U.S. patent application number 10/765579 was filed with the patent office on 2004-12-16 for gel pad arrays and methods and systems for making them.
Invention is credited to Croker, Kevin, Taylor, Seth, Weber, Shane.
Application Number | 20040253613 10/765579 |
Document ID | / |
Family ID | 27372003 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253613 |
Kind Code |
A1 |
Taylor, Seth ; et
al. |
December 16, 2004 |
Gel pad arrays and methods and systems for making them
Abstract
Gel pads and gel pad arrays, and methods for making and using
them, are disclosed. The gel pads preferably comprise an
intelligent gel.
Inventors: |
Taylor, Seth; (Cambridge,
MA) ; Croker, Kevin; (Cheshire, CT) ; Weber,
Shane; (Woodbridge, CT) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Family ID: |
27372003 |
Appl. No.: |
10/765579 |
Filed: |
January 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10765579 |
Jan 27, 2004 |
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09934420 |
Aug 21, 2001 |
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6682893 |
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09934420 |
Aug 21, 2001 |
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09234288 |
Jan 20, 1999 |
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60075698 |
Jan 21, 1998 |
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60071980 |
Jan 20, 1998 |
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60072089 |
Jan 21, 1998 |
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Current U.S.
Class: |
435/6.11 ;
427/2.11; 435/287.2 |
Current CPC
Class: |
B01J 19/0046 20130101;
B01J 2219/00644 20130101; C12Q 1/6837 20130101; B01J 2219/00621
20130101; B01J 2219/00659 20130101; B01J 2219/0061 20130101; B01J
2219/00612 20130101; B01J 2219/0063 20130101; B01J 2219/00743
20130101; B01J 2219/00626 20130101; B01J 2219/00637 20130101; G01N
27/44704 20130101; B01J 2219/00619 20130101; B01J 2219/00596
20130101; B01J 2219/00648 20130101; B01J 2219/00707 20130101; B01J
2219/00378 20130101; B01J 2219/00518 20130101; C40B 60/14 20130101;
B01J 2219/00605 20130101; B01J 2219/0072 20130101; B01J 2219/00585
20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 427/002.11 |
International
Class: |
C12Q 001/68; C12M
001/34; B05D 003/00 |
Claims
What is claimed is:
1. A method for preparing an array of gel pads, the method
comprising: providing a first gel layer on a substrate; selectively
removing portions of the first gel layer to create voids in the
first gel layer; providing a second gel in the voids; and removing
the first gel layer, such that an array of gel pads is
provided.
2. The method of claim 1, wherein the first gel layer comprises an
intelligent gel.
3. A gel pad comprising a living cell.
4. An array of the gel pads of claim 3.
5. A gel pad comprising a first gel layer and a second gel layer
adjacent to and in contact with said first gel layer.
6. The gel pad of claim 5, wherein at least one of the first gel
layer and the second gel layer comprises an intelligent gel.
7. A method for preparing a gel pad array, the method comprising:
preparing gel pads on a first substrate; and transferring the gel
pads from the first substrate to a second substrate in an array
format, thereby preparing a gel pad array.
8. The method of claim 7, wherein the first substrate is coated
with an intelligent gel.
9. A flexible tape having a gel pad array disposed on a surface of
the tape.
10. The flexible tape of claim 9, wherein the tape comprises means
for preventing compression of gel pads when the tape is wound on a
reel.
11. The flexible tape of claim 10, wherein the means for preventing
compression comprises at least one ridge which extends along a
length of the tape.
12. A carrier for a tape having gel pad arrays thereon, the carrier
comprising a housing, at least one tape reel for winding the tape,
and visible or machine-readable indicia for storing information
about the tape stored in the carrier.
13. A method of providing a gel having a substance disposed within
the gel comprising: (1) providing a substrate on which is disposed
a gel, and wherein said gel is an intelligent gel, capable of
existing in an expanded and a contracted state; (2) contacting the
intelligent gel, while in the expanded state, with the substance,
e.g., a solute in a solution, and allowing the substance to enter
the gel; (3) causing the expanded intelligent gel to contract,
wherein upon contraction molecules of the substance remain in the
gel, thereby forming a gel having a substance disposed, within the
gel.
14. A method of detecting analyte, comprising: (1) providing a gel
having a first layer which includes a molecule for detecting the
analyte and a second layer having a cell, which, e.g., releases,
produces, inactivates, modifies, or otherwise affects the level of
the analyte; (2) detecting the analyte.
15. A method of a polynucleotide sequence in a sample comprising:
providing a sample which includes a polynucleotide sequence to be
analyzed; providing an array of a plurality of capture probes,
wherein each of the capture probes is positionally distinguishable
from other capture probes of the plurality on the array, and
wherein each positionally distinguishable capture probe includes a
unique (i.e., not repeated in another capture probe) region
complementary to the plurality of selector probes and wherein the
array is a gel pad array described herein (each of the plurality of
probes can be in its own gel pad); hybridizing the selected nucleic
acid molecule with the array of capture probes, thereby detecting
or identifying a selected nucleic acid molecule which bound to the
polynucleotide sequence and thereby analyzing the polynucleotide
sequence.
16. A method of performing a reaction comprising: providing a first
reactant, disposed within a first intelligent gel which changes
porosity in response to an environmental parameter having a first
value,; providing a second reactant, disposed within a second
intelligent gel which changes porosity in response to an
environmental parameter having a second value; exposing the
intelligent gels to the parameter at a first value, thereby causing
a change in porosity of the first gel, and thereby modulating
exposure of the first reactant to a target,; exposing the
intelligent gels to the parameter at a second value, thereby
causing a change in porosity of the second gel (and preferably not
the first gel), and thereby modulating exposure of the second
reactant to a target, thereby performing a reaction.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
Provisional Ser. No. 60/075,698, filed Jan. 21, 1998; U.S.
Provisional Ser. No. 60/071,980, filed Jan. 20, 1998; and U.S.
Provisional Ser. No. 60/072,089, filed Jan. 21, 1998. The contents
of each of these provisional patent applications is hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Rapid advances in the ability to accurately determine
polynucleotide sequences, such as DNAs and RNAs from the genomes of
organisms, has made possible the sequencing of huge quantities of
polynucleotides. In recent years, the entire genomes of
microorganisms, such as Helicobacter pylori, have been
sequenced.
[0003] Traditional sequencing methods have relied on automated
sequencing equipment which processes a polynucleotide strand one
base at a time. A more recent approach, sequencing by hybridization
(SBH), which could potentially increase sequencing throughput,
relies on fragmenting a target polynucleotide into short segments;
these short segments can be captured, for example on an ordered
microarray of immobilized complementary single-stranded DNA probes,
and the sequences of the target polynucleotide determined by
analyzing the overlap of the sequences of the DNA probes bound to
fragments of the target polynucleotide. See, for example, U.S. Pat.
No. 5,525,464 to Drmanac et al. Microarrays of DNA attached to a
solid support have been prepared, see, for example, U.S. Pat. No.
5,445,934 to Fodor et al.
[0004] Often, however, DNA microarrays are limited to analyzing
nucleic acids in a single, fluid environment. An alternative to
conventional DNA microarrays on a solid support is a microarray
comprising biological molecules, such as DNA, attached to a matrix
of crosslinked polymers known as gel pads. See, e.g., U.S. Pat. No.
5,552,270 to Khrapko et al. Gel pads provide the ability to
customize the micro-environment surrounding the DNA in each
individual gel pad, which makes possible more sophisticated
experiments in micro-array format.
[0005] However, although gel pads have certain advantages over
convention micro-arrays, new types of arrays, and methods for
making them, are needed.
SUMMARY OF THE INVENTION
[0006] This invention features gel pad arrays, e.g., arrays on a
support, and methods for making and using them. The arrays can be
used for sequencing by hybridization (e.g., where the pads include
nucleic acid strands immobilized within the gel matrix), for cell
based assays (e.g., where the pads include, or are adjacent to and
contacting, living cells), and for other uses which will be
apparent to one of ordinary skill in the art.
[0007] In general, the invention features, a method of providing a
gel having a substance disposed within the gel. The method
includes:
[0008] (1) providing a substrate on which is disposed a gel, e.g.,
a gel pad or an array of gel pads, and wherein said gel is an
intelligent gel, capable of existing in an expanded and a
contracted state;
[0009] (2) contacting the intelligent gel, while in the expanded
state, with the substance, e.g., a solute in a solution, and
allowing the substance to enter the gel;
[0010] (3) causing the expanded intelligent gel to contract,
wherein upon contraction molecules of the substance remain in the
gel, thereby forming a gel having a substance disposed, e.g.,
concentrated or captured, within the gel.
[0011] In a preferred embodiment the substance can be: a nucleic
acid, e.g., DNA, RNA, or a probe; a protein, e.g., an enzyme which
modifies DNA, e.g., DNA polymerase; a particle; a cell; or a
reactant.
