U.S. patent application number 10/700990 was filed with the patent office on 2004-05-13 for porous silica substrates for polymer synthesis and assays.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Fidanza, Jacqueline A., Frank, Curtis W., Glazer, Marc I., McGall, Glenn, Vinci, Richard.
Application Number | 20040092396 10/700990 |
Document ID | / |
Family ID | 32232946 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040092396 |
Kind Code |
A1 |
Glazer, Marc I. ; et
al. |
May 13, 2004 |
Porous silica substrates for polymer synthesis and assays
Abstract
Methods are provided for making and using thin films of porous
silica substrates to synthesize arrays of polymers. Methods are
also provided for assaying such polymers on porous silica
substrates. The porous silica substrates offer an increase in array
density and signal enhancement over conventional flat glass
substrates. Examples of polymers that can be synthesized and
assayed include biological polymers such as nucleic acids,
polynucleotides, polypeptides, and polysaccharides. Arrays of
nucleic acids or polynucleotides can be used for a variety of
hybridization-based experiments such as nucleic acid sequence
analysis, nucleic acid expression monitoring, nucleic acid mutation
detection, speciation, effects of drug therapy on nucleic acid
expression, among others.
Inventors: |
Glazer, Marc I.; (Stanford,
CA) ; Fidanza, Jacqueline A.; (San Francisco, CA)
; McGall, Glenn; (Mountain View, CA) ; Frank,
Curtis W.; (Cupertino, CA) ; Vinci, Richard;
(Easton, PA) |
Correspondence
Address: |
John P. Iwanicki
BANNER & WITCOFF, LTD
28th Floor
28 State Street
Boston
MA
02109
US
|
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
32232946 |
Appl. No.: |
10/700990 |
Filed: |
November 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10700990 |
Nov 4, 2003 |
|
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09545207 |
Apr 7, 2000 |
|
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60128402 |
Apr 8, 1999 |
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Current U.S.
Class: |
502/439 ;
428/304.4; 65/17.2 |
Current CPC
Class: |
B01J 2219/00641
20130101; B01J 2219/00596 20130101; Y10T 428/252 20150115; B82Y
30/00 20130101; C40B 40/12 20130101; Y10T 428/249986 20150401; B01J
2219/00707 20130101; Y10T 428/249969 20150401; B01J 2219/00497
20130101; Y10T 428/249953 20150401; B01J 2219/00585 20130101; B01J
19/0046 20130101; B01J 2219/00605 20130101; C40B 40/06 20130101;
Y10T 428/249967 20150401; B01J 2219/00725 20130101; B01J 2219/00731
20130101; C40B 60/14 20130101; Y10T 428/249978 20150401; B01J
2219/00722 20130101; B01J 2219/00711 20130101; B01J 2219/00432
20130101; B01J 2219/0063 20130101; C40B 40/10 20130101; B01J
2219/0059 20130101; B01J 2219/00637 20130101; Y10T 428/249979
20150401; B01J 2219/00612 20130101; B01J 2219/00626 20130101; Y10T
428/24997 20150401; B01J 2219/00659 20130101 |
Class at
Publication: |
502/439 ;
065/017.2; 428/304.4 |
International
Class: |
B32B 003/26 |
Claims
What is claimed is:
1. A porous substrate comprising: a support region; and a porous
region on the support region, the porous region being primarily
inorganic and having a surface capable of forming a polymer array
thereon, the porous region comprising pores of a pore size of about
2 nm-500 nm, a porosity of about 10-90%, and a thickness of about
0.01 .mu.m to about 70 .mu.m.
2. The porous substrate of claim 1, wherein the porous region is
formed by an additive method.
3. The porous substrate of claim 2 wherein the additive method
includes the application of colloidal silica on the support
region.
4. The porous substrate of claim 2 wherein the additive method
includes the application of alkoxysilane on the support region.
5. The porous substrate of claim 1 wherein the porous region
comprises silica.
6. The porous substrate of claim 5 wherein the porous region
further comprises organic polymer of less than or equal to about
10% mole fraction.
7. The porous substrate of claim 5, wherein the porous region
comprises a plurality of pores, each of the plurality of pores
having a size of from about 2 to about 100 nm.
8. The porous substrate of claim 5, wherein the porous region
comprises a plurality of pores, each of the plurality of pores
having a size of from about 2 to about 50 nm.
9. The porous substrate of claim 1, wherein the porous region has a
porosity of from about 20 -80%.
10. The porous substrate of claim 1, wherein the porous region has
a porosity of from about 50-70%.
11. The porous substrate of claim 5, wherein the porous region
comprises a plurality of particles, each of the plurality of
particles having a size from about 5-500 nm.
12. The porous substrate of claim 5, wherein the porous region
comprises a plurality of particles, each of the plurality of
particles having a size from about 5-200 nm.
13. The porous substrate of claim 5, wherein the porous region
comprises a plurality of particles, each of the plurality of
particles having a size from about 70-100 nm.
14. The porous substrate of claim 2 wherein the porous region has a
thickness from about 0.1-1 microns.
15. The porous substrate of claim 2, wherein the porous region has
a thickness of from about 0.1 .mu.m to about 0.5 .mu.m.
16. The porous substrate of claim 2, wherein the porous region has
a thickness of from about 1 .mu.m to about 20 .mu.m.
17. The porous substrate of claim 6, wherein the organic polymer
coats silica particles of the porous region.
18. The porous substrate of claim 5, wherein the porous region is
silylated with a silyating agent.
19. The porous substrate of claim 18, wherein the silylating agent
is selected from the group consisting of
N,N-bis(hydroxyethylaminopropyl)tri- ethoxysilane and
glycidoxypropyl trimethoxy silane.
20. The porous substrate of claim 2, wherein the porous region is
formed by codepositing an organic template material with silica,
followed by removing the organic template material.
21. The porous substrate of claim 20 wherein the organic template
material comprises particles of about 10-100 nm and the silica
comprises particles of about 7-100 nm.
22. The porous substrate of claim 21 wherein an organic template
particle size is about equal to a silica particle size.
23. The porous substrate of claim 21 wherein a silica particle size
is less than or equal to about 2/3 an organic template particle
size.
24. The porous substrate of claim 21 wherein a silica particle size
is less than about 10% of an organic template particle size.
25. The porous substrate of claim 20 wherein the organic template
material is deposited in a volume ratio to the silica of about 10:1
to 1:10.
26. The porous substrate of claim 20 wherein the organic template
material is removed using a baking process at a temperature of
above about 150.degree. C.
27. The porous substrate of claim 26 wherein the silica is
densified using an annealing process.
28. The porous substrate of claim 20 wherein the porous region has
an effective surface area of about 15-40 times a flat substrate
with an equivalent two dimensional structure.
29. The porous substrate of claim 1 wherein the porous region is
formed by a subtractive method.
30. The porous substrate of claim 20, wherein the organic template
polymer is a latex polymer.
31. The porous substrate of claim 29 wherein the porous substrate
comprises phase-separable glass, a surface portion of the
phase-separable glass being treated to form the porous layer.
32. The porous substrate of claim 31 wherein the phase-separable
glass comprises a sodium borosilicate glass.
33. The porous substrate of claim 32 wherein the sodium
borosilicate glass has been annealed and leached to provide the
porous layer having a thickness of about 70 microns and comprised
of a plurality of pores, at least some of the plurality of pores
having a pore size greater than about 1000 .ANG..
34. The porous substrate of claim 29 wherein the porous region has
an effective surface area of about 50-400 times a flat substrate
with an equivalent two dimensional structure.
35. The substrate of claim 1, further comprising a high density
array of nucleic acids immobilized on the surface.
36. A porous substrate comprising: a support region; and a porous
region on the support region, said porous region of about 0.1-0.5
microns thick, wherein the porous layer comprises an unsintered
matrix formed from at least colloidal silica having a particle size
of about 70-100 microns, the unsintered matrix defining at least a
plurality of open pores having a pore size of about 10-20 nm, and
wherein the porous layer has a porosity of about 10-90%.
37. A method of forming a porous substrate, the method comprising:
providing a substrate material comprising a surface; dipping the
substrate material in a solution including colloidal silica and a
carrier, the colloidal silica having a particle size of about
12-100 nm; and withdrawing the substrate material to provide an
unsintered porous layer having a thickness of about 0.1-1 microns
and a porosity of about 10-90% on the substrate material.
38. A method of forming a porous substrate, the method comprising:
providing a substrate material comprising a surface; applying a
solution including colloidal silica and a carrier to the surface of
the substrate material, the colloidal silica having a particle size
of about 12-100 nm; spinning the substrate material and the applied
solution to achieve a spun layer on the substrate material; and
removing the carrier from the spun layer to provide an unsintered
porous layer having a thickness of about 0.1-1 microns and a
porosity of about 10-90% on the substrate material.
39. A method of forming a porous substrate comprising different
monomer sequences, the method comprising: immobilizing different
monomer sequences on a porous substrate of claim 1.
40. A method of synthesizing polymers on a porous substrate, the
method comprising: a) generating a pattern of light and dark areas
by selectively irradiating at least a first area of a surface of a
porous substrate of claim 1, said surface comprising immobilized
monomers on said surface, said monomers coupled to a photoremovable
protective group, without irradiating at least a second area of
said surface, to remove said protective group from said monomers in
said first area; b) simultaneously contacting said first area and
said second area of said surface with a first monomer to couple
said first monomer to said immobilized monomers in said first area,
and not in said second area, said first monomer having said
photoremovable protective group; c) generating another pattern of
light and dark areas by selectively irradiating with light at least
a part of said first area of said surface and at least a part of
said second area to remove said protective group in said at least a
part of said first area and said at least a part of said second
area; d) simultaneously contacting said first area and said second
area of said surface with a second monomer to couple said second
monomer to said immobilized monomers in at least a part of said
first area and at least a part of said second area; and e)
performing additional irradiating and monomer contacting and
coupling steps so that a matrix array of different polymers is
formed on said surface, whereby said different polymers have
sequences and locations on said surface defined by the patterns of
light and dark areas formed during the irradiating steps and the
monomers coupled in said contacting steps.
41. The method of claim 40, wherein the monomers are selected from
the group consisting of: nucleotides, amino acids, and
monosaccharides.
42. The method of claim 40, wherein the substrate has linker
molecules on its surface.
43. A method of forming polymers having different monomer sequences
on a porous substrate, the method comprising: providing a porous
substrate of claim 1 comprising a linker molecule layer thereon,
said linker molecule layer comprising a linker molecule and a
protective group; applying a barrier layer overlying said linker
molecule layer, said applying step forming selected exposed regions
of said linker molecule layer; exposing said selected exposed
regions of said linker molecule layer to a deprotecting agent to
remove the protective group; and coupling selected monomers to form
selected polymers on the substrate.
44. The method of claim 43, wherein the deprotection agent is in
the vapor phase.
45. The method of claim 43, wherein said deprotection agent is an
acid.
46. The method of claim 45, wherein the acid is selected from a
group consisting of trichloroacetic acid, dichloroacetic acid, and
HCl.
47. The method of claim 43, wherein the monomers are selected from
the group consisting of nucleotides, amino acids, and
monosaccharides.
48. A method for detecting a nucleic acid sequence, the method
comprising: (a) providing an array of nucleic acids bound to the
porous substrate of claim 1; (b) contacting the array of nucleic
acids with at least one labeled nucleic acid comprising a sequence
substantially complementary to a nucleic acid of said array, and
(c) detecting hybridization at least the labeled complementary
nucleic acid to nucleic acids of said array.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Serial No. 60/128,402, filed Apr. 8, 1999, the
disclosure of which is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0002] This invention pertains to preparation and use of very high
surface area porous substrates that can be used to synthesize high
density arrays of polymers.
BACKGROUND
[0003] Porous silica glass has been known for quite some time. U.S.
Pat. No. 4,220,461 provides a historical perspective and discussion
on the development of silica-rich phase-separable porous glass.
Various methods for the manufacture of phase-separable porous glass
are reviewed in U.S. Pat. No. 4,528,010. Both of these references
are incorporated by reference in their entireties for all
purposes.
BRIEF DESCRIPTION OF THE INVENTION
[0004] The present invention relates to a porous substrate and
methods for making and using the porous substrate. The porous
substrate provides an increased surface area for polymers to attach
to the substrate. Such porous substrates are often used to make an
array of polymers, such as for genetic diagnostic purposes. The
polymers may be placed or fabricated on the porous substrate by
various methods.
[0005] The polymers can include those of biological interest such
as nucleic acids, polynucleotides, proteins, polypeptides,
polysaccharides, oligosaccharides, mixtures of these or other
polymers on an array and combinations of the above polymer units in
individual polymers. The porous substrates thus are useful in, for
example, glass technology, polymer chemistry, molecular biology,
medicine, and medical diagnostics.
[0006] The porous substrate generally has at least two regions, a
support region and a porous region. The support region, which can
serve as an underlayer region, basically provides mechanical
support for ease of handling of a porous region. The porous region
may be for example a layer (film). The support region can be
selected or processed to provide additional features in the
finished porous substrate. One advantage of using a porous region
with higher surface area to make an array is that the array can be
functionalized with a much higher density of polymers for a given
two dimensional area without changing the spacing between polymers
on the surface of the porous substrate.
[0007] One embodiment of this invention provides a primarily
inorganic porous substrate including a support region, and a porous
region in contact with the support region. The porous region for
example includes pores with a pore size of 1-500 nm, or 2-500 nm,
the porous region having a porosity of, e.g., 10-90%, 20-80%, or
70-90%, and a porous surface thickness of 0.01-20 .mu.m, wherein
the porous region has a surface capable of forming arrays of
polymers thereon. The porosity is generally, for example, "open",
that is, some pores are connected to others to allow the infusion
of polymers or other fluids. Not all the pores need to connect to
another, that is, some of the pores may be closed. What is meant by
"primarily inorganic" is that a small amount of organic material
may remain in the porous region of the substrate, or may be
intentionally applied onto the surface(s) of the porous region.
