U.S. patent application number 11/022200 was filed with the patent office on 2005-07-21 for permeation layer attachment chemistry and method.
This patent application is currently assigned to Nanogen, Inc.. Invention is credited to Dan, Smolko, John, Havens R., Krotz, Jain, Onofrey, Thomas J., Winger, Theodore M..
Application Number | 20050158451 11/022200 |
Document ID | / |
Family ID | 23844823 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158451 |
Kind Code |
A1 |
John, Havens R. ; et
al. |
July 21, 2005 |
Permeation layer attachment chemistry and method
Abstract
Electronically addressable microchips having covalently bound
permeation layers and methods of making such covalently bonded
permeation layers to microchips are provided. The covalent bonding
is derived from combining the use of electrodes with silane
derivatives. Such chemistry provides the ability to apply an
electronic bias to the electrodes of the microchip while preventing
permeation layer delaminating from the electrode surface. Methods
for covalently attaching the permeation layer to the microchips are
also described.
Inventors: |
John, Havens R.; (San Diego,
CA) ; Winger, Theodore M.; (San Diego, CA) ;
Krotz, Jain; (San Diego, CA) ; Dan, Smolko;
(Jamul, CA) ; Onofrey, Thomas J.; (San Marcos,
CA) |
Correspondence
Address: |
O'MELVENY & MEYERS
114 PACIFICA, SUITE 100
IRVINE
CA
92618
US
|
Assignee: |
Nanogen, Inc.
|
Family ID: |
23844823 |
Appl. No.: |
11/022200 |
Filed: |
December 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11022200 |
Dec 23, 2004 |
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09922349 |
Aug 3, 2001 |
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6838053 |
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09922349 |
Aug 3, 2001 |
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09464670 |
Dec 15, 1999 |
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6303082 |
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Current U.S.
Class: |
427/2.11 |
Current CPC
Class: |
B01J 2219/00725
20130101; C40B 40/06 20130101; C40B 40/10 20130101; B01J 2219/00659
20130101; B01J 2219/00653 20130101; B01J 2219/00722 20130101; G01N
27/40 20130101; G01N 33/5438 20130101 |
Class at
Publication: |
427/002.11 |
International
Class: |
B05D 003/00; C12Q
001/68 |
Claims
We claim:
1. A method of covalently attaching a permeation layer having
reactive moieties to an electrode of an electronically addressable
microchip comprising: contacting the surface of the electrode with
a linker molecule by vapor deposition wherein the linker molecule
comprises a first reactive moiety that is capable of reacting with
the electrode surface to form a covalent bond with the electrode,
and a second reactive moiety that is capable of reacting with
monomers to form the permeation layer; reacting the first moiety of
the linker molecule with the electrode surface to form a covalent
bond between the linker molecule and the electrode surface;
synthesizing the permeation layer by reacting the linker molecule
with monomers under conditions where polymerization is conducted
between the monomers and the second reactive moiety of the linker;
wherein the resulting covalent attachment between the electrode and
the linker and the permeation layer material is stable at a current
density of at least 0.10 nA/.mu.m.sup.2.
2. The method of claim 1, wherein the electrode is a metal/silicide
electrode selected from the group consisting of platinum silicide,
tungsten silicide, titanium silicide, and gold silicide.
3. The method of claim 1, wherein the electrode is a metal/metal
electrode selected from the group consisting of platinum/titanium
and gold /titanium.
4. The method of claim 1, wherein the electrode is an organic
electrode selected from the group consisting of poly(phenylene
vinylene), polythiophene, and polyaniline.
5. The method of claim 1, wherein the linker has the formula
4wherein X is selected from the group consisting of acrylate,
methacrylate, acrylamide, methacrylamide, allyl, vinyl, acetyl,
amine, substituted amine, epoxy and thiol; SPACER is selected from
the group consisting of alkyl, aryl, mono- or polyalkoxy,
ethyleneglycol, polyethyleneglycol, mono- or polyalkylamine, mono-
or polyamide, thioether derivatives, and mono- or polydisulfides; A
and B are selected from the group consisting of Oxygen-R, Cl, Br,
X-SPACER moiety, and any combination thereof, wherein R is selected
from the group consisting of H, alkyl, methyl, ethyl, propyl,
isopropyl, and branched or linear alkyl of 4 to 10 carbon atoms;
and C is a hydrolyzable moiety selected from the group consisting
of Oxygen-R, Cl, and Br, wherein R is selected from the group
consisting of H, branched alkyl, methyl, ethyl, propyl, isopropyl,
and branched or linear alkyl. of 4 to 10 carbon atoms.
6. The method of claim 5, wherein the linker is selected from the
group consisting of
H.sub.2NCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3,
H.sub.2NCH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3,
H.sub.2NCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.-
2CH.sub.2Si(OCH.sub.3).sub.3,
CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH.sub.2C-
H.sub.2Si(OCH.sub.3).sub.3, and
CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.sub.-
2Si(OC.sub.2Hs).sub.3.
7. The method of claim 6, wherein the linker is
H.sub.2NCH.sub.2CH.sub.2CH- .sub.2Si(OCH.sub.3).sub.3.
8. The method of claim 6, wherein the linker is
H.sub.2NCH.sub.2CH.sub.2NH-
CH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3.
9. The method of claim 6, wherein the linker is
CH.sub.2.dbd.C(CH.sub.3)CO-
OCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3.
