U.S. patent application number 09/746840 was filed with the patent office on 2002-01-24 for combinatorial electrochemical synthesis.
This patent application is currently assigned to TheraSense, Inc.. Invention is credited to Caruana , Daren J., Heller , Adam.
Application Number | 20020008038 09/746840 |
Document ID | / |
Family ID | 22223147 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020008038 |
Kind Code |
A1 |
Heller , Adam ; et
al. |
January 24, 2002 |
Combinatorial Electrochemical Synthesis
Abstract
An array of selectively addressible microelectrodes for
combinatorial synthesis of complex polymers or alloys.
Inventors: |
Heller , Adam; ( Austin,
Texas) ; Caruana , Daren J.; ( Austin,
Texas) |
Correspondence
Address: |
Denise M. Kettelberger
Merchant & Gould P.C.
3200 IDS Center
80 South Eighth Street
Minneapolis
Minnesota
55402
US
612-371-5268
612-332-9081
|
Assignee: |
TheraSense, Inc.
1360 South Loop Road
Alameda
94502
California
|
Family ID: |
22223147 |
Appl. No.: |
09/746840 |
Filed: |
December 22, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09746840 |
Dec 22, 2000 |
|
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PCT/US99/14459 |
62, 199 |
|
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60/090,520 |
62, 199 |
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Current U.S.
Class: |
205/261 ;
204/280; 204/291; 204/292; 204/294; 205/413; 205/431; 205/455;
205/98 |
Current CPC
Class: |
B01J 2219/00637
20130101; C40B 40/18 20130101; C40B 60/14 20130101; B01J 2219/00612
20130101; C07H 21/00 20130101; B01J 2219/00713 20130101; B01J
2219/00353 20130101; C40B 80/00 20130101; B01J 2219/00389 20130101;
B01J 2219/00745 20130101; B01J 19/0046 20130101; B01J 2219/00752
20130101; B01J 2219/00653 20130101; B01J 2219/00628 20130101; B01J
2219/00659 20130101; B01J 2219/00626 20130101; B01J 2219/00605
20130101; B01J 2219/00689 20130101; C07B 2200/01 20130101; B01J
2219/00621 20130101 |
Class at
Publication: |
205/261 ;
204/280; 204/292; 204/294; 204/291; 205/431; 205/413; 205/455;
205/98 |
International
Class: |
C25D 003/00; C25C
007/02; C25B 011/00; C25D 017/10; C25B 011/04; C25B 011/12; C25B
003/00 |
Claims
Claims
28. A device comprising:a) a plurality of selectively adressable
microelectrodes;b) a conductive matrix disposed on each
microelectrode, the matrix comprising carbon, hydrogen and
functional reactive groups, wherein the functional reactive groups
are activated or deactivated by applying a current or a potential
to the conductive matrix; andc) a source of current or potential
arranged and configured to selectively provide a current or voltage
to each microelectrode,wherein each of the selectively addressable
microelectrodes has a smallest lateral dimension, and wherein each
microelectrode is separated from other microelectrodes of the
device by a distance of at least ten times the smallest lateral
dimension of the microelectrode.
29. The device according to claim 28, wherein the smallest lateral
dimension is a diameter.
30. The device according to claim 28, wherein the smallest lateral
dimension is measured from one edge of the microelectrode to an
opposite edge of the microelectrode.
31. The device according to claim 28, wherein each microelectrode
is separated from other microelectrodes of the device by a distance
of at least twenty times the smallest lateral dimension of the
electrode.
32. The device according to claim 28, wherein the smallest lateral
dimension of one or more of the microelectrodes is less than 100
m.
33. The device according to claim 32, wherein the smallest lateral
dimension of each of the microelectrodes of the device is the
same.
34. The device according to claim 28, wherein the smallest lateral
dimension of one or more of the microelectrodes is 0.1 m to 1
m.
35. The device according to claim 34, wherein the smallest lateral
dimension of each of the microelectrodes is the same.
36. The device according to claim 28, wherein the conductive matrix
comprises a redox polymer.
37. The device according to claim 36, wherein the redox polymer
comprises a transition metal, and wherein the transition metal is
osmium, ruthenium, iron, copper or cobalt.