[0012] Substances which can be disposed within a gel can include
the following: A molecule that is important for cell function, for
example: a molecule that mediates the expression of specific genes,
e.g., hormones, e.g., glucocorticoids; DNA subunits, e.g.,
nucleotides, e.g., dideoxy nucleotides; a molecule that donates a
phosphate group, e.g., ATP; a carbohydrate; a protein; a nucleic
acid; a lipid, e.g., a structure based in whole or in part on
lipids, e.g., bilayer membrane;
[0013] A protein that is generated by a living cell, for example: a
protein that interacts with the promoter of a gene, e.g., a
transcription factor; a protein that interacts with the origin of
replication, e.g., single-strand DNA binding protein; a protein
associated with the cytoskeleton of a cell, e.g., a matrix
attachment protein; a protein associated with the membrane of a
cell, e.g., a cell surface receptor; a protein associated with
signal transduction pathways within a cell, e.g., the RAS family of
proteins; a protein associated with RNA, e.g., heteronuclear RNA
binding protein (hnRNP); a protein associated with an immune
response, e.g., an antibody; a protein associated with the
contraction of muscle, e.g., actin or myosin; a protein that is
associated with the chromatin of a cell, e.g., a histone; a protein
that mediates protein folding, e.g., a chaperone; a protein
associated with cell cycle regulation, e.g., cyclin A;
[0014] Enzymes that are generated by living cells, for example: an
enzyme that links two nucleic acid molecules together, e.g., a DNA
or RNA ligase; an enzyme that cuts nucleic acids, e.g., a
restriction enzyme that cuts DNA at the binding site (e.g. EcoR1),
a type IIS restriction enzyme that cuts DNA 5' or 3' to the binding
site (e.g.); an enzyme that modifies the linking number of a closed
circular dsDNA molecule, e.g., a topoisomerase; an enzyme that
modifies the ends of a chromosome, e.g., a telomerase; an enzyme
comprised in whole or in part of RNA, e.g., a ribozyme; an enzyme
that generates proteins from amino acid sububits, e.g., a ribosome;
an enzyme that transfers phosphate groups onto a protein, e.g., a
kinase; an enzyme that removes a phosphate from a protein, e.g., a
dephosphorylase; an enzyme that generates a strand of RNA from a
template nucleic acid, e.g., an RNA polymerase; an enzyme that
generates a strand of DNA from a nucleic acid template, e.g., a DNA
polymerase or a reverse transcriptase; an enzyme that functions as
part of the DNA repair process, e.g., an enzyme that modifies
mismatched base pairs in double-stranded DNA, e.g., an endonuclease
or an exonuclease;
[0015] Cells, e.g., cells that can be cultured in vitro, for
example, living cells, e.g., bacterial cells, e.g., bacterial cells
that cause disease in humans (e.g. Stapholococcus Aureus, E. coli);
living cells, e.g., eukaryotic cells, e.g., fungal cells, e.g.,
yeast; living cells, e.g., eukaryotic cells, e.g., mammalian cells,
e.g., human cells, e.g., colon cancer cell, e.g., human cell lines
derived from colon cancer cells, e.g., colo320 cells; cells useful
in this assay include cells from nematodes, e.g., C. elegans;
flies, e.g., D. melanogaster; mouse, e.g., laboratory strains of
mouse; rat, e.g., laboratory strains of rat; chicken; cow; bovine;
fish, e.g., zebra fish; feline, e.g., house cat; canine; rabbit,
e.g., laboratory strains of rabbit; frogs, e.g., Xenopus laevis;
primates, e.g., humans or monkeys;
[0016] In preferred embodiments these cells will be modified with a
foreign piece of DNA, e.g., a foreign DNA that incorporates itself
into genomic DNA through the process of cloning. In other
embodiments, foreign DNA enters the cell but is not incorporated
into genomic DNA, e.g., the foreign DNA independently replicates in
the cytosol, e.g., a plasmid. For example, the cells are modified
with a foreign DNA that codes for a selective factor, e.g., a
protein that enables the cell to resist a toxic chemical, e.g., an
antibiotic, e.g., beta lactamase. Alternatively, the foreign DNA
codes for a recombinant molecule, e.g., a recombinant protein,
e.g., a fusion protein, e.g., an expressed fusion protein that
contains a tag at one end of the molecule, e.g., a FLAG tag
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) or a repeat of the amino acid
histidine (i.e. HIS tag) for capture of the recombinant protein.
Alternatively, the foreign DNA codes for a molecule that imparts a
property to the cell, e.g., a nutritive property, e.g., the ability
of the cell to grow in the absence of a molecule, such as an amino
acid, in the growth media, e.g., a gene coding for a protein
critical to the metabolic pathway leading to a specific amino
acid;
[0017] Viruses, for example, viruses that invade human cells, e.g.,
human immunodeficiency virus (HIV) or herpes simplex; viruses that
invade bacteria, e.g., bacteriophage; and
[0018] Proteinaceous agents that cause disease, for example,
proteins that are associated with disease in human cells, e.g.,
proteins that are associated with neurological disease, e.g., Jacob
Creutzfeld disease, e.g. prions.
[0019] In a preferred embodiment, the substance is a particle,
e.g., an insoluble particle, e.g., a polymeric particle. The
particle can be a magnetically responsive particle. Molecules,
e.g., nucleic acid molecules can be associated, e.g., adhered to or
coupled to the particle.
[0020] In a preferred embodiment the gel is caused to contract by
exposing it to environmental stimuli; this stimuli can include
changes in temperature, ionic composition, pH, light, electric
field, the presence of specific molecules, stress and solvent
composition.
[0021] In a preferred embodiment the gel is chosen from the group
of N-alkylacrylamides polymers, e.g., N-isopropylacrylamide (NIPA)
and N,N-Diethylacrylamide (DEAAm).
[0022] Steps 2 and 3 can be repeated, e.g., to provide further
concentration of the substance within the gel. They can be repeated
at least 2, 10, 50, 100, 250, or 500 times. Thus, in a preferred
embodiment the method further includes:
[0023] causing the gel of step 3 to expand, e.g., by exposing it
to, e.g., temperature, and contacting the gel, while in the
expanded state, with a substance, e.g., a solute in a solution, and
allowing the substance to enter the gel; causing the expanded
intelligent gel to contract, wherein upon contraction molecules of
the substance remain in the gel. The substance can be the same
substance as in step 3 (allowing a further concentration of the
substance) or can be a different substance.
[0024] In a preferred embodiment a plurality of substances to be
disposed inside the gel are brought into contact with the gel,
simultaneously, sequentially, or both simultaneously and
sequentially.
[0025] In a preferred embodiment, a step of the method is performed
in response to a signal generated by a computer.
[0026] In general, the invention features, a method for preparing
an array of gel pads. The method includes:
[0027] providing a first gel layer, e.g., an intelligent gel, on a
substrate;
[0028] selectively removing portions of the first gel layer to
create voids in the first gel layer,
[0029] providing a second gel in the voids; and
[0030] removing the first gel layer, such that an array of gel pads
is provided.
[0031] In a preferred embodiment, a step of the method is performed
in response to a signal generated by a computer.
[0032] In another aspect, the invention features, a method of
making a pattern in a gel layer on a substrate, e.g., forming a
mold, or forming an array of gel pads. The method includes:
[0033] (1) forming, e.g., by casting, a layer of a gel, e.g., an
intelligent gel on a substrate, e.g., on a non-porous substrate,
e.g., a glass or silicon plate, or a porous substrate, e.g., a
membrane or a glass or a silicon support with microchannels, e.g.,
with channels less than 10 micrometers in diameter,
[0034] (2) exposing a region of the gel layer to treatment which
causes the gel to liquefy. E.g., a laser rasters over the gel layer
and irradiates selected gel portions in the configuration of an
array (see also Patent Cooperation Treaty Publication WO95/04834).
Selective exposure can also be achieved, e.g. by protecting an area
of the gel layer, e.g., such that the unprotected area defines an
array configuration on the substrate (e.g., a 100.times.100 array
of gel pads). An area of the gel can be protected by masking the
gel layer, e.g., with a mask such as is conventionally used in
photolithography, the mask protects the masked gel from a phase
change; and
[0035] (3) removing the treated or untreated area of the gel, e.g.,
removing the area of the gel layer which has been exposed to the
treatment, thereby defining a pattern of gel on the substrate. By
way of example, gel in an area which are exposed to treatment,
e.g., the laser source, also become liquefied and the liquefied
portions are removed, e.g., by gentle washing. (The gel layer can
also be selectively heated by other means, such as an array of
heated wires or probes which are brought near to, or into contact
with, the surface of the gel layer.)
[0036] The method can be used to produce a gel layer having a
pattern, e.g., an array of voids, e.g., channels, grooves, holes,
or wells, or the like, formed by removal of the gel portion exposed
to the treatment, e.g., a laser source.
[0037] In a preferred embodiment the method includes exposing the
unprotected area of the gel to treatment which causes liquidation
and removing gel exposed to the treatment, to thereby form a
pattern of gel on the substrate. By way of example, the exposed
portions of the gel liquefy and are poured off or washed off with a
suitable solvent, without disturbing the remaining gel.