[0008] In one embodiment, a porous substrate is provided
comprising:
[0009] a support region; and
[0010] a porous region on the support region, the porous region
being primarily inorganic and having a surface capable of forming a
polymer array thereon, the porous region comprising pores of a pore
size of about 2 nm-500 nm or 1000 Angstroms to 500 nm, a porosity
of about 10-90%, and a thickness of about 0.01 .mu.m to about 70
.mu.m.
[0011] The porous region can be formed by an additive method, which
can include the application of colloidal silica on the support
region. The additive method also may include the application of
alkoxysilane on the support region. The porous region may comprise
silica. The porous region may further comprise organic polymer of
less than or equal to about 10% mole fraction. The porous region
may comprise a plurality of pores, each of the plurality of pores
having a size of from about 2 to about 100 nm. The porous region
may comprise a plurality of pores, each of the plurality of pores
having a size of from about 2 to about 50 nm. The porous region
has, for example, a porosity of from about 20 -80%, or 50-70%. The
porous region for example comprises a plurality of particles, each
of the plurality of particles having a size from about 5-500 nm,
5-200 nm, or 70-100 nm. The porous region has, for example, a
thickness from about 0.1-1 microns, or about 0.1 .mu.m to about 0.5
.mu.m, or about 1 .mu.m to about 20 .mu.m.
[0012] An organic polymer may coat silica particles of the porous
region. The porous region may be silylated with a silyating agent,
such as N,N-bis(hydroxyethylaminopropyl)triethoxysilane and
glycidoxypropyl trimethoxy silane. The porous region may be formed
by codepositing an organic template material with silica, followed
by removing the organic template material. The organic template
material for example comprises particles of about 10-100 nm and the
silica comprises particles of about 7-100 nm. The organic template
particle size can be about equal to a silica particle size. The
silica particle size is for example less than or equal to about 2/3
an organic template particle size. The silica particle size is in
one embodiment, less than about 10% of an organic template particle
size. The organic template material can be deposited in a volume
ratio to the silica of about 10:1 to 1:10, e.g., 2:1. The organic
template material is in one embodiment removed using a baking
process at a temperature of above about 150.degree. C. The silica
may be densified using an annealing process. The porous region has
in one embodiment an effective surface area about 15-40 times a
flat substrate with an equivalent two dimensional structure. In one
embodiment, the porous region is formed by a subtractive method.
The organic template polymer may be a latex polymer. The porous
substrate may comprise phase-separable glass, a surface portion of
the phase-separable glass being treated to form the porous layer.
The phase-separable glass may comprise for example a sodium
borosilicate glass. The sodium borosilicate glass may be been
annealed and leached to provide the porous layer having a thickness
of about 70 microns and comprised of a plurality of pores, at least
some of the plurality of pores having a pore size greater than
about 1000 .ANG.. The porous region has, e.g., an effective surface
area about 50-400 times a flat substrate with an equivalent two
dimensional structure.
[0013] The porous substrate may further comprise a high density
array of polymers, such as nucleic acids immobilized on the
surface.
[0014] In another embodiment, a porous substrate is provided
comprising:
[0015] a support region; and
[0016] a porous region on the support region, said porous region
being about 0.1-0.5 microns thick,
[0017] wherein the porous layer comprises an unsintered matrix
formed from at least colloidal silica having a particle size of
about 70-100 microns, the unsintered matrix defining at least a
plurality of open pores having a pore size of about 10-20 nm,
and
[0018] wherein the porous layer has a porosity of of about
10-90%.
[0019] In one embodiment, a method of forming a porous substrate is
provided, the method comprising:
[0020] providing a substrate material comprising a surface;
[0021] dipping the substrate material in a solution including
colloidal silica and a carrier, the colloidal silica having a
particle size of about 12-100 nm; and
[0022] withdrawing the substrate material to provide an unsintered
porous layer having a thickness of about 0.1-1 microns and a
porosity of of about 10-90% on the substrate material.
[0023] Also provided is a method of forming a porous substrate, the
method comprising:
[0024] providing a substrate material comprising a surface;
[0025] applying a solution including colloidal silica and a carrier
to the surface of the substrate material, the colloidal silica
having a particle size of about 12-100 nm;
[0026] spinning the substrate material and the applied solution to
achieve a spun layer on the substrate material; and
[0027] removing the carrier from the spun layer to provide an
unsintered porous layer having a thickness of about 0.1-1 microns
and a porosity of about 10-90% on the substrate material.
[0028] Another embodiment is a method of forming a porous substrate
comprising different monomer sequences, the method comprising:
[0029] immobilizing different monomer sequences on a porous
substrate.
[0030] In another embodiment, there is provided a method of
synthesizing polymers on a porous substrate, the method
comprising:
[0031] a) generating a pattern of light and dark areas by
selectively irradiating at least a first area of a surface of a
porous substrate, said surface comprising immobilized monomers on
said surface, said monomers coupled to a photoremovable protective
group, without irradiating at least a second area of said surface,
to remove said protective group from said monomers in said first
area;
[0032] b) simultaneously contacting said first area and said second
area of said surface with a first monomer to couple said first
monomer to said immobilized monomers in said first area, and not in
said second area, said first monomer having said photoremovable
protective group;
[0033] c) generating another pattern of light and dark areas by
selectively irradiating with light at least a part of said first
area of said surface and at least a part of said second area to
remove said protective group in said at least a part of said first
area and said at least a part of said second area;
[0034] d) simultaneously contacting said first area and said second
area of said surface with a second monomer to couple said second
monomer to said immobilized monomers in at least a part of said
first area and at least a part of said second area; and
[0035] e) performing additional irradiating and monomer contacting
and coupling steps so that a matrix array of different polymers is
formed on said surface, whereby said different polymers have
sequences and locations on said surface defined by the patterns of
light and dark areas formed during the irradiating steps and the
monomers coupled in said contacting steps.
[0036] The monomers are for example, nucleotides, amino acids, or
monosaccharides. The substrate may have linker molecules on its
surface.
[0037] There also is provided a method of forming polymers having
different monomer sequences on a porous substrate, the method
comprising:
[0038] providing a porous substrate comprising a linker molecule
layer thereon, said linker molecule layer comprising a linker
molecule and a protective group;
[0039] applying a barrier layer overlying said linker molecule
layer, said applying step forming selected exposed regions of said
linker molecule layer;
[0040] exposing said selected exposed regions of said linker
molecule layer to a deprotecting agent to remove the protective
group; and
[0041] coupling selected monomers to form selected polymers on the
substrate.
[0042] The deprotection agent may be, for example, in the vapor
phase or liquid phase, and may be, for example an acid, such as
trichloroacetic acid, dichloroacetic acid, or HCl. The monomers are
for example nucleotides, amino acids, or monosaccharides.
[0043] In another embodiment, there is provided a method for
detecting a nucleic acid sequence, the method comprising:
[0044] (a) providing an array of nucleic acids bound to the porous
substrate;
[0045] (b) contacting the array of nucleic acids with at least one
labeled nucleic acid comprising a sequence substantially
complementary to a nucleic acid of said array, and
[0046] (c) detecting hybridization at least the labeled
complementary nucleic acid to nucleic acids of said array.
[0047] In one embodiment, the porous substrates comprising arrays
may be used to screen for a previously identified polymorphic
variant in a target nucleic acid sequence, or for a target such as
a human immunodeficiency virus sequence. Nucleic acids such as a
p53 gene, an HIV RT gene, a CFTR gene, or a cytochrome p450 gene
can be screened for. The array may include, for example, at least
3200 polynucleotide probes, or, e.g., at least 10,000
polynucleotide probes, or at least 50,000 probes. The probes may
be, for example, 9 to 21 nucleotides in length.
BRIEF DESCRIPTION OF FIGURES
[0048] FIG. 1 is a simplified cross section of a portion of a
porous substrate with a porous region formed from particles
according to one embodiment of the invention.
[0049] FIG. 2 is a simplified cross section of a portion of a
porous substrate with a porous region formed by leaching according
to one embodiment of the invention.
[0050] FIG. 3a is a simplified cross section of a portion of a
substrate in an intermediate processing state with templating
particles that are substantially larger than interstitial silica
particles according to one embodiment of the invention.
[0051] FIG. 3b is a simplified cross section of the portion of the
substrate shown in FIG. 3a after the templating material has been
removed to form a porous substrate.
[0052] FIG. 4 is a simplified cross section of a portion of a
substrate in an intermediate processing state according to another
embodiment of the present invention with templating particles of
about the same size as silica particles.
DESCRIPTION OF THE INVENTION
[0053] The present invention relies on many patents, applications
and other references for details known to those of the art.
Therefore, when a patent, application or other reference is cited
or repeated below, it should be understood that it is incorporated
by reference in its entirety for all purposes as well as for the
proposition that is recited.
[0054] The present invention provides a porous substrate and
methods for making and using the porous substrate. The porous
substrate provides a large surface area for polymers to be attached
to make an array. The polymers may be placed or fabricated on the
array by various methods. A porous layer is formed on a substrate
material, and in some embodiments, the porosity, pore size, and
thickness of the porous layer is chosen according to desired
functionalization characteristics. Porous substrates are generated
by creating a 3D matrix to increase the surface area and therefore
increase the number of sites available for array synthesis in the
same lateral dimensions. One advantage in using a porous layer to
increase the effective surface area is to make an array that can be
functionalized with a much higher density of polymers for a given
two dimensional, or "flat" area without changing the spacing
between cells of the array on the surface of the substrate. The
effective surface area is the surface area of the porous region
that is available for adsorption of polymer molecules or for
polymer synthesis, of example.
[0055] The support region can be, for example sodalime glass,
borafloat glass, sodium borosilicate glass, fused silica, or a
polymer, such as plastic. When the porous layer is silica, it can
be manufactured by many means. Two exemplary ways to form the
porous region are by the addition of material (e.g. deposition),
and by removal of material (e.g. selective etching).
[0056] In additive methods, a porous region is formed on the
surface of the underlying substrate to increase the effective
surface area. The porous region can be formed from deposition of
any or all of the following with or without catalysts in
appropriate solvent and ratios. For example, the porous region can
be formed from colloidal silica, an organo-silicon compound, such
as tetramethoxysilane (TMOS), metal alkoxides, silsesquioxanes, or
other silanes, or-combinations of these materials typically used in
sol gel processes. See C. J. Brinker, Sol-Gel Science, Academic
Press, Boston, 1990. With these types of precursors, parameters
such as solution composition, concentration, pH, aging time, and
temperature can be used to tailor the morphology (pore size,
porosity, thickness) of the porous region that is formed.
Additionally, there can be combinations of the above techniques to
provide the same eventual result (and there can be combinations in
the additive and subtractive techniques to achieve similar results
for other purposes). Also, other inorganic materials can be used in
either of the same forms as above (such as aluminum or
titanium-based materials).
[0057] Furthermore, the porous region matrix can be "templated". In
a templating process, a sacrificial material, such as a polymer, is
deposited with the matrix and then burned out, leaving behind a
porous structure with selected characteristics. (Note that a porous
region can also be formed without templating). The template
material can be any of the following, a preformed polymer, such as
a polystyrene latex, polymers dissolved in solution, or a
combination of these materials. The bum-out process, typically done
by heating in air, can be carried out at temperatures above
150.degree. C. up to the melting point (or glass transition
temperature, if appropriate) of the material that will form the
matrix for the porous layer. After the templating material is
burned out, the matrix material can be sintered together. Those
skilled in the art will appreciate that the term "sintering" (or
"annealing" in some contexts) is used to describe a
time-temperature processes for heating glass or other particles to
cause them to join. Whether the process is strictly solid state, or
involves some amount of material liquefaction or softening, is not
essential if a porous structure results. Time and temperature of
the sintering process can be varied to achieve different amounts of
densification and pore characteristics. Post-treatments after
annealing can be used to clean the surface in preparation for array
synthesis, such as cleaning in a solution of sulfuric acid and
hydrogen peroxide ("piranha solution") or sodium hydroxide. The
porous region matrix particles and templating particles can be
applied to the surface of the underlying substrate individually, or
as a pre-agglomerated mass.
[0058] A porous region, such as a porous layer or film can be
formed on a surface of the underlying substrate by a variety of
processes, such as spin-coating, dip-coating, spraying (aerosol),
individual spots deposited on surface, use of barriers (physical or
chemical) to specifically deposit coatings into channels, pads,
spots, or patterned surfaces. It should be understood that the
porous region or layer can be created in various forms, shapes, or
areas, or over the entire surface.
[0059] In preferred embodiments, film thickness can be modulated by
altering either or both of the precursors or layer formation
conditions. For example, the weight percent of solids in the
reagents listed above (e.g., about 10 wt. % to 40 wt. % solution of
colloidal silica) can alter the layer thickness. Similarly,
performing multiple depositions can build up the layer thickness.
Additional processing might be done between application of
successive layers of reagents, such as baking the substrate to
remove solvents from the film before the next application of
reagents. Film thickness can also be altered by modulating the spin
or deposition speed, for example, slower speeds yielding thicker
films and faster speeds yielding thinner films. Also, altering the
pull speed out of the dip-coating bath can affect layer thickness,
as slower speeds yield thinner films and faster speeds yield
thicker films. One can also control the solution conditions to
affect the film thickness. For example, when using the TMOS
approach, the solution can be caused to begin gelling, which
increases the viscosity and therefore the thickness of the
deposited layer.
[0060] The coatings, particles or other components can be spun onto
the substrate surface, the substrate can be dipped in a solution
containing the above reagents, the reagents can be sprayed onto the
surface of the substrate, or applied by other methods. The
substrate can be treated to create areas of high porosity over the
entire surface of the substrate or just in select locations, such
as by spotting reagents in grids, circular spots, areas, cells or
any shape that is preferred (see U.S. Pat. Nos. 5,744,305;
5,445,934; and 6,040,138). When the porosity is high and the pores
or particles are small, it minimizes the light scattering
properties when read by an instrument that uses optical properties
to detect a reaction (see U.S. Pat. Nos. 5,744,305; 5,981,956; and
6,025,601).
