U.S. patent application number 10/131426 was filed with the patent office on 2002-08-22 for hydrogels and hydrogel arrays made from reactive prepolymers crosslinked by [2 + 2] cycloaddition.
This patent application is currently assigned to Motorola, Inc.. Invention is credited to Beuhler, Allyson, McGowen, John.
Application Number | 20020115740 10/131426 |
Document ID | / |
Family ID | 26807404 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115740 |
Kind Code |
A1 |
Beuhler, Allyson ; et
al. |
August 22, 2002 |
Hydrogels and hydrogel arrays made from reactive prepolymers
crosslinked by [2 + 2] cycloaddition
Abstract
Reactive prepolymers incorporating [2+2] photoreactive sites are
synthesized. Upon exposure to UV light, these prepolymers undergo
[2+2] cycloaddition to crosslink. When crosslinked, the reactive
prepolymers form a hydrogel. Selective hydrogel formation is
provided through selective exposure of the reactive prepolymer to
UV light. Supports and other molecules may be attached or
incorporated into the hydrogel through [2+2] cycloaddition with
uncrosslinked [2+2] photoreactive sites present in the
hydrogel.
Inventors: |
Beuhler, Allyson; (Downers
Grove, IL) ; McGowen, John; (Crystal Lake,
IL) |
Correspondence
Address: |
Jonathan Blanchard
c/o Brinks Hofer Gilson & Lione
P.O. Box 10395
Chicago
IL
60610
US
|
Assignee: |
Motorola, Inc.
|
Family ID: |
26807404 |
Appl. No.: |
10/131426 |
Filed: |
April 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10131426 |
Apr 23, 2002 |
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09344217 |
Jun 25, 1999 |
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6391937 |
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60109821 |
Nov 25, 1998 |
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Current U.S.
Class: |
522/152 |
Current CPC
Class: |
B01J 2219/00644
20130101; C08F 8/00 20130101; C08F 8/00 20130101; B01J 2219/00722
20130101; B01J 2219/00659 20130101; C08F 20/54 20130101; B01J
2219/00621 20130101; B01J 2219/00637 20130101; Y10T 428/31725
20150401 |
Class at
Publication: |
522/152 |
International
Class: |
C08J 003/28 |
Claims
What is claimed:
1. A reactive prepolymer comprising a copolymer of a first monomer
and a 2+2 photocyclizable monomer.
2. The reactive prepolymer of claim 1, wherein said copolymer
comprises between one and about 50 said first monomers for each
said 2+2 photocyclizable monomer.
3. The reactive prepolymer of claim 1, wherein said copolymer
comprises between about 10 and about 20 said first monomers for
each said 2+2 photocyclizable monomer.
4. The reactive prepolymer of claim 1, wherein said reactive
prepolymer has the structure: 6wherein x is an integer from 1 to
50; y is an integer from 1 to 50; and R is a moiety that comprises
a 2+2 photoreactive site.
5. The reactive prepolymer of claim 4, wherein x is between about
10 and about 20 and y is 1.
6. The reactive prepolymer of claim 4, wherein x is about 15 and y
is 1.
7. The reactive prepolymer of claim 1, wherein the first monomer is
a monomer that is water-soluble and can undergo chain-type
polymerization.
8. The reactive prepolymer of claim 7, wherein the first monomer is
selected from the group consisting of acrylamide, hydroxyethyl
acrylate, vinyl pyridine, acrylic acid, methacrylic acid, and vinyl
pyrrolidone, or mixtures thereof.
9. The reactive prepolymer of claim 7, wherein the first monomer is
acrylamide.
10. The reactive prepolymer of claim 1, wherein the 2+2
photocyclizable monomer comprises a 2+2 photoreactive site and a
polymerizable functionality.
11. The 2+2 photocyclizable monomer of claim 10, wherein a spacer
separates the polymerizable functionality and the 2+2 photoreactive
site.
12. The reactive prepolymer of claim 1, wherein the 2+2
photocyclizable monomer is selected from the group consisting of
N-(6-acryloylhexyl)-2,3-- dimethylmaleimide and vinyl cinnamate, or
mixtures thereof.
13. The reactive prepolymer of claim 1, wherein the 2+2
photocyclizable monomer comprises a second monomer and a 2+2
photocyclizable compound.
14. The reactive prepolymer of claim 13, wherein the second monomer
is selected from the group consisting of acrylic acid, glycidyl
methacrylate, and methacrylic acid, or mixtures thereof.
15. The reactive prepolymer of claim 13, wherein the second monomer
is glycidyl methacrylate.
16. The reactive prepolymer of claim 13, wherein the second monomer
is acrylic acid.
17. The reactive prepolymer of claim 13, wherein the 2+2
photocyclizable compound is selected from the group consisting of
glycidyl methacrylate, acrylic acid, hydroxyethyl acrylate,
hydroxypropyl acrylate, and acryloyl halides, or mixtures
thereof.
18. The reactive prepolymer of claim 15, wherein the 2+2
photocyclizable compound is acrylic acid.
19. The reactive prepolymer of claim 16, wherein the 2+2
photocyclizable compound is glycidyl methacrylate.
20. The reactive prepolymer of claim 1, wherein the 2+2
photocyclizable monomer is prepared by forming a copolymer of the
first monomer with a second monomer, and subsequently, condensing a
2+2 photocyclizable compound with the copolymer.
21. The reactive prepolymer of claim 20, wherein the second monomer
is selected from the group consisting of acrylic acid, glycidyl
methacrylate, and methacrylic acid, or mixtures thereof.
22. The reactive prepolymer of claim 20, wherein the 2+2
photocyclizable compound is selected from the group consisting of
glycidyl methacrylate, acrylic acid, hydroxyethyl acrylate,
hydroxypropyl acrylate, and acryloyl halides, or mixtures
thereof.
23. A hydrogel formed by an ultraviolet irradiation of the reactive
prepolymer of claim 1.
24. A hydrogel formed by an ultraviolet irradiation of the reactive
prepolymer of claim 2.
25. A hydrogel formed by ultraviolet irradiation of the reactive
prepolymer of claim 8.
26. A hydrogel formed by ultraviolet irradiation of the reactive
prepolymer of claim 10.
27. A hydrogel formed by ultraviolet irradiation of the reactive
prepolymer of claim 12.
28. A hydrogel formed by ultraviolet irradiation of the reactive
prepolymer of claim 13.
29. A hydrogel formed by ultraviolet irradiation of the reactive
prepolymer of claim 18.
30. A hydrogel formed by ultraviolet irradiation of the reactive
prepolymer of claim 19.
31. A hydrogel formed by ultraviolet irradiation of the reactive
prepolymer of claim 20.