[0038] In a preferred embodiment, a gel which liquefies in response
to UV irradiation is cast is in a thin film on a substrate such as
a glass plate. The masked gel layer is exposed to ultraviolet
light. The exposed portions of the gel liquefy and are poured off
or washed off with a suitable solvent, without disturbing the
array. After irradiation and removal of the mask, a pattern, e.g.,
an array of gel pads is obtained. Alternatively, conventional gels
can be used.
[0039] The gel can incorporate reagents, such as polynucleotide
probes for capturing fragments of DNA from a solution;
alternatively, such reagents can be added after the array has been
formed.
[0040] In preferred embodiments the method produces an array of
voids and the method further includes filling one or more void with
a gel, e.g., a different gel than the original gel. A void or voids
can be filled with, e.g., a second gel which is, e.g., a different
intelligent gel or a conventional gel, such as polyacrylamide. The
second gel can be allowed to solidify, to form a composite gel
layer, one having an area formed by the first gel and an area
formed by the second gel. By way of example, the method can be used
to form an array of conventional gel pads within a framework of an
intelligent gel layer. The method can be used to form an array of
intelligent gel pads within a framework of a conventional gel
layer. The method can also be used to form an array of intelligent
gel pads within a framework of an intelligent gel layer. The
composite gel layer can be exposed to a treatment which causes a
phase change to remove one of the gel components. By way of
example, the composite layer is then heated (e.g., by placing the
substrate in a warming bath or a warming oven) to liquefy the
intelligent gel layer, which is then removed by washing or pouring
off the liquefied material. An array of gel pads remains on the
substrate and can be further processed, if desired.
[0041] In a preferred embodiment, a step of the method is performed
in response to a signal generated by a computer.
[0042] In another aspect, the invention features, a device which
includes an intelligent gel which changes volume or size in
response to an analyte and a device, e.g., a piezo device, for
evaluating a change in the volume or size of the intelligent
gel.
[0043] In a preferred embodiment the intelligent gel swells in
response to a change, such as the presence of an analyte of
interest. For example, an intelligent gel which swells in response
to pH changes in provide in a gel pad on a support.
[0044] In a preferred embodiment the intelligent gel includes, an
enzyme, e.g., glucose oxidase, and the reaction of the enzyme with
its substrate, e.g., glucose oxidase with glucose, changes the pH
of the gel. Thus, in the presence of the analyte, e.g., glucose, in
a sample solution which is brought into contact with the gel pad,
the gel pad will shrink. A gel pad can be provided adjacent to a
piezocrystal, such that changes in gel pad swelling produce a
piezoelectric signal, which can be detected and correlated with the
glucose concentration.
[0045] In another aspect, the invention provides, a method of
forming a gel, e.g., an array of gel pads, on a substrate. The gel
can be an intelligent gel, e.g., an intelligent gel described
herein. The method includes:
[0046] (1) providing a first substrate having disposed thereon a
gel layer, e.g., a patterned layer of gel, e.g., an array of gel
pads, or a pattern of gel which defines one or an array of
voids;
[0047] (2) providing a second substrate;
[0048] (3) transferring the gel layer from the first substrate to
the second substrate, e.g., transferring one or more, e.g., an
array of gel pads, on the first substrate to in array of gel pads
on the second substrate.
[0049] In a preferred embodiment, the gel layer, e.g., a pattern of
gel, on the first substrate is formed by a method described
herein.
[0050] In a preferred embodiment: the gel is not covalently
attached to the first substrate; the gel is not covalently attached
to the second substrate; the gel is not covalently attached to the
first or second substrate; the gel is covalently attached to the
first substrate but not to the second; the gel is covalently
attached to the second substrate but not to the first.
[0051] In a preferred embodiment the gel on the first substrate is
inspected to determine if it possesses a quality, e.g., a defect,
before transfer to the second substrate. By way of example, the
quality of a gel pad can be evaluated. Undesirable gel pads (e.g.,
a pad of the wrong shape or size) can be removed before the final
array is prepared on the second substrate. This step can be
controlled by a computer. This procedure can prevent the formation
of arrays which contain faulty or non-standard gel pads.
[0052] In a preferred embodiment the gel on the first substrate is
contacted with a reagent prior to transfer to the second substrate.
By way of example, the gel pads can be further processed (e.g.,
washed, imparted with an additional component, such as a protein,
nucleic acid, label, buffer, or the like) prior to transfer of the
gel pads from the first substrate to the array format on the second
substrate.
[0053] In a preferred embodiment the first and second substrates
are the same material.
[0054] In a preferred embodiment the first and second substrates
differ, e.g., in size, flexibility, transparency, composition,
hydrophilicy, hydrophobicity, ability to adhere to a gel layer, or
state of derivitization, with e.g., a functional group. For
example, gel pads can be prepared on a first substrate, e.g., a
flexible substrate such as a tape, and then transferred to a second
substrate, e.g., a less flexible substrate such as a glass or
plastic plate, in an array format, to provide a gel pad array on
the second substrate.
[0055] In a preferred embodiment the gel layer is transferred by
bringing the first substrate into sufficiently close proximity to
the second such that the gel is transferred from the first to the
second, e.g., by contacting the second substrate with the gel on
the first substrate, such that the gel is transferred from the
first substrate to the second substrate. The transfer can be
facilitated by using first and second substrates which have
different surfaces, e.g., a hydrophobic first substrate and a
hydrophilic second substrate; in this example, a hydrophilic gel
pad will be more adherent to the second substrate and will be
transferred from the first substrate to the second substrate when
the two substrates are pressed together. The transfer can be
facilitated in other ways. For example, the gel pad can be
electrically charged, and the electric charge of the first and/or
second substrate can be adjusted such that the gel pad is repelled
from the first substrate and attracted to the second substrate.
[0056] In a preferred embodiment, a layer of an intelligent gel is
disposed between the substrate and the gel layer. A phase change
can be induced in the intelligent gel, e.g., to promote transfer of
the gel layer form the first substrate to the second substrate. For
example, the first substrate can be coated with a thin layer of an
intelligent gel such as described above, prior to the deposition of
the gel pads on the first substrate. When the first and second
substrates are placed into close contact, the intelligent gel can
be liquefied or otherwise modified to promote the release of the
gel. For example, for an intelligent gel, such as "Smart Hydrogel",
which liquefies at cooler temperatures, liquefaction can be
accomplished by cooling the first and/or second substrate. When the
intelligent gel is liquefied, the gel pads disposed on the
intelligent gel layer on the first substrate cannot adhere to the
first substrate, and are transferred to the second substrate.
Similarly, for other intelligent gels, the first and/or second
substrates (or selected portions thereof) can be heated, subjected
to an electric current, contacted with a solution having a high pH
or salt concentration, and the like, to liquefy or soften the
intelligent and thereby release the gel pads from the first
substrate.
[0057] In a preferred embodiment, a gel, e.g., one or more gel
pads, is transferred from a first substrate to the second
substrate, and a second gel, or second gel pad or second array of
gel pads is transferred from a third substrate to the first
substrate. Gel pads can be transferred to the second substrate in
groups, e.g., in a row or rows, or one at a time. Thus, a plurality
of first substrates can be used to transfer elements of a pattern
to a substrate.
[0058] In a preferred embodiment, substance which promotes adhesion
of the gel to the second substrate is delivered, e.g., by a piezo
dispenser, to a gel pad prior to transferring it to the second
substrate. The substance can activate sites which allow the gel to
bind to the second substrate. Alternatively, substance which
promotes adhesion of the gel to the second substrate is delivered,
e.g., by a piezo dispenser, to the second substrate prior to
transferring the pad to the second substrate.
[0059] In a preferred embodiment, a step of the method is performed
in response to a signal generated by a computer.
[0060] The invention also features multi-layered gel pad constructs
and methods of making and using them.
[0061] Accordingly, in one aspect, the invention provides a gel,
e.g., a gel pad, which includes at least two gel layers, preferably
in contact with each other, e.g., a first gel layer on which is
disposed a second gel layer, or first gel layer adjacent to and in
contact with a second gel layer. A multi-layer gel of the invention
can have two, three, four, or more layers. In a preferred
embodiment at least one of the first gel layer and the second gel
layer includes an intelligent gel.
[0062] In a preferred embodiment a first gel layer includes a first
reagent, e.g., any of a polynucleotide (e.g., a probe suitable for
performing sequencing by hybridization), a nucleic acid, e.g., DNA,
RNA, or a probe; a protein, e.g., an enzyme which modifies DNA; a
particle; a cell; or a reactant; and a second gel pad layer
includes a second reagent, e.g., any of a polynucleotide (e.g., a
probe suitable for performing sequencing by hybridization), a
nucleic acid, e.g., DNA, RNA, or a probe; a protein, e.g., an
enzyme which modifies DNA; a particle; a cell; or a reactant.