10. The method of claim 5, wherein the linker is an acrylate linker
selected from the group consisting of
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2C- H.sub.2Si(OCH.sub.3).sub.3,
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2SiCl- .sub.3,
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)(OCH.sub.3).-
sub.2,
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3).sub.2(OCH.su-
b.3),
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)Cl.sub.2, and
CH.sub.2.dbd.CHCOOCH.sub.2CH(OH)CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OC2H-
.sub.5).sub.3.
11. The method of claim 5, wherein the linker is a methacrylate
linker selected from the group consisting of
CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2-
CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3,
CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH- .sub.2CH.sub.2SiCl.sub.3,
CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH.sub.2CH.su-
b.2Si(CH.sub.3)(OCH.sub.3).sub.2,
CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH.su-
b.2CH.sub.2Si(CH.sub.3).sub.2(OCH.sub.3),
CH.sub.2.dbd.C(CH.sub.3)COOCH.su-
b.2CH.sub.2CH.sub.2Si(CH.sub.3)Cl.sub.2, and
CH.sub.2.dbd.C(CH.sub.3)COOCH-
.sub.2CH(OH)CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.5).sub.3.
12. The method of claim 5, wherein the linker is an acrylamide
linker selected from the group consisting of
CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2-
CH.sub.2Si(OC.sub.2H.sub.5).sub.3,
CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.s- ub.2SiCl.sub.3,
CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)(OC-
H.sub.3).sub.2,
CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3).su-
b.2(OCH.sub.3),
CH.sub.2.dbd.CHCONRCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)C1.- sub.2,
CH.sub.2.dbd.CHCONHCH.sub.2CH(OH)CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2-
Si(OC.sub.2H.sub.5).sub.3, and
CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CONHCH.s-
ub.2CH.sub.2CONHCH.sub.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.5).sub.3.
13. The method of claim 5, wherein the linker is a methacrylamide
linker selected from the group consisting of
CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.-
2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3,
CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.2- CH.sub.2CH.sub.2SiCl.sub.3,
CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.2CH.sub.2CH-
.sub.2Si(CH.sub.3)(OCH.sub.3).sub.2,
CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.2C-
H.sub.2CH.sub.2Si(CH.sub.3).sub.2(OCH.sub.3),
CH.sub.2.dbd.C(CH.sub.3)CONH-
CH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)Cl.sub.2, and
CH.sub.2.dbd.C(CH.sub.3)-
CONHCH.sub.2CH(OH)CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.5).su-
b.3.
14. The method of claim 5, wherein the linker is an allyl
derivative linker selected from the group consisting of
CH.sub.2.dbd.CHCH.sub.2NHCH.-
sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3,
CH.sub.2.dbd.CHCH.sub.2SiH(OCH.s- ub.3).sub.2,
CH.sub.2.dbd.CHCH.sub.2Si(CH.sub.3).sub.2Cl,
CH.sub.2.dbd.CHCH.sub.2SiHCl.sub.2, and
CH.sub.2.dbd.CHCH.sub.2Si(OCH.sub- .3).sub.3.
15. The method of claim 5, wherein the linker is an amino
derivative linker selected from the group consisting of
H.sub.2NCH.sub.2CH.sub.2NHCH-
.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3,
H.sub.2NCH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3,
H.sub.2NCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3, and
H.sub.2NCH.sub.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.5).sub.3.
16. The method of claim 5, wherein the linker is an epoxy
derivative linker selected from the group consisting of 5
17. The method according to claim 1, wherein the permeation layer
is a hydrogel comprising a material selected from the group
consisting of agarose, glyoxylagarose, acrylamide, methacrylamide,
polyacrylamide, and other synthetic polymers.
18. The method of claim 17, wherein the hydrogel comprises
glyoxylagarose.
19. The method of claim 17, wherein the hydrogel comprises
polyacrylamide.
20. The method of claim 1, wherein the step of reacting the linker
molecule with monomers is a Schiff base reduction or
polymerization.
21. The method of claim 1, wherein the second reactive moiety of
the linker comprises an amine group.
22. The method of claim 1, wherein steps (b) and (c) occur at
different times.
23. The method of claim 1, wherein the step of reacting the first
moiety of the linker molecule with the electrode surface comprises
heat curing of the linker molecule and the electrode surface.
24. The method of claim 1, wherein the resulting covalent
attachment between the electrode and the linker and the permeation
layer material is stable at a current density of at least 0.14
nA/.mu.m.sup.2.
25. The method of claim 1, wherein the resulting covalent
attachment between the electrode and the linker and the permeation
layer material is stable at a current density of at least 0.2
nA/.mu.m.sup.2.
26. The method of claim 1, wherein the resulting covalent
attachment between the electrode and the linker and the permeation
layer material is stable at a current density of at least 0.4
nA/.mu.m.sup.2.
27. The method of claim 1, wherein the step of reacting the linker
molecule with monomers is a free radical polymerization
reaction.
28. The method of claim 1, wherein the polymerization is conducted
in a solution phase reaction.
29. The method of claim 1, wherein the polymerization is conducted
between the monomers, and between the monomers and the second
reactive moiety of the linker.
Description
[0001] This is a divisional of U.S. application Ser. No.
09/922,349, filed on Aug. 3, 2001, which is a divisional of U.S.
application Ser. No. 09/464,670, filed on Dec. 15, 1999, now U.S.
Pat. No. 6,303,082, all of which are herein expressly incorporated
by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the attachment of a layer of
polymeric material to a substrate surface. More particularly, this
invention relates to chemistries and methods for covalently
attaching a porous polymeric material to an electrically conductive
substrate, such as a metal electrode of a microchip circuit.