32. A microarray comprising a solid support and the hydrogel of
claim 23.
33. A microarray comprising a solid support and the hydrogel of
claim 25.
34. A microarray comprising a solid support and the hydrogel of
claim 26.
35. A microarray comprising a solid support and the hydrogel of
claim 27.
36. A microarray comprising a solid support and the hydrogel of
claim 28.
37. A microarray comprising a solid support and the hydrogel of
claim 29.
38. A microarray comprising a solid support and the hydrogel of
claim 30.
39. A microarray comprising a solid support and the hydrogel of
claim 31.
40. A continuous film comprising a solid support and the hydrogel
of claim 23.
41. A continuous film comprising a solid support and the hydrogel
of claim 25.
42. A continuous film comprising a solid support and the hydrogel
of claim 26.
43. A continuous film comprising a solid support and the hydrogel
of claim 27.
44. A continuous film comprising a solid support and the hydrogel
of claim 28.
45. A continuous film comprising a solid support and the hydrogel
of claim 29.
46. A continuous film comprising a solid support and the hydrogel
of claim 30.
47. A continuous film comprising a solid support and the hydrogel
of claim 31.
48. A method of making a reactive prepolymer comprising
copolymerizing a first monomer with a 2+2 photocyclizable monomer,
wherein the 2+2 photocyclizable monomer comprises a polymerizable
functionality and a 2+2 photoreactive site.
49. A method of making a reactive prepolymer comprising: (a)
providing a first monomer; (b) copolymerizing said first monomer
with a second monomer; and (c) covalently attaching said second
monomer to a 2+2 photocyclizable compound to form a 2+2
photocyclizable monomer.
50. A method of making a hydrogel comprising: (a) providing a first
monomer; (b) providing a 2+2 photocyclizable monomer; (c)
polymerizing two or more first monomers with two or more 2+2
photocyclizable monomers, wherein the 2+2 photocyclizable monomers
comprise a polymerizable functionality and a 2+2 photoreactive
site; and (d) cyclizing at least two of the 2+2 photocyclizable
monomers with ultraviolet light to form the hydrogel.
51. The method of claim 50, wherein said hydrogel has a ratio
between about one 2+2 photocyclizable monomer to about every 20
first monomers and about one 2+2 photocyclizable monomer to about
every 30 first monomers.
52. The method of claim 51, wherein between 20% and 65% of said 2+2
photocyclizable monomer is crosslinked.
53. The method of claim 51, wherein between 30% and 50% of said 2+2
photocyclizable monomer is crosslinked.
54. The method of claim 50, wherein said hydrogel is between about
2 nanometers and about 5 micrometers in thickness.
55. The method of claim 50, wherein said hydrogel is between about
2 nanometers and about 100 nanometers in thickness.
56. The method of claim 50, wherein said hydrogel is cyclized to a
solid support.
57. A method of making a hydrogel comprising: (a) providing a first
monomer; (b) providing a second monomer; (c) polymerizing at least
two first monomers to at least two second monomers; (d) covalently
attaching a 2+2 photocyclizable compound to at least two of the
second monomers, wherein said covalent attachment is by a
condensation reaction; (e) providing an additional crosslinking
agent; and (f) cyclizing at least two of the 2+2 photocyclizable
compounds with at least one additional crosslinking agent using
ultraviolet light to form the hydrogel.
58. The method of claim 57, wherein said additional crosslinking
agent is pentaerythritol tetraacrylate.
59. The method of claim 57, wherein said hydrogel has a ratio
between about one 2+2 photocyclizable compound to about every 20
first monomers and about one 2+2 photocyclizable compound to about
every 30 first monomers.
60. The method of claim 59, wherein between 20% and 65% of said 2+2
photocyclizable compound is crosslinked.
61. The method of claim 59, wherein between 30% and 50% of said 2+2
photocyclizable compound is crosslinked.
62. The method of claim 57, wherein said hydrogel is between about
2 nanometers and about 5 micrometers in thickness.
63. The method of claim 57, wherein said hydrogel is between about
2 nanometers and about 100 nanometers in thickness.
64. The method of claim 57, wherein said hydrogel is cyclized to a
solid support.
65. A method of making a hydrogel comprising: (a) providing a first
monomer; (b) providing a second monomer; (c) polymerizing at least
two first monomers to at least two second monomers; (d) covalently
attaching a 2+2 photocyclizable compound to at least two of the
second monomers, wherein said covalent attachment is by a
condensation reaction; (e) providing the reactive prepolymer of
claim 48; and (f) cyclizing at least one of the 2+2 photocyclizable
compounds with at least one reactive prepolymer of claim 48 using
ultraviolet light to form the hydrogel.
66. The method of claim 65, wherein said hydrogel has a ratio
between about one 2+2 photocyclizable compound to about every 20
first monomers and about one 2+2 photocyclizable compound to about
every 30 first monomers.
67. The method of claim 66, wherein between 30% and 50% of said 2+2
photocyclizable compound is crosslinked.
68. The method of claim 66, wherein said hydrogel is between about
2 nanometers and about 5 micrometers in thickness.
69. The method of claim 65, wherein said hydrogel is cyclized to a
solid support.
70. A method of making a hydrogel array comprising: (a) placing a
reactive prepolymer including at least two 2+2 photocyclizable
sites on a solid support; and (b) cyclizing at least two of the 2+2
photocyclizable sites present in the reactive prepolymer to form a
hydrogel array, wherein said hydrogel array is formed by selective
irradiation with ultraviolet light.
71. The method of claim 70, wherein said selective irradiation is
with ultraviolet light having a wavelength of about 365
nanometers.
72. The method of claim 70, wherein said selective irradiation
occurs at areas on an array that are not blocked to ultraviolet
irradiation by a mask.
73. The method of claim 70, wherein said selective irradiation
occurs at areas on an array irradiated by an ultraviolet laser.
74. The method of claim 70, wherein a hydrogel location of said
hydrogel array is between about 2 nanometers and about 5
micrometers in thickness.
75. The method of claim 70, wherein a hydrogel location of said
hydrogel array is about 200 micrometers in diameter.
76. The method of claim 70, wherein a hydrogel location of said
hydrogel array is about 100 micrometers in diameter.
77. The method of claim 70, wherein a hydrogel location of said
hydrogel array is about 50 micrometers in diameter.
78. A method of making a continuous hydrogel film comprising: (a)
placing a reactive prepolymer including at least two 2+2
photocyclizable sites on a solid support; and (b) cyclizing at
least two of the 2+2 photocyclizable sites present in the reactive
prepolymer to form a continuous hydrogel film, wherein said
continuous hydrogel film is formed by irradiation with ultraviolet
light.
79. The method of claim 78, wherein said irradiation is with
ultraviolet light having a wavelength of about 365 nanometers.
