U.S. patent application number 10/029472 was filed with the patent office on 2002-07-04 for inorganic permeation layer for micro-electric device.
This patent application is currently assigned to Nanogen, Inc.. Invention is credited to Greef, Charles H., Havens, John R., Heller, Michael J., Krihak, Michael K., Raymond, Daniel E..
Application Number | 20020085954 10/029472 |
Document ID | / |
Family ID | 23395504 |
Filed Date | 2002-07-04 |
United States Patent
Application |
20020085954 |
Kind Code |
A1 |
Havens, John R. ; et
al. |
July 4, 2002 |
Inorganic permeation layer for micro-electric device
Abstract
The present invention pertains to a method of, and a device
created by, depositing an inorganic permeation layer on a
micro-electronic device for molecular biological reactions. The
permeation layer is preferably sol-gel. The sol-gel permeation
layer can be created with pre-defined porosity, pore size
distribution, pore morphology, and surface area. The sol-gel
permeation layer may also function as the attachment layer of the
micro-electric device.
Inventors: |
Havens, John R.; (San Diego,
CA) ; Krihak, Michael K.; (San Diego, CA) ;
Greef, Charles H.; (Ramona, CA) ; Raymond, Daniel
E.; (San Diego, CA) ; Heller, Michael J.;
(Encinitas, CA) |
Correspondence
Address: |
LYON & LYON LLP
633 WEST FIFTH STREET
SUITE 4700
LOS ANGELES
CA
90071
US
|
Assignee: |
Nanogen, Inc.
10398 Pacific Center Court
San Diego
CA
92121
|
Family ID: |
23395504 |
Appl. No.: |
10/029472 |
Filed: |
October 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10029472 |
Oct 22, 2001 |
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09354931 |
Jul 15, 1999 |
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09354931 |
Jul 15, 1999 |
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08986065 |
Dec 5, 1997 |
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08986065 |
Dec 5, 1997 |
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08534454 |
Sep 27, 1995 |
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08534454 |
Sep 27, 1995 |
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08304657 |
Sep 9, 1994 |
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08304657 |
Sep 9, 1994 |
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08271882 |
Jul 7, 1994 |
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08271882 |
Jul 7, 1994 |
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08146504 |
Nov 1, 1993 |
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08271882 |
Jul 7, 1994 |
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08708262 |
Sep 6, 1996 |
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Current U.S.
Class: |
422/63 ;
257/E21.705; 435/287.2 |
Current CPC
Class: |
B01J 2219/00527
20130101; C40B 40/10 20130101; B01J 2219/00637 20130101; B82Y 5/00
20130101; B01J 2219/00317 20130101; B01J 2219/00659 20130101; B01L
2400/0421 20130101; B01J 2219/00686 20130101; B01L 3/502761
20130101; B01J 2219/00653 20130101; B01J 2219/00689 20130101; G11C
19/00 20130101; H01L 51/0595 20130101; B01J 2219/0072 20130101;
C07K 1/24 20130101; B01J 2219/00315 20130101; B01J 2219/00722
20130101; B01L 2300/0645 20130101; B01J 2219/00626 20130101; B01L
3/50273 20130101; B01L 2300/069 20130101; B01J 2219/0063 20130101;
B01J 2219/00641 20130101; C40B 60/14 20130101; C07B 2200/11
20130101; C07K 1/047 20130101; B01L 3/508 20130101; B01L 2200/0647
20130101; B01J 19/0046 20130101; B01J 2219/00605 20130101; B01L
2300/0636 20130101; C12Q 1/6837 20130101; H01L 25/50 20130101; B01J
2219/00731 20130101; C07H 21/00 20130101; B01J 2219/00644 20130101;
B01L 2400/0415 20130101; C07K 1/045 20130101; B01J 2219/00713
20130101; B01J 2219/00725 20130101; C40B 40/06 20130101; B01J
19/0093 20130101; B01J 2219/00596 20130101; B01J 2219/00853
20130101; B01L 3/502707 20130101; B82Y 10/00 20130101; C07K 1/04
20130101; C40B 40/12 20130101; B01J 2219/0061 20130101; G11C
13/0014 20130101; H01L 2924/14 20130101; C12Q 1/6825 20130101; H01L
2924/10253 20130101; G11C 13/0019 20130101; B01J 2219/00585
20130101; B01L 3/5085 20130101; B01J 2219/00621 20130101; G11C
13/04 20130101; B01J 2219/0059 20130101; B01J 2219/00612 20130101;
H01L 24/95 20130101; C12Q 1/6825 20130101; C12Q 2565/515 20130101;
C12Q 2565/607 20130101; C12Q 1/6837 20130101; C12Q 2565/515
20130101; C12Q 2565/607 20130101; H01L 2924/10253 20130101; H01L
2924/00 20130101; H01L 2924/14 20130101; H01L 2924/00 20130101 |
Class at
Publication: |
422/63 ;
435/287.2 |
International
Class: |
C12M 001/34; B32B
005/02 |
Claims
We claim:
1. An electronic device adapted to received a solution comprising:
a substrate; a plurality of selectively addressable electrodes on
the substrate; and a permeation layer adjacent the electrodes, the
permeation layer being a sol-gel composition; and an electric
source for selectively addressing the electrodes.