38. The device according to claim 36 wherein the redox polymer
comprises a hydrogel.
39. The device according to claim 28 wherein the conductive matrix
comprises a polycation.
40. The device according to claim 28 wherein the conductive matrix
has a thickness of 3 nm to 20 m.
41. The device according to claim 28, wherein the functional
reactive groups are independently selected from amines, aldehydes,
carboxylic acids, or active esters.
42. The device according to claim 28 further comprising one or more
reference electrodes.
43. The device according to claim 28 further comprising one or more
counter-electrodes.
44. A method for selective synthesis of an array of compounds, the
method comprising steps of:a) providing a device comprising:(i) a
plurality of selectively addressable microelectrodes;(ii) a
conductive matrix disposed on each microelectrode, the matrix
comprising carbon, hydrogen and functional reactive groups, wherein
the functional reactive groups are activated or deactivated by
applying a current or a potential to the conductive matrix;
and(iii) a source of current or potential configured and arranged
to selectively provide a current or voltage to each
microelectrode,wherein each of the selectively addressable
microelectrodes has a smallest lateral dimension, and wherein each
microelectrode is separated from other microelectrodes of the
device by a distance of at least ten times the smallest lateral
dimension of the microelectrode;b) providing a first reactant;
andc) selectively applying to one or more microelectrodes a current
or potential sufficient to cause a faradaic reaction in the
immediate vicinity of the one or more microelectrodes to induce
binding of the first reactant to the conductive matrix.
45. The method according to claim 44, further comprising repeating
the step of selectively applying to one or more microelectrodes a
potential sufficient to cause a faradaic reaction in the immediate
vicinity of the one or more microelectrodes to induce binding of an
additional reactant to form an array of compounds.
46. The method according to claim 44, further comprising
selectively applying to one or more microelectrodes a potential
sufficient to cause a faradaic reaction in the immediate vicinity
of the one or more microelectrodes to induce binding of a second
reactant to the first reactant.
47. The method according to claim 44, wherein the faradaic reaction
causes a chemical change in one or more of the functional reactive
groups, the reactant, or a chemical species, in the immediate
vicinity of the microelectrode.
48. The method according to claim 47, wherein the chemical change
is a change in ionic concentration or an oxidation or a reduction
of the functional reactive groups, the reactant, or the chemical
species.
49. The method according to claim 48 wherein the change in ionic
concentration is a change in pH.
50. The method according to claim 48, further comprising providing
an enzyme, wherein the chemical change is a change in ionic
concentration, and wherein adjustment of the ionic concentration in
the immediate vicinity of the microelectrode modulates activity of
the enzyme in the immediate vicinity of the microelectrode.
51. The method according to claim 44 wherein the reactant comprises
a nucleotide.
52. The method according to claim 44 wherein the reactant comprises
an amino acid.
53. The method according to claim 44 wherein the reactant comprises
an organic compound, an inorganic compound or a metal-organic
ion.
54. The method according to claim 53 wherein the organic compound
is ascorbic acid or benzoquinone.
55. The method according to claim 53 wherein the inorganic compound
is iron, cobalt, ruthenium, osmium or copper.
56. The method according to claim 44 wherein the method
comprises:a) providing a device comprising:(i) a plurality of
selectively addressable microelectrodes;(ii) a redox polymer
comprising poly(4-vinyl pyridine), osmium and amine reactive
groups; and(iii) a source of current or potential configured and
arranged to selectively apply a current or voltage to each
microelectrode;b) providing a first nucleotide; andc) selectively
applying to one or more microelectrodes a current or potential
sufficient to cause a faradaic reaction in the immediate vicinity
of the one or more microelectrodes to induce binding of the first
nucleotide to the redox polymer.
57. The method according to claim 56 further comprising:d)
providing a second nucleotide; ande) selectively applying to one or
more microelectrodes a current or potential sufficient to cause a
faradaic reaction in the immediate vicinity of the one or more
microelectrodes to induce binding of the second nucleotide to the
redox polymer or to one or more of the first nucleotides.
58. The method according to claim 56 wherein the step of
selectively applying to one or more microelectrodes a current or
potential sufficient to cause a faradaic reaction in the immediate
vicinity of the one or more microelectrodes induces binding of the
first nucleotide to one or more of the amine reactive groups.