BACKGROUND OF THE INVENTION
[0003] The following description provides a summary of information
relevant to the present invention. It is not an admission that any
of the information provided herein is prior art to the presently
claimed invention, nor that any of the publications specifically or
implicitly referenced are prior art to the invention.
[0004] In the art of electronically addressable microchips that are
used to direct biomaterials, such as nucleic acids and proteins,
from one point in a solution to another, the microchips should be
designed so that electric potential from the microchip electrodes
will translate to the solution overlying the microchip such that
any electrochemistry occurring from the electrode surface will
neither damage the electrodes themselves, nor any biomaterials in
the solution. Generally, protection from such damage is provided by
the use of a porous membrane layer deposited over the microchip
electrodes. Usually, such layer comprises materials derived from
natural or synthetic polymers such as agarose or polyacrylamide,
respectively. These types of materials allow electrochemical
products generated at the electrode surface to travel through their
porous matrix or `permeation layer` and into the solution
immediately above the electrodes.
[0005] Although materials such as those noted above have been found
useful in the role of a porous membrane having desired qualities,
it has been found that because of the methodologies commonly used
to layer such membranes onto the microchip substrate, the membranes
are prone to separate or `delaminate` from the electrode surface.
It is believed this delamination is caused by a change in the
chemical make-up at the interface between the permeation layer and
the electrode resulting from the application of electronic
potential at the electrode and by physical disruption from charged
ions and gases emanating from the electrode. Such delamination can
be viewed from the standpoint of `microdelamination` and
`macrodelamination`.
[0006] Microdelamination involves the electrochemical degradation
of the chemical interface between the permeation layer and the
electrode itself. It is observed by the formation of raised bulges
in the permeation layer, or by ringlets visible due to defraction
of light from the delaminated layer when appropriately viewed by a
confocal microscope and results in the loss of consistency in
permeation layer performance (possibly due to the loss of control
over the electric field uniformity). Macrodelamination, on the
other hand, is caused by a mismatch of the surface energies between
the permeation layer and the chip substrate and results in
permeation layer peeling (lift-off) which can extend across the
entire microchip surface. Since the permeation layer provides a
means for chemical anchorage of analytes present in the liquid
overlay, its physical loss by macrodelamination results in
catastrophic chip failure during bioassays.
[0007] Electronically addressable systems such as the microchips
considered herein follow Ohm's law which establishes the
relationship between the voltage drop (V) between two electrodes
(i.e., the anode, placed at a positive potential and the other, the
cathode, placed at a negative potential), and the electric current
(I) which flows between these electrodes, as follows:
V=R.times.I (1)
[0008] where R is the electrical resistance of the medium between
the anode and the cathode. In systems where a permeation layer is
present over such electrodes, the value of R is greatly determined
by the physical and chemical nature of said permeation layer. Thus,
according to formula (1), the difference between the electronic
potentials applied to the electrodes is directly proportional to
the intensity or density of the electric current which flows
through them. The invention described in this Letters Patent uses a
relationship between electric current and voltage wherein electric
current densities are at least 0.04 nA/.quadrature.m.sup.2 and/or
voltage drops are between 1 and 3 V. The electric current density
is defined as the electric current divided by the area of the
electrode used to support it.
[0009] Additionally, the effectiveness of the translocation of
charged biomolecules such as nucleotide oligomers within an
electronically-driven system such as that described herein depends
on the generation of the proper gradient of positively and
negatively charged electrochemical species by the anode and
cathode, respectively. For example, effective nucleic acid (i.e.
either DNA or RNA) transport may be accomplished by generation of
protons and hydroxyl anions when the potential at the anode is
greater than +1.29 V with respect to a `saturated calomel
electrode` (SCE). When subjected to such demanding operating
conditions, noncovalently-attached permeation layers prove to be
unsatisfactory since such systems are likely to experience micro-
and sometimes macrodelamination. Moreover, the transport efficiency
of charged molecules increases with increasing current density,
thus driving the desire for operation at higher voltage drops and
current densities and, thus, the need for evermore robust
permeation layers.
[0010] Therefore, a need still remains for methodologies for
keeping permeation layers from delaminating from electronic
microchip substrates and particularly from the electrode pads
themselves. We have discovered an improvement in permeation layer
attachment chemistry that provides a significant increase in
permeation layer performance. Specifically, we have solved the
problem of micro- and macrodelamination by discovery of a covalent
chemistry linkage system that, as applied to electronically
addressable microchip art, can be incorporated between the
microchip and the permeation layer matrix. This chemistry is
applicable to a variety of permeation layer compositions, including
polymers, hydrogels, glyoxylagarose, polyacrylamide, polymers of
methacrylamide, materials made from other synthetic monomers, and
porous inorganic oxides created through a sol-gel process, and is
able to withstand current densities of at least 0.04
nA/.quadrature.m.sup.2 and/or voltage drops between 1 and 3 V.
SUMMARY OF THE INVENTION
[0011] The current invention provides a unique system for the
covalent attachment of a porous `permeation layer` to the surface
of electronically addressable microchips. In a preferred
embodiment, the covalent attachment is between chemical moieties of
the permeation layer and metal/silicide, metal/metal, or organic
electrodes. Preferred metal/silicide electrodes include platinum
silicide (PtSi), tungsten silicide (WTi), titanium silicide (TiSi),
and gold silicide (AuSi). Preferred metal/metal electrodes include
platinum/titanium (PtTi) and gold/titanium (AuTi). Preferred
organic electrodes include materials such as poly(phenylene
vinylene), polythiophene, and polyaniline.