[0063] In a preferred embodiment the gel layers have different
porosities. For example, the first layer has a larger porosity than
does the second, e.g., the first layer allows free passage of a
substance, e.g., molecule, e.g., a nucleic acid molecule, or cell,
but the second layer has a porosity which, when compared with the
first, does not allow free passage of the substance. Such a second
gel layer can be disposed over and covering the first gel layer;
the second gel layer can be a gel having an effective pore size
small enough to prevent the diffusion of high-molecular-weight
substances, such as nucleic acids or proteins. The second layer
thus serves as an effective barrier to prevent diffusion of
substances, e.g., proteins, from a sample solution into the first
gel layer, or from the first gel layer into solution. The
multi-layer gel pad can prevent interference from sample
constituents, or can prevent the loss of valuable components from
the first gel layer.
[0064] In another embodiment a layered gel has a first layer having
a first ionic strength and a second layer having a second ionic
strength, e.g., a first gel layer has a relatively, low ionic
strength, e.g., an ionic strength lower than the ionic strength of
a sample solution to be applied to the gel pad array, and the
second layer has a, relative to the first layer or a solution to be
applied, a high ionic strength. The second, protective or filtering
gel layer can cover or encapsulate the first gel pad layer. The
difference in ionic strength can promote transfer of a component
into or out of the layers. E.g., the low ionic strength of the
first gel layer can promote osmotic movement of sample components
into the first gel layer, e.g., increasing the sensitivity of the
first gel layer for a sample component of interest.
[0065] In another aspect, the invention features, a gel described
herein, wherein a cell, e.g., a living cell, is disposed with in
the gel. Such gels are sometimes referred to herein as "cell
pads".
[0066] In a preferred embodiment, the gel is a multi-layered gel
described herein, having a first layer without cells, and second
layer which includes cells (e.g., bacterial or eukaryotic cells).
(Alternatively, cells can be grown on top of a gel layer, without
being immobilized within a second gel layer).
[0067] In preferred embodiments: a first gel layer not having a
cell is disposed adjacent a second, cell-containing gel layer; a
cell is immobilized in a second gel layer which encapsulates a
first gel layer.
[0068] In a preferred embodiment a cell is disposed on the surface
of a gel layer.
[0069] In preferred embodiments a first gel layer includes
detection means for detecting the presence (or absence) of a cell
constituent (such as DNA) or a product of cellular metabolism (such
as proteins, or products of transcription). The cell can be
provided in the second layer or on the surface of the first or
second layer.
[0070] In another aspect, the invention features, a method of
detecting analyte, e.g., cell constituent (such as DNA) or a
product of cellular metabolism (such as proteins, or products of
transcription). The method includes:
[0071] (1) providing a gel having a first layer which includes a
molecule for detecting the analyte and a second layer having a
cell, which, e.g., releases, produces, inactivates, modifies, or
otherwise affects the level of the analyte;
[0072] (2) detecting the analyte.
[0073] In a preferred embodiment, the first, the second, or both
layers is an intelligent gel, e.g., an intelligent gel described
herein.
[0074] In a preferred embodiment a biological molecule is attached
to the first layer, e.g., a protein or nucleic acid; the biological
molecule interacts with a second molecule, e.g., a biological
molecule, e.g., a protein that forms a multimeric complex with the
immobilized protein, e.g., a protein dimer; or a complex between
the immobilized protein and a nucleic acid molecule, e.g., single
or double stranded DNA, e.g., the nucleic acid binding site for a
transcription factor, replication factor, structural protein, e.g.,
a matrix attachment protein or a histone; the attached biological
molecule interacts with a small molecule, e.g., a drug candidate;
the protein attached to the gel contains a tag, e.g., a nucleic
acid tag, that can be used to identify the protein, e.g., by the
process of polymerase chain reaction (PCR), or by binding to a
molecule that emits a strong signal, e.g., a fluorescent
signal.
[0075] In a preferred embodiment a cell in one layer can secrete or
release molecules, such as growth factors, which can be monitored,
e.g., by the use of capture molecules in another layer of the
multi-layer gel pad.
[0076] In a preferred embodiment the cell is lysed and cellular
components measured.
[0077] In a preferred embodiment the method evaluates the response
of a cell to a stimulus, such as addition of a growth factor, a
toxin, a drug, or the like.
[0078] In a preferred embodiment the gel is configured to permit
cells in one layer (or pad) to secrete, release, or otherwise
modulate the level of a molecule which influences the growth of
other cells a second layer or pad, e.g., in an adjacent pads. Thus,
complex cell-based assays, e.g., autocrine system assays, or
developmental assays, can be reduced to microscale format. The
cells in the first and second component can be the same or
different.
[0079] In a preferred embodiment, a step of the method is performed
in response to a signal generated by a computer.
[0080] In another aspect, the invention features, a method of
detecting an analyte, e.g., cell constituent (such as DNA) or a
product of cellular metabolism (such as proteins, or products of
transcription). The method includes:
[0081] (1) providing a gel having a first layer which includes a
molecule for detecting the analyte and a second layer having a
cell, which, e.g., retains, releases, produces, inactivates,
modifies, or otherwise affects the level of the analyte;
[0082] (2) detecting the analyte.
[0083] In preferred embodiments the molecule for detecting is an
antibody or a functional variant of an antibody, e.g., an aptamer.
The antibody is either expose to the second layer or an analyte
released from the second layer. Preferably, the analyte contains an
antigen recognized by the antibody. Alternatively, the analyte acts
an antigen to antibodies that are either in solution or are present
on the second layer.
[0084] In a preferred embodiment a population of molecules are
attached to the first layer. For example, the first layer
represents and array of gel pads, e.g., intelligent gel pads, that
each are individually addressable and contain a unique population
of attached molecules. The population of molecules in each gel pad
can be either homogenous (i.e. all the same molecule), or under
certain embodiments a heterogenous population of molecules (i.e.
many different molecules). The molecules can be derived from cells,
e.g., protein or nucleic acid, or derived from chemical synthesis,
e.g., hormones or small molecules. The chemical synthesis process
may represent many combinations of molecules, e.g., a combinatorial
library of chemicals. In preferred embodiments, the gel pads
containing molecules described herein can be exposed to phage that
display a unique protein on the solvent exposed surface of the
phage, e.g., phage used in the technique called phage display. In
preferred embodiments, the phage is present in a population, where
the phage, each expressing a unique protein, as a group provides
many unique proteins on their solvent exposed surfaces. In
preferred embodiments the phage population interacts with the
molecules immobilized in the gel pads. In preferred embodiments,
the gel pads are intelligent and are present in the expanded state.
In preferred embodiments unbound phage are removed from the first
layer and any surrounding chamber. In preferred embodiments a
population of cells are presented to the first layer with a gel pad
array that contains molecules associated with a select group of
phage.
[0085] The invention also provides gel pad arrays on a flexible
support, such as a flexible tape, and methods for making and using
them, and carriers for storing gel pad arrays on tapes. The gel can
be an intelligent gel, e.g., an intelligent gel described herein.
The arrays can be used for sequencing by hybridization (e.g., where
the pads include nucleic acid strands immobilized within the gel
matrix), for cell based assays (e.g., where the pads include, or
are adjacent to and contacting, living cells), and for other uses
which will be apparent to one of ordinary skill in the art.
[0086] In one aspect, the invention provides flexible tape having a
gel pad array disposed on a surface of the tape. In preferred
embodiments: the tape comprises means for preventing compression of
gel pads when the tape is wound on a reel more preferably, the
means includes at least one ridge which extends along a length of
the tape.
[0087] In another aspect, the invention provides a carrier for a
tape having gel pad arrays thereon. The carrier includes a housing,
at least one tape reel for winding the tape, and visible or
machine-readable indicia for storing information about the tape
stored in the carrier.
[0088] In one aspect, the invention provides gel pad arrays on
flexible substrates, such as tapes, A variety of tapes can be
employed as substrates for the gel pad arrays. Preferred tapes are
biocompatible and/or compatible with test conditions, e.g., as are
used for performing assays (to avoid interference with such
assays). In addition, preferred tapes are relatively resistant to
stretching, to reduce distortion of gel pad arrays deposited on the
tape, e.g., during manufacture or storage of the tape. One
preferred material for a tape substrate is polystyrene tape, which
is commercially available from several suppliers.
[0089] A tape substrate can be transparent or translucent, and
optionally includes a magnetic coating for information storage. The
film can optionally be optically encoded.
[0090] If a tape having gel pad arrays disposed on a tape surface
is wound up, gel material could potentially be transferred from one
tape surface (e.g., the top surface) to another tape surface (e.g.,
the tape back) which is pressed against the first surface when the
tape is wound. To prevent such transfer and consequent loss of gel
pad integrity, the tape can be shaped or formed to have ridges or
other structure along the length of the tape web. For example, as
shown at the top of FIG. 1, a tape can be provided with ridges
along each edge, running along the length of the tape, to prevent
contact between a gel pad and the layer of tape which is wound
above the pad. This configuration ensures that the integrity of the
gel pad will not be disturbed during storage of the tape.