59. A method for selective synthesis of an array of compounds, the
method comprising steps of:a) providing a device comprising:(i) a
plurality of selectively addressable microelectrodes;(ii) a
conductive matrix disposed on each microelectrode, the matrix
comprising carbon, hydrogen and functional reactive groups, wherein
the functional reactive groups are activated or deactivated by
applying a current or a potential to the conductive matrix;
and(iii) a source of current or potential providing a selective
current or voltage to each microelectrode,wherein each of the
selectively addressable microelectrodes has a smallest lateral
dimension, and wherein each microelectrode is separated from other
microelectrodes of the device by a distance of at least ten times
the smallest lateral dimension of the microelectrode; andb)
selectively applying to one or more microelectrodes a current or
potential sufficient to cause a faradaic reaction in the immediate
vicinity of the microelectrode to induce deposit of a metal onto
the microelectrode.
60. The method according to claim 59 further comprising repeating
the step of selectively applying to one or more microelectrodes a
potential sufficient to cause a faradaic reaction in the immediate
vicinity of the microelectrode to induce deposit of a second metal
onto the microelectrode to synthesize a non-stoichiometric
inorganic compound or metal alloy on the microelectrode.
61. The method according to claim 59 further comprising the step of
inducing etching or dissolution of a portion of one or more metals
deposited onto the microelectrode.
62. The method according to claim 61 further comprising reacting by
heating, oxidation, sulfidation, or consolidation to form an alloy
or non-stoichiometric inorganic compound.
63. A method for selective synthesis of an array of compounds, the
method comprising steps of:a) providing a device comprising:(i) a
plurality of selectively addressable microelectrodes;(ii) a
conductive matrix disposed on each microelectrode, the matrix
comprising carbon, hydrogen and functional reactive groups;
and(iii) a source of current or potential configured and arranged
to selectively provide a current or voltage to each
microelectrode,wherein each of the selectively addressable
microelectrodes has a smallest lateral dimension, and wherein each
microelectrode is separated from other microelectrodes of the
device by a distance of at least ten times the smallest lateral
dimension of the microelectrode;b) providing a first reactant;c)
providing an enzyme; andd) selectively applying to one or more
microelectrodes a current or potential sufficient to cause a
faradaic reaction in the immediate vicinity of the one or more
microelectrodes to change ionic concentration in the immediate
vicinity of the one or more microelectrodes,wherein change of the
ionic concentration in the immediate vicinity of the one or more
microelectrodes modulates activity of the enzyme, andwherein the
enzyme catalyzes reaction of the first reactant with the functional
reactive groups.
Description
Cross Reference to Related Applications
[0001] This application is a continuation application of PCT
Application No. PCT/US99/14459 A1, which claims priority to
Provisional Patent Application Serial No. 60/090,520, which
applications are hereby incorporated for all purposes.
Background of Invention
[0002] Field of the Invention The present invention is generally
related to the combinatorial syntheses of compounds, particularly
of biological compounds and metal alloys, on microelectrode arrays.
The invention relates in particular to the combinatorial syntheses
of oligonucleotides, peptides and biologically active compounds on
microelectrode arrays under electrochemical control.
[0003] Background of the Invention: The simultaneous synthesis of
massive numbers of different oligonucleotides, peptides, and
biologically active compounds has applications in the
identification of new drugs, in the mapping of genes, and in
testing for interactions between biological molecules. Arrays of
different genes are applied, for example, in diagnosing various
diseases, such as cancer and hereditary diseases, in sequencing the
human genome, and identification of drugs capable of blocking
bioconjugation reactions.
[0004] Conventional synthetic methods are time consuming and the
step-by-step preparation of individual potentially bioactive agents
consumes the largest fraction of the capital invested in the
development of new drugs. To address these problems, a variety of
methods for combinatorial syntheses has been developed. One
commonly used method involves the definition of pixels where a
nucleotide is added to an existing sequence through photochemical
methods. Pixels for the occurrence or the avoidance of a chemically
synthetic step can be defined by photolithographic methods.
Alternatively, reactions in a particular pixel can be driven by
light when photochemically active reactants are used.