[0061] A subtractive approach can also be used to increase the
porosity of the substrate. For example, a porous region can be
etched into the surface of the substrate (i.e. the material at the
surface of what will become the underlying substrate). The surface
can be prepared as etched glass, such as phase-separating sodium
borosilicate glass, techniques for which are known in the art. In a
particular embodiment of the subtractive approach to forming a
porous substrate, the pore size can be further controlled by the
annealing time and temperature of an annealing step performed
before the etch (longer annealing, higher temperatures increase
pore size). Also, the depth of the porous region can be controlled
by etching parameters (solution concentration, composition, time,
etc.) in accordance with the substrate material.
[0062] In some embodiments, the silica substrate has an organic
polymer content of less than or equal to 10% mole fraction. In some
embodiments, an organic polymer coats the porous substrate. In some
embodiments, the porous substrate is coated with a silane compound
capable of linking with a polymer, such as
glycidoxypropyl-trimethoxysilane or with
N,N-bis(hydroxyethylaminopropyl)triethoxysilane.
[0063] In either the subtractive or additive techniques, it is
desirable to increase the effective surface area of the underlying
substrate so that more polymers can be attached to the surface. In
one embodiment, the pore size is at least 2, 5, 10, 15, 20, 25, 30,
50, 75, 100, 200, 250, or 500 nanometers. In one embodiment, the
largest pore size is 750, 800, 900, or 1,000 nanometers. It is
understood that not all pores will be of precisely the same size,
but rather will fall within a range in a given porous layer. The
numbers used are merely examples of the approximate average pore
diameter. The effective surface area can be expressed in units of
meters of surface area per gram of layer deposited. In one
embodiment, at least 15, 20, 25, 30, 50, 75, 100, 200, 250, 500
meters of surface area is per gram of layer deposited is formed and
not more than 750, 800, 900, or 1,000 meters of surface area per
gram of layer deposited.
[0064] The effective surface area can also be expressed as a factor
of area enhancement over the flat surface area of the underlying
substrate. For example, if the flat surface area is expressed as
"1", the increase in the effective surface area is for example at
least 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, 200, 250, 500, 750, or
1,000 times the flat surface area. Porosity is for example
expressed as at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the
surface of the substrate actually treated. Note that when a film is
deposited on a support material, the "porosity" refers to the
porosity of the film. The porosity of the layer is defined as the
space occupied by void divided by the sum of the space occupied by
both solid material and void within the layer. The array may
contain areas that have not been treated to increase the surface
area or the whole surface can be treated, or selected areas might
be treated differently. A porous region formed by depositing
colloidal silica for example has a thickness of about 0.01-20
microns, or about 0.1-1.0 microns, about 0.1 -0.5 microns, or about
0.1 to 10 microns. It was found that this layer thickness provided
good surface area enhancement for processes such as bioassay and
biosynthesis, and could be reliably fabricated according to methods
of the present invention.
[0065] In a preferred embodiment, the present invention is a porous
silica substrate that is useful for the manufacture of an array of
polymers. Polymer arrays and their uses are well described in many
patents and applications. Example uses of polymer arrays include
gene expression monitoring, discovery of polymorphisms, genotyping,
diagnostics, and affinity columns. See the following, which are
incorporated by reference in their entireties for all purposes. See
U.S. Pat. Nos. 5,677,195, 5,631,734, 5,624,711, 5,599,695,
5,510,270, 5,445,934, 5,451,683, 5,424,186, 5,412,087, 5,405,783,
5,384,261, 5,252,743 and 5,143,854; and U.S. application Ser. No.
08/388,321, filed Feb. 14, 1995, the disclosures of each of which
are incorporated herein. Uses of these polymer arrays are described
in U.S. Pat. Nos. 5,795,716 5,800,992, and 6,040,138.
[0066] Porous silica has been produced for other uses, such as an
intermediate form of a silica glass article. A process according to
the present invention utilizes a similar removal method with a
phase-separable glass body (substrate) in combination with an
etching process and possibly post-etch thermal treatment(s) to form
a porous layer on the underlying substrate. The removal method
involves (1) forming an article of desired shape from a parent
sodium borosilicate glass; (2) thermally treating the glass article
at a temperature of 500-600.degree. C. to separate the glass into a
silica-rich phase and a silica-poor phase; (3) dissolving or
leaching the silica-poor phase with acid to provide a porous
structure composed of the silica-rich phase; and (4) washing to
remove leaching residue, and then drying.
[0067] Another method according to the present invention uses
"sol-gel" in a deposition process to prepare a porous substrate at
moderate temperatures. Production of porous inorganic oxide glass
by the sol-gel process are described in U.S. Pat. Nos. 3,640,093
and 4,426,216. See also Scholze et al., J. Non-Crystalline Solids,
73: 669 (1985). The sol-gel procedure involves the formation of a
three-dimensional network of metal oxide bonds at room temperature
by a hydrolysis-condensation polymerization reaction of metal
alkoxides, followed by low temperature dehydration. The resultant
porous glass structure optionally can be sintered at elevated
temperatures. More recently, U.S. Pat. No. 4,765,818 described the
sol-gel preparation of microporous glass monoliths having 0.1-2
moles of trioxane per mole of tetraalkoxysilane, and reported that
the glass displayed superior optical properties.
[0068] Advantages of creating porous films through sol-gel
processing include that the films can be deposited and processed
easily, are inert to most chemicals, and can be created with a wide
range of morphology and surface chemistry.
[0069] Methods of synthesis of arrays of polymers, each polymer
comprising a plurality of monomers, are described in U.S. Pat. No.
5,744,305 ('305) or 5,831,070. "Monomer" may be, for example, a
member of the set of individual molecules which can be joined
together to form a larger polymer. Monomers can include individual
units of a polymer (such as one nucleotide) or can be larger
individual units (such as dimers, trimers, and higher) to make up a
larger polymer by sequential addition of these larger units.
Polymers of all types include analogous or mimics of the natural
polymer units. Predefined or known region means a localized area on
a surface that contains a polymer. The region may have a convenient
shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
For the sake of brevity herein, "predefined or known regions" are
sometimes referred to simply as "regions." Many synthesis methods
can be used to apply polymers to these regions. These regions are
the sized as shown in '305 and 5,445,934 ('934). Exemplary region
sizes are between 1, 5, 10, 20, 25, 30, 40 and 50 microns square.
Densities of the regions per square centimeter are shown in '305
and '934. For example, there are 10, 50, 100, 200, 400, 500, 700,
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, and 10.sup.7 different
regions per square centimeter. Primarily in one embodiment refers
to about 90% of the adjective it modifies. Thus, in one embodiment,
a "primarily inorganic substrate" means a substrate that has about
90% inorganic component such as silica. The remaining portion of
the substrate can have organic materials, or in some cases, trace
impurities, or both. As used in the specification and claims, the
singular form "a", "an" and "the" include plural references unless
the context clearly dictates otherwise. For example, the term "an
agent" includes a plurality of agents, including mixtures
thereof.
[0070] Porous Substrate
[0071] The porous substrate is useful as an article of manufacture
having a rigid or semi-rigid surface on which polymers can be
synthesized and or various applications (such as hybridization,
ligand-binding assays) using polymers can be performed. In some
aspects, the porous region comprises a primarily inorganic porous
material providing an enhanced surface area greater than the flat
surface area. In some embodiments, the primarily inorganic porous
region comprises silica. In some embodiments, at least one surface
of the substrate will be substantially flat, although in some
embodiments it may be desirable to physically separate synthesis
regions for different polymers with, for example, wells, raised
regions, etched trenches, large beads, light transmitting fibers,
or the like. According to other embodiments, small beads may be
provided on the surface of the substrate itself, which may be
released upon completion of the synthesis. The porous region is
formed on an support material that can be of a similar or different
material than the material of the porous region. Suitable materials
include those recited herein as support, such as all types of glass
materials, plastics, polymers, fused silica and other rigid and
semi-rigid materials.
[0072] The porous region may be made of silica or other material or
materials. The term "silica" represents silica compounds such as
silicon dioxide, although the exact stoichiometric ratio of oxygen
to silicon may vary and the silica may include modifying elements.
The silica may be in a colloidal form (which is known as colloidal
silica) or in a noncolloidal form. Silica can be made from an
organic compound or compounds comprising the silicon atom such as
an alkoxysilane, an example of which is tetramethoxysilane
("TMOS"). Colloidal silica, which is a form of very fine silica
particles, can be suspended in water (commonly called a "sol") or
in an organic solvent.
[0073] The porous substrate has a porous region wherein a
substantial number of the pores of the layer are connected to each
other and eventually to the free surface of the substrate. This
allows the infusion of the porous layer with a fluid or fluids,
such as a gas, a liquid, including liquid solutions, or a fluid
polymer, and can provide a substantial increase in surface area
(compared to the flat area of the substrate) for molecules to
attach to the surface of porous layer, as well as providing surface
area for reactions to occur. The porous substrate provides a
three-dimensional matrix that can be functionalized with reactive
groups, such as silylating agents, that serve as starting points
for polymer synthesis. The porous films provide a large number of
synthesis sites per unit area of the substrate. Additionally, the
porous substrates hold the potential to greatly increase the
binding of "target" molecules to immobilized polynucleotide or
nucleic acid sequences, which would thereby enhance detection.
Additionally, the multiplicity of binding sites may provide
additional kinetic enhancement.
[0074] FIG. 1 is a simplified cross section of a portion of a
porous substrate 100 showing a porous region 102 formed on a
support region 104. The support region can be one of several
different materials, such as silica, glass, silicon, or other
material that forms a suitable mechanical support for the porous
region, can withstand processing, and will not significantly effect
the intended use of the substrate, such as through chemical
reaction with assay or synthesis materials. The porous substrate is
made up of a plurality of particles 106, 108, and 110. In this
view, the diameters vary because of the nature of taking a cross
section, and also because there is typically some distribution of
particle size. A free surface ("surface") 112 has opening, or pores
114, that allow the entry of fluids, such as liquids or gas, into
the porous layer. In this example the particles are nominally 70 nm
across silica particles. This section view represents particles of
essentially the same size that intersect the section plane. The
various diameters shown in the figure represent sections of
particles, some of which are not sectioned through their center. It
is understood that, generally, each particle touches several other
particles, and thus a silica matrix is formed.
[0075] Sodium Borosilicate Porous Silica Substrate
[0076] The sodium borosilicate porous silica to be used herein has
the composition of about 65-70% SiO.sub.2, about 24-27%
B.sub.2O.sub.3, and about 6-8% Na.sub.2O (by weight). In one
embodiment, the composition is 67.4% SiO.sub.2, 25.7%
B.sub.2O.sub.3, and 6.9% Na.sub.2O (by weight). The glass is
prepared by a variety of methods, including by a modification of
what is annealed, causing phases to separate. The soluble phase,
which is rich in sodium and boron, is then removed by leaching with
hydrochloric or hydrofluoric acid, leaving behind a porous phase
that is nearly pure silica. After leaching, a thermal treatment, or
anneal, can be used to modify the porous structure. If a silica
product is desired, the porous silica glass can then be sintered at
high temperatures to full density and extremely high purity. This
allows one to fabricate an article out of sodium borosilicate
glass, which has a lower softening point and is easier to form than
pure silica, and end up with an article that is nearly pure silica.
In the present application, the initial porosity is desired. The
pore size can be controlled by the time of annealing, while the
layer depth can be controlled by the time of leaching with
acid.
[0077] FIG. 2 is a simplified cross section of a portion of a
porous substrate 120 showing a porous region 122 formed on a
support region 124. The support region is generally phase separable
glass, but could be other material bonded to the porous region, for
example. In one preferred embodiment, the porous region is etched
in a phase separable glass that also provides the support region
(i.e. the porous substrate is formed from a blank of phase
separable glass). The porous region is formed by preferential
leaching, as described above, and forms a "sponge-like" or
"coral-like" matrix 126 with pores 128 accessible from the surface
130 by fluids. In contrast to the deposited porous region shown in
FIG. 1, the leached porous region has a less-defined transition
between the porous region 122 and the support region. It is
understood that the figures are not to scale and are not scaled
relative to each other.
[0078] Sol-Gel Type Porous Silica Substrate
[0079] The sol-gel method can use colloidal silica with, or without
an alkoxysilane. A preferred embodiment uses a tetraalkoxysilane,
for example, Si(OCH.sub.3).sub.4 as a starting material, which is
mixed and stirred with CH.sub.3OH and H.sub.2O. The resulting
mixture is transferred into a desired vessel. The vessel is allowed
to stand to subject the mixture to hydrolysis and condensation
reactions.
[0080] Illustrative of tetraalkoxysilanes and other metal and
metalloid alkoxides that can be used in this invention are methoxy
and ethoxy derivatives of silicon, lithium, sodium, potassium,
rubidium, cesium, magnesium, calcium, strontium, barium, titanium,
zirconium, vanadium, tantalum, chromium, molybdenum, tungsten,
manganese, iron, nickel, cobalt, copper, zinc, cadmium, boron,
aluminum, phosphorus, gallium, germanium, tin, arsenic, antimony,
bismuth, selenium, and the like. Aryloxy derivatives such as
trimethoxyphenoxysilane also can be utilized in the sol-gel
process.
[0081] Illustrative of water-miscible solvents which can be
employed in a sol-gel process embodiment are alcohols such as
methanol and ethanol; ketones such as acetone and methyl ethyl
ketone; esters such as methyl acetate and ethyl formate; ethers
such as dibutyl ether and tetrahydrofuran; amides such as
formamide, dimethylformamide, dimethylacetamide and
1-methyl-2-pyrrolidinone; and the like.
[0082] Acidic pH conditions in the sol-gel process can be provided
by the addition of mineral acids such as hydrochloric acid, and
basic pH conditions can be provided by the addition of bases such
as ammonium hydroxide. Hydrogen fluoride is a particularly
preferred acidic pH reagent, because the fluoride anions have a
catalytic effect on the hydrolysis and condensation reactions of
the sol-gel process.