80. The method of claim 78, wherein said continuous hydrogel film
is between about 2 nanometers and about 5 micrometers in thickness.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
Nonprovisional Application No. 09/344,217, filed Jun. 25, 1999,
entitled "Polyacrylamide Hydrogels and Hydrogel Arrays Made from
Polyacrylamide Reactive Prepolymers," which claimed the benefit of
U.S. Provisional Application No. 60/109,821, filed Nov. 25, 1998
entitled "Polyacrylamide Hydrogels and Hydrogel Arrays Made from
Polyacrylamide Reactive Prepolymers."
BACKGROUND
[0002] Acrylamide (CH.sub.2.dbd.CHC(O)NH.sub.2; C.A.S. 79-06-1;
also known as acrylamide monomer, acrylic amide, propenamide, and
2-propenamide) is an odorless, free-flowing white crystalline
substance that may be polymerized to form polyacrylamides. The
resulting high molecular weight polymers have a variety of uses and
further can be modified to optimize nonionic, anionic, or cationic
properties for specified uses.
[0003] Polyacrylamide hydrogels are used as molecular sieves for
the separation of nucleic acids, proteins, peptides,
oligonucleotides, polynucleotides, and other biological materials.
They are also used as binding layers for biological materials. When
used as binding layers, the gels currently are produced as thin
sheets or slabs, typically by depositing a solution of acrylamide
monomer, a crosslinker such methylene bisacrylamide, and an
initiator such as N,N,N',N'-tetramethylethylendiami- ne (TEMED)
between two glass surfaces (e.g., glass plates or microscope
slides) using a spacer to obtain the desired thickness of
polyacrylamide.
[0004] Generally, the acrylamide polymerization solution is a 4-5%
solution (acrylamide/bisacrylamide 19/1) in water/glycerol, with a
nominal amount of initiator. The acrylamide is chain-polymerized
and crosslinked by radical initiation from ultraviolet (UV)
radiation (e.g., 254 nm for about 15 minutes) or heat (e.g., about
400.degree. C.). Following polymerization and crosslinking, the top
glass slide is removed from the surface to uncover the gel.
Changing the amount of crosslinker and the % solids in the monomer
solution controls the pore size of the gel. Changing the
polymerization temperature also can control the pore size.
[0005] The current approach of making polyacrylamide hydrogels
starting from acrylamide monomer has several disadvantages, some of
which are described below:
[0006] 1. The coating process is difficult and expensive to
automate because the film thickness is controlled with a spacer and
top glass plate. The removal of the top glass plate must be done
manually. Due to difficulties automating the process (e.g.,
viscosity too low for commercial coating methods), the coating of
the monomer solution currently is done manually.
[0007] 2. The reaction time of the acrylamide is excessively long
(e.g., typically from about 15 to about 90 minutes at a short
wavelength of about 254 nm), making the UV polymerization and
crosslinking step incompatible with standard imaging equipment such
as mask aligners and photoprinters.
[0008] 3. The acrylamide monomer is a neurotoxin and a carcinogen
which makes coating, handling, and waste disposal of the material
hazardous and expensive.
[0009] Added to these disadvantages are the further problems that
crystallization of monomer frequently occurs on commonly used
equipment and laboratory surfaces, and exothermic polymerization
can occur in coating reservoirs, necessitating the use of
stabilizers or inhibitors. The present invention overcomes at least
one of these disadvantages.
BRIEF SUMMARY
[0010] Polyacrylamide hydrogels that incorporate [2+2]
photoreactive sites are disclosed. The hydrogels are especially
useful for microarray formation and are made from prepolymers,
including polyacrylamide reactive prepolymers. The photoreactive
sites allow use of [2+2] cycloaddition reactions to not only
crosslink the polyacrylamide reactive prepolymers forming the
hydrogel, but also for later attachment of any other molecules
incorporating additional photoreactive sites. The disclosed
hydrogels provide a more uniform pore size, likely resulting from
an improved control of crosslinking, making them preferable for use
with DNA based probes. Additionally, because higher viscosity
prepolymers, as opposed to low viscosity monomer solutions, are
used to form the arrays, manufacturing is simplified.
[0011] The scope of the present invention is defined solely by the
appended claims, and is not affected to any degree by the
statements within this summary.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts a DMI based polyacrylamide reactive
prepolymer formed by a "one-step" process. The polyacrylamide
reactive prepolymer (PRP) was formed by thermally polymerizing
monomers of acrylamide and
N-(6-acryloylhexyl)-2,3-dimethylmaleimide. Because
N-(6-acryloylhexyl)-2,3-dimethylmaleimide is "bifunctional,"
(having both a polymerizable functionality and a [2+2]
photoreactive site in a single molecule) it forms a PRP directly
upon copolymerization with acrylamide. Upon irradiation with
ultraviolet light, the PRP is crosslinked to form a hydrogel by
cycloaddition at the [2+2] photoreactive sites. Substituents x and
y can be varied between 1 and 50. The wavy line (~~~~~) between
oxygen and nitrogen represents the hexyl spacer group. The dashed
bonds (------) indicate attachment points for additional
monomers.
[0013] FIG. 2 depicts a "two-step" reaction scheme for reactive
prepolymer formation. First, a copolymer of acrylamide and acrylic
acid is formed. Second, the copolymer is treated with a [2+2]
photoreactive compound, such as acryloyl chloride. The acrylic acid
monomer then condenses with the acryloyl chloride to give a PRP
with an acrylate provided [2+2] photoreactive site. Substituents x
and y can be varied between 1 and 50. The dashed bonds indicate
attachment points for additional monomers.
[0014] FIG. 3 depicts a Glycidyl Methacrylate Based PRP resulting
from a two-step process in which a copolymer of acrylamide and
acrylic acid is reacted with glycidyl methacrylate. An acrylate
provided [2+2] photoreactive site results. Because [2+2]
photoreactive sites provided by acrylate groups will not
photocyclize with themselves, but will undergo undesirable
chain-type polymerization if irradiated with UV light, a mixed
hydrogel is formed by ultraviolet exposure in the presence of a DMI
based PRP. Alternatively, an additional crosslinking agent can be
used to form a hydrogel from the acrylate based PRP. Substituents x
and y can be varied between 1 and 50. The dashed bonds indicate
attachment points for additional monomers.