Non-photochemical methods of combinatorial syntheses involve valved
grids of microfluidic channels, such that a reaction occurs in
microreactors at the intersection points of the channels with open
valves. In general, these methods have resulted in pixels or
microreactors that were as small as about 35 m x 35 m in their
cross-sectional area. The syntheses involved small but still
significant amounts of expensive reactants, typically more than a
billion molecules in each reaction in each pixel.
[0005] Michael Heller et.al, (U.S. Patent No. 5, 605,662) discloses
a procedure for producing arrays of selectively addressable
microelectrodes. To transport and concentrate specific charged
oligonucleotides, an electrophoretic field is used. For example, a
positive potential is applied to a specific microelectrode in order
to attract a negatively charged oligonucleotide and to concentrate
the charged moiety at the site.
[0006] The present invention provides a method for parallel
syntheses where a specific reaction is induced and controlled at
the microelectrode.
Summary of Invention
[0007] The present invention provides an electronic device and
method for the combinatorial synthesis of biopolymers, alloys, and
non-stochiometric inorganic compounds. The device includes an array
of selectively addressable microelecrodes which preferably includes
on its surface a conductive matrix, which may be a redox polymer or
hydrogel. The matrix further comprises functional reactive groups,
such as amines, aldehydes, carboxyllic acids, active esters, and
the like, useful for the sequential addition of monomeric units to
form polymeric compounds. For example, in one embodiment of the
invention, the array includes amines in the matrix, to which
nucleotides or oligonucleotides may be sequentially added by
chemical reaction.
[0008] In the method of the invention, a potential sufficient to
induce a Faradaic reaction is selectively applied to each
microelectrode to induce binding of a reactant to one or more of
the functional reactive groups in the matrix. A complex compound is
synthesized on the microelectrode by repeated Faradaic reactions,
i.e., by repeated applications of a potential sufficient to induce
the desired reactions.
[0009] In an alternative embodiment, the method of the invention
includes application of a potential to induce a Faradaic reaction
and selectively deposit metals in layers onto a microelectrode.
Optionally, a portion of one or more of the metal layers is etched
or dissolved via subsequent Faradaic reaction at the
microelectrode. Heating, oxadation, sulfadation, or consolidation
of two or more layers causes formation of an alloy or
non-stochiometric inorganic compound.
Brief Description of Drawings
[0010] Figure 1is a diagram showing the arrangement of the
electrode or array surface bearing the reactive groups as a
monolayer or in a polymeric matrix. The electrode surface is
schematically drawn as a flat surface but the surface roughness
(geometric area/actual area) may vary between 1 and 1000.
[0011] Figure 2 is a flow diagrams for the syntheses of
oligopeptides (poly amino acids, peptide nucleic acids or other
oligopeptides). The coupling is carried out with carbodiimide in
conjunction with N-hydroxysuccinimide. The solid bar connecting the
COOH function to the electrode is made of condensed amino acids.
The amino acids may be natural amino acids or amino acids not found
in nature.
[0012] Figure 3 is a flow diagram for the synthesis of
oligonucleotides. The syntheses is modulated by the
electrochemically generated protons. At potentials where protons
are produced the local pH drops and the protecting dimethoxytrityl
(DMT) on the 5" end of the oligonucleotide is cleaved. The cleavage
enables the extension of the oligonucleotide.
[0013] Figure 4 is a flow diagram showing the synthesis of an
oligopeptide with an electrochemically pH modulated enzyme
catalyzed step.
[0014] Figure 5 is a schematic diagram showing the computerized
automated potentiostatic and liquid delivery control to the
microelectrode array.
Detailed Description
[0015] Microelectrode ArrayThe electronic device of the invention
includes an array of selectively addressable microelectrodes. A
matrix comprising at least carbon (C) and hydrogen (H), and
preferably capable of conducting electrons or holes, and also of
conducting ions, is disposed on a surface of the electrodes. The
conductive matrix is disposed on the microelectrode with a
thickness greater than 3 nanometers, and preferably in the range of
3 nanometers to 20 microns, most preferably with a thickness of 5
or more nanometers. Most preferably, the matrix comprises a redox
polymer or redox hydrogel, having a molecular weight of more than
10.sup.4 daltons. Most preferably, the redox polymer or hydrogel
comprises a fast redox couple, such as complexes of transition
metals such as osmium, ruthenium, iron, copper, and cobalt.