2. The electronic device of claim 1 wherein the sol-gel composition
is comprised of silicon dioxide.
3. The electronic device of claim 2 wherein the silicon dioxide
sol-gel composition is formed from tetraethyl orthosilicate,
ethanol, de-ionized water, hydrochloric acid and a surfactant.
4. The electronic device of claim 3 wherein the surfactant is
cetyltrimethylammonium bromide.
5. The electronic device of claim 3 wherein the concentration of
the surfactant is selected from 1 weight percent to 5 weight
percent to generate a predetermined pore size in the sol-gel.
6. The electronic device of claim 1 further comprising: an
attachment layer adjacent the permeation layer and having selective
binding properties for specific binding entities.
7. The electronic device of claim 1 further comprising: an
attachment layer integral with the permeation layer and having
selective binding properties for specific binding entities.
8. An electronic device adapted to receive a solution comprising: a
substrate; a plurality of selectively addressable electrodes on the
substrate; and a permeation layer adjacent the electrodes, the
permeation layer being a silicon dioxide composition.
9. The electronic device of claim 8 wherein the silicon dioxide
composition is formed from tetraethyl orthosilicate, ethanol,
de-ionized water, hydrochloric acid and a surfactant.
10. The electronic device of claim 9 wherein the surfactant is
cetyltrimethylammonium bromide.
11. The electronic device of claim 9 wherein the concentration of
the surfactant is selected from 1 weight percent to 5 weight
percent to generate a predetermined pore size in the silicon
dioxide composition.
12. The electronic device of claim 8 further comprising: an
attachment layer adjacent the permeation layer with selective
binding properties for specific binding entities.
13. The electronic device of claim 8 further comprising: an
attachment layer integral with the permeation layer and having
selective binding properties for specific binding entities.
14. A method for forming an electronic device adapted to receive a
solution comprising: providing a substrate; locating a plurality of
selectively addressable electrodes on the substrate; and forming a
permeation layer adjacent the electrodes, the permeation layer
being a sol-gel composition.
15. The method of claim 14 wherein the sol-gel composition is
comprised of silicon dioxide.
16. The method of claim 15 wherein the silicon dioxide sol-gel
composition is formed from tetraethyl orthosilicate, ethanol,
de-ionized water, hydrochloric acid and a surfactant.
17. The method of claim 16 wherein the surfactant is
cetyltrimethylammonium bromide.
18. The method of claim 16 wherein the concentration of the
surfactant is selected from 1 weight percent to 5 weight percent to
generate a predetermined pore size in the sol-gel.
19. The method of claim 14 further comprising: forming an
attachment layer adjacent the permeation layer with selective
binding properties for specific binding entities.
20. A method of forming a permeation layer for use on an electronic
device comprising: mixing tetraethylorthosilicate, an alcohol,
water and an acid to form a stock solution; mixing the stock
solution with additional water and additional acid; adding
additional alcohol; adding a surfactant to form a sol-gel solution;
depositing the sol-gel solution on a substrate; spinning the
substrate; and heating the substrate.
21. The method of claim 20 wherein the surfactant is
cetyltrimethylammonium bromide, the acid is hydrochloric acid and
the alcohol is ethanol.
22. The method of claim 21 wherein the final molar ratio is
tetraethylorthosilicate=about 1.0, water=about 0.0 to about 40.0,
ethanol=about 0.0 to about 40.0 and hydrochloric acid=about 0.0001
to about 0.1.
23. The method of claim 20 wherein the weight percent of the
surfactant is from 1 weight percent to 5 weight percent.
24. The method of claim 20 wherein the amount of surfactant is
varied to vary the pore size in the permeation layer.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/354,931, filed Jul. 15, 1999, which is a
continuation-in-part of U.S. application Ser. No. 08/986,065, filed
Dec. 5, 1997, which is a continuation-in-part of U.S. application
Ser. No. 08/534,454, filed Sep. 27, 1995, which is a
continuation-in-part of U.S. application Ser. No. 08/304,657, filed
Sep. 9, 1994, now U.S. Pat. No. 5,632,957 (which has been continued
as application Ser. No. 08/859,644, filed May 20, 1997), which is a
continuation-in-part of Ser. No. 08/271,882, filed Jul. 7, 1994,
which is a continuation-in-part of Ser. No. 08/146,504, filed Nov.
1, 1993, now U.S. Pat. No. 5,605,662, and a continuation-in-part of
Ser. No. 08/708,262, filed Sep. 6, 1996.
FIELD OF THE INVENTION
[0002] This invention pertains to the design, fabrication, and uses
of a self-addressable, self-assembling microelectronic system which
can actively carry out and control multi-step and multiplex
reactions in microscopic formats. In particular, these reactions
include molecular biological reactions, such as nucleic acid
hybridizations, antibody/antigen reactions, clinical diagnostics,
and biopolymer synthesis. More specifically, the invention relates
to an inorganic permeation layer for the micro-electric device.