[0012] In an example of this embodiment, the covalent attachment
comprises a linking moiety that provides an attachment mechanism
for bonding the linker to the silanol moiety of a metal/Si surface
and a separate moiety for bonding the linker to the permeation
layer. Where metal/metal and organic electrodes are employed, the
attachment mechanism of the linker to the electrode is the same in
that the moiety of the linker attaching to the electrode will react
with specific metals and reactive centers on organic molecules to
form covalent bonds.
[0013] In a particularly preferred embodiment, the linking moiety
is defined by the formula: 1
[0014] where X=acrylate, methacrylate, acrylamide, methacrylamide,
allyl, vinyl, acetyl, amine (substituted or not), epoxy or
thiol;
[0015] SPACER=alkyl, aryl, mono- or polyalkoxy (such as
ethyleneglycol or polyethyleneglycol), mono- or polyalkylamine,
mono- or polyamide, thioether derivatives, or mono- or
polydisulfides;
[0016] A and B=any combination of Oxygen-R, where R.dbd.H, alkyl
such as methyl, ethyl, propyl, isopropyl or other linear or
branched hydrocarbon, Cl, Br or a moiety functionality similar to
that of X-SPACER; and
[0017] C=Oxygen-R, where R.dbd.H, alkyl such as methyl, ethyl,
propyl, isopropyl or other linear or branched hydrocarbon, Cl, Br,
or any other hydrolyzable moiety.
[0018] In the example of the metal/Si electrodes, these linkage
groups, which contain a silicide group can react with hydroxyl
groups bonded to an oxygen moiety of the electrode surface. On the
other end of the linker, the X moiety comprises chemical groups
that are available to covalently react with reactive centers of the
permeation layer polymer.
[0019] In another embodiment, the permeation layer is a material
suitable for transmitting electronic charge from an electrode to a
solution overlaying the electrode. Materials contemplated for
constructing polymers used for the permeation layer may include,
but are not limited to, agarose, glyoxylagarose, acrylamide,
methacrylamide, polyacrylamide, materials made from other synthetic
monomers, hydrogels; and porous inorganic oxides created through a
sol-gel process (Brinker et al., Sol-Gel Science, Academic Press,
San Diego, 1990).
[0020] Synthetic monomers used to make polymeric permeation layers
may include those selected from the group consisting of epoxides,
alkenyl moieties including, but not limited to, substituted or
unsubstituted .alpha., .beta. unsaturated carbonyls wherein the
double bond is directly attached to a carbon which is double bonded
to an oxygen and single bonded to another oxygen, nitrogen, sulfur,
halogen, or carbon; vinyl, wherein the double bond is singly bonded
to an oxygen, nitrogen, halogen, phosphorus or sulfur; allyl,
wherein the double bond is singly bonded to a carbon which is
bonded to an oxygen, nitrogen, halogen, phosphorus or sulfur;
homoallyl, wherein the double bond is singly bonded to a carbon
which is singly bonded to another carbon which is then singly
bonded to an oxygen, nitrogen, halogen, phosphorus or sulfur; and
alkynyl moieties wherein a triple bond exists between two carbon
atoms.
[0021] In another embodiment, the covalently attached permeation
layer is kept from delaminating while the anode is charged with an
electronic potential above +1.29V/SCE and/or the cathode with a
potential below -0.89 V/SCE. In a particularly preferred
embodiment, the current flow between the electrodes has a density
sufficient to induce the transport of molecules in the solution
above the electrodes of the microchip. Such density is preferably
at least 0.04 nA/.mu.m.sup.2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee. The invention will be further
described with reference to the accompanying drawings in which:
[0023] FIG. 1 is a chemical structure schematic showing attachment
of a linker moiety to the electrode surface.
[0024] FIG. 2 is a schematic diagram showing a process for covalent
attachment of the permeation layer to the electrode. In the example
of the figure, the electrode is treated with an argon plasma for 5
minutes at 250 mTorr (250 W). This cleans the electrode, which has
hydroxyl functionalities at its surface. Linker is then attached to
the electrode such as by vapor deposition for 5 minutes at room
temperature followed by curing at 90.degree. C. for 2 hours. This
process leaves reactive moieties that can bond to the permeation
layer. In the example of the figure, a linker having reactive amine
groups is used wherein the amine moieties are available for bonding
to reactive moieties of the permeation layer matrix. The bonding
between the linker and permeation layer reactive centers can be
accomplished using a Schiff base reaction.
[0025] FIGS. 3A and B are confocal microscope photos of partial
images of individual electrodes wherein the permeable membrane
attached to the electrode surface without use of a linker moiety is
shown before (A) and after (B) delamination.
[0026] FIG. 4A-D are confocal microscope photos of partial images
of individual electrodes wherein the permeable membrane attached to
the electrode surface using AEAPS deposited by vapor is shown at
various degrees of delamination.
[0027] FIGS. 5A and B are confocal microscope photos of partial
images of 80 .mu.m diameter Pt (A) and a PtSi (B) electrodes
wherein the permeable membrane was attached to the electrode
surface using AEAPS. In FIG. 5A, the Pt electrode began to
delaminate at the second direct current impulse of 500 nA (0.1
nA/.mu.m.sup.2) for 2 min. In contrast, (FIG. 5B) the PtSi
electrode showed no delamination after the second direct current
impulse of 500 nA (0.1 nA/.mu.m.sup.2).
