applying a suction at a vent 64 of the waste chamber 60 to
pull the liquid out of the channel 12. A pressure to the liquid in
the incubation chamber 10 may be generated by applying a pressure
through the dome valve 50 (e.g., using a pipette or a syringe). If
the incubation chamber 10 is covered only with a hydrophilic tape
or a hydrophilic film, a pressure to the liquid inside the
incubation chamber 10 may be generated by simply pressing the
hydrophilic tape or film that form the top surface of the
incubation chamber 10. Alternatively, the contact between the
liquid in the channel 12 and the absorbent 62 may be established by
advancing the absorbent 62 towards the channel 12 until the
absorbent 62 touches the liquid inside the channel 12.
[0042] Once a contact is established, the liquid in the incubation
chamber 10 is wicked into the absorbents 62 in the waste chamber 60
through the channel 12. The flow rate of the liquid is determined
by the size of the channel 12, the surface tension and viscosity of
the liquid, and the wicking rate of the absorbent 62. In addition,
the flow rate decreases as the absorbent becomes more saturated.
The flow rate can also be controlled by the placement of the
absorbent 62 in the waste chamber 60. An absorbent placed close to
the outlet of the channel 12 result in higher flow rates than an
absorbent placed further away. Therefore, cutting a corner off of
the absorbent 62 results in a slower flow rate because of the
increased distance between the outlet of the channel 12 and the
absorbent 62.
[0043] In the event that an air bubble is introduced into the
incubation chamber 10, the air bubble may be lodged in the channel
12 and partially or completely block liquid flow in the channel 12.
The air bubble may also stop the wicking action of the absorbent 62
if the air bubble is located right at the interface of the liquid
and the absorbent 62. This problem can be overcome with a channel
design shown in FIG. 4. In this embodiment, the channel 12 includes
three sections: an inlet section 15, a funnel shape connecting
section 16 and an outlet section 17. The outlet section 17 has a
diameter that is smaller than the diameter of the inlet section 15.
The smaller diameter results in a stronger capillary pressure in
the outlet section 17 compared to the pressure in the inlet section
15. The pressure difference leads to liquid movement towards the
outlet section 17. In operation, the liquid already in the outlet
section 17 is pushed out of the outlet section 17 and passed around
the air pocket at the interface of the liquid and the absorbent 62.
The funnel shape connecting section 16 offers an overflow region
that prevents premature wicking due to the capillary action of the
channel. In another embodiment, the outlet section 17 is further
divided into two subsections, a larger diameter first section
(corresponding to the horizontal portion of section 17 in FIG. 4)
and a smaller diameter second section (corresponding to the
vertical portion of section 17 that enters the waste chamber
60).
[0044] If the hybridization or amplification process in the
incubation chamber 10 involves a heating step, such as the
denaturing step of thermal cycling in a polymerase chain reaction
(PCR), the liquid inside the incubation chamber 10 may be pushed
out of the channel 12 and make a premature contact with the
absorbent 62 due to increased pressure in the incubation chamber
10. Under these circumstances, air may be intentionally left in the
channel 12 (at the time when incubation chamber 10 is filled) to
prevent premature wicking by the absorbent 62. Alternatively, a
hydrophobic stop may be placed inside the channel 12 to prevent
premature wicking by the absorbent 62. In one embodiment, the
hydrophobic stop comprises a channel section with a hydrophobic
interior surface. In one embodiment, the hydrophobic surface is
formed by coating or treating the native channel surface with a
hydrophobic material such as Teflon.RTM., silicone or silane. In
another embodiment, the interior surface of channel 12 is coated
with a hydrophilic material and the hydrophobic stop comprises a
section of channel 12 that has a non-coated surface exposing the
native hydrophobic plastic material.
[0045] In another embodiment, the incubation chamber 10 is
connected to multiple waste chambers 62 to ensure that wicking
occurs at the appropriate interval.