BACKGROUND OF THE INVENTION
[0003] Sol-gel has been employed as a monolithic gel deposition on
a variety of substrates. See, for example, U.S. Pat. No. 4,652,467
and U.S. Pat. No. 5,224,972, both issued to Brinker et al. In this
process, metal alkoxides of network forming cations, e.g., Si, Al,
B, Ti, P, and optionally soluble salts of modifying cations, are
used as glass precursors. In alcoholic solutions catalyzed by
additions of acid or base, the alkoxides are partially or
completely hydrolyzed and then polymerized to form molecules of
glass-like oxide networks linked by bridging oxygen atoms. This
technique is readily adapted to preparation of multicomponent oxide
solutions as well as single component systems.
[0004] The net reactions which describe this process are generally
represented as:
M(OR).sub.n+xH.sub.2O.fwdarw.M(OH.sub.x (OR).sub.n-x+x ROH (1)
M(OH).sub.x(OR).sub.n-x.fwdarw.MO.sub.n/2+x/2H.sub.2O+(n-x) (2)
[0005] where x in reaction 1 can be varied, e.g., from about 1-20.
Generally, reaction 2 does not go to completion, i.e., colloidal
particles of anhydrous oxides do not result. When the growing
polymers link together to form an infinite network, the solution
stiffens to a gel.
[0006] The chemistry involved in the formation of these monolithic
gels is well documented in the prior art. See, e.g., (1) Brinker et
al, "Sol-gel Transition in Simple Silicates", J. Non-Cryst. Solids,
48 (1982) 47-64; (2) Brinker et al, "Sol-gel Transition in Simple
Silicates II", J. Non-Cryst. Solids, 63 (1984) 45-59; (3) Schaefer
et al, "Characterization of Polymers and Gels by Intermediate Angle
X-ray Scattering", presented at the International Union of Pure and
Applied Chemists MAC-RO'82, Amherst, Mass., Jul. 12, 1982; (4)
Pettit et al, Sol-Gel Protective Coatings for Black Chrome Solar
Selective Films, SPIE Vol. 324, Optical Coatings for Energy
Efficiency and Solar Applications, (pub. by the Society of
Photo-Optical Instrumentation Engineers, Bellingham, Wash.) (1982)
176-183; (5) Brinker et al, "Relationships Between the Sol to Gel
and Gel to Glass Conversions", Proceedings of the International
Conference on Ultrastructure Processing of Ceramics, Glasses, and
Composites, (John Wiley and Sons, N.Y.) (1984); (6) Brinker et al,
"Conversion of Monolithic Gels to Glasses in a Multicomponent
Silicate Glass System", J. Materials Sci., 16 (1981) 1980-1988; (7)
Brinker et al, "A Comparison Between the Densification Kinetics of
Colloidal and Polymeric Silica Gels", Mat. Res. Soc. Symp. Proc.
Vol. 32 (1984), 25-32; all of which disclosures are entirely
incorporated by reference herein. For example, much work has been
done in characterizing the relationship between the properties of a
monolithic, bulk gel prepared by these systems and of the
properties of the solution from which such gel is made. For
instance, the relationship between solution characteristics such as
pH and water content for a given solution chemical composition and
the size and nature of the polymer which results in solution, and
the relationship between such polymer properties and the
characteristics of the finally produced gel, e.g., the degree of
crosslinking, the porosity of the gel, etc., have been well studied
and discussed in these references.
[0007] The fact that gel formation can be retarded by making the
solutions sufficiently dilute, e.g., less than 10% equivalent
oxides, is known. In such dilutions, more typically 2-5% equivalent
oxides, the solution can be applied to various substrates by
conventional processes. Under such circumstances, the partially
hydrolyzed glass-like polymers react chemically with the substrate
surface, thereby achieving complete wetting.
[0008] The physical properties of sol-gel materials are tailored
through stoichiometry, aging, drying conditions and method of
deposition. Emphasis for examining these parameters has been on
silicate-based systems, which has led to microporous monoliths and
thin films (pore size<2 nm). The most prominent applications of
sol-gel synthesis have been the development of mesoporous (pore
size from 2 nm to 50 nm) materials that possess well-defined pore
morphology. To generate this pore morphology, a method known as
surfactant templating has been devised. This approach is based on
the ability for a ternary system, consisting of water, ethanol and
surfactant, to develop a three dimensional structure (or a
lyotropic phase) that may be described as cubic, hexagonal,
lamellar or isotropic, depending upon the molar ratio of the three
components. The formation of these phases is sometimes referred as
liquid crystal templating. In general, the introduction of a
hyrdolyzed silicon alkoxide precursor, once hyrolyzed, infiltrates
the water rich regions and forms in inorganic `shell` around the
hydrophobic surfactant. Upon drying and heating in excess of
400.degree. C., the organic surfactant phase is removed, leaving
behind the inorganic, silica shell with porosity defined by the
once present surfactant phase. The pore sizes range from 2 nm to
100 nm depending upon the nature of the surfactant. The silica wall
thickness ranges from 1 nm to 10 nm, which relies on processing
parameters such as aging, pH and temperature.