[0083] Characteristics of the Porous Silica Substrate
[0084] In one embodiment, the porous region has the following
preferred characteristics: a pore size of 2-500 nm, e.g., 2-100 nm,
2-200 nm, or 2-50 nm; a porosity of 10-90%, e.g., 10-30%, 20-80%,
40-60%, 70-90%, or 50-70%; and a thickness of 0.01-20 .mu.m,
0.1-0.5 .mu.m, 0.1-1.0 .mu.m, or 1-20 .mu.m. When the substrate is
prepared from a colloidal silica deposition, the average particle
diameter is in one embodiment 5-500 nm, 5-100 nm, or 70-100 nm. It
is well-known in the the art how to measure and determine the above
characteristics of porosity, pore size, thickness and particle
diameter. Additionally, the substrate can be made of an
alkoxysilane, or colloidal silicon dioxide or both in varying
concentrations. It is also known in the art that some tolerance for
trace impurities is allowed. The alkoxysilanes include
trialkoxysilanes and tetraalkoxysilanes.
[0085] Functionalization/Silylation
[0086] The porous silica substrate of the present invention can be
silylated to provide many functionalized attachments.
Alternatively, the colloidal silica particles, which are used in
preparing the porous silica substrate, can be functionalized so
that the porous silica substrate that is formed is already
functionalized. The silylation can be accomplished by using any
number of silylating agents. Many silylating agents are known in
the art. For example, N-(3-(triethoxysilyl)-propyl)-4-hydroxybut-
yramide (PCR Inc., Gainesville, Fla.) has been used to silylate a
glass substrate prior to photochemical synthesis of arrays of
polynucleotides on the substrate, as described in McGall et al., J.
Am. Chem. Soc., 119:5081-5090 (1997), the disclosure of which is
incorporated herein by reference.
[0087] Hydroxyalkylsilyl compounds that have been used to prepare
hydroxyalkylated substances, such as glass substrates.
N,N-bis(hydroxyethyl) aminopropyltriethoxysilane has been used to
treat glass substrates to permit the synthesis of high-density
polynucleotide arrays. See McGall et al., Proc. Natl. Acad. Sci.,
93:13555-13560 (1996); and Pease et al., Proc. Natl. Acad. Sci.,
91:5022-5026 (1994), the disclosures of which are incorporated
herein. Acetoxypropyl-triethoxysila- ne and 3-Glycidoxy
propyltrimethoxysilane have been used to treat glass substrates to
provide a linker for the synthesis of polynucleotides. See EP
Patent Application No. 89 120696.3.
[0088] The functionalized silicon compounds include an activated
silicon group and a derivatizable functional group. Exemplary
derivatizable functional groups include hydroxyl, amino, carboxyl
and thiol, as well as modified forms thereof, such as activated or
protected forms. The functionalized silicon compounds may be
covalently attached to surfaces to form functionalized surfaces
that may be used in a wide range of different applications. The
silicon compounds are attached to the surface of a substrate
comprising silica, such as a glass substrate, to provide a
functionalized surface on the silica containing substrate, to which
molecules, including polypeptides and nucleic acids, may be
attached. After covalent attachment of a functionalized silicon
compound to the surface of a solid silica substrate to form a
functionalized coating on the substrate, an array of nucleic acids
may be covalently attached to the substrate or synthesized off of
the functional groups. Thus, the method permits the formation of
high density arrays of nucleic acids immobilized on a substrate,
which may be used in conducting high volume nucleic acid
hybridization assays.
[0089] As used herein, the term "silicon compound" refers to a
compound comprising a silicon atom. In a preferred embodiment, the
silicon compound is a silylating agent comprising an activated
silicon group, wherein the activated silicon group comprises a
silicon atom covalently linked to at least one reactive group, such
as an alkoxy or halide, such that the silicon group is capable of
reacting with a functional group, for example on a surface of a
substrate, to form a covalent bond with the surface. Exemplary
activated silicon groups include --Si(OMe).sub.3;
--SiMe(OMe).sub.2; --SiMeCl.sub.2; SiMe(OEt).sub.2; SiCl.sub.3 and
--Si(OEt).sub.3.
[0090] As used herein, the term "functionalized silicon compound"
refers to a silicon compound comprising a silicon atom and a
derivatizable functional group. In a preferred embodiment, the
functionalized silicon compound has an activated silicon group and
a derivatizable functional group. "Derivatizable functional group"
refers to a functional group that is capable of reacting to permit
the formation of a covalent bond between the silicon compound and
another substance, such as a polymer or a polymer building block.
Exemplary derivatizable functional groups include hydroxyl, amino,
carboxyl and thiol, as well as modified forms thereof, such as
activated or protected forms. Derivatizable functional groups also
include substitutable leaving groups such as halo or sulfonate. In
one preferred embodiment, the derivatizable functional group is a
group, such as a hydroxyl group, that is capable of reacting with
activated nucleotides to permit nucleic acid synthesis.
[0091] The surface can be functionalized by covalently attaching to
the surface a functionalized silicon compound, wherein the
functionalized silicon compound comprises at least one
derivatizable functional group and a plurality of activated silicon
groups, for example, 2, 3, 4 or more activated silicon groups. An
array of nucleic acids can be covalently attached to the
functionalized silicon compounds on the surface. The number of
silicon groups and the number of derivatizable functional groups in
the silicon compound may be modified for different applications, to
increase or decrease the number of bonds to a support such as a
glass support.
[0092] Further description of several silylating agents and methods
for their preparation can be found in U.S. Pat. No. 5,624,711 and
in U.S. Ser. No. 09/172,190, filed Oct. 13, 1998, which are hereby
incorporated by reference. Commercially available silicon compounds
and a review of silicon compounds is provided in Arkles, Ed.,
"Silicon, Germanium, Tin and Lead Compounds, Metal Alkoxides,
Diketonates and Carboxylates, A Survey of Properties and
Chemistry," Gelest, Inc., Tullytown, Pa. (1995), the disclosure of
which is incorporated herein. Functionalized silicon compounds may
be synthesized using methods available in the art of organic
chemistry, for example, as described in March, Advanced Organic
Chemistry, John Wiley & Sons, New York (1985).
[0093] Polymer Coated Porous Substrate
[0094] The porous substrate of the present invention can be
polymer-coated. The substrate can be polymer-coated using dip
coating, covalent polymer attachment, in situ polymerization, or
combinations thereof. In yet another aspect, the substrate can be
glycan-coated. While similar to the polymer-coated supports, the
properties of glycan-coated supports can be quite different and
provide extremely hydrophilic surfaces that are useful in binding
assays and diagnostic applications. A detailed description of
polymer and glycan coating materials and methods is given in the
U.S. Pat. No. 5,624,711, which is hereby incorporated by
reference.
[0095] In any of these methods, the choice of available surface
polymers is extensive. Suitable polymers include chloromethylated
styrene-divinylbenzene (Merrifield resin),
phenylacetamidomethylated styrene-divinylbenzene (PAM resin), and
crosslinked polyethylene glycol-polystyrene grafts (TentaGel
resin). The polymers which are used to coat the solid support can
also be selected based upon their functional groups which will
serve as synthesis initiation sites. Typically, polymers having
primary amine, carboxyl or hydroxyl functional groups will be
selected.
[0096] Polymers having primary amine functional groups are of
interest as these polymers can be readily adapted to coupling
chemistry currently used in the high density array synthesis.
Suitable polymers having primary amine functional groups include
polyethyleneimine linear or branched polymers, polyacrylamide, and
polyallylamine which are all commercially available. Other
polymers, such as polydimethylacrylamide (or other polymers in this
genus), can be synthesized according to published procedures (see
Atherton, E. et al. in "Solid Phase Peptide Synthesis: A Practical
Approach," Chapter 4, pp. 39-45, IRL Press (1989); and Arshady, R.
et al., J. Chem. Soc. Perkin. Trans. 1:529 (1981)).
[0097] Polymers having carboxyl functional groups are also useful
as the resulting surfaces are very hydrophilic. Furthermore, the
synthesis initiation sites (i.e. the carboxylic acid groups) are
useful in peptide synthesis which proceeds from the amino terminus
of the peptide to the carboxylic acid terminus. Suitable polymers
having carboxylic acid functional groups include poly(acrylic
acid), poly(ethylene/maleic anhydride), and poly(methylvinyl
ether/maleic anhydride).
[0098] Polymers having hydroxyl functional groups are also useful
as the resulting surfaces are extremely wettable. Examples of
suitable polymers include polyethyleneglycol (PEG), polyvinyl
alcohol and carbohydrates.
[0099] In general, the glycan-coated surfaces can be prepared in a
manner analogous to the preparation of polymer-coated surfaces
using covalent attachment. Thus, a glass surface can be modified
(silanized) with reagents such as aminopropyltriethoxysilane to
provide a glass surface having attached functional groups (in this
case, aminopropyl groups). The modified surface is then treated
with a solution of a modified dextran to provide a surface having a
layer of dextran which is covalently attached.
[0100] Linking Molecules
[0101] After derivatization of the porous substrate, the
derivatized surface may be contacted with a mixture of linking
molecules and diluent molecules (the diluent molecules are optional
and are not included in preferred embodiments). The diluent
molecules for example have only one center which is reactive with
the reactive sites on the derivatized substrate surface. All the
other reactive centers on the diluent molecules are protected,
capped or otherwise rendered inert. The linking molecules will
similarly have one center which is reactive with the reactive sites
on the derivatized substrate surface. Additionally, the linking
molecules will have a functional group which is optionally
protected and which can later serve as a synthesis initiation site.
The linking and diluent molecules are present in the mixture in a
ratio which is selected to control the functional site density on
the surface. The ratio of linking molecules to diluent molecules is
for example from about 1:2 to about 1:200, e.g., from about 1:10 to
about 1:50. Alternatively, the ratio of linking molecules to
diluent molecules can be from about 200:1, or from about 100:1, or
from about 10:1, as desired for adjusting the density of polymers
on the surface.
[0102] The linking molecules should be of sufficient length to
permit polymers synthesized thereon to interact freely with
molecules exposed to the polymers. The linking molecules should be
3-50 atoms long to provide sufficient exposure of ligands to their
receptors. Typically, the linking molecules will be aryl acetylene,
ethylene glycol oligomers containing 2-14 monomer units, diamines,
diacids, amino acids, peptides, or combinations thereof. In some
embodiments, the linking molecule can be a nucleotide or a
polynucleotide. The particular linking molecule used can be
selected based upon its hydrophilic/hydrophobic properties to
improve presentation of the polymer synthesized thereon to certain
receptors, proteins or drugs.
[0103] The linking molecules can be attached to the substrate by
siloxane bonds (using, for example, glass or silicon oxide
surfaces). Siloxane bonds with the surface of the substrate may be
formed in one embodiment via reactions of linking molecules bearing
traditional aminopropyl silane groups such as trichlorosilyl,
trimethoxy or triethoxy silyl groups. The linking molecules may
optionally be attached in an ordered array, i.e., as parts of the
head groups. In some aspects, the linking molecules are absorbed to
the surface of the substrate. In addition, linking molecules may
also be present in case of nucleic acid synthesis and hybridization
assays.
[0104] As noted above, the linking molecule, prior to attachment to
the derivatized surface has an appropriate functional group at each
end, one group appropriate for attachment to the reactive sites on
a derivatized surface and the other group appropriate as a
synthesis initiation site. For example, groups appropriate for
attachment to the derivatized surface would include amino, hydroxy,
thiol, carboxylic acid, ester, amide, isocyanate and
isothiocyanate. Additionally, for subsequent use in synthesis of
polymer arrays or libraries, the linking molecules used herein will
typically have a protecting group attached to the functional group
on the distal or terminal end of the linking molecule (opposite the
solid support).
[0105] The linking molecule contributes to the net hydrophobic or
hydrophilic nature of the surface. For example, when the linking
molecules comprise a hydrocarbon chain, such as
----(CH.sub.2).sub.n----, the effect is to decrease wettability.
Linking molecules like polyoxyethylene
(----(CH.sub.2CH.sub.2O.sub.n)--, or polyamide
(----(CH.sub.2CONH).sub.n----) chains tend to make the surface more
hydrophilic (i.e., increase wettability).
[0106] The diluent molecules can be any of a variety of molecules
which can react with the reactive sites present on the derivatized
substrate and which generally have remaining functional groups
capped or protected. The diluent molecules can also be selected to
impart hydrophobic or hydrophilic properties to the substrate
surface. For example, the diluent molecules are alkanoic acids,
which impart hydrophobic properties to the surface. In other cases,
the diluent molecules are amino acids, wherein the amine and any
side chain functionality which is present are protected. In these
instances, the diluent molecules can contain functionality which is
altered upon treatment with various reagents such as acid, base or
light, to generate a surface having other desired properties. For
example, use of O-t-Butyl serine as a diluent molecule provides a
hydrophobic surface during polymer synthesis, but upon treatment
with acid (cleaving the t-butyl ether), a more hydrophilic surface
is produced for assays.
[0107] Thus, after reacting the mixture of linking molecules and
diluent molecules with the surface and subsequently synthesizing a
desired polymer onto the functional sites on the linking group, the
protecting groups on the surface-attached diluent molecules are
removed to provide a more hydrophilic (i.e. "wettable") surface. In
preferred embodiments, the diluent molecules are protected glycine,
protected serine, glutamic acid or protected lysine. Dimethyl
N,N-diiosopropylphosphoramidite can be used to phosphorylate
surface hydroxyls, to alter the surface characteristics. Further
description of linking molecules and diluent molecules are given in
the U.S. Pat. No. 5,624,711, which is hereby incorporated by
reference.
[0108] Substrates with Acidic Surfaces
[0109] The present invention also provides porous substrates which
are derivatized to provide acidic surfaces, or "carboxy chips." The
carboxy chips can be considered as "reverse polarity" surfaces (as
compared with the more typical aminopropylsilane derivatized
surfaces). Such reverse polarity surfaces will find application in
combinatorial synthesis strategies which require a carboxylic acid
initiation site. For example, peptide synthesis which is carried
out from the N-terminal end to the C-terminal end can be carried
out on a carboxy chip. Additionally, small molecules such as
prostaglandins, .beta.-turn mimetics and benzodiazepines can also
be synthesized on a carboxy chip. Carboxy chips will also find
application in the preparation of chips having synthesis initiation
sites which are amines. In this aspect, the carboxy chips will be
reacted with a suitably protected alkylenediamine to generate an
amino surface.