[0028] FIGS. 6A and B, and 7A and B are confocal microscope photos
of partial images of electrode arrays wherein the permeable
membrane was either deposited on a Pt electrode without chemical
attachment (6A and 7A) or was attached to a PtSi electrode surface
using AEAPS (6B and 7B). The images show the levels of repeated
biasing that result in delamination for Pt without covalent bonding
of the permeation layer and PtSi microchips with covalent
bonding.
[0029] FIGS. 8, 9, and 10 are confocal microscope photos of partial
images of a Pt electrode overlaid with agarose. In FIG. 8 the focal
plane of the image is set at 3 .mu.m above the electrode prior to
electronic biasing. This indicates the permeation layer surface is
3 .mu.m above the electrode as indicated by the beads being in
focus. In FIG. 9 an unchanged focal plane during electronic biasing
is shown. In FIG. 10, a focal plane 4 .mu.m above the electrode is
shown indicating that delamination occurred causing the permeation
layer to rise so that the beads resting on top of the layer come
into focus at a greater distance from the electrode.
[0030] FIG. 11 is a confocal microscope photo showing confirmation
that the permeation layer of FIG. 10 delaminated as indicated by
the presence of concentric rings.
[0031] FIGS. 12 and 13 are confocal microscope photos of partial
images of electrodes showing PtSi electrode with an acrylamide
permeation layer covalently attached. FIG. 12 shows that the focal
plane remained unchanged after a two-minute bias at +2 .mu.A (0.4
nA/.mu.m.sup.2). FIG. 13 confirms that no delamination occurred
with this electrode.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] In the art of electronically addressable microchips used for
transporting charged molecules from one point in a solution to
another, the transported molecules must be protected from direct
contact with the electrodes of the microchip and ions produced at
the electrode when the electrodes are biased to impart an electric
field to the solution. Protection is provided by an insulating
membrane, i.e., the permeation layer, which also allows for the
flow of charge from the electrode to the solution without damaging
the transported molecules. Typically, the insulating membrane is a
polymeric material such as agarose or cross-linked polyacrylamide.
These materials are ideal in that they are porous and allow
electrochemical products created at the electrode to escape to the
overlying solution.
[0033] More specifically, such insulating membrane materials can
comprise, but are not limited to, agarose, glyoxylagarose,
acrylamide, methacrylamide, polyacrylamide, materials made from
other synthetic monomers, and porous inorganic oxides created
through a sol-gel process. Synthetic monomers used to make
polymeric permeation layers may also include those selected from
the group consisting of epoxides, alkenyl moieties including, but
not limited to, substituted or unsubstituted .alpha., .beta.
unsaturated carbonyls wherein the double bond is directly attached
to a carbon which is double bonded to an oxygen and single bonded
to another oxygen, nitrogen, sulfur, halogen, or carbon; vinyl,
wherein the double bond is singly bonded to an oxygen, nitrogen,
halogen, phosphorus or sulfur; allyl, wherein the double bond is
singly bonded to a carbon which is bonded to an oxygen, nitrogen,
halogen, phosphorus or sulfur; homoallyl, wherein the double bond
is singly bonded to a carbon which is singly bonded to another
carbon which is then singly bonded to an oxygen, nitrogen, halogen,
phosphorus or sulfur; and alkynyl moieties wherein a triple bond
exists between two carbon atoms.
[0034] As described above, for optimal functionality of
electronically addressable microchips, it is important that the
porous insulating layer or permeation layer remain in contact with
the electrode in order to enhance uniformity and consistency of the
electronic potential from one pad to the other. As shown in FIG. 1
the permeation layer may be linked to the electrode by a linking
moiety that has at least two reactive centers. Linkers having
suitable characteristics such as that shown in FIG. 1 are provided
in Table I.
1TABLE I CHEMICAL TYPE FORMULA ACRYLATES:
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2SiCl.sub.3
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)(OCH.sub.3).sub.2
CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3).sub.2(OCH.su-
b.3) CH.sub.2.dbd.CHCOOCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)Cl.sub.-
2 CH.sub.2.dbd.CHCOOCH.sub.2CH(OH)CH.sub.2NHCH.sub.2CH.sub.2CH.sub-
.2Si(OC.sub.2H.sub.5).sub.3 METHACRYLATES:
CH.sub.2C(CH.sub.3)COOCH- .sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3
(MOTS)
CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH.sub.2CH.sub.2SiCl.sub.3
CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)(OCH.sub.3-
).sub.2 CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH.sub.2CH.sub.2Si(CH.s-
ub.3).sub.2(OCH.sub.3) CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH.sub.2-
CH.sub.2Si(CH.sub.3)Cl.sub.2 CH.sub.2.dbd.C(CH.sub.3)COOCH.sub.2CH-
(OH)CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.5).sub.3
ACRYLAMIDES:
CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.-
5).sub.3 (AMPTS) CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.sub.2SiCl.s-
ub.3 CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)(OCH.s-
ub.3).sub.2 CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3-
).sub.2(OCH.sub.3) CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CH.sub.2Si(C-
H.sub.3)Cl.sub.2 CH.sub.2.dbd.CHCONHCH.sub.2CH(OH)CH.sub.2NHCH.sub-