[0009] However, none of the known uses of sol-gel chemistry in thin
film deposition contemplates the use of sol-gel as a permeation
layer for an electrical micro-array devices. Current permeation
layers for electric micro-arrays are organic monomers or polymers
with undefined pure size and porosity that swell when exposed to an
aqueous solution. The previously not contemplated use of sol-gel as
a permeation layer for an electrical micro-assay device solves the
above limitations of organic permeation layers by providing a
permeation layer that has controllable porosity and pore size and
is not susceptible to swelling when exposed to an aqueous
solution.
SUMMARY OF THE INVENTION
[0010] Current methods for synthesizing permeation layers involve
the utilization of monomers or polymers to form a membrane with
undefined pore size and porosity. Furthermore, these permeation
layers (i.e. agarose and synthetic polymers) may swell when exposed
to an aqueous solution.
[0011] To circumvent these obstacles, sol-gel processing provides a
means for fabricating thin films (up to 1 micron) with
pre-determined pore size, pore size distribution, pore morphology,
surface area and porosity. With these capabilities the sol-gel
support may be tailored to achieve a variety of porous
characteristics, suited for a specified application or assay. Since
sol-gel materials are based on metal alkoxide precursor chemistry
or metal oxide colloidal suspensions, the resulting material is
inorganic. Thus, a rigid support is formed that will maintain its
physical properties when immersed in aqueous solutions (resistance
to swelling) and remain chemically resistant to biological and
electrochemically generated products.
[0012] Typically, sol-gel chemistry is based upon silicate
precursor chemistry, but may be applied to other inorganic systems
that include alumina, titania, zirconia, hafnia, germania, borates
and phosphates. These systems alone or in combination with silica
may be implemented to yield a robust, yet porous sol-gel permeation
layer. In addition, sol-gel chemistry is amenable for large-scale
manufacturing in which coatings may be applied at the wafer level
rather than on the individual chip.
[0013] Inorganic membranes synthesized by sol-gel chemistry have
been applied as a permeation layer and as a support for attachment
chemistry. In both instances, the sol-gel layer acted as a
base-layer for the subsequent attachment layer. Attachment layer
chemistry includes at least two methods: agarose/streptavidin and
direct-attachment to the permeation layer. In the first example, a
thin layer of agarose/streptavidin was directly deposited on the
sol-gel film. Passive attachment, electronic attachment and reverse
dot blot hybridizations were achieved with this permeation and
attachment layer configuration. In a second example, the direct
attachment of oligonucleotides was attained by bonding the capture
probes to the sol-gel, followed by passive hybridization.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is the cross-section of three self-addressable
micro-locations fabricated using microlithographic techniques;
[0015] FIG. 2 is the cross-section of a microlithographically
fabricated micro-location;
[0016] FIG. 3 is a schematic representation of a self-addressable
64 micro-location chip which was actually fabricated, addressed
with oligonucleotides, and tested;
[0017] FIGS. 4a and 4b show the mechanism the device uses to
electronically concentrate analyte or reactant molecules at a
specific micro-location;
[0018] FIGS. 5a, 5b, 5c and 5d show the self-directed assembly of a
device with three specific oligonucleotide binding entities (SSO-A,
SSO-B, and SSO-C);
[0019] FIG. 6 is a schematic of a sol-gel permeation layer and an
agarose/streptavidin attachment layer;
[0020] FIG. 7 is a first micro photograph of the capture of
oligonucleotides to the agarose/streptavidin attachment layer of
FIG. 6;
[0021] FIG. 8 is a second micro photograph of the capture of
oligonucleotides to the agarose/streptavidin attachment layer of
FIG. 6.
[0022] FIG. 9 is a schematic of a sol-gel permeation layer also
functioning as an attachment layer;
[0023] FIG. 10 is a micro photograph of the binding of a
ribo-uridine capture probe to the sol-gel layer of FIG. 9; and
[0024] FIG. 11 is a graphical representation of the passive
hybridization of the sol-gel permeation layer/attachment layer of
FIG. 9.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The devices and the related methodologies of this invention
allow important molecular biology and diagnostic reactions to be
carried out under complete electronic control. The basic concept of
this invention is a microelectronic device with specially designed
addressable microscopic locations. Each micro-location has a
derivatized surface for the attachment of specific binding entities
(i.e., an attachment layer), a permeation layer, and an underlying
direct current (DC) micro-electrode. After the initial fabrication
of the basic microelectronic structure, the device is able to
self-direct the addressing of each specific micro-location with
specific binding entities. The self-addressed device is
subsequently able to actively carry out multi-step, combinatorial,
and multiplex reactions at any of its micro-locations. The device
is able to electronically direct and control the rapid movement and
concentration of analytes and reactants to or from any of its
micro-locations. The ability of the device to electronically
control the dynamic aspects of various reactions provides a number
of new and important advantages and improvements.
[0026] In order for a device to carry out multi-step and multiplex
reactions, its crucial electronic components must be able to
maintain active operation in aqueous solutions. To satisfy this
requirement, each micro-location must have an underlying
functioning DC mode micro-electrode. Other considerations for the
design and fabrication of a device include, but are not limited to,
materials compatibilities, nature of the specific binding entities
and the subsequent reactants and analytes, and the number of
micro-locations.