[0110] Carboxy chips can be prepared by a variety of methods. For
example, a solid support is derivatized with an aminoalkylsilane to
provide a surface of attached amino groups. The derivatized surface
is then treated with an anhydride such as glutaric anhydride to
acylate the amino group and provide a surface of carboxylic acid
functionalities. Alternatively, the aminoalkylsilane is first
reacted with an anhydride (i.e., glutaric anhydride) to generate a
carboxylic acid silane which can then be coupled to the porous
substrate, and similarly provide a surface of carboxylic acid
residues. Further description of carboxy chips can be found in the
U.S. Pat. No. 5,624,711, which is hereby incorporated by
reference.
[0111] Array Synthesis
[0112] Large scale chemical diversity on primarily inorganic porous
substrates can be achieved by synthetic strategies and devices
presented herein. The preferred substrates, solid-phase chemistry,
photolabile protecting groups, deprotection techniques, and
photolithography, when brought together, achieve very high density,
spatially-addressable, parallel chemical synthesis. Thus, the
preferred substrates provided herein can be used in a number of
applications, including light-directed methods, flow channel and
spotting methods, pin-based methods and bead-based methods (see the
patents and references cited above).
[0113] Alternatively, the primarily inorganic porous substrates of
the present invention can be used to prepare high density arrays of
polymers using conventional linkage chemistry-based synthetic
methods, also known as phosphoramidite-based synthesis methods. One
example of conventional linkage chemistry-based polynucleotide
synthesis is known as standard dimethoxytrityl (DMT) method.
Examples of this and additional phosphoramidite synthesis methods
are described in the "User Manual for Applied Biosystems Model
391," pp. 6-1 to 6-24, available from Applied Biosystems, 850
Lincoln Center Dr., Foster City, Calif. 94404, and are generally
known by those skilled in the art. See also M. Gait,
Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press,
London.
[0114] Light-Directed Methods
[0115] "Light-directed" methods (which are one technique in a
family of methods known as VLSIPS.TM. methods) are described in
U.S. Pat. No. 5,143,854, '305, '934 and other patents above all of
which are incorporated by reference. The light directed methods
discussed in these patents involve activating known locations or
predefined regions of a substrate or solid support and then
contacting the substrate with a preselected solution of monomers or
polymers. The known locations or predefined regions can be
activated with a light source, typically shown through a mask (much
in the manner of photolithography techniques used in integrated
circuit fabrication). Other regions of the substrate remain
inactive because they are blocked by the mask from illumination and
remain chemically protected. Thus, a light pattern defines which
regions of the substrate react with a given monomer. By repeatedly
activating different sets of predefined regions and contacting
different monomer solutions with the substrate, a diverse array of
polymers is produced on the substrate. Of course, other steps such
as washing unreacted monomer solution from the substrate can be
used as necessary.
[0116] The porous silica substrate and the optionally provided
linker molecules thereon can be the same as described infra in the
context of conventional linkage chemistry-based synthesis. The
linker molecules may each include a protecting group. In
light-directed polymer synthesis, the protecting group is a
photocleavable (photoreactive) protecting group. Photocleavable
protecting groups, addition, binary synthesis strategy, and other
processes associated with light directed methods are shown in U.S.
Pat. No. 5,744,305.
[0117] Flow Channel or Spotting Methods
[0118] Additional methods applicable to library synthesis on a
single substrate are described in U.S. Pat. Nos. 5,677,195,
5,384,261, and 6,040,138 incorporated herein by reference for all
purposes. In the methods disclosed in these applications, reagents
are delivered to the substrate by either (1) flowing within a
channel defined on predefined regions or (2) "spotting" on
predefined regions. However, other approaches, as well as
combinations of spotting and flowing, may be employed. In each
instance, certain activated regions of the substrate are
mechanically separated from other regions when the monomer
solutions are delivered to the various reaction sites. One of
ordinary skill in the art would also appreciate that this method
can also be used to deposit pre-synthesized oligomers or polymers
for further polymerization.
[0119] The "spotting" methods of preparing arrays of the present
invention can be implemented in much the same manner as the flow
channel methods. For example, a monomer A can be delivered to and
coupled with a first group of reaction regions which have been
appropriately activated. Thereafter, a monomer B can be delivered
to and reacted with a second group of activated reaction regions.
Unlike the flow channel embodiments described above, reactants are
delivered by directly depositing (rather than flowing) relatively
small quantities of them in selected regions. In some steps, of
course, the entire substrate surface can be sprayed or otherwise
coated with a solution. In preferred embodiments, a dispenser moves
from region to region, depositing only as much monomer as necessary
at each stop. Typical dispensers include a micropipette, a quill,
or a pin and ring to deliver the polymer solution to the substrate
and a robotic system to control the position of the micropipette
with respect to the substrate, or an ink-jet printer. In other
embodiments, the dispenser includes a series of tubes, a manifold,
an array of pipettes, or the like so that various reagents can be
delivered to the reaction regions simultaneously.
[0120] Pin-Based Methods
[0121] Another method which is useful for the preparation of
compounds and libraries of the present invention involves "pin
based synthesis." This method is described in detail in U.S. Pat.
No. 5,288,514, previously incorporated herein by reference. The
method utilizes a substrate having a plurality of pins or other
extensions. The pins are each inserted simultaneously into
individual reagent containers in a tray. In a common embodiment, an
array of 96 pins/containers is utilized.
[0122] Each tray is filled with a particular reagent for coupling
in a particular chemical reaction on an individual pin.
Accordingly, the trays will often contain different reagents. Since
the chemistry disclosed herein has been established such that a
relatively similar set of reaction conditions may be utilized to
perform each of the reactions, it becomes possible to conduct
multiple chemical coupling steps simultaneously. In the first step
of the process the invention provides for the use of substrate(s)
on which the chemical coupling steps are conducted. The substrate
is optionally provided with a spacer having active sites. In the
particular case of polynucleotides, for example, the spacer may be
selected from a wide variety of molecules which can be used in
organic environments associated with synthesis as well as aqueous
environments associated with binding studies. Examples of suitable
spacers are polyethyleneglycols, dicarboxylic acids, polyamines and
alkylenes, substituted with, for example, methoxy and ethoxy
groups.
[0123] Additionally, the spacers will have an active site on the
distal end. The active sites are optionally protected initially by
protecting groups. Among a wide variety of protecting groups which
are useful are FMOC, BOC, t-butyl esters, t-butyl ethers, and the
like. Various exemplary protecting groups are described in, for
example, Atherton et al., "Solid Phase Peptide Synthesis," IRL
Press (1989), incorporated herein by reference. In some
embodiments, the spacer may provide for a cleavable function by way
of, for example, exposure to acid or base.
[0124] Bead Based Methods
[0125] A general approach for bead based synthesis is described in
U.S. Pat. Nos. 5,770,358, 5,639,603, and 5,541,061 the disclosures
of which are incorporated herein by reference. For the synthesis of
molecules such as polynucleotides on beads, a large 5 plurality of
beads are suspended in a suitable carrier (such as water) in a
container. The beads are provided with optional spacer molecules
having an active site. The active site is protected by an optional
protecting group.
[0126] In a preferred embodiment, the beads are tagged with an
identifying tag which is unique to the particular double-stranded
polynucleotide or probe which is present on each bead. A complete
description of identifier tags for use in synthetic libraries is
provided in U.S. Pat. No. 5,639,603.
[0127] Conventional Linkage Chemistry Methods
[0128] In addition to the above-described light-directed
methodology, high density arrays of polymers can be synthesized
using conventional linkage-based chemistry and using protecting
groups that are chemically cleaved using solution or vapor-phase
deprotection agents. This methodology is described in greater
detail in the U.S. Pat. No. 5,599,695, which is incorporated by
reference.
[0129] The above methodology can be applied to the synthesis of
several types of polymer, including those of biological interest
such as polynucleotides, nucleic acids, polypeptides, proteins,
oligosaccharides and polysaccharides. Chemical synthesis of
polypeptides is known in the art and are described further in
Merrifield, J., J. Am. Chem. Soc., 91: 501 (1969); Chaiken I. M.,
CRC Crit. Rev. Biochem., 11: 255 (1981); Kaiser et al., Science,
243:187 (1989); Merrifield, B., Science, 232:342 (1986); Kent, Ann.
Rev. Biochem., 57: 957 (1988); and Offord, R. E., Semisynthetic
Proteins, Wiley Publishing (1980), which are incorporated herein by
reference). In addition, methods for chemical synthesis of peptide,
polycarbamate, and polynucleotide arrays have been reported (see
Fodor et al., Science, 251:767-773 (1991); Cho et al., Science,
261:1303-1305 (1993), each of which is incorporated herein by
reference).
[0130] Data Collection
[0131] Devices to detect regions of a substrate which contain
fluorescent markers are known in the art. See e.g., U.S. Pat. Nos.
5,631,734; 5,744,305; 5,981,956 and 6,025,601, incorporated by
reference. These devices would be used, for example, to detect the
presence or absence of a labeled receptor such as an antibody which
has bound to a synthesized polymer on a substrate.
[0132] U.S. Pat. No. 5,527,681, the disclosure of which is
incorporated herein, describes use of computer tools for forming
arrays. For example, a computer system may be used to select
nucleic acid or other polymer probes on the substrate, and design
the layout of the array as described in U.S. Pat. No. 5,571,639,
the disclosure of which is incorporated herein.
[0133] Further understanding of the nature and advantages of the
inventions herein may be realized by reference to the remaining
portions of the specification and the attached drawings.
[0134] All publications cited herein are incorporated herein by
reference in their entirety.
APPLICATIONS
[0135] The above-described arrays of polymers such as polypeptides,
or nucleic acids or polysaccharides prepared on the porous
substrates of this invention can be used in a variety of
applications including biological binding assays and nucleic acid
hybridization assays. For example, polynucleotide or nucleic acid
arrays can be used to detect specific nucleic acid sequences in a
target nucleic acid. See, e.g., PCT patent publication Nos. WO
89/10977 and 89/11548. General hybridization and detection of
nucleic acids is shown in U.S. Pat. Nos. 5,631,734, 5,510,270 and
5,324,633.
[0136] Methods for screening target molecules for specific binding
to arrays of polymers, such as nucleic acids, immobilized on a
solid substrate, are disclosed, for example, in U.S. Pat. Nos.
5,677,195, 5,631,734, 5,624,711, 5,599,695, 5,510,270, 5,445,934,
5,451,683, 5,424,186, 5,412,087, 5,405,783, 5,384,261, 5,252,743
and 5,143,854; 5,800,992, 5,795,716, 6,040,138 and U.S. application
Ser. No. 08/388,321, filed Feb. 14, 1995. Accessing genetic
information using high density DNA arrays is further described in
Chee, Science 274:610-614 (1996). Arrays to detect mutations in
reference. Arrays to detect mutations in HIV genes are described in
detail in the U.S. Pat. No. 5,861,242. Arrays to detect nucleic
acid from nonviral pathogens infecting AIDS patients are shown in
U.S. Pat. No. 5,861,242. Devices for concurrently processing
multiple biological chip assays may be used as described in U.S.
Pat. No. 5,545,531. The arrays of polynucleotides or nucleic acids
prepared on porous silica substrates provided herein can be used to
screen polymorphisms in samples of genomic material. The detailed
methodology is provided in the U.S. Pat. No. 5,858,659. Tiling
strategies are discussed in detail in the U.S. Pat. No. 5,837,832.
Hybridization and scanning are generally carried out by methods
described in, e.g., Published PCT Application Nos. WO 92/10092 and
WO 95/11995, and U.S. Pat. No. 5,424,186, incorporated herein by
reference. Gene expression may be monitored by hybridization of
large numbers of mRNAs in parallel using high density arrays of
nucleic acids in cells, such as in microorganisms such as yeast, as
described in Lockhart et al., Nature Biotechnology, 14:1675-1680
(1996), and U.S. Pat. Nos. 5,800,992 and 6,040,138, PCT WO
97/10365. Bacterial transcript imaging by hybridization of total
RNA to nucleic acid arrays may be conducted as described in Saizieu
et al., Nature Biotechnology, 16:45-48 (1998), the disclosure of
which is incorporated herein. Additional examples of applications
in biomedical and genetic research and clinical diagnostics are
disclosed in U.S. Pat. Nos. 5,547,839, 5,710,000 (using Type-IIs
restriction endonucleases), and in U.S. patent application Ser. No.
08/143,312. Other applications include chip based genotyping,
species identification and phenotypic characterization, as
described in U.S. patent application Ser. No. 08/797,812, filed
Feb. 7, 1997, and U.S. application Ser. No. 08/629,031, filed Apr.
8, 1996.
[0137] Arrays of nucleic acids for use in gene expression
monitoring are described in PCT WO 97/10365, the disclosure of
which is incorporated herein. In one embodiment, arrays of nucleic
acid probes or other polymer probes are immobilized on a surface,
wherein the array comprises more than 100 different nucleic acids
and wherein each different nucleic acid is localized in a
predetermined area of the surface, and the density of the different
nucleic acids is greater than about 60 different nucleic acids per
1 cm.sup.2.