[0046] Also described herein is an integrated microarray system
having a hydrophilic incubation chamber for uniform filling, a
one-way valve to prevent sample contamination, and a waste chamber
for liquid removal from the incubation chamber. Referring now to
FIG. 5, an embodiment of the integrated microarray system 100
includes a microarray 20 printed or formed on a substrate 30, a
hydrophilic incubation chamber 10 formed around the microarray 20,
a dome valve 50 in fluid communication with the incubation chamber
10 through a channel (not shown), and a waste chamber 60 connected
to the incubation chamber 10 through a channel 12. An absorbent 62
is incorporated in the waste chamber 60, which is vented to the
atmosphere through a vent 64. A transparent hydrophilic cover 70
forms the top surface of the incubation chamber 10 and the waste
chamber 60. In one embodiment, the vent 64 is created by simply
punching a hole in the cover of the waste chamber 60.
[0047] One advantage of covering the incubation chamber 10 and the
waste chamber 60 with a hydrophilic tape or film is that the thin
film or tape is capable of deforming under pressure. It is
therefore possible to mix the liquid in the incubation chamber 10
by applying modest pressure to the waste chamber, which would cause
slight deformation to the incubation chamber 10 and hence movement
of liquid inside the incubation chamber 10.
EXAMPLES
Example 1
Covering Incubation Chamber with Hydrophilic Tape Resulted in
Complete Filling of the Chamber
[0048] FIG. 6 shows the geometry of an embodiment of a microarray
slide. The circle is a filling inlet port 11, the square is the
microarray incubation chamber 10, and the long rectangle is the
waste chamber 60. A channel 14 of 0.5 mm in width connects the
filling inlet port 11 to the microarray incubation chamber 10, a
2.0 mm channel 12 connects the microarray incubation chamber 10 to
the waste chamber 60, and a 1.0 mm channel 64 from the waste
chamber 60 to the outside serves as a vent. The microarray
incubation chamber 10 has a size of 10 mm.times.10 mm. An inner
gasket tape, with a thickness of 0.25 mm (available from 3M, Part
No. 9087), was laser cut to form a gasket with the geometry
described above. The gasket was placed on a hydrophobic surface
with a contact angle that is similar to slides used for the gel
spot printing process. The top of the gasket was sealed with a
hydrophilic tape (AR 90128) to provide a hydrophilic surface.
Thirty microliters of water filled the chamber uniformly without
leaving air bubbles or air pockets. Thirty microliters of
hybridization buffer (3 M guanidine thiocyanate, 150 mM HEPES pH
7.5, and 15 mM EDTA) also filled the chamber uniformly without air
bubbles. A similar test with a hydrophobic tape (AR 8192) left air
pockets in the microarray chamber due to non-uniform filling.
[0049] This experiment demonstrated that the hydrophilic surface of
the chamber overcomes the surface tension of the liquid and allows
complete filling of the chamber, including the square edges. This
result is surprising since square corners typically trap air
pockets as liquid fills the chamber.
Example 2
Evaluation of the Wicking Efficiency of the Waste Chamber
[0050] FIG. 7A shows four test microarray slides, each having a
hydrophilic incubation chamber connected to a waste chamber
containing an absorbent. The waste chambers were vented to
atmosphere. The chambers were formed by placing a gasket (laser cut
from double sided tape provided by Grace Biolab) on top of a
microarray supporting slide. The hydrophilic surface in the
incubation chamber was produced by covering the incubation chamber
space with a hydrophilic tape (AR 90469). The absorbent was from
Millipore (C048). Ninety-five microliters of sample containing
amplified product from Yersinia pestis, hybridization markers, BSA
and a hybridization buffer were denatured at 95.degree. C. for 5
minutes and introduced into the incubation chamber through an inlet
port. The inlet port was then sealed with tape (AR90697). The
reaction was incubated at 50.degree. C. for one hour in an MJ
Research PTC-200 DNA Engine thermalcycler with attached slide
tower. The microarray slides were removed from the tower and washed
at room temperature with 150 uL of water. As water was added into
the incubation chamber through the inlet port, the liquid in the
incubation chamber was pushed into the waste chamber through the
channel connecting the incubation chamber with the waste chamber.