DETAILED DESCRIPTION
[0015] Overview
[0016] Hydrogels suitable for forming microarrays and continuous
films to which probes may be attached by [2+2] photocycloaddition
chemistry are described. The arrays or films are formed by first
synthesizing a reactive prepolymer that incorporates [2+2]
photocyclizable sites. A thin layer of prepolymer solution is then
placed on a solid support and exposed to ultraviolet light. If
microarrays are desired, the hydrogel layer is selectively exposed
to ultraviolet light. During exposure, a portion of the [2+2]
photocyclizable sites exposed to the light undergoes cyclization to
crosslink the reactive prepolymer, thus forming a hydrogel. The
uncyclized reactive prepolymer is then washed away with an aqueous
solution. Unlike the reactive prepolymer, the crosslinked hydrogel
is not water-soluble. A continuous hydrogel film or microarray of
hydrogel sites attached to the solid support results.
[0017] Probes or other molecules containing [2+2] photoreactive
sites may undergo cycloaddition with the hydrogel through further
UV exposure. Sufficient [2+2] photoreactive sites are incorporated
into the reactive prepolymer to crosslink the polymer to form the
hydrogel and to remain available for later probe and other molecule
attachment.
[0018] For arrays, the hydrogel pattern results from selective
irradiation with ultraviolet light. Selective irradiation may be
accomplished in multiple ways known to those of skill in the art.
Preferable methods include (1) using an opaque mask to shield
portions of the reactive prepolymer from ultraviolet exposure and
(2) using a laser beam to only irradiate selected positions on the
reactive prepolymer. Either method results in only selected
portions of the reactive polymer being crosslinked. The
uncrosslinked reactive polymer can then be removed to leave the
array grid on the support.
[0019] Reactive Prepolymer
[0020] While a variety of compounds or molecules may be used to
form the reactive prepolymer, two basic procedures are preferred.
In the first, a bifunctional molecule with a polymerizable end and
a [2+2] photocyclizable end is copolymerized with a hydrophilic
first monomer. In the second, a second monomer that may be attached
to a compound having a [2+2] photoreactive site is copolymerized
with the hydrophilic first monomer. This copolymer is then reacted
with a compound having a [2+2] photoreactive site to produce the
desired reactive prepolymer. Either method results in a reactive
prepolymer that may be crosslinked into a hydrogel by [2+2]
photocycloaddition.
[0021] Monomers
[0022] [0019] Monomers are the individual molecular units that are
repeated to form polymers. Multiple monomers covalently attached
form the backbone of a polymer. Polymers that are made from at
least two different monomer units are referred to as copolymers.
Polymerizing or copolymerizing describes the process by which
multiple monomers are covalently linked to form polymers or
copolymers, respectively. A discussion of polymers and monomers
from which they are made may be found in Stevens, Polymer
Chemistry: An Introduction, 3.sup.rd ed., Oxford University Press,
1999. The preferred embodiments rely on three general classes of
monomers to form reactive prepolymers.
[0023] The monomers used in the preferred embodiments may be
covalently attached by chain-type polymerization chemistry to form
the backbone of the reactive prepolymer. Thus, these monomers are
"polymerizable."
[0024] Chain-type Polymerization
[0025] Chain-type polymerization is a polymerization reaction that
is radical initiated. An energetic radical is formed in the
presence of the monomers, which then join to form a polymer. Many
methods of radical initiation are known, but most rely on thermal
or light energy to generate the reactive radicals. In one
embodiment, thermal radical initiation is most preferred to form a
polymer backbone incorporating acrylamide first monomers.
[0026] If the radicals are light generated, preferred initiators
include N,N,N', N'-tetramethylethylendiamine (TEMED) and
benzophenones. Preferable thermal initiators include peroxides,
such as benzoyl peroxide. A chain-type polymerization reaction is
depicted below. 1
[0027] In order to undergo chain-type polymerization, a monomer
must have "polymerizable functionality," as provided by the olefins
in the butylenes depicted above. During this butylene
polymerization, for example, the unsaturated monomers covalently
bond to form a saturated polybutylene polymer. The monomers are
covalently attached through their polymerizable groups.
[0028] First Monomers
[0029] The first type of monomer, or "first monomer," is preferably
water-soluble and polymerizable. First monomers may be
copolymerized by chain-type methods with other polymerizable
monomers and impart hydrophilicity to the resultant copolymer.
After polymerization, the first monomers are covalently bonded to
themselves or other polymerizable monomers. They are also resistant
to [2+2] cyclization reactions as described below. By cyclization
resistant, it is meant that when heated or exposed to ultraviolet
light, first monomers will preferentially undergo chain-type
polymerization in relation to [2+2] cyclization.
[0030] Monomers that undergo chain-type radical polymerization,
resist [2+2] photocyclization, and solubilize in water are
preferred first monomers. Examples of preferred first monomers
include acrylamide, hydroxyethyl acrylate, vinyl pyridine, acrylic
acid, methacrylic acid, and vinyl pyrrolidone, or mixtures thereof.
Most preferred is acrylamide.
[0031] Second Monomer
[0032] The second type of monomer, or "second monomer," may be
copolymerized by chain-type methods with other polymerizable
monomers and includes a heteroatom (e.g. oxygen, nitrogen, sulfur,
phosphorous). After copolymerization, the heteroatom may be used to
covalently attach the second monomer to a [2+2] photocyclizable
compound by a condensation reaction.
[0033] A condensation reaction occurs when two molecules are
covalently joined through a non-radical pathway. The condensation
of acrylic acid with glycidyl methacrylate is shown below. In this
reaction the oxygen heteroatom of acrylic acid opens the epoxide
ring of glycidyl acrylate to form a covalent bond. 2
[0034] Preferred second monomers can be any monomer that is
polymerizable and has a heteroatom suitable for bonding with a
compound having a [2+2] photoreactive site, as described below.
More preferred second monomers include acrylic acid, glycidyl
methacrylate, and methacrylic acid, or mixtures thereof. Acrylic
acid is most preferred.
[0035] Preferable [2+2] photocyclizable compounds include any
compound incorporating a [2+2] photoreactive site that can undergo
a condensation reaction with a second monomer. More preferred [2+2]
photocyclizable compounds include glycidyl methacrylate, acrylic
acid, hydroxyethyl acrylate, hydroxypropyl acrylate, and acryloyl
halides, or mixtures thereof.
[0036] Bifunctional Monomers
[0037] The third type of monomers used to form reactive prepolymers
includes both polymerizable functionality and [2+2] photoreactive
sites in a single monomer. By tuning the electron density and
steric parameters of a monomer to favor polymerization (greater
electron density, less sterics) at one end and to favor [2+2]
cycloaddition (less electron density, greater sterics) at the
other, bifunctional monomers may be formed. When copolymerized with
first monomers, bifunctional monomers form reactive prepolymers
without requiring further incorporation of [2+2] photocyclizable
compounds. Preferably, bifunctional monomers are thermally
copolymerized with a first monomer and then exposed to ultraviolet
light to crosslink a portion of the [2+2] photoreactive sites to
form a hydrogel.