[0138] Arrays of nucleic acids or other polymers immobilized on a
surface also are described in detail in U.S. Pat. No. 5,744,305,
the disclosure of which is incorporated herein. As disclosed
therein, on a substrate, nucleic acids or other polymers with
different sequences can be immobilized each in a predefined area on
a surface. For example, 10, 50, 60, 100, 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7, or 10.sup.8 different monomer
sequences may be provided on the substrate. The nucleic acids of a
particular sequence are provided within a predefined region of a
substrate, having a surface area, for example, of about 1 cm.sup.2
to 10.sup.-10 cm.sup.2. In some embodiments, the regions have areas
of less than about 10.sup.-1, 10.sup.-2, 10.sup.-3, 10.sup.-4,
10.sup.-5, 10.sup.-6, 10.sup.-7, 10.sup.-8, 10.sup.-9, or
10.sup.-10 cm.sup.2. For example, in one embodiment, there is
provided a planar, non-porous support having at least a first
surface, and a plurality of different nucleic acids attached to the
first surface at a density exceeding about 400 different nucleic
acids/cm.sup.2, wherein each of the different nucleic acids is
attached to the surface of the solid support in a different
predefined region, has a different determinable sequence, and is,
for example, at least 4 nucleotides in length. The nucleic acids
may be, for example, about 4 to 20 nucleotides in length. The
number of different nucleic acids may be, for example, 1000 or
more.
[0139] Since the application of a porous glass is considerably
broader than its use in the examples discussed and provided herein,
these examples are used for illustrative purposes only and should
not be construed as a limitation on the full scope of the invention
disclosed herein.
EXAMPLES
Example 1
[0140] Sol-Gel Silica Deposition
[0141] Methods and Materials
[0142] Glass microscope slides (2 in.times.3 in.times.0.027 in,
from ERIE SCIENTIFIC) were cleaned in piranha solution (30% v/v
hydrogen peroxide, 70% v/v sulfuric acid) for 30 minutes with
gentle stirring. They were then transferred immediately to clean
water in which they were stored. The slides were removed and blown
dry with dry nitrogen immediately prior to film deposition.
Long-term storage in water is not necessary for The starting point
for the film preparation is to mix the precursors in solution.
Colloidal silica, tetramethoxysilane (TMOS), hydrochloric acid,
water, and methanol are mixed in solution. The particle size of the
colloidal silica ranged from about 12 nm to >100 nm and was
deposited on a glass surface. In addition to the glass slides
discussed above, other exemplary glass providing a surface for the
deposition of colloidal silica is soda lime glass (Erie
Scientific), as well as borofloat glass or fused silica glass (U.S.
Precision Glass, Elgin, Ill.). The resultant pores from such
particles are in the 2-40 nm range. Layers having a thickness of
about 0.1 .mu.m-2 .mu.m were investigated, but the thickness was
chosen for experimental purposes and actual devices may have
thicker or thinner layers. TMOS may be added to the colloidal
silica to strengthen the resulting silica matrix.
[0143] Two sources of colloidal silica were used. LUDOX.TM. was
purchased from E. I. Dupont de Nemoirs, and includes silica spheres
suspended in water, stabilized by sodium counterions. For HS-40,
the spheres are nominally 12nm diameter, whereas for TM-40 they are
22 nm. SNOWTEX.TM. colloidal silica was purchased in from NISSAN
CHEMICALS in three sizes (listed size range in parenthesis):
SNOWTEX-50 (20-30 nm), SNOWTEX-20L (40-50 nm), and SNOWTEX-ZL
(70-100 nm).
[0144] Tetramethoxysilane was purchased from ALDRICH, with
specification of 98% purity. The pH of the total mixture was
adjusted to 3.75+/-0.25, measured with a VWR benchtop pH meter
Model 8015.TM. (VWR SCIENTIFIC).
[0145] Formation of Films
[0146] Equal weights of colloidal silica were used in each
solution, and then water was added as necessary to keep the
solution volume constant. Table 1 shows the compositions that were
used. 1.2M HCl was used to titrate the solutions to the appropriate
pH, with different amounts being required in each case. It should
be noted that the glass composition listed in the Table below may
also have trace amounts of
1TABLE 1 Representative Colloidal Silica Solution Compositions
(amounts in mL) Size (nominal) 12 nm 2 nm 20-30 nm 40-50 nm 70-100
nm Brand (Ludox HS-40) (Ludox TM-40) (Snowtex-50) (Snowtex-20L)
(Snowtex-ZL) Water 13.4 13.4 14.9 5.2 13.4 Methanol 12.6 12.6 12.6
12.6 12.6 Colloidal Silica 6.6 6. 5.2 14.8 6.6 TMOS 1.36 1.36 1.36
1.36 1.36
[0147] The components were mixed in solution and stirred gently for
approximately 10 minutes. Stirring was discontinued, and the
mixture was left to react for approximately one hour. The solution
was then filtered with a 0.45 micron filter (either with a filter
attached to the syringe or by filtering the solution separately and
then dispensing). Films were also created without the use of TMOS
in the precursors. For those films, using the SNOWTEX-20L and ZL,
the solution was typically diluted to 10-20 wt. % with pure water,
and then filtered with a syringe filter (or HPLC-grade nylon 0.45 u
filter paper for large batches). These cases will be noted in later
sections. Prior to deposition, dynamic light scattering was
performed on the solutions. Light scattering analysis gives the
size distribution of the particles in solution.
[0148] The substrate was then spun at various speeds, ranging from
500 to 2500 rpm on a Headway Research photo-resist spin coater
(Headway Research) to distribute the solution into a thin film. For
the data reported, the films were deposited in a lab environment
with no explicit control of air flow, temperature, or humidity.
[0149] The substrates were then allowed to dry at room temperature
conditions for several hours. They were then transferred to an oven
were they were fired at 75.degree. C. for one hour, 100.degree. C.
for 2 hours, and then 500.degree. C. for 5 hours. The first two
steps are to remove residual moisture, the last to cause mild
sintering of the matrix for mechanical stability. Some films were
also created without the use of this procedure, and will be noted
in later sections. In a preferred embodiment, no sintering
(annealing) step is performed. Such a porous layer also has
improved effective surface area because sintering or similar
high-temperature processing generally causes particles to at least
partially combine or densify. It has been found that the
un-sintered colloidal silica layers are sufficiently robust for a
variety of intended uses, such as polymer synthesis or
bioassay.
[0150] Thickness of the films was measured by scratching the films
and then measuring the depth of scratch with a surface
profilometer, such as a DEKTAK II.TM. available from VEECO.
Surfaces for ellipsometric measurements were made by depositing the
films on silicon rather than glass to provide a contrast in the
index of refraction. The silicon dioxide surface layer on the
silicon substrate should provide a substrate similar to the glass
surface. Ellipsometric measurements were made with a Gaertner
Ellipsometer Model L116C.TM. with a light wavelength of 632.8 nm
(Gaertner Scientific Corporation).
[0151] A desirable feature of the porous layers of the present
invention formed on the surface of the underlying substrate is that
they do not appreciably scatter light, such as may be used in
scanning arrays of polymer or genetic materials. In some prior art
processes, relatively large holes have been etched into the surface
of a substrate, such as a glass substrate. Such large holes are
distinguishable from the pores of the present invention in that a
process mask or similar method is typically used to define the
location of the holes. Furthermore, such holes can be large enough
to scatter light and interfere with scanning or other light-based
use of the substrate.
[0152] After the porous surfaces were prepared they needed to be
silanated to appropriately prepare the surface for oligomer
synthesis. Prior to silanation, the dried and annealed slides were
soaked in water for approximately 15 minutes. They were then
transferred to a bath of reagent alcohol for 3 minutes, and then to
the silane bath for 15 minutes. The silane bath consists of 94%
reagent alcohol, 5% water, and 1% bis (2
hydroxyethyl)-3-aminopropyltriethoxysilane in 62% ethanol (Gelest
Inc.) by volume. Slides were then transferred to two successive
isopropanol baths (5 minutes each), blown dry with dry nitrogen,
and placed in a 35-50.degree. C. oven for 3 minutes. The above
silanation conditions were used for most applications. However
other silanes and silylation conditions were also used.
[0153] Fluoreprime Stain Assay: Qualitative/Quantitative Surface
Performance using Photochemical Polynucleotide Synthesis
[0154] Quantitative studies of the synthesis, density and
uniformity of the porous silica substrates was conducted using
methods based on surface fluorescence as described in McGall et al;
J. Am. Chem. Soc.; 119: 5081-5090 (1997). Fluorescent "staining" of
the surface was performed as described, with the exception that a
fluorescein concentration of 0.5 mM in a solution containing 50 mM
DMT-T-CEP in acetonitrile was used. The fluorescein phosphoramidite
is coupled to the free hydroxyl groups with the standard protocol.
Substrates are then deprotected for a minimum of one hour in a 1:1
solution of ethylenediamine/ethanol, rinsed with deionized water,
and blown dry with dry nitrogen. The substrate is then scanned.
using a confocal microscopy. The signal obtained is a function of
the number of available hydroxyl groups on the surface. In this
case, the relative values as compared to other types of similarly
treated glass is an indication of the relative density and capacity
of the surface. This technique also provides a visual picture of
the surface with respect to quality and unformity of the surface.
The technique is not limited to hydroxyl groups but may be modified
to measure other groups of interest for support of polymer of
interest on the surface by using the appropriately functionalized
molecule for detection.
[0155] HPLC Quantitation Assay
[0156] This technique is described in U.S. Pat. No. 5,843,655,
"Methods for Testing Oligonucleotide Arrays," also by Affymetrix.
HPLC (high performance liquid chromatography) analyses were
performed on a Beckman System Gold ion exchange column using
fluorescence detection at 520 nm. Elution is performed with a
linear gradient of 0.4M NaClO.sub.4 in 20 mM Tris pH 8, at a flow
rate of 1 mL/min, or other suitable buffer system.
[0157] The HPLC quantitation assay was used to measure the site
density available for generating polymers, the coupling efficiency
of each subsequent addition of monomer to the growing chain, and
the extent of adsorption or entrapment of the reagents within the
porous matrix.
[0158] In this technique, MeNPOC PEG is attached to the surface,
followed by a capping step, a cleavable (SO.sub.2) linker, a spacer
molecule ("C3," a three carbon chain), and a fluorophore. The
linker is 5'-phosphate-ON reagent (ChemGenes Corporation), and the
fluorophore is 5-carboxyfluorescein-CX CED phosphoramidite (Flam)
(BioGenex). The purpose of the spacer molecule is to discriminate
between fluorescent molecules that have attached to the intended
synthesis sites vs. those that have remained on the surface without
chemical attachment. It should be noted that the cleavable linker
can be attached directly to the glass surface without the use of a
PEG- type linker. Synthesis was also accomplished on the surfaces
using traditional acid-based polynucleotide chemistry (trityl
chemistry). Similar chemistries can be applied for the synthesis of
polynucleotide, peptide, oligosaccarides, peptide nucleic acids,
and other polymers. The description relating to the peptide nucleic
acids can be found in the PCT publication WO92/20702, published
Nov. 26, 1992, which is incorporated by reference in its
entirety.
[0159] After synthesis the surface is treated with a known solution
volume of reagent necessary to cleave the linker to release
3'-C.sub.3-flam-5', and this is typically cleaved in solution
overnight (1:1 by volume ethylenediamine/water). The resulting
solution is diluted and coinjected with an internal standard onto
and analyzed by HPLC. The internal standard is a
3'-C.sub.3-C.sub.3-Flam-5' chain prepared separately on an ABI
synthesizer. Concentration is determined by UV-Vis spectra on a
Varian Cary 3E spectrophotometer (Varian). Integration of HPLC peak
areas can be used to determine total site density and cleanliness
of coupling.
[0160] In an analogous experiment step by step coupling yields can
be determined by coupling the cleavable linker amidite and
Fluorescein amidite to the surface followed by synthesis of a
polymer of interest using MeNPOC chemistry. Typically a 6-mer
homopolymer such as poly (A).sub.6 is synthesized. The probes are
then cleaved in solution, diluted with an appropriate corresponding
internal standard, and run through the HPLC column. Peaks are
observed for the probes of lengths 1-6, and give indication of the
relative coupling yield of successively added bases.
[0161] Synthesis of Full-length Probes for Functional Assessment of
Porous Surface under Hybridization Conditions
[0162] Full length probes capable of hybridization, typically
20-mer probes, were synthesized using Affymetrix synthesizers as
described in U.S. Pat. No. 5,405,783, using nucleoside
phosphoramidites equipped with 5'- photolabile MeNPOC protecting
groups. The sequence used for the majority of the preliminary
experiments is (3')-AGG TCT TCT GGT CTC CTT TA (5'), with the 3'
end attached to the surface. The non-photolabile protecting groups
were removed post synthesis in 1:1 ethylenediamine/ethanol (v/v)
for a minimum of 4 hours.
[0163] Hybridization assays were performed on the 2.times.3" slides
without further processing. Each slide was placed in 10-15 mls of
10-50 nM target oligonucleotide in hybridization buffer with gentle
stirring. The two hybridization buffers commonly used are
6.times.SSPE and MES (sodium chloride, sodium phosphate, EDTA and
2-[N-morpholino]ethanesulfon- ic acid respectively). The target
sequence is the exact complement of the probe sequence: (5')
Fluorophore-TCC AGA AGA CCA GAG GAA AT.
[0164] The pattern and intensity of surface fluorescence was imaged
with a specially constructed scanning laser confocal fluorescence
microscope, as described in McGall, supra. Where necessary, the
photon multiplier tube gain was adjusted to keep signals within
range for the detector.
[0165] Results
[0166] Initial testing of the porous films was conducted with the
smaller particle sizes (Ludox) and including the TMOS and annealing
steps. Deposition of the particles on the surface of the substrate
has been confirmed by using scanning electron microscopy and atomic
force microscopy. For films deposited at 2500, 1500, and 500 rpm
with the TM-40 solution, the ratio of the hybridization signal on
porous glass to flat glass reaches factors of approximately 5, 7
and 18 times that of flat glass respectively. The ratios are
calculated by dividing the hybridization signal from the porous
surface (with background subtracted) by the hybridization signal
from the flat surface (also with background subtracted). This data
shows the actual ability both to synthesize full 20-mer probes and
hybridize target DNA to the probes, and thus the actual functional
improvement which can be obtained with using these surfaces for DNA
arrays. The porous surfaces yield signals many times greater than
the flat glass. It has been found that the hybridization signal
increases with films spun under decreasing spin speed. This
corresponds to deposition of a thicker layers at slower speeds and
indicates that fluorescence signal obtained is proportional to film
thickness.