.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.5).sub.3
CH.sub.2.dbd.CHCONHCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2CONHCH.sub.2CH.sub-
.2CH.sub.2Si(OC.sub.2H.sub.5).sub.3 METHACRYLAMIDES:
CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3
CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.2CH.sub.2CH.sub.2SiCl.sub.3
CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.2CH.sub.2CH.sub.2Si(CH.sub.3)(OCH.s-
ub.3).sub.2 CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.2CH.sub.2CH.sub.2Si-
(CH.sub.3).sub.2(OCH.sub.3) CH.sub.2.dbd.C(CH.sub.3)CONHCH.sub.2CH-
.sub.2CH.sub.2Si(CH.sub.3)Cl.sub.2 CH.sub.2.dbd.C(CH.sub.3)CONHCH.-
sub.2CH(OH)CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.5).sub.3
ALLYL DERIVATIVES:
CH.sub.2CHCH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OCH.- sub.3).sub.3
CH.sub.2.dbd.CHCH.sub.2SiH(OCH.sub.3).sub.2
CH.sub.2.dbd.CHCH.sub.2Si(CH.sub.3).sub.2Cl
CH.sub.2.dbd.CHCH.sub.2SiHCl.sub.2 CH.sub.2.dbd.CHCH.sub.2Si(OCH.-
sub.3).sub.3 AMINO DERIVATIVES:
H.sub.2NCH.sub.2CH.sub.2NHCH.sub.2C-
H.sub.2CH.sub.2Si(OCH.sub.3).sub.3 (AEAPS) H.sub.2NCH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).s-
ub.3 (AHAPS) H.sub.2NCH.sub.2CH.sub.2CH.sub.2Si(OCH.sub.3).sub.3
(APS) H.sub.2NCH.sub.2CH.sub.2CH.sub.2Si(OC.sub.2H.sub.5).sub.3
EPOXY DERIVATIVES: 2 3
[0035] In a particularly preferred embodiment, microchips having
covalent attachment chemistry of the current invention use linkers
denoted APS, AEAPS, AHAPS, MOTS, and AMPTS.
[0036] FIG. 2 shows a schematic of one embodiment wherein AEAPS is
used to bond the electrode to the permeation layer. In this
example, the PtSi electrode microchip is first treated with an
argon plasma for 5 minutes at 250 mTorr and 250 Watts. The chip is
then treated with AEAPS by vapor deposition over 5 minutes at room
temperature then cured onto the chip by heating for 2 hours at
90.degree. C. This causes the linker to covalently bind to the
hydroxyl groups of the silicide moiety in the PtSi electrode. Once
the linker is attached to the microchip, the permeation polymer
(for example glyoxylagarose) is overlaid onto the electrode surface
and treated in the presence of NaBH.sub.3CN so that a Schiff base
reaction and reduction can occur and cause the amine groups of the
AEAPS linker to bond to the aldehyde functionality available on the
permeation polymer (e.g., glyoxylagarose). Where polyacrylamide is
employed as the permeation layer polymer, a UV-initiated free
radical polymerization reaction can be conducted between the
monomers which will make up the permeation layer and the vinyl
moieties present at the surface of MOTS- or AMPTS linker-derived
electrodes, thereby synthesizing the permeation layer and
covalently anchoring it to the electrode in a single step.
[0037] Examples are provided below showing various delamination
threasholds after attachment of the permeation layer using various
linkers and attachment reaction conditions.
EXAMPLE 1
[0038] Agarose permeation layer matrix was attached to a PtSi
electrode microchip following deposition of either APS or AEAPS by
one of two methodologies.
[0039] APS and AEAPS were deposited by exposure of the chip to a
0.1 wt % silane/dry MeOH solution for 1 hour at room temperature.
The chips were rinsed in EtOH and cured at 90.degree. C. for 1
hour. In parallel experiments, APS and AEAPS linkers were deposited
onto microchips by a. vapor of neat silane in humid atmosphere for
5 min. at room temperature followed by a two hour cure at
90.degree. C.
[0040] After the agarose permeation layer was attached, the
microchips were subjected to electronic assays wherein the
electrodes were biased with three direct current (DC) impulses for
2 minutes each at 200, 500, 700, and 1000 nAmps/80 .mu.m pad (i.e.,
0.04, 0.10, 0.14 and 0.20 nA/.mu.m.sup.2) using a model 236
Source-Measure unit (Keithley Instruments Inc., Cleveland, Ohio).
Following the set of three DC impulses, the electrodes were biased
with a sequence of 150 negative pulses, each comprised of a 0.1
sec. ON state at -0.2 nA/.mu.m.sup.2, followed by a 0.2 sec. OFF
state at 0 nA/.mu.m.sup.2. As shown in Table II, the attachment
schemes using vapor deposition of the linkers provided protection
from delamination up to DC impulses of 700 nA for an 80 .mu.m
electrode (0.14 nA/.mu.m.sup.2).
2TABLE II 200 nA 200 nA 200 nA -1uA 500 nA 500 nA 500 nA -1uA 700
nA 700 nA samp DC1 DC2 DC3 AC DC1 DC2 DC3 AC DC1 DC2 A PtSi (no + +
+ + + + + + + + permlayer) B PtSi/perm + +/- +/- - - - - - layer(no
linker) C PtSi/APS/perm + + +/- - - - layer(dry MeOH deposited)* D
PtSi/AEAPS/ + + + + + +/- - - - - perm layer(dry MeOH deposited)* E
PtSi/APS/perm + + + + + +/- layer(vapor deposited)* F PtSi/AEAPS/ +
+ + + + +/- perm layer(vapor deposited)* + = no delamination +/- =
initial indication of delamination - = delamination resulting in
decoupling of layer from pad. *= the method of deposition applies
to the silane, not the permeation layer
[0041] As shown in FIGS. 3 and 4, delamination will occur at low
levels of DC (200 nA (0.04 nA/.mu.m.sup.2) after second DC pulse)
where no covalent linker attachment is used to anneal the
permeation layer to the electrode (FIGS. 3A and B). Conversely,
where AEAPS is used that has been applied to the electrode using
vapor deposition, the delamination does not appear until the
electrode has been exposed to the second DC pulse at 700 nA (0.14
nA/.mu.m.sup.2) (delamination extended to 25% of the pad area at 3
min. past shut-off) with complete delamination by 2 minutes past
third DC shut-off (FIGS. 4A-D).