[0027] By "a functioning DC mode micro-electrode" is meant a
micro-electrode biased either positively or negatively, operating
in a direct current mode (either continuous or pulse), which can
affect or cause the free field electrophoretic transport of charged
specific binding entities, reactants, or analytes to or from any
location on the device, or in the sample solution.
[0028] Within the scope of this invention, the free field
electrophoretic transport of molecules is not dependent on the
electric field produced being bounded or confined by dielectrical
material.
[0029] A device can be designed to have as few as two addressable
micro-locations or as many as hundreds of thousands of
micro-locations. In general, a complex device with a large number
of micro-locations is fabricated using microlithography techniques.
Fabrication is carried out on silicon or other suitable substrate
materials, such as glass, silicon dioxide, plastic, or ceramic
materials. These microelectronic "chip" designs would be considered
large scale array or multiplex analysis devices. A device with a
small number of micro-locations would be fabricated using
micro-machining techniques.
[0030] Addressable micro-locations can be of any shape, preferably
round, square, or rectangular. The size of an addressable
micro-location can be of any size, preferably range from sub-micron
(.about.0.5 .mu.m) to several centimeters (cm), with 5 .mu.m to 100
.mu.m being the most preferred size range for devices fabricated
using microlithographic techniques, and 100 .mu.m to 5 millimeters
being the most preferred size range for devices fabricated using
the micro-machining techniques. To make micro-locations smaller
than the resolution of microlithographic methods would require
techniques such as electron beam lithography, ion beam lithography,
or molecular beam epitaxy. While microscopic locations are
desirable for analytical and diagnostic type applications, larger
addressable locations (e.g., larger than 2 mm) are desirable for
preparative scale biopolymer synthesis.
[0031] After micro-locations have been created by using
microlithographic and/or micro-machining techniques, chemical
techniques are used to create the specialized attachment and
permeation layers which would allow the DC mode micro-electrodes
under the micro-locations to: (1) affect or cause the free field
electrophoretic transport of specific (charged) binding entities
from any location; (2) concentrate and covalently attach the
specific binding entities to the specially modified surface of the
specific micro-location; and (3) continue to actively function in
the DC mode after the attachment of specific binding entities so
that other reactants and analytes can be transported to or from the
micro-locations.
[0032] A. DESIGN PARAMETERS
[0033] FIG. 1 shows a basic design of self-addressable
micro-locations fabricated using microlithographic techniques. The
three micro-locations (10) (ML-1, ML-2, ML-3) are formed on the
surface of metal sites (12) which have been deposited on an
insulator layer/base material. The metal sites (12) serve as the
underlying micro-electrode structures (10). An insulator material
separates the metal sites (12) from each other. Insulator materials
include, but are not limited to, silicon dioxide, glass, resist,
rubber, plastic, or ceramic materials.
[0034] FIG. 2 shows the basic features of an individual
micro-location (10) formed on a microlithographically produced
metal site (12). The addressable micro-location is formed on the
metal site (12), and incorporates an oxidation layer (20), a
permeation layer (22), an attachment layer (24), and a binding
entity layer (26). The metal oxide layer provides a base for the
coupling of the permeation layer. The permeation layer provides
spacing between the metal surface and the attachment/binding entity
layers and allows solvent molecules, small counter-ions, and gases
to freely pass to and from the metal surface. The thickness of the
permeation layer for microlithographically produced devices can
range from approximately 1 nanometer (nm) to 10 microns (.mu.m),
with 2 nm to 1 .mu.m being the most preferred. The attachment layer
provides a base for the binding of the binding entities. The
thickness of the attachment layer for microlithographically
produced devices can range from 0.5 nm to 1 .mu.m, with 1 nm to 200
nm being the most preferred. In some cases, the permeation and
attachment layers can be formed from the same material. The
specific binding entities are covalently coupled to the attachment
layer, and form the specific binding entity layer. The specific
binding entity layer is usually a mono-layer of the specific
binding molecules. However, in some cases the binding entity layer
can have several or even many layers of binding molecules.
[0035] Certain design and functional aspects of the permeation and
attachment layer are dictated by the physical (e.g., size and
shape) and the chemical properties of the specific binding entity
molecules. They are also dictated to some extent by the physical
and chemical properties of the reactant and analyte molecules,
which will be subsequently transported and bound to the
micro-location. For example, oligonucleotide binding entities can
be attached to one type of micro-location surface without causing a
loss of the DC mode function, i.e., the underlying micro-electrode
can still cause the rapid free field electrophoretic transport of
other analyte molecules to or from the surface to which the
oligonucleotide binding entities are attached. However, if large
globular protein binding entities (e.g., antibodies) are attached
to the same type of surface, they may effectively insulate the
surface and cause a decrease or a complete loss of the DC mode
function. Appropriate modification of the attachment layer would
have to be carried out so as to either reduce the number of large
binding entities (e.g., large globular proteins) or provide spacing
between the binding entities on the surface.
[0036] The spacing between micro-locations is determined by the
ease of fabrication, the requirement for detector resolution
between micro-locations, and the number of micro-locations desired
on a device. However, particular spacings between micro-locations,
or special arrangement or geometry of the micro-locations is not
necessary for device function, in that any combination of
micro-locations (i.e., underlying micro-electrodes) can operate
over the complete device area. Nor is it necessary to enclose the
device or confine the micro-locations with dielectric boundaries.