[0167] Thicker films can also be achieved by depositing multiple
layers. A multilayer film was created by depositing a layer at 1500
rpm, allowing the film to dry for approximately 15 minutes, and
then repeating the process. The hybridization signal at the 20 and
40 hour time points is approximately 2 times higher for the
multilayer film than for the single layer. Film thickness can be
modulated by depositing multiple layers or by increasing the weight
percent of the colloidal silica in the solution prior to deposition
on the glass surface. This again demonstrates the tremendous
flexibility of sol-gel deposition and its use for this
application.
[0168] It should be noted that the sol-gel layers were formed
without explicit control of temperature, humidity, or air flow.
This demonstrates the robustness of the process. Controlling these
variables and other environmental factors to refine the properties
of the system is within the capability of one of ordinary skill in
the art.
[0169] Ellipsometric measurements on the 500 rpm surface gave an
index of refraction for the film of approximately 1.3. The
refractive index of a porous film is the volume average of the
solid phase and the pore space, and thus gives an estimate of the
porosity. Using this technique, it is estimated that the above film
is approximately 30-40% porous. Film thickness for the 500 rpm film
was measured as 6400+/-300 .ANG.. Combining these measurements with
the nominal particle size, it is estimated that the porous surface
will have a maximum surface area of approximately 100 times that of
a flat surface of comparable lateral dimensions. However, this
estimate does not take into account factors such as particle
contacts and TMOS coverage, so the actual area available is smaller
than this multiple.
[0170] To analyze how much of the surface is being accessed by
probe synthesis, an HPLC quantitation analysis of the surface was
performed to determine the site density and this value was compared
to the fluorescence signal attained by scanning. For the films spun
at 500 rpm with the TM-40 solution, both procedures give signals
which agree within experimental error, and indicate that the porous
surface has approximately 40 times more sites than a flat surface.
Furthermore, since the site density and fluorescence agree,
quenching is not significant or an issue when evaluating surfaces
of this density.
[0171] The HPLC results also showed that there are not a
significant number of fluorescent molecules which are not attached
to activated synthesis sites, as the only large peaks in the
chromatogram were from the activated synthesis sites and the
internal standard. This is an important result because molecules
getting "stuck" in the matrix could be a concern. Given the 40-fold
factor improvement (out of a theoretical 100-fold maximum), it is
clear that the films offer substantial advantages over flat glass,
and also that there is room for further improvement if even more of
the surface can be accessed.
[0172] Pore size in these films is estimated as the smallest spaces
between particles through which a potential target would have to
diffuse. For the current system, this is estimated by assuming
three same-sized particles in a triangular formation. In this
configuration, the pore size is approximately 0.15 times the
particle diameter. Thus for 22 nm particles, the smallest pores
would be on the order of 4 nm.
[0173] The use of larger particles (and thus larger pores) may be
one route to access more of the surface. An additional advantage of
depositing films comprised of larger particle sizes, is that there
is a potential to use larger target molecules. As will be described
in following sections, various staining and antibody amplification
techniques are often used for signal amplification in hybridization
to complex arrays. These molecules are often much larger than the
fluorescein-labeled polynucleotides. For this reason, experiments
were conducted with the larger Snowtex particles before proceeding
to complex arrays.
[0174] Using the same procedures as described previously (TMOS,
annealing of substrate), films were deposited at 500 and 2500 rpm
with Snowtex-20L (referred to as "40 nm") and Snowtex-ZL (referred
to as "70 nm") solutions and hybridized to a target solution (50 nM
target, 6.times.SSPE). These films show significant improvement
over the flat glass, especially under stringent assay conditions of
long hybridization times at elevated temperature. With these porous
surfaces, signal enhancements of 15 to 45 times that of flat glass
were reached, with higher signals for smaller particles (higher
surface area to volume ratio per particle) and slower spin speeds
(thicker films).
[0175] Edge diffraction studies revealed that the porous silica
surface did not affect light absorbance through the glass substrate
or cause light scattering, thus rendering the surface amenable to
photochemical methods of synthesis. For photochemical methods to be
employed on such surfaces for the printing of small features, the
edge resolution must be very sharp. The methodology was as
described in McGall, et al, supra. The results indicate that there
was no discernible distinction (i.e., diffraction effects) between
the flat glass and the porous silica, which was verified down to
features sizes of 24.times.24 microns.
[0176] Experiments were conducted to determine if several of the
steps of the original procedure, use of the TMOS and annealing
steps, could be eliminated. The primary effect of removing these
steps could be a resultant decrease in the stability of the films.
This film stability was tested in several ways.
[0177] Films originating from a 10 wt. % ZL solution were deposited
and fluoroprime stained as described above. Flat "standard" glass
controls was run as well. The arrays were scanned at intervals and
the signal was tracked over the course of several days under harsh
assay-type conditions: MES buffer or 6.times.SSPE buffer at
45.degree. C. In MES buffer, the fluoreprime stain signal intensity
on the porous surfaces decreased slightly more slowly than the flat
glass. The porous films exhibited similar rates of signal decay
when compared to the flat glass in 6.times.SSPE buffer, The rate
signal decrease in the 6.times.SSPE buffer assay was quicker for
both the porous and flat glasses.
[0178] To further test stability, a functional hybridization assay
was performed under typical "gene expression" conditions of an
overnight (16-18 h) hour hybridization at 45.degree. C. Films were
deposited with a 10 wt. % ZL solution and a concentrated ZL
solution (equivalent to 4-5 layers deposited at 10 wt %), yielding
films of approximately 1500 and 6000 angstroms thick respectively.
20 mer probes were synthesized and hybridized to 10 nM labeled
target. The films achieved fluorescence hybridization signals of 6
and 29 times that of the flat glass respectively, which shows that
the signal increase is approximately linear with film
thickness.
[0179] These films provide examples for determining surface
accessibility in the larger pore films. The thickness, index of
refraction, and light scattering (prior to deposition) analysis are
combined to estimate the surface area of these films. The thinner
films have a surface area approximately 10 times that of flat
glass, and the thicker films are approximately 44 times flat glass.
These values are within reasonable tolerances of the observed
6-fold and 29-fold respective increase detected hybridization
signals.
[0180] Since the films achieve a signal comparable to their
expected area, it is expected that chemical coupling is proceeding
efficiently. This was tested by the 6-mer coupling method on
several films. Flat glass was tested vs. films deposited from 10
wt. % 20L and ZL solutions. Coupling yield is nearly identical to
the flat glass on both of the porous substrates.
[0181] Kinetics of hybridization can be determined by real time
scanning confocal microscopy of the to follow the annealing of the
probe sequence to a 5'-fluorescein labeled complementary
oligonucleotide target. (Forman, J. E., Walton, I. D., Stern, D.,
Rava, R. P., Trulson, M. O", Thermodynamics of Duplex Formation and
Mismatch Discrimination Onphotolithographically Synthesized
Oligonucleotide Arrays Acs Symposium Series 682:206-228,1998)
[0182] With a 1500 Angstom porous glass layer of derived from the
70-100 nm particles, hybridization equilibrium at room temperature
is typically reached in approximately 2 h. The time to reach
equilibrium increased to 3-5 h for a 3000 Angstom layer as compared
to 40-60 minutes for standard flat surface.
[0183] The substrates provided herein can be used with any type of
array formation pattern. It can be readily appreciated that by
varying the experimental conditions such as viscosity, thickness of
layers, size of the colloidal particles, aging time and pH of the
colloidal mixture, one can control layer thickness, porosity,
morphology, and surface chemistry in order to optimize the system.
For example, raising the viscosity (such as by adding less water)
or lowering the spin speed can increase film thickness. Depositing
multiple layers of the same thickness or different layers of
varying thickness would also result in porous layers of the desired
thickness. Additionally, other properties such as particle size
could be varied on different layers. Controlling the environment,
such as humidity, partial pressures of other solvents (such as
methanol), air flow, temperature can also lead to porous glass of
desired pore size and layer thickness.
[0184] Since varying the colloid size changes pore size, one can
obtain the desired pore size by appropriately formulating the
colloidal mixture of proper particle size initially. Colloid
aggregation can be controlled by varying the "aging" time and pH
before deposition. Other ceramic colloids, such as titania or
alumina, which may have different surface chemistry and/or
aggregation properties can also be used to prepare colloids of
desired composition and size. Additionally, surface chemistry of
the colloidal particles can be controlled for example, by
silanating the colloidal particles before deposition, in order to
tailor the chemistry of the final film.
[0185] Optical scattering can be reduced by using refractive
index-matched fluids. These fluids reduce optical scattering during
patterning and scanning of the chips. Water has a refractive index
of 1.33, whereas that of glass is typically 1.45-1.52. An example
of an index-matched fluid is an aqueous solution of 64% by weight
sucrose which has an index of 1.45, or dioxane with index of
1.42.
[0186] The preparation and synthesis of porous glass substrates is
amenable to scale up. Preliminary studies were performed on
2.times.3 slides. Current manufacturing of "real" GeneChip.RTM.
arrays requires synthesis to be performed on a 5".times.5" fused
silica wafer and synthesized on an AFFYMETRIX MOS synthesizer.
Moving to this scale posed several new challenges for porous
surfaces, all which have been met so far.
[0187] Preliminary evaluation of wafer uniformity showed no
difficulties in scaling up. The wafers can be coated uniformly by
spin coating and most tests that were performed in initial
evaluations of the porous surfaces were also used to monitor
uniformity across the wafer. These tests involve functional
assessment of chip performance as a function of location on the
wafer.
[0188] There are many mechanical and chemical demands during wafer
scale synthesis of DNA arrays. A synthesis cycle is comprised of
chemical delivery of reagents on the MOS, followed by removal of
the wafer then alignment on a photolysis station. Typical
GeneChip.RTM. array synthesis involves 70-80 such cycles, consuming
up to 18 hrs. of processing and handling, followed then by
deprotection, dicing the wafer into individual arrays, and
packaging into cartridges. Wafers coated with films comprised of
colloidal silica particles survived these various steps and showed
higher functional performance that flat glass.
[0189] The porous silica substrates of the present invention also
possess excellent array feature characteristics. Checkerboard
patterns that contain 400.times.400 micron features as well as the
24 micron features present on the Human 6800 arrays exhibited no
defects resulting light scattering- or diffraction-related
difficulties during either the photolithographic synthesis or
scanning microscopy on the experiments as described herein.
[0190] Porous inorganic colloidal silica particle size ranging from
12 nm-greater than 100 nm was deposited on a glass surface. The
glass surface was made of but limited to either soda lime,
borofloat or fused silica glass. The resultant pores from such
particles are in the 2-40 nm range. Layers from 0.1 micron or 2
micron were actively investigated. The layers could be thicker or
thinner.
[0191] The porous silica substrates of the present invention allow
the synthesis of high-density 3-dimensional arrays. High-density
arrays were prepared on layers of silica particles of approximately
0.25-0.3 microns thickness which are prepared from a 20% (by
weight) colloidal silica solution. Two representative complex
high-density DNA arrays will be discussed in detail here to
exemplify the capabilities of the present porous glass substrates:
one representing a sequence analysis (disease management) type
array and one representing a gene expression array. A 0.3 micron
porous surface was selected for synthesis of GeneChip.RTM. arrays
for evaluation in both disease management and gene expression type
assays.
[0192] Sequence Analysis Array
[0193] Sequence analysis or disease management arrays are typically
but not limited to "tiling type-arrays." Assay times are relatively
short because target concentrations are not limited. The target may
be labeled with either fluorescein for direct detection or may be
labeled with biotin for detection via signal amplification. A
GeneChip.RTM. test array containing an HIV sequence was synthesized
on a porous glass substrate and a representative HIV assay was
performed. The array on this test vehicle is comprised of probes
representing the HIV protease and reverse transcriptase genes. The
sequence analysis assay was performed on fragmented
fluorescein-labeled HIV cRNA target. The surface was scanned at
regular intervals and was approaching equilibrium at 6 hours, at
which time there was a 4-6 fold increase in hybridization signal
intensity over flat glass. Furthermore, at this time the base call
discrimination on the porous surface was comparable to the flat
surface. The assay time is somewhat longer than typical assays of
this type, but as a practical matter, these longer assay times are
not material. This assay further verifies that fragmented RNA
target can indeed access the probe sites within the porous matrix.
Large RNA fragments or reagents do not get trapped within the
matrix as there is no increase background signal and there is no
reduced dicrimination by probes for target.
[0194] Gene Expression Monitoring
[0195] A Human 6800 array (HuF1) was synthesized (24 micron
features; 16-20 probe pairs /gene) on a 0.3 micron porous silica
substrate coated with either BIS or GPS silane (BIS[2-
hydroxyethyl]-3-aminopropyl-trietho- xysilane or 3-glycidoxylpropyl
trimethoxy silane respectively) (Gelest, Tullytown, Pa.) and
compared to the appropriately silanated flat glass control. The GPS
silane can be deposited in the vapor or solution phase followed by
ring opening with acid. Standard quality control assay, which
involves hybridization of four biotinylated control gene
transcripts as well as twelve biotinylated polynucleotide targets
in 6.times.SSPE buffer for 16 h at 45.degree. C. followed by
staining with streptavidin-phycoerythryn complex (SAPE) (Molecular
Probes, Eugene Oreg.), showed that the BIS and GPS silanes yield
similar hybridization results on a given surface type. A 1-3-fold
enhancement in hybridization signal intensity and average
difference (perfect match minus mismatch divided by the total
number of probes in a gene) was observed on the porous silica
substrates relative to the flat glass with the control gene probe
pairs (RNA target). The porous silica substrate surface exhibited a
7-fold increase in the average difference data with the DNA target
polynucleotides.
[0196] Additional assays involved detection of 9
biotinylated-control genes spiked into the hybridization mixture
that contained a background of complex labeled human RNA target.
Assays were conducted and materials were obtained as described in
The Affymetrix Gene Chip Expression Analysis Manual, 1999. Assays
were performed in MES buffer at 45.degree. C. for 16 h followed by
staining with SAPE. These assays show that the porous silica
substrate results in an enhancement in signal of four to six fold
with respect to the flat glass surface. Similar values were
obtained by looking at the average difference for the control
genes. Scatter plots comparing all the genes on the wafer reveal
that this trend holds with all the probe sets on the surface and
indicate uniform surface response to target.