EXAMPLE 2
[0042] In this example, delamination of the permeation layer from
the electrode was tested using a multilayer permeation layer
wherein the layers were applied using spin coating techniques then
reacted to cause the linking moieties to covalently bond the layers
together and to the electrode.
[0043] Specifically, microchips having PtSi electrodes were cleaned
with oxygen plasma for 10 minutes followed by argon plasma for 10
minutes. AEAPS was then vapor-deposited for 5 minutes followed by
curing at 90.degree. C. under vacuum. Subsequently, a first layer
solution comprising 2.5% glyoxylagarose solution (NuFix) which had
been stirred for 10 minutes at room temperature then boiled 7
minutes followed by filtering at 1.2 .mu.m into the ASC device
reservoir at 65.degree. C., was spin-deposited onto the microchips
with an automatic spin-coating device (ASC). Following deposition
of the first layer, a second layer, comprising streptavidin
(Scripps Laboratory, San Diego) at 5 mg/ml in 10 mM sodium
phosphate, 250 mM NaCl (pH 7.2) which was filtered at 0.2 .mu.m
into the ASC reservoir and maintained at room temperature, was
deposited similarly. The bottom layer was spin-coated at either
1500 or 2500 rpm, while the top layer was spin-coated at 5,000 rpm.
The reaction for the reduction of the Schiff bases generated
between streptavidin and glyoxylagarose, and between the AEAPS
surface and glyoxylagarose was carried out by treating the coated
microchip with 0.2 M NaBH.sub.3CN 0.1 M sodium phosphate (pH 7.4)
for 1 hr. at room temperature. Capping of the unreacted sites was
performed by application of 0.1 M Gly/0.1 M NaBH.sub.3CN, 0.1 M
sodium phosphate (pH 7.4) to the chip for 30 minutes at room
temperature. Finally, the treated microchip was exhaustively rinsed
and soaked in deionized water for 30 minutes and then air dried
overnight at room temperature.
[0044] As shown in Table III below, the thickness of the double
permeation layer was examined where the substrate contained either
plain platinum electrodes or PtSi electrodes using two different
rotational speeds for the bottom layer deposition. The results
indicate that spin-coating results in deposition of permeation
layers of variable thicknesses.
3 TABLE III Bottom layer Bottom layer spun at 1.5K rpm, spun at
2.5K rpm, bilayer bilayer thickness in thickness in Microchip type
nanometers nanometers Pt/AEAPS/agarose 587 .+-. 4 465 .+-. 4 668
.+-. 4 465 .+-. 4 668 .+-. 3 -- PtSi/AEAPS/agarose 744 .+-. 17 511
.+-. 4 685 .+-. 1 620 .+-. 5 494 .+-. 90
[0045] The chips as fabricated in this example, were tested for
resistance to delamination. For the platinum electrode microchips,
9 electrode pads were individually addressed from two separate
chips in 50 mM fresh Histidine buffer. These pads showed consistent
delamination past the second two-minute direct current pulse of 500
nA/80 .mu.m pad (0.1 nA/,.mu.m.sup.2) (FIG. 5A). In contrast, 6
pads were individually addressed from 2 of the PtSi microchips
under the same conditions. These PtSi pads had no delamination up
to several .mu.A/pad (FIG. 5B). Thus, the PtSi electrode using the
AEAPS attachment linker provided protection from delamination.
EXAMPLE 3
[0046] In this example, data is presented showing that the covalent
attachment method of the invention using PtSi electrodes, agarose
and aminopropylsilanes also protects against delamination of the
permeation layer under alternating current conditions. Here, Pt and
PtSi microchips bonded to the permeation layer with AEAPS were
tested using two pulsed biasing protocols.
[0047] Both protocols were carried out using 50 mM L-Histidine
buffer. Specifically, in protocol A, the microchips were biased at
+800 nA/pad (0.16 nA/.mu.m.sup.2) for 38 milliseconds (ms), -800
nA/pad for 25 ms, cycled for a total of 25 seconds using 3 pads
each pulse. In protocol B, the microchips were biased at +1.6
.mu.A/pad (0.32 nA/.mu.m.sup.2) for 19 ms, -1.6 .mu.A/pad for 12
ms, and cycled for a total of 14 seconds each on 3 pads addressed
simultaneously. Images were taken using an INM 100 confocal
microscope (Leica).
[0048] FIG. 6A shows Pt chips that were biased using protocol A,
followed by 0, 4, 8, 12 or 16 repeats of protocol B. The images
show that delamination begins after 8 repeats of protocol B. In
contrast, the PtSi chips (FIG. 6B) showed delamination to a much
less extent at the 8.sup.th biasing. In order to more accurately
define the delamination threshold, the chips were assayed with
smaller stringency increments using biasing repeats of 2, 4, 6, and
8 times. On Pt electrodes, delamination began to occur at bias
repeat number 6 (FIG. 7A). In contrast, the PtSi chip showed less
delamination effect at the same level of electrodynamic stress
(FIG. 7B).