This is because complex electronic field patterns or dielectric
boundaries are not required to selectively move, separate, hold, or
orient specific molecules in the space or medium between any of the
electrodes. The device accomplishes this by attaching the specific
binding molecules and subsequent analytes and reactants to the
surface of an addressable micro-location. Free field
electrophoretic propulsion provides for the rapid and direct
transport of any charged molecule between any and all locations on
the device.
[0037] As the number of micro-locations increases beyond several
hundred, the complexity of the underlying circuitry of the
micro-locations increases. In this case the micro-location grouping
patterns have to be changed and spacing distances increased
proportionally, or multi-layer circuitry can be fabricated into the
basic device.
[0038] In addition to micro-locations which have been addressed
with specific binding entities, a device will contain some
un-addressed, or plain micro-locations which serve other functions.
These micro-locations can be used to store reagents, to temporarily
hold reactants or analytes, and as disposal units for excess
reactants, analytes, or other interfering components in samples.
Other unaddressed micro-locations can be used in combination with
the addressed micro-locations to affect or influence the reactions
that are occurring at these specific micro-locations. These
micro-locations add to intra-device activity and control. It is
also possible for the micro-locations to interact and transport
molecules between two separate devices. This provides a mechanism
for loading a working device with binding entities or reactants
from a storage device, and for copying or replicating a device.
[0039] FIG. 3 shows a matrix type device containing 64 addressable
micro-locations (30). A 64 micro-location device is a convenient
design, which fits with standard microelectronic chip packaging
components. Such a device is fabricated on a silicon chip
approximately 1 cm.times.1 cm, with a central area approximately
750 .mu.m.times.750 .mu.m containing the 64 micro-locations. Each
micro-location (32) is approximately 50 .mu.m square with 50 .mu.m
spacing between neighboring micro-locations. Connective circuitry
for each individual underlying micro-electrode runs to an outside
perimeter (10 mm.times.10 mm) of metal contact pads (300 .mu.m
square) (34). A raised inner perimeter can be formed between the
area with the micro-locations and the contact pads, producing a
cavity which can hold approximately 2 to 10 microliters (.mu.l) of
a sample solution. The "chip" can be mounted in a standard quad
package, and the chip contact pads (34) wired to the quad package
pins. The packaged chip can then be plugged into a microprocessor
controlled DC power supply and multimeter apparatus which can
control and operate the device.
[0040] B. FABRICATION PROCEDURES
[0041] 1. Microlithography Fabrication Steps
[0042] General microlithographic or photolithographic techniques
can be used for fabrication of the complex "chip" type device,
which has a large number of individually addressable
microelectrodes. The conventional electronics for addressing these
electrodes can be located on the chip in the form of an integrated
circuit or off the chip on a printed circuit board. While the
fabrication of an array of microelectrodes does not require complex
photolithography, the selection of materials requires special
considerations in order for such electrodes to operate in an
aqueous environment.
[0043] The devices like the sixty-four microelectrode device (30)
shown in FIG. 3 can be fabricated using relatively simple mask
designs and standard microlithographic techniques. Generally, the
base substrate material would be a 4-inch diameter silicon wafer,
approximately 20 mils thick. For fabricating microelectrode arrays
whose electronic addressing is controlled off chip, the first
processing step is to grow an insulating thermal silicon dioxide
0.5 to 1.0 microns into the wafer. In the case of fabricating
platinum silicide (PtSi) electrodes a thin layer (.about.50 nm) of
amorphous silicon (a-Si) is deposited over the surface of the wafer
by means of a sputter deposition system. Using standard
optolithography techniques, photo resist would be spun onto the
wafer (i.e., Shippley Photo Resist 3612) and then exposed with the
negative image of the metal wiring defining the electrodes, the
wire bond pads, and the metal traces connecting the electrodes to
the wire bond pads. After the photo resist is removed and a thin
layer (.about.50 nm) of platinum (Pt) is sputter deposited over the
entire surface of the wafer. The Pt and patterned a-Si are alloyed
together in a tube furnace, forming PtSi. The unalloyed Pt is then
removed using an aqua regia etch, leaving only the patterned PtSi.
At this point an electronically insulating top dielectric (either
silicon dioxide (SiO.sub.2) or silicon nitride (Si.sub.xN.sub.y) or
a combination of the two) is deposited over the entire wafer by
means of a Plasma Enhanced Vapor Deposition (PECVD) system. Again
standard photolithography techniques are used to pattern openings
in photo resist above electrodes and the wire bond pads, and again
a plasma etcher is used to etch down through the top dielectric to
the PtSi. At this point the wafer can be diced into individual
chips.