[0038] Preferable bifunctional monomers incorporate vinyl and
dimethylmaleimide or cinnamate groups. Most preferred are
N-(6-acryloylhexyl)-2,3-dimethylmaleimide (as shown below) and
vinyl cinnamate. 3
[0039] [2+2] Photocyclizable Monomer
[0040] [0032] [2+2] photoreactive sites can undergo [2+2]
photocycloaddition and are therefore [2+2] photocyclizable. [2+2]
photocyclizable monomers are preferably any monomer unit that
includes a [2+2] photoreactive site and a polymerizable site.
Therefore, [2+2] photocyclizable monomers can be either
bifunctional, like DMI, or made through condensation of a
polymerizable compound with a [2+2] photocyclizable compound.
[0041] A monomer or compound incorporating a [2+2] photoreactive
site can undergo [2+2] cycloaddition with other monomers or
compounds that possess a [2+2] photoreactive site. If a monomer or
compound incorporating a [2+2] photoreactive site is irradiated
with ultraviolet light, the [2+2] reactive portion of the molecule
will preferentially undergo cycloaddition or "cyclize" with other
[2+2] photoreactive sites, instead of undergoing chain-type
polymerization. Preferably, polymerizable sites have a greater
electron density and are less sterically hindered than [2+2]
photoreactive sites.
[0042] While second monomers and [2+2] photocyclizable compounds
serve different chemical functions in the present embodiments, some
molecules can provide either functionality depending on reaction
order. For example, if acrylamide is copolymerized with acrylic
acid and then condensed with glycidyl methacrylate, acrylic acid
provides second monomer functionality and glycidyl methacrylate
provides [2+2] photocyclizable compound functionality.
Alternatively, if acrylamide is copolymerized with glycidyl
methacrylate and then condensed with acrylic acid, glycidyl
methacrylate provides second monomer functionality and acrylic acid
provides [2+2] photocyclizable compound functionality. In either
reaction sequence, a PRP is formed with an acrylate based [2+2]
photoreactive site. This reversibility holds true for the reaction
of acrylic acid with glycidyl methacrylate, hydroxyethyl acrylate,
or hydroxypropyl acrylate. Hence, reaction order can determine
whether a compound serves as a second monomer or a [2+2]
photoreactive compound during two-step reactive prepolymer
synthesis.
[0043] [2+21] Cyclization
[0044] In the disclosed embodiments, [2+2] cyclization or
cycloaddition is a light-induced reaction between two photoreactive
sites, at least one of which is electronically excited.
Advantageously, [2+2] cycloaddition reactions can proceed with high
efficiency. While it is chemical convention to write cycloaddition
centers in brackets, such as "[2+2]" or "[4+2]," the brackets were
omitted from the claims to prevent confusion with the patent
convention of deleting bracketed material. Hence, in the claims
"[2+2]" is written as "2+2."
[0045] Most preferably, cyclization is of the [2+2] variety,
wherein two carbon-carbon or a carbon-carbon and a
carbon-heteroatom single bond are formed in a single step. The
[2+2] cycloaddition involves addition of a 2.pi.-component of a
double bond to the 2.pi.-component of a second double bond, as
shown below. 4
[0046] Alternatively, the reaction may proceed by way of a
2.pi.-component of triple bonds. Under the rules of orbital
symmetry, such [2+2] cycloadditions are thermally forbidden, but
photochemically allowed. Such reactions typically proceed with a
high degree of stereospecificity and regiospecificity.
[0047] Photochemical [2+2] cycloaddition of monomers on opposing
copolymer chains results in crosslinking of the copolymer
backbones. In addition to cyclizing with other photocyclizable
monomers, [2+2] photocyclizable monomers can cyclize with other
molecules, such as DNA probes, which incorporate [2+2]
photoreactive sites. In this manner, other molecules may be
attached to the preferred hydrogels. The additional [2+2]
cyclizable molecules may be cyclized with the monomers during or
after the cyclization reaction that crosslinks the monomers to form
the hydrogel. The cyclization between monomers results in covalent
crosslinking to form hydrogels, while cyclization between monomers
and other molecules results in covalent attachment of the other
molecules to the hydrogel. In a likewise fashion, the reactive
prepolymer may be cyclized to a solid support, such as glass or
plastic, which incorporates [2+2] photoreactive sites.
[0048] Preferred [2+2] cycloadditions include those between two
carbon-carbon double bonds to form cyclobutanes and those between
alkenes and carbonyl groups to form oxetanes. Cycloadditions
between two alkenes to form cyclobutanes can be carried out by
photo-sensitization with mercury or directly with short wavelength
light, as described in Yamazaki et al., J. Am. Chem. Soc., 91, 520
(1969). The reaction works particularly well with
electron-deficient double bonds because electron-poor olefins are
less likely to undergo undesirable side reactions. Cycloadditions
between carbon-carbon and carbon-oxygen double bonds, such as
.alpha.,.beta.-unsaturated ketones, form oxetanes (Weeden, In
Synthetic Organic Photochemistry, Chapter 2, W. M. Hoorspool (ed.)
Plenum, New York, 1984) and enone addition to alkynes (Cargill et
al., J. Org. Chem., 36, 1423 (1971)).
[0049] Photoreactive Sites
[0050] Photoreactive sites are defined as chemical bonds capable of
undergoing [2+2] cycloaddition (cyclization) to form a ring
structure when exposed to light of an appropriate wavelength.
Photoreactive sites can yield homologous linking, where a monomer
or other molecule photoreactive site cyclizes with a monomer or
other molecule photoreactive site having the same chemical
structure, or for heterologous linking, where a monomer or other
molecule photoreactive site cyclizes with a monomer or other
molecule photoreactive site having a different chemical
structure.
[0051] In the current embodiments, DMI and vinyl cinnamate [2+2]
photoreactive sites can cyclize with themselves, each other, or
acrylate provided [2+2] photoreactive sites. Unlike the
photoreactive sites of DMI and vinyl cinnamate, however, acrylate
provided [2+2] photoreactive sites do not undergo [2+2]
photocycloaddition with themselves. Preferred homologous linking
occurs between dimethyl maleimide (DMI) photoreactive sites, while
preferred heterologous linking occurs between acrylate and DMI
photoreactive sites. A detailed discussion of photoreactive sites
may be found in Guillet, Polymer Photophysics and Photochemistry,
Ch. 12 (Cambridge University Press: Cambridge, London). Generally,
double bonds that are not part of a highly conjugated system (e.g.
benzene will not work) are preferred. Sterically-hindered, electron
deficient double bonds, such as found in maleimide, are most
preferred.