[0091] The invention also provides a carrier for tapes which
includes gel pad arrays. The carrier includes a housing, and at
least one tape reel (more preferably two reels) for winding the
tape. As shown in FIG. 5, the tape carrier can resemble a
conventional videotape housing, although the dimensions will vary
depending upon factors such as the width, thickness, and length of
the tape employed. In preferred embodiments, the housing includes a
cover for closing the carrier, to thereby exclude light, moisture,
dust, or other contaminants which could degrade the tape or the gel
pads disposed thereon. The housing can optionally include visible
or machine-readable indicia, such as a bar code or magnetic
recording stripe, for storing information (such as date of
manufacture, type of gel pad array, and the like) about the tape
stored within the enclosure.
[0092] In general, the invention features, a method of analyzing,
e.g., sequencing all or a part, e.g., a single nucleotide of, a
polynucleotide sequence in a sample. The method includes:
[0093] providing a sample which includes a polynucleotide sequence
to be analyzed;
[0094] providing an array of a plurality of capture probes, wherein
each of the capture probes is positionally distinguishable from
other capture probes of the plurality on the array, and wherein
each positionally distinguishable capture probe includes a unique
(i.e., not repeated in another capture probe) region complementary
to the plurality of selector probes and wherein the array is a gel
pad array described herein (each of the plurality of probes can be
in its own gel pad);
[0095] hybridizing the selected nucleic acid molecule with the
array of capture probes, thereby detecting or identifying a
selected nucleic acid molecule which bound to the polynucleotide
sequence and thereby analyzing the polynucleotide sequence.
[0096] In preferred embodiments the method includes one or more
enzyme mediated reactions in which a nucleic acid used in the
method, a capture probe, a sequence to be analyzed, and or a
molecule which hybridizes thereto, is the substrate or template for
the enzyme mediated reaction. The enzyme mediated reaction can be:
an extension reaction, e.g., a reaction catalyzed by a polymerase,
e.g., for DNA amplification (e.g. Polymerase Chain Reaction (PCR))
or a base extension in the presence of labeled dideoxy nucleotides
(e.g. Genetic Bit Analysis); a linking reaction, e.g., a ligation,
e.g., a reaction catalyzed by a ligase (e.g., a ligation Chain
Reaction (LCR) for DNA amplification); or a nucleic acid cleavage
reaction, e.g., a cleavage catalyzed by a restriction enzyme, e.g.,
a Type IIs enzyme, or a cleavage reaction catalyzed by a ribozyme.
The nucleic acid which hybridizes with the capture probe can be the
substrate in an enzyme mediated reaction, e.g., it can be ligated
to a strand of the capture probe or it can be extended along a
strand of the capture probe. Alternatively, the capture probe can
be extended along the hybridized selected nucleic acid. (Any of the
extension reactors discussed herein can be performed with labeled,
or chain terminating, subunits.) These reactions can be used to
increase specificity of the method or to otherwise aid in
detection, e.g., by providing a signal.
[0097] In preferred embodiments, the capture probe bound to a
target becomes the substrate for an isothermal amplification
reaction. In certain embodiments the target is an RNA molecule and
the probe is a nucleic acid primer in a process known as Nucleic
Acid Sequence-Based Amplification (NASBA), where a primer (Primer
I) attached to the RNA target is extended with a reverse
transcriptase to form a cDNA copy of the RNA target, RNase degrades
the RNA portion of the DNA:RNA hybrid to form single-stranded DNA,
a second primer (Primer 2) anneals to the DNA and is extended by
reverse transcriptase, T7 RNA polymerase recognizes the
double-stranded DNA target and produces many copies of
complementary RNA, and the process is repeated on these new
molecules of RNA. In other embodiments, the capture probe bound to
a target is used to prime a single-stranded, circular DNA molecule
in a process known as Rolling Circle Amplification (RCA), e.g., a
primer can be attached to a protein molecule that binds to a
capture probe attached to the gel pad. In certain embodiments an
endonuclease is used to nick the unmodified strand of a
hemiphosphorothioate formed at its recognition site, thereby
creating a site for nick translation by a DNA polymerase that
generates a new target, a process that is known in the art as a
Strand Displacement Assay (SDA).
[0098] In preferred embodiments the polynucleotide sequence is: a
DNA molecule: all or part of a known gene; wild type DNA; mutant
DNA; a genomic fragment, particularly a human genomic fragment; a
cDNA, particularly a human cDNA.
[0099] In preferred embodiments the polynucleotide sequence is: an
RNA molecule: nucleic acids derived from RNA transcripts; wild type
RNA: mutant RNA, particularly a human RNA.
[0100] In preferred embodiments the polynucleotide sequence is: a
human sequence; a non-human sequence, e.g., a mouse, rat, pig,
primate.
[0101] In preferred embodiments the selector probes are coupled to
a support prior to hybridizing with the target.
[0102] In preferred embodiments the selector or capture probes are
coupled to a light transmissable support, e.g., an optical fiber or
fiber optic rod. The fiber optic rod may contain a single selector
or capture probe or an array of such probes. In preferred
embodiments the selector or capture probes are attached to a gel,
e.g., an intelligent gel. The
[0103] In preferred embodiments the method is performed: on a
sample from a human subject; and a sample from a prenatal subject;
as part of genetic counseling; to determine if the individual from
which the target nucleic acid is taken should receive a drug or
other treatment; to diagnose an individual for a disorder or for
predisposition to a disorder; to stage a disease or disorder.
[0104] In preferred embodiments the capture probes are single
stranded probes in an array.
[0105] In preferred embodiments the capture probes have a structure
comprising a double stranded portion and a single stranded portion
in an array.
[0106] In preferred embodiments, the capture probe forms a double
stranded region with the target that contains a DNA base pair
mismatch, e.g., a bubble in the double stranded DNA or an
intrastrand secondary structure, that is the subtrate for an
enzymatic reaction, e.g., an enzymatic reaction mediated by
resolvase or mutS or cleavase I, with the product of the first
enzymatic reaction acting as the substrate for a second enzymatic
reaction, e.g., a reaction with DNA polymerase in the presence of a
nucleotide termoinator, e.g., a dideoxy nucleotide labeled with a
fluorescent dye. Diagnostic assays incorporating some or all of
these steps are known in the trade as enzymatic Mutation Detection
(EMD) or Cleavase Fragment Length Polymorphism (CFLP).
[0107] In preferred embodiments hybridization is detected by mass
spectrophotometry, e.g., by MALDI-TOF mass spectrophotometry.
[0108] In preferred embodiments hybridization is detected by a
signal amplification molecule, e.g., hyperbranch chain DNA (bDNA);
e.g. bDNA that binds to a probe linked to an enzyme that generates
a colorimetric, fluorescent or proximity detection assay, e.g.,
probes linked to alkaline phosphatase. In certain embodiments the
target to be amplified first binds to a branched DNA molecule
called a capture extender that binds, in turn, to the bDNA which,
in turn, binds to alkaline phoshatase probes. In other embodiments,
the probe is a fluorescently labeled DNA molecule that is known to
those skilled in the art as a molecular beacon.
[0109] In a preferred embodiment, a step of the method is performed
in response to a signal generated by a computer.
[0110] In another aspect, the invention features, a method of
performing a reaction. The method includes
[0111] providing a first reactant, e.g., a protein, e.g., an
enzyme, disposed within a first intelligent gel which changes
porosity in response to an environmental parameter having a first
value, e.g, a first temperature, ionic strength, or pH;
[0112] providing a second reactant, e.g., a protein, e.g., an
enzyme, disposed within a second intelligent gel which changes
porosity in response to an environmental parameter having a second
value, e.g, a second temperature, ionic strength, or pH;
[0113] exposing the intelligent gels to the parameter at a first
value, thereby causing a change in porosity of the first gel (and
preferably not the second gel), and thereby modulating exposure of
the first reactant to a target, e.g., a molecule or a cell or other
biological structure;
[0114] exposing the intelligent gels to the parameter at a second
value, thereby causing a change in porosity of the second gel (and
preferably not the first gel), and thereby modulating exposure of
the second reactant to a target,
[0115] thereby performing a reaction.
[0116] In a preferred embodiment, the first exposure and the second
exposure are performed: sequentially or simultaneously.
[0117] In a preferred embodiment, the first exposure and or the
second exposure is repeated.
[0118] Targets can be disposed in intelligent gels and their
release controlled analagously to reactant release, thus in a
preferred embodiment a first target is disposed in a first target
intelligent gel. In a preferred a second target is disposed in a
second target intelligent gel.
[0119] In a preferred embodiment a target gel and a reactant gel (a
target-reactant pair) are disposed such that upon modulation of a
parameter which causes one or more of the gels to change volume,
the target gel and reactant gel are brought into or out of
proximity or contact, thus modulating the ability of target and
reactant to come into contact. In preferred embodiments a plurality
of such target-reactant pairs are provided. They can be
individually activated by choosing intellingent gels which respond
at different parameter values, e.g., different temperatures.
[0120] Members of a target-reactant pair can be on the same surface
or on different surfaces. When on two different surfaces the
surfaces can be manipulated to bring the pair closer or more
distant to one another.
[0121] A target-reactant pair can be physically separated, such
that molecules released form it do not interact with other
target-reactant pairs.