Once the contact was established between the liquid inside the
incubation chamber and the absorbent in the waste chamber, the
absorbent was able to wick out the liquid (including the washing
volume) from the incubation chamber. The microarray slide was then
heated at 95.degree. C. for 20 minutes to thoroughly dry the
incubation chamber. The microarray was imaged on an Aurora
Photonics Port Array 5000.TM. without any manipulation to the
device. The image was taken through the hydrophilic tape that
covers the incubation chamber.
[0051] FIG. 7B shows the image of an example microarray after the
hybridization, washing, and drying step. Product spots are shown as
dark black dots. Control spots include Cy3 spots and hybridization
markers. Each array is a replicate of four subarrays, hence the
four sets of Yp product spots. Uniform hybridization was achieved
in all test slides.
Example 3
Microarray System Containing a Dome Valve, a Hydrophilic Incubation
Chamber and a Waste Chamber
[0052] FIG. 8A shows an embodiment of an integrated microarray
system having an incubation chamber covered with a hydrophilic
tape, a waste chamber with incorporated absorbent, and a dome valve
connected to the incubation chamber. Ninety-five microliters of
sample consisting of hybridization master mix and Yersinia pestis
product were denatured at 95.degree. C. for 5 minutes and
introduced into the incubation chamber through the dome valve with
a Rainin P200 uL pipettor. The sample uniformly flowed into the
microarray chamber without leaving air bubbles or air pockets. The
incubation chamber was heated for 60 minutes at 50.degree. C. on an
MJ Research PTC-200 DNA Engine thermalcycler with attached slide
tower without any changes to the flow cell device. The microarray
slide was removed from the slide tower and washed with 150 uL of
water. As the water was introduced into the incubation chamber, the
hybridization mixture inside the incubation chamber was pushed into
the waste chamber and the Millipore C048 absorbent wicked out the
entire volume of liquid from the incubation chamber. The microarray
slide was then heated at 95.degree. C. for 20 minutes to thoroughly
dry the incubation chamber. The microarray was imaged on an Aurora
Photonics Port Array 5000.TM. without any manipulation to the
device. The image was taken through the hydrophilic tape that
covers the incubation chamber.
[0053] FIG. 8B shows the image of an exemplary microarray after the
hybridization, washing, and drying step. Product spots are shown as
dark black dots. Control spots include Cy3 spots and hybridization
markers. Each array is a replicate of four subarrays, hence the
four sets of Yp product spots. As shown in FIG. 8B, uniform
hybridization was achieved in all subarrays.
Example 4
Microarray System Containing a Hydrophilic Incubation Chamber and a
Waste Chamber
[0054] FIG. 9A shows another embodiment of a microarray system
having a hydrophilic chamber 10 and a waste chamber 60. The two
chambers are connected by a channel 12 having an inlet section 15,
a funnel shape connecting section 16, a large diameter outlet
section 17 and a small diameter outlet section 172. Liquid sample
is added to the incubation chamber 10 through inlet port 11 and
channel 14.
[0055] The microarray system shown in FIG. 9A was constructed on a
glass slide using a gasket which was laser cut from double sided
tape (Grace Biolabs). The waste chamber 60 contained a filter paper
absorbent (CF4, Millipore) and was vented to the atmosphere. The
resulting microarray assembly was covered with Lexan HPFAF
(0.007''/175 .mu.m) antifog tape (GE Plastics). Twenty microliters
(20 .mu.l) of sample containing amplified product from
Streptococcus pyrogenase, hybridization markers (positive control),
BSA and a hybridization buffer were denatured at 95.degree. C. for
5 minutes and introduced into the incubation chamber 10 through the
inlet port 11 using a Rainin P200uL pipettor. The inlet port 11 was
sealed with tape and the entire slide was allowed to incubate at
55.degree. C. for 30 minutes in an MJ Research PTC-200 DNA Engine
thermalcycler with attached slide tower. The slides were removed
from the tower and washed at room temperature with 150 uL of water.
The array was imaged on an Aurora Photonics Port Array 5000.TM.
microarray reader (2 second exposure time). FIG. 9B shows the
hybridization result. FIG. 9C is a chip map showing the layout of
array spots. As shown in FIG. 9B, strong positive results were
obtained from the hybridization control and Streptococcus specific
probes.