[0052] Additionally, molecules having a structure similar to
dimethyl maleimide may be used as photoreactive sites, including
maleimide/N-hydroxysuccinimide (NHS) ester derivatives. Such
preferred maleimide/NHS esters include 3-maleimidoproprionic acid
hydroxysuccinimide ester; 3-maleimidobenzoic acid N-hydroxy
succinimide; N-succinimidyl 4-malimidobutyrate; N-succinimidyl
6-maleimidocaproate; N-succinimidyl 8-maleimidocaprylate;
N-succinimidyl 11-maleimidoundecaoate. These esters can be obtained
from a variety of commercial vendors, such as ALDRICH (Milwaukee,
Wis).
[0053] Ultraviolet Irradiation
[0054] Crosslinking of the [2+2] photoreactive sites of the
photocyclizable monomers in the reactive prepolymer is most
preferably done with ultraviolet irradiation. Optionally, a
photosensitiser may be added to the reactive prepolymer to increase
the efficiency of the cycloaddition reaction. Preferred
photosensitisers include water-soluble quinones and xanthones,
including anthroquinone, thioxanthone, sulfonic acid quinone,
benzoin ethers, acetophenones, benzoyl oximes, acylphosphines,
benzophenones, and TEMED (N,N,N',N'-tetramethylethylendia- mine).
Anthroquinone-2-sulfonic acid is most preferred and is available
from ALDRICH, Milwaukee, Wis.
[0055] While irradiation by light in the ultraviolet spectrum
between 250 and 450 nanometers is preferred, longer wavelength
ultraviolet radiation is more preferred. Light of about 365
nanometers in wavelength is most preferred. By using longer
wavelength ultraviolet light, possible damage of other molecules,
such as DNA probes that degrade at 256 nanometers, is avoided.
Irradiation is preferably carried out between 22 and 300.degree.
C.
[0056] The reactive prepolymer is irradiated with ultraviolet light
for preferably between 10 and 100 seconds, most preferably for
about 30 seconds. Longer irradiation times result in thicker
hydrogels, while shorter irradiation times yield thinner hydrogels.
Useful hydrogels are preferably from about 2 nanometers to about 5
micrometers in thickness, more preferably from about 2 nanometers
to about 100 nanometers in thickness, and most preferably from
about 2 nanometers to about 50 nanometers in thickness. Thickness
is defined as the dimension perpendicular to the support.
[0057] While irradiation may be carried out with any device capable
of producing light at the preferred wavelengths, a photolithography
tool, such as a metal halide lamp equipped Calibre ORC, available
from Mentor Graphics (Wilsonville, Oreg.), is most preferred.
[0058] Microarray Formation
[0059] To form a microarray, it is preferable to selectively
crosslink only a portion of the reactive prepolymer present on the
solid support, thus forming a pattern or grid of separate hydrogel
locations. One method of accomplishing this goal is after
application of the reactive prepolymer to the support, the reactive
prepolymer is covered with a mask having only a portion of its
surface transparent to the irradiation light. When the mask is then
irradiated, only the portion exposed to the light crosslinks to
form a hydrogel. The uncrosslinked reactive polymer is then
removed, preferably with aqueous solutions. In this fashion, the
pattern of the mask is transferred to the hydrogel microarray. See
Sze, VLSI Technology, McGraw-Hill (1983).
[0060] Patterned hydrogel microarrays can also be formed on the
support by using an ultraviolet laser to only irradiate the
portions of the reactive prepolymer where a hydrogel is desired.
See U.S. Pat. No. 4,719,615. After this targeted irradiation, the
excess reactive polymer is removed, leaving a microarray of
individual hydrogel locations. For either method, the hydrogel
locations may preferably be arranged in rows and columns.
[0061] Preferably each location of hydrogel extends from about 2
nanometers to about 5 microns, more preferably from about 2
nanometers to about 100 nanometers, and most preferably from about
2 nanometers to about 50 nanometers in the direction perpendicular
to the solid support. Each hydrogel location is preferably about
200 micrometers in diameter, more preferably about 100 micrometers
in diameter, and most preferably about 50 micrometers in diameter.
Diameter is defined as the dimension parallel to the support.
[0062] Spacers
[0063] Spacers are preferably groups that neither polymerize nor
undergo [2+2] cyclization during reactive prepolymer and hydrogel
formation and separate the polymerizable and [2+2] photoreactive
sites of bifunctional monomers. Spacers may also be used to
physically separate second monomers from [2+2] photoreactive sites
before condensation of the second monomer with the [2+2]
photocyclizable compound. More preferred spacers are
--CH.sub.2).sub.n-- (methylene) or --(OCH.sub.2CH.sub.2).sub.n--
(ethylene oxide), most preferably where n=1-10.
A--(CH.sub.2).sub.6-- spacer is present in
N-(6-acryloylhexyl)-2,3-dimethylmaleimide, as shown above.
[0064] Reactive Prepolymers
[0065] Reactive prepolymers are water-soluble copolymers that
incorporate [2+2] photoreactive sites that are available for [2+2]
cycloaddition type crosslinking. Water-solubility is measured by
determining the clarity of an aqueous solution containing a
specific weight percent of the reactive prepolymer. Preferable
reactive prepolymer solutions are clear and contain from about 0.5%
to about 22% reactive prepolymer, on a weight basis. The clarity of
the reactive prepolymer when coated on the solid support is also a
measure of solubility, since if precipitation occurs and
water-solubility is lost, an opaque or non-uniform film results.
Preferable water-soluble reactive prepolymers form a clear solution
when an aqueous solution contains about 22% by weight, or less, of
the reactive prepolymer. The aqueous solution to which the reactive
prepolymer is added is defined as including at least 80% water by
weight.
[0066] Reactive prepolymers preferably have a weight average
molecular weight of from about 1,000 to about 300,000 g/mole, more
preferably about 5,000 to about 100,000 g/mole, and most preferably
about 5,000 to about 50,000 g/mole. However, reactive prepolymers
also can be modified from the structures described with other
molecules that do not interfere with copolymerization or [2+2]
cyclization.
[0067] Generally, if the reactive prepolymer's weight average
molecular weight is less than about 1,000 g/mole it can become too
brittle to properly coat the solid support. Alternatively, if the
weight average molecular weight exceeds about 300,000 g/mole, the
reactive prepolymer can become too thick to coat the solid support.
Thus, the reactive prepolymer's viscosity is preferably from about
25 centiPoise to about 500,000 centiPoise, more preferably from
about 50 centiPoise to about 500,000 centipoise, and most
preferably about 200 centiPoise, as measured in deionized water at
about 270.degree. C.