[0016] The matrix is modified to include functional reactive
groups, such as amines, aldehydes, carboxyllic acids, active
esters, and the like.
[0017] The arrays of electrodes on which the combinatorial
syntheses is carried out are usually large. Typically the number of
electrodes in the array is greater than 4, greater than 100, and is
preferably greater 1000 and it is most preferably greater than
10,000.
[0018] The longest dimension of a microelectrode is preferably less
than 100 m, and is more preferably between 0.1 and 10 m. The shape
of a microelectrode may be, for example, oval, rectangular or
preferably circular. Fine line electrodes, that are typically
longer than 100 m and have typical widths between 0.1 m and 20 m,
can also be used. It is not necessary that the microelectrode
surface be flat. A metal or carbon microelectrode surface may be,
for example, concave or convex relative to the plane of the
surrounding insulating layer. Preferably all the microelectrodes in
the array are of the same dimensions and are spaced individually,
or grouped in elements of the array, those elements forming
organized patterns. It is preferred that the pattern have repeating
elements and it is particularly preferred that the elements have
rotational and/or translational symmetry. An example of a preferred
pattern is one where all electrodes except those at the periphery
of the array are equidistant from each other and have the same
number of nearest neighbors.
[0019] Typically it is desirable to space the electrodes at
distances greater than 10 times the diameter or smallest dimension
of the microelectrodes and it is most preferred to space the
electrodes at distances greater than 20 times the diameter or
smallest dimension of the microelectrodes. It is generally
desirable to make the electrodes small, so as to minimize the
amount of material consumed in the reaction driven at a subset of
electrodes. Preferably fewer than one hundred million molecules are
reacted at an electrode. It is more preferred that fewer than one
million be reacted and it is most preferred that fewer than one
hundred thousand be reacted. Reduction of electrode size also
allows closer spacing of electrodes in an array, i.e. denser
packing, meaning more electrodes per unit area. Typically the
density of microelectrodes is greater than 100/cm.sup.2; and most
preferably it is greater than 100,000/cm.sup.2.
[0020] The electrodes are made of a conductor which is preferably
non-corroding and may comprise gold, carbon, tantalum, ruthenium
dioxide, a conductive metal carbide, a conductive metal nitride, or
a conductive metal oxide, or a Group VIII metal such as platinum,
iridium, rhodium, rhenium, palladium or ruthenium. The electrodes
are insulated from each other by an insulator that may be organic
or inorganic. Examples of inorganic insulators are silicon dioxide
and silicon nitride and insulating doped silicon. Examples of
organic insulators are polymers, such as poly(methyl methacrylate),
polyimides, polyesters, or polyamides. The substrate of the
structure can be any material having the desired mechanical
properties such as glass, ceramic, silicon, plastic, or a metal
coated with an insulating film. The electrical contacts to each
element in the array may be in the plane of the electrodes, or in
the plane of the electrodes and in a second plane, or in multiple
planes including or excluding the plane of the electrodes. Contacts
to the electrodes may be formed individually to each electrode, to
groups of electrodes, or to rows and columns of electrodes in a
grid.
[0021] The elements of the array, i.e. its microelectrodes, can be
connected to potential or current sources by either hard wiring or
by light. Hard wiring means that the connection involves a
continuous electrical path through electrical conductors,
particularly metallic conductors or carbon. Connection by light is
possible when a photoconductor is used, so that the illuminated
areas conduct electrons or holes, while the non-illuminated areas
do not conduct. Instead of applying an external potential or
passing a current from an external current source, the potential at
or the current passing through a microelectrode of the array can
also be photogenerated. For generation of a photopotential or a
photocurrent it is preferred that the microelectrode material be or
comprise a semiconductor. The semiconductor may be an inorganic
semiconductor, such as silicon or a III-V compound. It may also be
an organic compound, such as a polymeric or non-polymeric organic
compound used in light emitting diodes or photodiodes or
photovoltaic cells.