[0044] The bottom dielectric (thermal SiO.sub.2) electrically
insulates the PtSi from the silicon substrate, while the top
dielectric (PECVD SiO.sub.2 and/or Si.sub.xN.sub.y) electrically
insulates the wire traces from the aqueous solution. Other metal
systems other than PtSi can be used to fabricate the electrodes
(i.e., Ti--Pt, TiW--Pt, Ti--Au, Ti--Pd, C) and would have
processing steps consistent with patterning techniques for those
material systems. In the case of the electro-deposited permeation
layers, the ideal material system is a PtSi metalization and a
layer of PECVD SiO.sub.2 covered by a layer of PECVD
Si.sub.xN.sub.y for the top dielectric. The PtSi provides
Si/SiO.sub.2 attachment sites on the surface of the electrode for
the permeation layer. The PECVD SiO.sub.2 provides attachment sites
to the dielectric well walls while the PECVD Si.sub.xN.sub.y
provides a dense ion barrier that inhibits the DNA attachment
chemistry used on the permeation layer.
[0045] 2. Permeation and Attachment Layer Formation Steps
[0046] At this point the micro-electrode locations on the device
are ready to be modified with specialized permeation and attachment
layers. The objective is to create on the micro-electrode an
intermediate permeation layer with selective diffusion properties
and an attachment surface layer with optimal binding properties.
The attachment layer should have from 10.sup.5 to 10.sup.7
functionalized locations per square micron (.mu.m.sup.2) for the
optimal attachment of specific binding entities. However, the
attachment of specific binding entities must not overcoat or
insulate the surface so as to prevent the underlying
micro-electrode from functioning. A functional device requires some
fraction (.about.5% to 25%) of the actual metal micro-electrode
surface to remain accessible to solvent (H.sub.2O) molecules, and
to allow the diffusion of ions (e.g., H.sup.+ and OH.sup.-) and
electrolysis gases (e.g., O.sub.2 and H.sub.2) to occur.
[0047] The intermediate permeation layer must also allow diffusion
to occur. Additionally, the permeation layer should have a pore
limit property which inhibits or impedes the larger binding
entities, reactants, and analytes from physical contact with the
micro-electrode surface. The permeation layer keeps the active
micro-electrode surface physically distinct from the binding entity
layer of the micro-location device.
[0048] In terms of the primary device function, this design allows
the electrolysis reactions required for electrophoretic transport
to occur on micro-electrode surface, but avoids adverse
electrochemical effects to the binding entities, reactants, and
analytes. Sol-gel has been found to have benefits as a permeation
layer not present in organic compounds, including pre-defined
porosity, pore size, por size distribution, pore morphology and
surface area.
[0049] The sol-gel compositions are comprised of tetraethyl
orthosilicate, ethanol, de-ionized water, hydrochloric acid and
surfactant. Specifically, tetraethyl orthosilicate,
sub-stoichiometric concentration of water, 200 proof ethanol, and
hydrochloric acid are added to a boiling flask in the above listed
order:
Preparation of Stock Solution
[0050]
1 Volume Molar Ratio 61 mL Tetraethyl orthosilicate (Aldrich) 1.0
61 mL absolute ethanol (200 proof, Quantum) 4.0 4.87 mL de-ionized
water (Milli-Q) 1.0 0.2 mL 0.07 M HCl 5 .times. 10.sup.-5
[0051] The solution is refluxed at 60.degree. C. for 90 minutes
while magnetically stirring. After cooling this "stock solution" to
room temperature, a portion of the partially hydrolyzed metal
alkoxide solution may be extracted and mixed with additional
de-ionized water and HCl:
2 Volume 34.5 mL stock solution 1.38 mL de-ionized water 4.14 mL
0.07 M HCl Final Preferred Molar Ratio Final Molar Ratio Range TEOS
1.0 TEOS 1.0 H.sub.2O 5.1 H.sub.2O 1.0-40.0 EtOH 22 EtOH 0.0-40.0
HCl 0.0039 HCl 0.0001-0.1
[0052] After these components are mixed for 15 minutes, the
solution is diluted with ethanol in a ratio of 2:1 (2 parts ethanol
to 1 part sol-gel solution). To generate the appropriate pore size,
a surfactant such as cetyltrimethylammonium bromide (or CTAB) may
be added to the solution. The concentration of CTAB ranges from 1
wt. % to 5 wt. % depending upon the desired pore morphology. Once
the surfactant has completely dissolved, the sol is ready for
deposition by spin coating. The chips are spin coated for 20 sec.
to 30 sec. at a rate that ranges from 1500 rpm to 6000 rpm. Prior
deposition of the liquid onto the chip, however, the solution is
passed through a 0.2 .mu.m filter. After spin coating, the chips
are placed in a furnace and heated at a rate of 1.degree. C./min
until 450.degree. C. is attained. The temperature is held at this
point for 3 hours before slowly cooling to room temperature. The
sol-gel film that remains consists of more than 99% SiO.sub.2. The
average pore size of the sol-gel films was estimated to be 25 .ANG.
according to TEM evaluation of films prepared with similar
compositions.
[0053] Subsequently, the surface of this material may be
functionalized by silanization techniques to provide favorable
attachment chemistries. In a first iteration, as shown in FIG. 6, a
thin layer of agarose/streptavidin was deposited onto a .about.500
nm thick sol-gel coating. As shown in FIG. 7, by applying the
established biotin-streptavidin attachment chemistry BODIPY-Texas
Red labeled oligonucleotides (T.sub.12) were electronically bound
to the agarose layer in a capture loading experiment with a 20
nanomolar biotinylated capture probe. Columns 1, 2 and 5 of FIG. 7
show specific hybridization, columns 2 and 4 show non-specific
hybridization. In an ensuing experiment as shown in FIG. 8, a
reverse dot blot assay was performed with ATA5-RCA5, thus
demonstrating that this dual layer of sol-gel/agarose is feasible.