[0122] Other embodiments are within the clams and the following
description.
DETAILED DESCRIPTION
BRIEF DESCRIPTION OF THE DRAWINGS
[0123] FIG. 1 depicts gel pads of the invention which include
living cells.
[0124] FIG. 2 depicts apparatus for preparing gel pads and
transferring the pads to a substrate to form an array of gel
pads.
[0125] FIG. 3 depicts a system for manufacturing and testing a tape
substrate with gel pads disposed thereon.
[0126] FIG. 4 depicts an imager for use in quality assurance of a
tape substrate having gel pads deposited thereon.
[0127] FIG. 5 depicts a tape and a tape carrier.
[0128] This invention provides gel pads and gel pad arrays having a
variety of uses, some of which are known in the art. The invention
also provides methods for making the gel pads and gel pad arrays of
the invention.
[0129] Gel Pads and Gel Arrays
[0130] The term "gel pad" as used herein refers to a discrete
portion of a gel disposed on a substrate, such as a solid support,
e.g., a plastic, glass, or metal substrate. The substrate can be
any support suitable for supporting a gel pad, and can be rigid
(e.g., a glass or plastic plate or sheet) or flexible (e.g., a
tape), transparent (e.g., for performing optical measurements
through the pad and substrate) or opaque. The properties of the
support can be readily selected for use in any particular
application. In preferred embodiments, the solid support is
substantially non-reactive under conditions used to perform an
assay or test procedure with the gel pad or gel pad array. An
"array" can be any pattern of spaced-apart gel pads disposed on a
substrate; arrays can be conveniently provided in a grid pattern,
but other patterns can also be used. In preferred embodiments, a
gel pad array according to the invention includes at least about 10
gel pads, more preferably at least about 50, 100, 500, 1000, 5000,
or 10000 gel pads. In certain embodiments, the array is an array of
gel pads of substantially equal size, thickness, density, and the
like, e.g., to ensure that each gel pad behaves consistently when
contacted with a test mixture. In certain embodiments, however, the
pads of a gel pad array can differ from one another; e.g., a mixed
gel pad array can be constructed which includes more than one size
or type of gel pad, e.g., gel pads made of different gel materials,
or which entrap different species such as reagents or
polynucleotide probes. In certain preferred embodiments, gel pads
in an array are less than about 1 mm in diameter (or along a side,
e.g., in the case of square gel pads), more preferably less than
about 500 microns, still more preferably less than about 100, 75,
50, 25, 10, 5, 1, or 0.1 micron in diameter.
[0131] A gel pad can have any convenient dimension for use in a
particular assay. In preferred embodiments, a gel pad is thin
enough, and porous enough, to permit rapid diffusion of at least
certain reaction components into the gel pad when a solution or
suspension is place din contact with the gel pad. For example, in
one embodiment, a gel pad array for use in sequencing by
hybridization permits polynucleotide fragments from a sample
mixture to diffuse (within a conveniently short time period) into
the gel pads and hybridize to oligonucleotide capture sequences
disposed within the gel pads. In certain preferred embodiments, a
gel pad (e.g., in an array of gel pads) has a thickness of at least
about 0.1, 1, 5, 10, 20, 30, 40, 50 or 100 microns. In certain
preferred embodiments, a gel pad (e.g., in an array of gel pads)
has a thickness of less than about 1 millimeter, 500 microns, 200,
100, 50, 40, 30, 20, 10, 5, 1 or 0.1 microns.
[0132] It will be appreciated from the foregoing that a gel pad can
entrap additional chemical species, if desired, e.g., to perform
assays with or within the gel pad. For example, gels which include
DNA probes have been used for SBH (for example, U.S. Pat. No.
5,552,270 to Khrapko et al. and U.S. Pat. No. 5,525,464 to Drmanac
et al.). Thus, a gel pad can be prepared such that a chemical
species is trapped within the gel pad, or a desired species can be
added after the gel pad has been prepared. e.g., by contacting a
preformed gel pad with a solution of the reagent and allowing the
reagent to diffuse into the gel pad. Examples of reagents which can
be entrapped, suspended or dissolved in a gel pad include proteins,
such as enzymes (e.g., ligases, which can be useful for positional
SBH (see, e.g., Cantor, U.S. Pat. Nos. 5,503,980 and 5,631,134)),
polynucleotides, growth factors (e.g., for use with cells, e.g.,
see infra), salts and the like.
[0133] Derivatized Gels
[0134] Gels described herein can be made with derivatized subunits,
e.g., subunits with are coupled to a molecule of interest. A gel
component, e.g., layer or pad (e.g., in an array) can be prepared
through the use of a derivatized monomer unit, followed by
formation of the gel component by polymerization of the monomer.
For example, acrylic acid can be readily derivatized with a
polynucleotide (e.g., a probe for use in SBH); for example, a
polynucleotide can be coupled to acrylic acid through the use of a
conventional coupling reagent such as dicyclohexylcarbodiimide
(DCC) (or a water-soluble derivative thereof such as
1-(3-dimethylaminopropyl)-- 3-ethylcarbodiimide, EDC).
Alternatively, amino or aldehyde groups in the gel can couple to
oligonucleotides bearing aldehyde or amino groups, respectively, in
the presence of a reducing agent, e.g., as described in Timofev et
al., 1996, Nucleic Acids Research, 24:3142-3148. A spacer or linker
moiety can be used to increase the distance between the acrylate
monomer and the polynucleotide, if desired, e.g., to increase
mobility of the polynucleotide in the polymer). The resulting
acrylic ester of the polynucleotide can then be disposed in an
array format on a substrate, e.g., by dispensing a solution of the
acrylic ester through a nozzle or array of nozzles (such as
conventional piezoelectric inkjet printing nozzles; see also Patent
Cooperation treaty Publication WO95/04594). Alternatively, an array
format can be provided by using a cast or mold. The array of
droplets, e.g., dispensing to a mold containing an array of voids,
is then polymerized in situ to provide an array of gel pads which
incorporate a polynucleotide covalently bound to the gel
polymer.
[0135] Gel Arrays Using Intelligent Gels
[0136] In one aspect, the invention provides methods for making gel
pads and gel pad arrays. In certain preferred embodiments, gel pads
and gel pad arrays can be conveniently prepared by use of
"intelligent gels."
[0137] An intelligent gel, as used herein, can be a gel having an
internal lattice which defines pores in the gel structure. In
preferred embodiments the lattice is covalently stabilized. An
intelligent gel can exist in two states: a first state in which
pore size is, relative to that of the second state, small, and a
second state in which the pore size is, relative to the first
state, large. Although the pore size changes between states, the
lattice maintains its integrity and the pore-phase change is
generally reversible. Intelligent gels can fall into one of two
major classes, "Expandable intelligent gels," see, Li and Tanaka
1990, J. Chem Phys, 92:1365; Matsuo and Tanaka, 1988, J. Chem Phys,
89:1695-1703; Matsuo and Tanaka, 1992, Nature, 358:482; Kokutata et
al., 1991, Nature, 351:302; and Annaka and Tanaka, 1992, Nature,
335:430, in which pore seize change is accompanied by a change in
the volume of the gel, and "lattice constant" intelligent gels in
which pore size change is accompanied by a change in internal
condensation, see Tokita and Tanaka, 1991, Science, 253:1121-1123.
See, also e.g., Kajiwara et al., "Synthetic Gels on the Move",
Nature, vol. 355, pp. 208-209 (1992); Kwon et al., "Electrically
Erodible Polymer Gel for Controlled Release of Drugs", Nature, vol.
354, pp. 291-293 (1991); Suzuki et al., "Phase Transition in
Polymer Gels Induced by Visible Light", Nature, vol. 346, pp.
345-347 (1990); Osada et al., "Intelligent Gels", Scientific
American, pp. 82-87 (1993); R. Dagani, "Intelligent Gels," Chem.
Eng. News., Jun. 9, 1997).
[0138] In a preferred embodiment, the intelligent gel has an
expanded pore state and a minimized pore state. The expanded pore
state will allow passage of a molecule which is up to 5, 10, 50,
100, 500 or 1,000 times the molecular weight of the largest
molecule which is allowed passage by the minimized pore state. In a
preferred embodiment the gel allows, e.g., when the pores are
expanded, passage of molecules (or particles) of at least 0.1, 0.5,
1, 5, 10, 50, 100, 200, 500, or 1,000 kilodilations to enter the
gel.
[0139] Expandable intelligent gels undergo an isotropic swelling
and shrinking process where the gel either expands or contracts
equally in both length and width. The process of swelling and
shrinking can be either continuous or discontinuous, based on the
balance between molecular forces, e.g., electrostatic, osmotic,
hydrophobic, Van der Waals, hydrogen bonding and ion-ion
interactions. Examples of intelligent gels include gels which
become softer or firmer (e.g., solidify or liquefy) in response to
changes in temperature, salt concentration (e.g., ionic strength),
pH, exposure to radiation (e.g., ultraviolet (UV) radiation),
presence or absence of a selected metal ion, electrical current,
magnetic field, and the like. For example, a copolymer of
poly(acrylic acid) and poly(N-isopropylacrylamide) has been
reported to be temperature-sensitive, swelling at lower
temperatures and collapsing at higher temperatures (Tanaka et al.,
Faraday Discuss. 101:201 (1995)). One of ordinary skill in the art
will be able to select an intelligent gel with the desired
properties for a selected application using no more than routine
experimentation. In certain preferred embodiments, an intelligent
gel for use in the present invention is responsive (e.g.,
liquefies) in response to an increase in temperature or irradiation
with ultraviolet light.