[0049] The overall results indicate that damage begins to occur
during the sixth application of the above protocol B and that the
delamination increased with increasing cycle repeats. This
delamination effect was less prominent in the PtSi chips.
EXAMPLE 4
[0050] In this example, methacryloylsilanes are employed as linkers
for attaching synthetic permeation layers such as acrylamide-based
hydrogels to Pt and PtSi chips. Additionally, the integrity of the
permeation layer was examined using a technique wherein glass beads
are applied to the surface of the permeation layer as a reference
upon which the confocal microscope can focus. This enables
permeation layer thickness determination and facilitates the
monitoring of permeation layer distortions due to such things as
delamination.
[0051] FIG. 8 shows a Pt microchip having an agarose permeation
layer wherein the thickness of the layer before electronic biasing
was determined to be 3.0.+-.0.5 .mu.m. The figure shows the focal
point at the position of the beads above the electrode. Thus, the
underlying electrode is slightly out of focus. FIG. 9, the same
electrode during a bias at .+-.200 nA (0.04 nA/.mu.m.sup.2) with
direct current without observable distortion of the permeation
layer. The beads migrate to the electrode due to the positive bias.
Following this two-minute bias, the impulse was terminated and the
electrode observed for changes in its appearance. As seen in FIG.
10, the beads resting over the center of the electrode moved to a
location 4.0.+-.0.5 .mu.m above the electrode based on the vertical
shift required to bring said beads back into the focal plane. Thus,
the permeation layer underwent a 1 .mu.m expansion. As shown in
FIG. 11, this expansion appears to be related to the delamination
of the permeation layer from the electrode (microdelamination) as
indicated by the presence of concentric rings visible at the edges
of the electrode pad. Additionally, in other experiments, not
shown, we have observed permeation layer thickness distortions from
2 to 6 .mu.m occurring with delamination.
[0052] In another experiment, acrylamide-based hydrogel permeation
layers anchored to PtSi electrodes via the MOTS linker were exposed
to a .+-.200 nA (0.04 nA/.mu.m.sup.2) bias for 2 minutes and
examined for delamination. FIG. 12 shows beads resting atop the
permeation layer 6 .mu.m above the electrode surface. The beads
remained at the same position above the electrode after bias
shut-off, indicating that no distortion of the permeation layer
occurred. FIG. 13 shows the same pad with the focal point
positioned at the electrode. No delamination ringlets were
observed. When the electrodynamic stress was increased to +5 uA (1
nA/.mu.m.sup.2) for 2 mins., the permeation layer was observed to
distort such that the layer seemed to swell.
[0053] However, no delamination from the electrode was observed.
The results of the above experiments are shown in Table IV.
4TABLE IV bias conditions initial post (current dry wet address
integrity of electrode/ chip type densities) thickness thickness
distortion permeation layer bond Pt/agarose 200 nA, 2 min 0.80 .+-.
0.01 3.0 .+-. 0.5 4.0 .+-. 0.5 delamination & (0.04
nA/.mu.m.sup.2) distortion 200 nA, 2 min 0.80 .+-. 0.01 3.0 .+-.
0.5 6.0 .+-. 0.5 delamination & (0.04 nA/.mu.m.sup.2)
distortion Pt/polyacrylamide 200 nA, 2 min 2.0 .+-. 0.1 5.0 .+-.
0.5 9.0 .+-. 0.5 delamination & (0.04 nA/.mu.m.sup.2)
distortion 200 nA, 2 min 1.9 .+-. 0.1 5.0 .+-. 0.5 9.0 .+-. 0.5
delamination & (0.04 nA/.mu.m.sup.2) distortion
PtSi/polyacrylamide 200 nA, 2 min 2.0 .+-. 0.1 5.0 .+-. 0.5 5.0
.+-. 0.5 intact (0.04 nA/.mu.m.sup.2) 500 nA, 1 min 2.0 .+-. 0.1
6.0 .+-. 0.5 6.0 .+-. 0.5 intact (0.1 nA/.mu.m.sup.2) 1 uA, 2 min
2.0 .+-. 0.1 6.0 .+-. 0.5 6.0 .+-. 0.5 intact (0.2 nA/.mu.m.sup.2)
2 uA, 2 min 2.0 .+-. 0.1 6.0 .+-. 0.5 6.0 .+-. 0.5 intact (0.4
nA/.mu.m.sup.2) 5 uA, 2 min 2.0 .+-. 0.1 6.0 .+-. 0.5 12.0 .+-. 0.5
distortion without (1 nA/.mu.m.sup.2) delamination
[0054] Given that these results show that current densities in the
range of 1 nA/.mu.m.sup.2 are useful in the operation of microchips
having bonding chemistry resistant to delamination, we further
contemplate that current densities in the range of at least 10
nA/.mu.m.sup.2 may be used with microchips having permeation layers
which are bound to the electrodes using the bonding chemistry of
the present invention without delamination.
[0055] Modifications and other embodiments of the invention will be
apparent to those skilled in the art to which this invention
relates having the benefit of the foregoing teachings,
descriptions, and associated drawings. The present invention is
therefore not to be limited to the specific embodiments disclosed
but is to include modifications and other embodiments which are
within the scope of the appended claims. All references are herein
incorporated by reference.
* * * * *