Example 5
One-Step Protein Microarray System
[0056] A one-step, integrated protein microarray system, such as
one of the embodiments shown in FIG. 5 and FIG. 9, is constructed
using gel drop elements containing capture antibodies. The capture
antibodies are antibodies that bind specifically to a panel of
biological warfare agents (BWAs). Each gel spot contains an
antibody that binds to a specific BWA. A set of colloidal gold
labeled secondary antibodies are placed at a location near the
inlet channel. The secondary antibodies recognize the same panel of
BWAs. When a liquid sample is loaded into the incubation chamber
through the inlet channel, the colloidal gold labeled secondary
antibodies are mixed with the sample as the sample enters the
incubation chamber. During incubation, the BWAs of interest are
captured by the antibodies in the array gel spot and the secondary
antibodies bind to the captured BWAs. After the incubation period,
unbound secondary antibodies are washed away. The secondary
antibodies bind to the BWAs captured on the gel spots and produce
positive signals in the microarray.
[0057] The term "antibody" as used herein, is used in the broadest
possible sense and may include but is not limited to an antibody, a
recombinant antibody, a genetically engineered antibody, a chimeric
antibody, a monospecific antibody, a bispecific antibody, a
multispecific antibody, a diabody, a chimeric antibody, a humanized
antibody, a human antibody, a heteroantibody, a monoclonal
antibody, a polyclonal antibody, a camelized antibody, a
deimmunized antibody, an anti-idiotypic antibody, and/or an
antibody fragment. The term "antibody" may also include but is not
limited to types of antibodies such as IgA, IgD, IgE, IgG and/or
IgM, and/or the subtypes IgG1, IgG2, IgG3, IgG4, IgA1 and/or IgA2.
The term "antibody" may also include but is not limited to an
antibody fragment such as at least a portion of an intact antibody,
for instance, the antigen binding variable region. Examples of
antibody fragments include Fv, Fab, Fab', F(ab'), F(ab').sub.2, Fv
fragment, diabody, linear antibody, single-chain antibody molecule,
multispecific antibody, andlor other antigen binding sequences of
an antibody. Additional information may be found in U.S. Pat. No.
5,641,870, U.S. Pat. No. 4,816,567, WO 93/11161, Holliger et al.,
Diabodies: small bivalent and bispecific antibody fragments, PNAS,
90: 6444-6448 (1993), Zapata et al., Engineering linear F(ab')2
fragments for efficient production in Escherichia coli and enhanced
antiproliferative activity, Protein Eng. 8(10): 1057-1062 (1995),
which are incorporated herein by reference.
Example 6
Two-Step Protein Microarray System
[0058] A two-step, integrated protein microarray system, such as
one of the embodiments shown in FIG. 5 and FIG. 9, is constructed
using gel drop elements containing antibodies. Each gel spot
contains an antibody that binds to a specific target. A sample is
introduced into the incubation chamber and incubated in the chamber
for a fixed period of time. A wash buffer is added to remove the
unbound sample. The wash buffer is wicked into the waste chamber,
thus removing all of the liquid from the incubation chamber. In the
next step, a secondary antibody or antibodies is added to the
incubation chamber and incubated for a fixed period of time. After
the incubation period, unbound secondary antibodies are washed
away. The secondary antibodies that bind to the targets captured on
the gel spots produce positive signals in the microarray.
[0059] In this embodiment, an air bubble is left in the channel,
connecting the incubation chamber to the waste chamber to separate
the liquid in the incubation chamber from the waste and prevent
premature wicking. When the additional wash volume is added to the
incubation chamber, the unbound antibody is pushed out of the
incubation chamber and wicks into the waste chamber. Multiple waste
chambers ensure that wicking occurs at the appropriate
interval.
[0060] The terms and descriptions used herein are set forth by way
of illustration only and are not meant as limitations. Those
skilled in the art will recognize that many variations are possible
within the spirit and scope of the invention as defined in the
following claims, and their equivalents, in which all terms are to
be understood in their broadest possible sense unless otherwise
indicated.
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