[0068] The reactive prepolymer preferably includes between 1 and
about 50 first monomers to each bifunctional monomer or second
monomer condensed with a [2+2] photocyclizable compound. More
preferably, the reactive prepolymer includes between about 10 and
about 20 first monomers to each bifunctional monomer or second
monomer condensed with a [2+2] photocyclizable compound. Most
preferably, the reactive prepolymer includes about 15 first
monomers to each bifunctional monomer or second monomer condensed
with a [2+2] photocyclizable compound. By varying the amount of
first monomer to the bifunctional or second monomer, the number of
sites available for [2+2] cyclization may be optimized.
[0069] A preferred reactive prepolymer has the structure: 5
[0070] where x is an integer from 1 to 50, y is an integer from 1
to 50, and R is a moiety that comprises a 2+2 photoreactive
site.
[0071] Hydrogels
[0072] Hydrogels are formed from reactive prepolymers that have had
at least a portion of their [2+2] photoreactive sites
photochemically crosslinked. While hydrogels are hydrophilic and
tend to entrap water, they are not appreciably soluble in water.
Preferably, reactive prepolymers that incorporate water-soluble
monomers and monomers incorporating [2+2] photoreactive sites are
crosslinked to form hydrogels. Generally, homologous hydrogels can
be formed from DMI or vinyl cinnamate and heterologous hydrogels
can be formed from DMI and vinyl cinnamate, DMI and an acrylate, or
vinyl cinnamate and an acrylate. While these molecular pairings are
preferred, any alternative compounds that provide the required
functionality for polymerization and [2+2] cyclization may be
used.
[0073] While not necessary, hydrogels may also be formed by adding
an additional crosslinking agent to the reactive prepolymer before
UV irradiation. The inclusion of an additional crosslinking agent
increases the amount of crosslinking between the reactive
prepolymers. Preferable additional crosslinking agents include
pentaerythritol tetraacrylate. Additionally, crosslinking agents
can be [2+2] cyclized with acrylate based reactive prepolymers to
form hydrogels. Thus, while acrylate based reactive prepolymers
will not cyclize with themselves, they can be cyclized into
hydrogels through the addition of an additional crosslinking
agent.
[0074] Preferably, hydrogels have a ratio of cyclized or
crosslinked monomers to first monomers of between about one
cyclized [2+2] photocyclizable monomer or compound to about every
one first monomer and about one cyclized [2+2] photocyclizable
monomer or compound to about every 50 first monomers. More
preferably, hydrogels have a ratio of cyclized or crosslinked
monomers to first monomers of between about one cyclized [2+2]
photocyclizable monomer or compound to about every one first
monomer and about one cyclized [2+2] photocyclizable monomer or
compound to about every 30 first monomers. Most preferably,
hydrogels have a ratio of about one cyclized or crosslinked [2+2]
photocyclizable monomer or compound to about every 15 first
monomers.
[0075] Of the total [2+2] photocyclizable monomers or compounds
present in the reactive prepolymer, preferably between 10% and 80%
are crosslinked to form the hydrogel, more preferably between 20%
and 65% are crosslinked to form the hydrogel, and most preferably
between 30% and 50% are crosslinked to form the hydrogel. The
remaining uncrosslinked [2+2] photocyclizable monomers or compounds
are available for cyclization with probes, solid supports, or other
molecules. The approximate percent of crosslinked [2+2]
photocyclizable monomers or compounds may be determined by
FTIR.
[0076] Solid Supports
[0077] The "solid support" is any surface, including glass,
silicon, modified silicon, ceramic, plastic, or polymer, such as
(poly)tetrafluoroethylene, or (poly)vinylidenedifluoride, upon
which the reactive prepolymer may be placed and attached.
Preferable methods of spreading the reactive prepolymer on the
solid support include, but are not limited to, roller coating,
curtain coating, extrusion coating, offset printing, and spin
coating. Most preferably, attachment is by a [2+2] cycloaddition of
the reactive prepolymer to the solid support. A preferred solid
support is glass.
[0078] The solid support can be any shape or size, and can exist as
a separate entity or as an integral part of any apparatus (e.g.,
bead, curvette, plate, or vessel). The solid support may inherently
provide an attachment surface for the reactive prepolymer, or the
solid support may be modified to provide adherence of
polyacrylamide to the solid support. If the solid support is glass,
it is preferably modified with with
.gamma.-methacryl-oxypropyl-trimethoxysilane ("Bind Silane,"
Pharmacia).
[0079] More preferably, covalent linkage of the polyacrylamide
hydrogel to the solid support is performed as described in European
Patent Application 0 226 470 or through [2+2] cyclization. The
solid support may optionally contain electronic circuitry useful in
the detection of bit molecules, or microfluidics used in the
transport of micromolecules.
[0080] The preceding description is not intended to limit the scope
of the invention to the preferred embodiments described, but rather
to enable any person skilled in the art of chemistry to make and
use the invention.
EXAMPLES
[0081] Solvents are analytical or HPLC grade. General reagents can
be purchased from a variety of commercial suppliers (e.g., Fluka,
Aldrich, and Sigma Chemical Co.). Glass slides can be obtained from
commercial suppliers (e.g., Corning Glass Works).
Example 1
Method For Synthesizing a 20:1 DMI PRP
[0082] The 20:1 dimethyl maleimide (DMI) based PRP (as depicted in
FIG. 1) is a copolymer of acrylamide and bifunctional
N-(6-acroloylhexyl)-2,3-dim- ethyl-maleimide co-monomers. Thus, the
PRP is polyacrylamide
co-N-(6-acryloylhexyl)-2,3-dimethyl-maleimide.
[0083] First, 17.06 gram (0.24 mol.) of acrylamide (Fluka
BioChemica, electrophoresis grade), 3.35 gram (0.012 mol.) of
N-(6-acroloylhexyl)-2,3- -dimethyl-maleimide, 0.39 gram (0.00156
mol.) of copper(II)sulfate pentahydrate, and 0.3 gram (0.00111
mol.) of potassium peroxodisulfate were dissolved in 81.6 gram of
n-propanol/water 2:1 in a 250 mL-3-neck flask equipped with a
condenser, a stirrer, and a gas inlet/outlet. The solution was
deoxygenated with argon gas for 15 minutes, and then heated to
65.degree. C. and stirred for 4 hours. After cooling to room
temperature, the salts were removed from the solution by filtration
over a column filled with ion exchange resin (Dowex Monosphere
450).
[0084] The PRP was obtained by adding 0.5 gram anthroquinone
2-sulfonic acid sodium salt as a photosensitiser to 49.5 gram of
the above solution recovered from the column. The solid content of
this solution was 19.9% by weight. The molecular weight of the PRP
was approximately 138,000 on a weight average basis and
approximately 3,000 on a number average basis. UV crosslinking of a
5 .mu.m coating of the DMI based PRP followed by imaging and
developing operations resulted in formation of a crosslinked
polyacrylamide hydrogel array.