[0140] Examples of suitable intelligent gels include:
[0141] I. N-ackylacrylamide group, e.g., N-isopropylaerylamide and
N,N-Diethylaerylamide.
[0142] II. Independent interpenetrating polymer networks (IPNs) in
which one cross-linked network is intertwined with another, e.g.,
poly(acrylic acid) and poly(N,N-Dimethylacrylamide), or
poly(ethylene oxide) and poly (N-Acryloylpyprolidine).
[0143] The IPNs are particularly suitable for pulsated gels pads
(gel pads that exhibit rapid expansion/contraction cycles).
[0144] Gels can be liquefied at ambient temperature (25.degree. C.)
and solid at higher (i.e., body) temperatures (37.degree. C.).
[0145] Other intelligent gels are described, e.g., in Bromberg and
Ron, 1998, Advanced Drug Delivery Reviews, 31:197-221; Schild,
1992, Prog. Polym. Sci. 17:163-249; Irie, 1993, Adv. Polym. Sci.
110:49-65; Okano, 1993, Adv. Polym. Sci., 110:179-200; Sen et al.,
1998, Polymer, 40:913-917. Also useful are bilayer membrane gels,
e.g.; as described in Tsujii et al., 1997, Macromolecules,
30:7397-74029; and Hayakawa et al., 1997, Langmuir, 13:3595-3597;
intelligent gels that can recognize and recover molecules, e.g., as
described in Tanaka et al., 1996, Faraday Discuss., 102-206; and
Umeno et al., 1998, Bioconjugate Chem, 9:719-724; intelligent gels
that can function as a detachable cell culture substrate, e.g., as
described in von Reum et al., 1998, J. Biomed Matter Res,
40:631-639; Intelligent gels used for drug delivery that are
comprised, e.g., of block co-polymers of poly(ethylene) oxide and
poly(propylene) oxide, otherwise known in the trade as Pluronics or
Poloxamers, e.g., as described in Alexandridis and Hatton, 1995,
Colloid Surfaces A, 96:1-46; and Wang and Johnson, 1991, J. Appl.
Polym. Sci., 43: 283-292; intelligent gels attached to a fibor
optic rod, e.g., as described in McCurley, 1994, Biosensors &
Bioelectronics, 9: 527-533; polymerized colloidal crystal hydrogels
used as chemical sensors, e.g., as described in Holtz and Asher,
1997, Nature, 389: 829-832; intelligent gels used to encapsulate
proteins, e.g., as described in Serres et al., 1996, Pharm Res,
13:196-201; and Baudys et al., 1996, Drug Delivery Systems,
Springer, Tokyo, pp 112-115.
[0146] In an illustrative embodiment, gel, e.g., an intelligent
gel, can be used to prepare a gel pad array. The gel pads can
comprise an intelligent gel, or the intelligent gel can be used as
a form or mold to prepare a gel pad array. For example, in one
embodiment, a gel which liquefies in response to UV irradiation is
cast is in a thin film on a substrate such as a glass plate. The
gel can incorporate reagents, such as polynucleotide probes for
capturing fragments of DNA from a solution; alternatively, such
reagents can be added after the array has been formed. The gel is
allowed to cool and solidify. The gel layer is then masked, e.g.,
with a mask such as is conventionally used in photolithography; the
mask protects gel portions in an array configuration on the
substrate (e.g., a 100.times.100 array of gel pads). The masked gel
layer is exposed to ultraviolet light. The exposed portions of the
gel liquefy and are poured off or washed off with a suitable
solvent, without disturbing the array. After irradiation and
removal of the mask, an array of gel pads is obtained.
[0147] Examples of the use of photolithographic masks in the
generation of arrays of gel, e.g., as described in Guschin et al.,
1997, Analytical Biochemistry, 250:203-211.
[0148] Alternatively, a gel, e.g., an intelligent gel can be used
as a mold or form for preparing a gel pad array. A gel which is
temperature-responsive is cast on a substrate. The gel layer is
then exposed to a laser, which is rasters over the gel layer and
irradiates selected gel portions in the configuration of an array
(see also Patent Cooperation Treaty Publication WO95/04834). The
portions of the gel pad which are exposed to the laser source are
heated and become liquefied; the liquefied portions are removed,
e.g., by gentle washing. (The gel layer could be selectively heated
by other means, such as an array of heated wires or probes which
are brought near to, or into contact with, the surface of the gel
layer.) The gel layer now has an array of "holes" formed by removal
of the gel portions exposed to the laser source. These "holes" can
be filled with a second gel (which can be a different intelligent
gel or a conventional gel, such as polyacrylamide); the second gel
is permitted to solidify, forming an array of gel pads within the
intelligent gel layer. The slide is then heated (e.g., by placing
the substrate in a warming bath or a warming oven) to liquefy the
intelligent gel layer, which is then removed by washing or pouring
off the liquefied material. An array of gel pads remains on the
substrate and can be further processed, if desired.
[0149] It will be appreciated that the methods of using intelligent
gels to prepare gel pad arrays will have many applications. The
mild conditions employed can be tailored to the preparation of a
wide variety of intelligent and conventional gel pad arrays,
preferably without degradation of sensitive reagents, such as
polynucleotide probes, which may be present in the gel layer.
Methods for preparing gel pads, e.g., such as conventionally known
or described herein, can be combined, if desired.
[0150] Furthermore, the use of intelligent gels in gel pad arrays
provides additional advantages. For example, an intelligent gel pad
can be provided which swells in response to a change, such as the
presence of an analyte of interest. For example, an intelligent gel
which swells in response to pH changes in provide in a gel pad on a
support. The gel pad includes glucose oxidase. The reaction of
glucose oxidase with glucose produces gluconic acid, lowering the
pH of the gel. Thus, in the presence of glucose in a sample
solution which is brought into contact with the gel pad, the gel
pad will shrink. A gel pad can be provided adjacent to a
piezocrystal, such that changes in gel pad swelling produce a
piezoelectric signal, which can be detected and correlated with the
glucose concentration.
[0151] Gel pad arrays can also be prepared by treating the surface
of the substrate to create a pattern of alternating hydrophobic and
hydrophilic sites on the surface. For example, a glass surface can
be silated with a conventional silating reagent to prepare a
patterned surface having hydrophobic and hydrophilic portions. A
gel, such as an intelligent gel, is then poured onto the surface. A
hydrophobic gel will be repelled by a hydrophilic surface, while a
hydrophilic gel will be repelled by a hydrophobic surface. A
patterned surface can be used to urge the liquefied gel into a
pre-selected pattern on the substrate, thereby forming a gel pad
array.
[0152] Another method for preparing a gel pad array comprises
preparing individual gel pads, or sub-arrays of gel pads, on a
first substrate, and then transferring the individual gel pads to a
second substrate, in array format, to prepare a gel pad array on
the second substrate. This method can be used to substantially
avoid covalent attachment of the gel pad to the second substrate.
In addition, the gel pads prepared on the first substrate can be
examined to ensure quality of the individual gel pads, and faulty
gel pads (e.g., of the wrong shape or size) can be removed before
the final array is prepared on the second substrate. This procedure
can prevent the formation of arrays which contain faulty or
non-standard gel pads. Moreover, the gel pads can be further
processed (e.g., washed, imparted with an additional component such
as a protein, and the like) prior to transfer of the gel pads from
the first substrate to the array format on the second
substrate.
[0153] For example, gel pads can be prepared on a first substrate,
such as a tape, and then transferred to a second substrate, such as
a glass or plastic plate, in an array format, to provide a gel pad
array on the second substrate. The gel pads can be transferred by
contacting the first and second substrates, e.g., by pressing the
first substrate against the second substrate, such that the gel
pads are transferred from the first substrate to the second
substrate. The transfer can be facilitated by making using first
and second substrates which have different surfaces, e.g., a
hydrophobic first substrate and a hydrophilic second substrate; in
this example, a hydrophilic gel pad will be more adherent to the
second substrate and will be transferred from the first substrate
to the second substrate when the two substrates are pressed
together. Alternatively, gel pads can be modified prior to the
transfer process by a reagent, e.g., a reagent dispensed from a
piezo-electric fluidic dispensing robot, said reagent creating a
change in the gel pad in the tape, e.g., activating chemical groups
present in the gel pad on the first substrate such that the gel pad
binds to the second substrate.