In this example, fluorescently labeled capture oligonucleotides
(modified with biotin) were electronically addressed to specified
electrodes, which attached to the agarose/streptavidin layer. The
resulting reverse dot hybridizations with ATA5-RCA5 yielded
specific to non-specific ratios that ranges from 2 to 5.
[0054] In FIG. 9, direct attachment of oligonucleotides at either
the 3' or 5' end has also been achieved on the sol-gel permeation
layer, itself. In FIG. 10, an example of direct attachment is
provided. Treatment of the sol-gel layer with
aminopropyltrimethoxysilane yields a surface covered with amines
that can readily bind a fluorescently labeled capture probe
modified with ribo-uridine. In this instance, an ATA5-riboU capture
probe was attached to the sol-gel surface and then passively
hybridized to RCA5-BTR (10 .mu.M). The best results rendered an
average of 6760 MFI/sec. FIG. 11 shows a bar graph comparing the
passive hybridization (measured by fluorescence) of
oligonucleotides directly to the sol-gel permeation layer as a
function of concentration, time and pH.
[0055] The above data demonstrate the first electric field assisted
biological assays performed on a sol-gel substrate, complete with
attachment chemistry. In the examples cited above, the sol-gel
layer may act as a membrane that permits ionic conduction (agarose)
or as an ionic conducting membrane that doubles as a support for
the binding of an attachment layer. Surfactant templated sol-gel
materials have not been previously employed as a membrane on
electrodes for electrochemically addressed reactions or assays. In
either case, the porous nature of the sol-gel layer is of utmost
importance is controlled via processing conditions and the
lyotropic phase formed upon the addition of surfactant. The sol-gel
chemistry is not limited to the composition, components and
synthesis procedure listed. Instead, numerous formulations are
possible and are attributed to the versatility of sol-gel
processing. For example, the cited composition is easily modified
by altering the following parameters: (1) water to TEOS ration, (2)
HCl concentration, (3) type of catalyst (acid or base), (4)
concentration of solvent (EtOH), (5) type of precursor, (6) method
of synthesis (i.e., use a one step catalysis procedure instead of
the two-step procedure) and (7) pH value. Since sol-gel synthesis
is performed in the liquid phase, the addition of components such
as surfactants, drying control agents, organic/inorganic dopants,
organically modified precursors, non-silicate based precursors and
polymers may be included in the batch process.
[0056] The modification of sol-gel materials is not limited to
inorganic precursors (alumina, titania, etc.). If additional
mechanical and chemical properties, such as flexibility and
hydrophobicity, respectively, are sought then organically modified
silicate precursors may be introduced. This class of compounds
includes metal alkoxide or metal halide precursors that have at
least one moiety that is a non-oxide group (i.e., a Si--C bond).
Most of the organically modified precursors employ an alkyl group
bonded to the Si atom. This alkyl group may stand alone as an alkyl
group such as ethyltrimethoxysilane or may provide an additional
functional group such as an epoxy in 3-glycidoxypropyltrimetho-
xysilane. If these organic groups are introduced, however, the
heating temperature will be greatly reduced to preserve these
functionalities.
[0057] 3. Self-directed addressing of the devices
[0058] The devices are able to electronically self-address each
micro-location with a specific binding entity. The device itself
directly affects or causes the transport and attachment of specific
binding entities to specific micro-locations. The device
self-assembles itself in the sense that no outside process,
mechanism, or equipment is needed to physically direct, position,
or place a specific binding entity at a specific micro-location.
This self-addressing process is both rapid and specific, and can be
carried out in either a serial or parallel manner.
[0059] A device can be serially addressed with specific binding
entities by maintaining the selected micro-location in a DC mode
and at the opposite charge (potential) to that of a specific
binding entity. All other micro-locations are maintained at the
same charge as the specific binding entity. In cases where the
binding entity is not in excess of the attachment sites on the
micro-location, it is necessary to activate only one other
micro-electrode to affect the electrophoretic transport to the
specific micro-location. The specific binding entity is rapidly
transported (in a few seconds, or preferably less than a second)
through the solution, and concentrated directly at the specific
micro-location where it immediately becomes bonded to the special
surface. The ability to electronically concentrate reactants or
analytes (70) on a specific micro-location (72) is shown in FIGS.
4a and 4b. All other micro-locations remain unaffected by that
specific binding entity. Any unreacted binding entity is removed by
reversing the polarity of that specific micro-location, and
electrophoresing it to a disposal location. The cycle is repeated
until all desired micro-locations are addressed with their specific
binding entities. FIGS. 5a through 5b show the serial process for
addressing specific micro-locations (81, 83, 85) with specific
oligonucleotide binding entities (82, 84, 86).
[0060] The parallel process for addressing micro-locations simply
involves simultaneously activating a large number (particular group
or line) of micro-electrodes so that the same specific binding
entity is transported, concentrated, and reacted with more than one
specific micro-locations.
* * * * *