[0197] Further signal amplification by using a second staining step
as is commonly done in complex gene expression analysis assays
involves treating the SAPE stained surface with an antibody (IgG)
followed by a second round of SAPE staining. Again this led to
another six fold increase in signal on both surfaces and a four to
six fold increase in signal on the porous silica substrates
relative to the flat glass. The discrimination on both surfaces is
the same. Furthermore, the discrimination on the porous surfaces
can be additionally improved by employing more stringent wash
conditions.
[0198] The results thus show that gene expression monitoroing type
assays on porous silica substrates have yielded very high signal
intensities under the standard conditions without any assay
optimization. Assay optomization which is underway may improve this
further. This is significant because, it is generally known that
more sensitivity is needed in expression monitoring assays.
Additionally, because the signal is much stronger after the first
stain, it may be possible to eliminate further staining/signal
amplification or cut back the time necessary to run the assay. No
increase in nonspecific binding has been observed. Clearly the
pores generated between the particles enable diffusion of target,
label (SAPE) and antibodies to access the sites. Thus, the porous
silica substrates of the present invention appear to be superior to
the traditional flat glass substrates in many important aspects and
appear to provide many advantages in high-density array synthesis
and assays.
Example 2
[0199] Particle Templating
[0200] The approach of depositing colloidal silica to form a porous
layer for DNA arrays shows a tremendous potential for using porous
inorganic layers as supports for biosensing devices. Furthermore,
it demonstrates a very simple and reproducible technique that can
be effective. However, this technique can be further improved with
a templating process to further control pore size and porosity, two
key aspects of the film morphology. Control of pore size is
desirable for at least two aspects of hybridization. First of all,
larger pores allow for larger molecules to penetrate the matrix.
Thus, arbitrary targets can be selected and the morphology of the
porous layer (surface) altered to match. Second it is known from
literature that flow in a porous structure is affected by pore
size. Larger pores provide the potential advantage of increasing
flow rates and thus decreasing hybridization (processing) times,
thereby boosting assay performance.
[0201] In the technique described, pore size and porosity are
controlled by co-depositing sacrificial organic particles, such as
polymer (e.g. polymer latex) spheres, with the silica particles,
and then burning out the organic material at high temperatures or
otherwise removing it. Templating using a sacrificial material is
not a new technique in itself. Studies have been conducted where
latex particles served as pore templates in gelled silica networks
formed from alkoxysilanes. The current approach is novel at least
because only particles are used for both the template and the
silica matrix that remains after pyrolysis. As is described in the
following section, simple modificiations of the size, charge, and
concentration of the latex can provide a wide range of film
morphologies. In typical deposition with alkoxysilanes, more
complicated changes in the chemistry and processing are necessary
to alter the morphology. Additionally, an important advantage of
this approach for the application of polymer synthesis and
hybridization is that the voids inherent in the particle system
ensure open porosity, whereas films formed from alkoxysilanes with
templating can often results in closed pores. The open porosity
results in at least some pores being connected to each other, thus
allowing fluids to pass into the porous layer of the substrate
through the free surface of the substrate.
[0202] The polymer to be co-deposited with the silica can be any
suitable polymer that achieves the objective of providing the
desirable porous structure. One of ordinary skill in the art would
understand that several such polymers are available for the present
purposes, for example, polystyrene latex can be used.
[0203] By the term "co-depositing", it is meant here that the
silica and the polymer need not be presented simultaneously so long
as the polymer and the silica occupy the surface of the substrate
to provide the desired porosity upon the removal of the
polymer.
[0204] FIG. 3a is a simplified cross section of a portion of a
substrate 140 being processed to form a porous region according to
a templating method. Templating particles 142 have been mixed with
smaller silica particles 144 (shown without cross hatching for
purposes of clarity) and applied to an underlying substrate or
support region 104. It is believed that the silica particles coat
the templating particles (e.g., latex spheres). After the silica
and templating particles are applied to the underlying substrate,
the templating material can be burned off, leaving behind a matrix
of the silica particles. The particles can be of the same or
different size. In one embodiment, the small silica particles
further enhance the effective area of the porous layer. Porosity
and pore size are increased by voids left from the removal of the
templating material. In other embodiments, the remaining silica
particle can be sintered or annealed to strengthen the remaining
matrix.
[0205] FIG. 3b is a simplified cross section of the portion of a
porous substrate 141 after the templating particles have been
removed, such as in a burn-off process. The burn-off process has
removed the templating particles (compare FIG. 3b, reference
numeral 142) to leave behind a matrix of silica particles 144. The
voids left by the templating particles provide increased effective
surface area for the porous substrate. The silica particles may be
further processed, such as in an annealing process, to further
densify the matrix 146 of silica particles.
[0206] FIG. 4 is a simplified cross section of a portion of a
substrate 150 being processed to form a porous layer according to
another embodiment of the present invention. Templating particles
152 are mixed with silica particles 154 and applied to the
underlying substrate 104, such as by spin coating. The templating
particles are then removed, as discussed above in reference to
FIGS. 3a and 3b, leaving behind a matrix of silica particles. As
discussed above in relation to FIG. 1, the section viewed
represents particles of essentially the same size that intersect
the section plane. The various diameters shown in the figure
represent sections of the particles, some of which are not
sectioned through their center. It is understood that, generally,
each particle touches several other particles, thus when the
templating material is removed a silica matrix forms. It is further
understood that particles other than silica could be used to form
the resulting matrix.
[0207] Experimental
[0208] Polystyrene- latex dispersions (hereinafter referred to as
"latex" or "latex particles") were purchased from INTERFACIAL
DYNAMICS CORPORATION of Portland, Oreg. The solutions are
stabilized either by negatively charged sulfate groups on the
surface or positive amidine groups. Particles with other surface
groups could also be used.
[0209] In a typical film deposition, the latex and silica solutions
are mixed and diluted with pure water to the desired
concentrations. Exemplary concentrations include 10:1 by volume
latex to 1:1 by volume latex. The solution was then filtered
through a 450 nm syringe filter and dispensed onto either glass or
a silicon wafer as described previously. Thick yet homogeneous
films could be obtained at spin speeds of 1000 rpm and greater (70
second spin time). Thicker films can be obtained by using slower
spin speeds, more concentrated solutions, or multiple
depositions.
[0210] Following spin coating, the films were annealed to remove
polymer. Typical conditions were to ramp 2.degree. C./minute to
400.degree. C., dwell 4 hours, then cool to 20.degree. C. at rate
of 20.degree. C./minute. Following annealing, a final cleaning step
was applied to some samples. This step consisted of immersing the
annealed substrate in either piranha solution (30 minutes) and/or
etching in 1M sodium hydroxide at 70.degree. C. (3 minutes).
[0211] Results
[0212] Atomic force microscopy images of co-deposited 40 nm silica
and 60 nm latex particles have been taken before annealing in
"phase contrast mode," which shows the distribution of the two
types of particles. From the images it can be observed that the
deposition is relatively homogeneous. In an ideal distribution,
silica and latex particles would alternate in an "array" manner.
While the distribution does not reach this level of homogeneity,
extensive inter-mingling of the phases can be observed.
[0213] The uniqueness and flexibility of using particles to create
the final films was demonstrated by two examples. In a first
example, 7 nm silica particles were mixed in solution with 100 nm,
positively charged latex particles. The charge difference causes
the relatively small silica particles to coat the larger latex
particles. This sample was dipcoated by withdrawing slowly from
solution with the use a variable speed motor. Following annealing,
large pores on the order of 100 nm were left behind, with a
relatively dense matrix formed from the remaining silica.
[0214] Very different structures were created by mixing same-sized,
same-charge particles, such as negatively charged 40 nm latex and
silica particles of the same size. As silica is typically
negatively charged in this environment, the latex and silica do not
substantially aggregate. A potential advantage of these films is
that the pores left behind by the annealed latex are better
connected to each other by the passages between the larger silica
particles than in non-templated films. In comparison to the films
created with the larger particles and no templating (such as
SNOWTEX-ZL), the templated films have pores which are as large (or
larger) with surface area that is as high (or higher).
[0215] The techniques of light scattering, ellipsometry, and
thickness profiling were combined to characterize and estimate the
surface area per unit thickness for the templated films. The
statistics shown in Table 2 compare a templated film made from 20
nm Latex/SNOWTEX-50 2:1 v/v film with a pure silica film made from
SNOWTEX-ZL. This table demonstrates the potential benefit of
templated films. Several factors should be noted. First of all, for
approximately the same film thickness, the templated film has
nearly 3 times the area. Second, the pores in the templated film
are actually as large or larger than the pure silica film. The
pores in the film made with SNOTEX-ZL (70-100 nm particles) are on
the order of 15 nm, whereas the templated film formed with latex
particles has 20 nm pores; thus, even though the starting particle
size of the pure silica film is approximately 5 times larger than
the starting particle size of the templated film, the templated
film provides pores that are greater than about 30% larger.
Finally, the porosity is much higher, which gives much more room
for a potential target to diffuse into the matrix. Thus, the
templated film provides a much more efficient substrate for some
applications.
2TABLE 2 Comparison of Pure and Templated Films Templated 20 nm
silica (2:1 Pure 70 nm silica v/v latex:silica) Thickness 2200 2500
Index 1.37 1.16 Porosity 20% 65% Area factor 14.5 35.4
Area/thickness 6.6 14.2 (per 0.1 micron)
[0216] Full 20 mer probes were synthesized on the films described
in Table 2. A kinetic scan of the adsorbed target vs. time was
performed. The conditions used were room temperature, 10 nM target,
flow of 4 ml/min, MES buffer. The kinetic scan demonstrates the
advantage of using templated films. For a film of similar
thickness, a higher signal is reached. The pure non-templated film
reaches a signal 14 times that of the flat glass, where the
templated glass reaches a signal 40 times that of flat glass. These
signals agree well with the area factors estimated in Table 2.
Additionally, the templated film reaches equilibrium more quickly
(3 hrs vs. 5 hrs.).
Example 3
[0217] Etched Sodium Borosilicate Glass
[0218] Glass samples of sodium borosilicate glass, suitable for use
in a VYCOR.TM. process, were obtained from Dow CORNING, in the form
of 150.times.150.times.0.7 mm sheets. The specifications are for a
composition of 67.4% SiO.sub.2, 25.7% B.sub.2O.sub.3, 6.9%
Na.sub.2O (by weight), but the precise ratios of components may
vary within known ranges. These sheets where then diced into
10.times.10 mm pieces for testing. The test pieces were annealed at
650.degree. C. for 4 hours to separate the glass into regions of a
sodium and boron-rich phase and regions of a silica-rich phase. The
samples were then placed in 4% rich phase, preferentially leaving a
matrix of the silica-rich phase.After leaching, the samples were
soaked in methanol for approximately 15 minutes. The glass was then
cleaned in a sodium hydroxide solution (20 g/liter) that to
dissolve silica that might remain in the pores following the
leaching step. Finally, the glass was again soaked in methanol for
15 minutes, and allowed to dry at room temperature.
[0219] Immediately before silanation, the test pieces were placed
in sodium hydroxide solution (20 g/liter) at 70.degree. C. for 3
minutes, followed by water for 15 minutes. This step was used to
further insure that the surface would be covered with hydroxyl
groups that may have been removed during the HF treatment.
[0220] The films are silanated with the same procedures described
above as in the case of sol-gel porous silica. Fluorescent staining
was performed with 5 mM concentrations of fluorescein, in a manner
similar to that described above in the case of sol-gel silica.
Exceptions are that the test pieces were mounted on 2.times.3 in
slides using room-temperature vulcanizing ("RTV") silicone glue
available from DOW CORNING, and a 20 mil fluoropolymer tape
(Polyken) was used as a spacer. This tape showed minimal
degradation by solvents over the period for fluorescent
staining.
[0221] Results
[0222] Cross sectional scanning electron microscopy reveals an
etched surface layer of approximately 70 microns. It is not clear
whether the porosity is interconnected through the depth of the
entire layer, although it can be assumed since the etch solution
must penetrate into the matrix. Large pores can be observed, on the
order of 1000 .ANG. and larger, although rigorous measurements of
pore size were not made. Using these measurements, it was estimated
that a layer of 70 microns thick could increase the surface area by
as much as 400 times over the flat area of the glass.
[0223] Staining the surface with fluorescene, as described above,
revealed that the etched porous surface yielded a fluorescence
signal gain 200 times that of flat glass. Additionally, for the
process examined, the etched porous surface actually had a lower
background, that is as a percent of the total signal, resulting in
an overall increase in the number of sites of over 300 times the
flat glass after correction for background.
[0224] Additionally, site density was examined using the HPLC
quantitation procedure. The HPLC analysis confirmed a factor of 200
increase in accessible sites. However, the chromatogram from the
HPLC also showed that there was a significant fraction of
fluorescent molecules removed from the surface which were not
attached to activated sites (i.e. fluorescent molecules not
attached to C.sub.3 spacers molecules). These may be molecules
which were "stuck" in the matrix during the staining procedure.
This result differs from the sol-gel system in which all the
observed signal was from covalently attached molecules. This
trapping of molecules within the matrix is most likely due to the
extremely thick surface layer, and using shorter etch time to
obtain a thinner layer could decrease the portion of molecules not
attached to synthesis sites.
[0225] The above experiments show that the etched borosilicate
glass layer of the above-described composition holds significant
promise for use as a substrate in a variety of polymer
applications, including high-density polymer synthesis and assays.
When the polymer is a polynucleotide or a nucleic acid, a number of
assays comprising hybridization can be performed, as pointed out in
Detailed Description above. When the polymer is a polypeptide or a
protein, their kinetic functions and antigen-antibody reactions,
can be performed. The borosilicate system can be tailored to suit
the application by simply modifying the annealing time (e.g.,
longer annealing increases pore size) and etching time (e.g.,
longer etch creates thicker layer).
* * * * *