Example 2
Method for Synthesizing a 15:1 Glycidyl Acrylate PRP
[0085] 15:1 glycidyl acrylate based PRP was formed by using acrylic
acid as a second monomer and glycidyl acrylate as a [2+2]
photocyclizable compound (as depicted in FIG. 3) or by using
glycidyl acrylate as a second monomer and acrylic acid as a [2+2]
photocyclizable compound. In the latter case, the polymer backbone
was initially a copolymer of acrylamide and glycidyl methacrylate.
Thus, the first copolymer formed was polyacrylamide co-glycidyl
methacrylate. This first copolymer was further modified with
acrylic acid to produce the PRP.
[0086] First, 15.99 gram (0.225 mol.) of acrylamide (Fluka
BioChemica, electrophoresis grade), 2.13 gram (0.015 mol.) of
glycidyl methacrylate, 0.39 gram (0.00156 mol.) of
copper(II)sulfate pentahydrate, and 0.3 gram (0.00111 mol.) of
potassium peroxodisulfate were dissolved in 82.5 gram of
n-propanole/water 2:1 in a 250 mL-3-neck flask equipped with a
condenser, a stirrer, and a gas inlet/outlet. The solution was
deoxygenated with argon gas for 15 minutes, and then was heated up
to 65.degree. C. and stirred at this temperature for 4 hours. After
cooling to room temperature, the salts were removed from the
solution by filtration over a column filled with ion exchange resin
(Dowex Monosphere 450).
[0087] Forty-five grams of the solution recovered from the column
and 0.47 gram (0.0065 mol.) of acrylic acid were combined in a 100
mL flask equipped with a condenser and stirred (magnetic stirrer
bar) for 20 hours at 90.degree. C. with external heating. The PRP
was obtained after adding 0.45 gram anthroquinone 2-sulfonic acid
sodium salt and 0.25 gram triethanol amine to the acrylated
acrylamide solution. The solid content of this solution was 23.8%
by weight.
Example 3
Photocrosslinking the 20:1 DMI PRP
[0088] A 20% by weight solids aqueous solution (range of from about
2% to about 40% solids) of 20:1 DMI PRP and 1% by weight
anthroquinone 2-sulfonic acid sodium salt was coated on a solid
support to a wet thickness of about 25 .mu.m (range of from about 2
nanometers to about 5 .mu.m). The coating was then exposed with UV
radiation (less than about 1,000 milliJoules/cm.sup.2) through a
photomask containing a grid array pattern of pads (pad size of from
about 1 .mu.m to about 500 .mu.m) to cyclize the exposed PRP into a
water insoluble, crosslinked, hydrogel (FIG. 1). Although not shown
in FIG. 1, the PRP was simultaneously crosslinked to a glass solid
support modified with [2+2] photoreactive sites. The unexposed, and
therefore still water-soluble PRP, was then selectively removed by
an aqueous developer solution, leaving an array pattern of the
crosslinked, porous, hydrogel. Optionally, the solid support was
then diced into individual biochips, each containing from about 500
to about 100,000 pads.
Example 4
Photocrosslinking the 15:1 Glycidyl Acrylate PRP
[0089] A 20% by weight solids aqueous solution (range of from 2% to
about 40% solids) of 15:1 Glycidyl Acrylate PRP containing 1% by
weight pentaerythritol tetraacrylate (range of from about 0.05% to
1.5%) and 0.05% by weight anthroquinone 2-sulfonic acid sodium salt
was coated on a solid support to a wet thickness of 25 .mu.m (range
of from about 1 .mu.m to about 50 .mu.m). The coating was then
exposed with UV radiation through a photomask containing a grid
array pattern of pads (pad size of from about 1 .mu.m to about 500
.mu.m) to cyclize the exposed PRP into a water insoluble,
crosslinked hydrogel. The unexposed, and therefore still
water-soluble PRP was then removed by an aqueous developer solution
which left an array pattern of the crosslinked, porous, hydrogel.
Optionally, the solid support was diced into individual biochips,
each containing from about 500 to about 100,000 hydrogel pads.
Example 5
Forming a Continuous Film Hydrogel
[0090] A 20% by weight solids aqueous solution (range of from about
0.5% to about 40% solids) of 20:1 DMI PRP and 1% by weight
anthroquinone 2-sulfonic acid sodium salt was coated on a solid
support to a wet thickness of about 25 .mu.m (range of from about 2
nanometers to about 5 .mu.m). The coating was then exposed with UV
radiation (less than about 1,000 milliJoules/cm.sup.2) to cyclize
the exposed PRP into a water insoluble, crosslinked, hydrogel (FIG.
1). Although not shown in FIG. 1, the PRP was simultaneously
crosslinked to a glass solid support modified with [2+2]
photoreactive sites. The excess, and therefore still water-soluble,
PRP was then removed by an aqueous developer solution, leaving a
continuous film of the crosslinked, porous, hydrogel.
Example 6
Use of an Additional Crosslinking Agent with an Acrylate PRP and
Offset Printing to Form a Pattern.
[0091] A 5% by weight solids aqueous solution (range of from about
2% to about 25% solids) of an acryloyl based polyacrylamide
reactive prepolymer containing 1% by weight pentaerythritol
tetraacrylate and 1% by weight of an anthroquinone 2-sulfonic acid
sodium salt photosensitiser was coated in a grid array pattern (pad
size from about 50.mu.m to about 500 .mu.m) by offset printing on a
solid support to a wet thickness of 25 .mu.m (range of from about 1
.mu.m to about 50 .mu.m).
[0092] The patterned coating was then exposed with UV radiation to
cyclize the exposed PRP into a water insoluble hydrogel, leaving an
array pattern of crosslinked, hydrogel material. Optionally, the
solid support was diced into individual biochips, each containing
from about 500 to about 100,000 hydrogel pads.
Prophetic Example 1
Two-step Synthesis of an Acrylate Based Polyacrylamide Reactive
Prepolymer
[0093] A solution of acrylamide and acrylic acid (from about 3% to
about 50% acrylic acid) is thermally polymerized between 40 and
50.degree. C. A copolymer of acrylamide and acrylic acid is formed
as depicted in FIG. 2. This resultant copolymer is condensed with
acryloyl chloride to obtain an acrylate based polyacrylamide
reactive prepolymer (PRP) ready for crosslinking. This reaction
sequence graphically depicted in FIG. 2.
[0094] As any person skilled in the art of chemistry will recognize
from the previous description, figures, and examples that
modifications and changes can be made to the preferred embodiments
of the invention without departing from the scope of the invention
defined by the following claims.
* * * * *