[0154] The transfer can be facilitated in other ways. For example,
the gel pad can be electrically charged, and the electric charge of
the first and/or second substrate can be adjusted such that the gel
pad is repelled from the first substrate and attracted to the
second substrate. In another embodiment, an intelligent gel can be
employed to facilitate the transfer. For example, the first
substrate can be coated with a thin layer of an intelligent gel
such as described above, prior to the deposition of the gel pads on
the first substrate. When the first and second substrates are
placed into close contact, the intelligent gel can be liquefied.
For example, for an intelligent gel, such as "Smart Hydrogel",
which liquefies at cooler temperatures, liquefaction can be
accomplished by cooling the first and/or second substrate. When the
intelligent gel is liquefied, the gel pads disposed on the
intelligent gel layer on the first substrate cannot adhere to the
first substrate, and are transferred to the second substrate.
Similarly, for other intelligent gels, the first and/or second
substrates (or selected portions thereof) can be heated, subjected
to an electric current, contacted with a solution having a high pH
or salt concentration, and the like, to liquefy or soften the
intelligent and thereby release the gal pads from the first
substrate and adhere the pads to the second substrate.
[0155] In one exemplary embodiment of this method of the invention,
illustrated in FIG. 2, a system for creating gel pads on a first
substrate and transferring the pads to an array format on a second
substrate includes tape winding reels 10, 12 between which a tape
15 (e.g., a polystyrene tape) (first substrate) is passed. The tape
can optionally be used for information storage, e.g., by coating
with a conventional magnetic oxide layer. As the tape passes from
the first tape reel 10, it is guided along a tape path by guide
wheel 20, past a gel pad dispenser (e.g., a nozzle, not shown,
which deposits a solution of the gel monomer, which can be
polymerized in situ) which deposits gel pads along the tape 15. The
gel pads can optionally be washed, e.g., by spraying the pads with
a buffer solution followed by air-drying, if desired. As the tape
moves beyond the gel pad dispenser, each pad can be assayed to
ensure the quality of the pads deposited (see infra and FIGS. 3 and
4). Defective pads are noted and the location stored and tracked,
e.g., in a computer memory. The tape passes over a
temperature-controlled head 30, which can be heated (or cooled)
according to the properties of the gel pad employed. The tape head
is controlled by a microcomputer which first ensures that the gel
pad currently under the head is not defective; defective pads are
passed unchanged over the tape head and toward tape takeup reel 12,
where they are collected. If the gel pad under the tape head is of
acceptable quality, the microcomputer positions the tape head over
the second substrate 40 (or moves the second substrate 40 under the
tape head) to the correct location for the next pad in the array on
second substrate 40. The tape head is then urged against second
substrate 40 and the temperature is changed (e.g., the tip of the
tape head is heated). In response to the change in temperature, the
gel pad is dissociated from the tape 15 as the tape head is pressed
against the surface of a second substrate 40, and the gel pad is
transferred to the substrate 40. Used tape is collected past guide
wheel 22 to takeup reel 12, and can be cleaned (e.g., to remove
defective pads and any residue) and reused for further array
preparations.
[0156] Gel pads can be transferred from the first substrate in
groups, e.g., in a row or rows, rather than one at a time, as
illustrated above. Furthermore, the second substrate can also be a
tape, rather than a rigid substrate; in this case, the second
substrate tape could be urged against the first (tape) substrate by
means of a roller or other tape transport mechanism. It will also
be appreciated by the ordinarily skilled artisan, in light of the
disclosure herein, that systems such as described herein can be
used to effect the transfer of arrays of nucleotides (including,
but not limited to DNA, RNA, and peptide nucleic acids) from one
substrate to another, with or with concomitant transfer of a gel
pad. Thus, for example, DNA can be transferred from one substrate
to another, e.g., electrostatically, as described above.
[0157] A manufacturing system for preparing a first (tape)
substrate with gel pads deposited thereon is shown in FIG. 3. The
system includes tape reels which serve to pass tape, at the urging
of a precision stepper, under a dispenser, which provides pads on
the first substrate. The pads are polymerized at a polymerization
station, followed by washing at a wash station to remove
impurities, unpolymerized monomers, and the like. The QA station
can include a spectrophotometric instrument for determining the
size, shape, and quality of the pads deposited on the substrate. In
one embodiment, a charge-coupled device camera can be used to
detect fluorescence, e.g., in response to a laser source, of a
fluorescent molecule which is incorporated in the gel layer (and
can optionally be removed at a later processing step, e.g., a
second washing station). An exemplary imaging system is shown in
more detail in FIG. 4. If a magnetic layer is included in the tape
substrate, the tape can be encoded with information such as date of
manufacture, location of defective pads, and the like. The tape is
taken up on a take-up reel and stored for later transfer of the
pads to an array on a second substrate, e.g., as described above.
To prevent destruction of the pads as the tape is wound on the
take-up reel, the tape can be ridged, for example as shown in the
upper inset of FIG. 2, to prevent crushing of the pads.
[0158] In another embodiment, the first substrate can be a roller,
e.g., a cylindrical element. Gel pads can be provided on the
surface of the roller as the roller is rotated by a motor, the gel
pads on the roller can be washed and assayed as described above.
The gel pads arc then transferred from the roller surface to the
second substrate (which could be rigid or a flexible tape) as
described above. After the gel pads are transferred to the second
substrate, the roller surface as it rotates preferably passes
through a cleaning apparatus. The roller surface can then be
cleaned with each revolution to prevent contamination of gel pads
with residue from preceding preparations. This system
advantageously can provide continuous, rather than batch,
operation.
[0159] The invention also provides multi-layered gel pad
constructs. For example, in one aspect, the invention provides a
gel pad which comprises at least two gel layers in contact with
each other, e.g., a first gel layer on which is disposed a second
gel layer, or first gel layer adjacent to and in contact with a
second gel layer. A multi-layer gel pad of the invention can have
two, three, four, or more layers, although greater numbers of
layers will generally require more effort to prepare. The
multi-layer gel pads of the invention can be configured to provide
a variety of functions. For example, a first gel pad layer can
include a polynucleotide (e.g., a probe for performing SBH) within
the first gel matrix. A second gel layer can be disposed over and
covering the first gel layer-, the second gel layer can be a gel
having an effective pore size small enough to prevent the diffusion
of high-molecular-weight substances, such as proteins. The second
layer thus serves as an effective barrier to prevent diffusion of
substances, e.g., proteins, from a sample solution into the first
gel layer, or from the first gel layer into solution. The
multi-layer gel pad can prevent interference from sample
constituents, or can prevent the loss of valuable components from
the first gel layer.
[0160] In another embodiment, a first gel layer can be formed with
low ionic strength, e.g., an ionic strength lower than the ionic
strength of a sample solution to be applied to the gel pad array. A
second, protective or filtering gel layer covers and encapsulates
the first gel pad layer. The low ionic strength of the first gel
layer promotes osmotic movement of sample components into the first
gel layer, thereby increasing the sensitivity of the first gel
layer for a sample component of interest.
[0161] A multi-layer gel pad can be constructed by methods known in
the art for the preparation of single-layer gel pads, or by the
methods described herein. It will be appreciated that in certain
embodiments, it is preferred to maintain registration between the
layers of the multi-layer gel pad, e.g., in certain embodiments, it
is preferred to place a second gel layer directly atop a first gel
layer. The use of a mold or form can be useful in this embodiment,
because molds can provide good registration between layers. A
particularly useful method for preparing a multi-layer gel pad
array is the intelligent gel "molding" or "forming" layer
methodology described above.
[0162] In still another embodiment, the invention provides gel pads
which include living cells (referred to herein as "cell pads"). In
one embodiment, the gel pad of the invention is a multi-layered gel
pad, having a first layer without cells, and second layer which
includes cells (e.g., bacterial or eukaryotic cells).
(Alternatively, cells can be Frown on top of a gel layer, without
being immobilized within a second gel layer). Exemplary embodiments
are shown in FIG. 1. FIG. 1A depicts a first gel layer disposed
adjacent a second, cell-containing gel layer. FIG. 1B depicts cells
immobilized in a second gel layer which encapsulates a first gel
layer. FIG. 1C shows cells maintained on the surface of a gel
layer. The cells can be maintained in culture. This embodiment,
provides a useful assay format for performing cell based assays in
an array format. For example, the first gel layer could include
detection means for detecting the presence (or absence) of a cell
constituent (such as DNA) or a product of cellular metabolism (such
as proteins, or products of transcription). For example, the cells
in one layer can secrete molecules, such as growth factors, which
can be monitored by the use of capture molecules in another layer
of the multi-layer gel pad. The cells can also be lysed and
cellular components measured. Thus, the response of the cells to a
stimulus, such as addition of a growth factor, a toxin, a drug, or
the like, can be monitored in a convenient and easily handled
format.
[0163] Cell pads can also be configured to permit cells in one pad
to secrete molecules which influence the growth of other cells in
adjacent pads, e.g., an autocrine system. Thus, complex cell-based
assays can be reduces to microscale format.
[0164] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the following claims.
[0165] The contents of all publications and patent applications
described herein are hereby incorporated by reference.
[0166] Other embodiments are within the following claims.
* * * * *