[0022] The electrochemical circuits require, in addition to the
microelectrodes on which the syntheses are carried out, known as
working electrodes, at least one reference electrode and may
require at least one counter-electrode, that may or may not be the
reference electrode. These added electrodes may be part of the
array or may not be part of it. The reference electrode may be a
conventional reference electrode such as a saturated calomel or
silver/silver chloride electrode or it may be a pseudo-reference
electrode consisting of a conducting material in contact with the
solution. The counter electrode can be a conducting material with a
surface area which is preferably to at least twice the sum of the
combined surfaces area of the microelectrodes in the working
electrode array. There may only be one reference electrode and one
counter electrode for the entire array.
[0023] Reactive groups on the microelectrode surface: The electrode
surface may be modified with reactive groups in a variety of ways
depending in the electrode material of choice. Two examples are
shown in Figure 1. On the left, a matrix with remote reactive
functions R, such as a carboxylic acid functions, extending into
the solution is shown. On the right, a polymeric gel/hydrogel
support incorporating reactive functions R, such as carboxylic acid
groups, attached to the back bone of the polymer is shown.
Preferably the matrix should not be detached under the conditions
of the reaction that is to be carried out. If the reaction is
carried out under potentiostatic control, it is preferred that the
potential which is applied should affect only the reactive groups
or their reactions, not their attachment to the electrode.
[0024] The conducting microelectrode, or group of microelectrodes,
or the surface of the entire microelectrode array, including the
surface of the insulating material between the electrodes, may be
modified with a matrix. This matrix can, for example, be a matrix
having affinity for a reactant, such as a polycation when the
reactant is a nucleotide or a nucleotide derivative which is an
anion. It can also be a matrix comprising a redox polymer or a
redox hydrogel, the polymer or gel containing attached redox
centers. When a redox hydrogel or conductive polymer is used, it is
preferred that only the conducting region of the array be
covered.
[0025] Method of the InventionIn the method of the invention, an
array of desired chemical compounds is selectively synthesized on
selectively addressable microelectrodes. A potential sufficient to
cause a Faradaic reaction in the immediate vicinity of the
microelectrode is selectively applied to each microelectrode to
induce binding of a first reactant to one or more funcional
reactive groups present in the matrix disposed on the
microelectrode. After binding of a first reactant to a functional
reactive group in the matrix, the selective application of
potential is repeated to cause a second Faradaic reaction and
induce binding of a second reactant to the first bound reactant.
This procedure is repeated a sufficient number of times to
synthesize a desired compound on the microelectrode, thus forming
an array of individual compounds.
[0026] The reactants may be, for example, nucleotides, amino acids,
chemical moieties, or other molecular subunits such as
oligonucleotides or peptides that are induced to bind amines,
aldehydes, carboxylic acids, active esters, and the present in the
matrix.
[0027] The Faradaic reaction preferably causes a chemcial change in
one or more of the fuctional reactive groups in the matrix, in a
reactant supplied to the microelectrode, for example in a solution
bathing the electrode, or in a further chemical species present in
the solution. The chemical change can result in a change in pH, in
the oxidation or reduction of a functional reactive group on the
matrix, of the supplied reactant, or of a further chemical species
in the reaction solution.
[0028] Control through a faradaic process: A Faradaic current is a
current that passes when a species is electrooxidized or
electroreduced at an electrode. This current is contrasted with a
capacitive or polarization current where a current will flow for a
period of time when a potential is applied without an
electrooxidation or electroreduction reaction taking place.
[0029] The capacitive or polarization current flows as a result of
the redistribution of ions in the solution (or film) near the
electrode. The electrophoretic process, where a macromolecular
polyelectrolyte migrates to an electrode is such a non-Faradaic
process. After some time this current stops because of the
accumulation of cations near a negative electrode or of anions near
a positive electrode.
[0030] Upon flow of a Faradaic current, a substance is
electrolyzed. At least one species in the immediate vicinity of the
microelectrode (that is, the distance from the microelectrode where
the microelectrode can cause redox reactions) is electrooxidized
and at least one species is electroreduced. As a result, there is a
net change in the chemical composition in the immediate vicinity of
the microelectrode, not a mere re-distribution of ions in the
cell.
[0031] Faradaic reactions, unlike capacitive or polarization
processes, take place only when sufficient potential is applied to
an electrode. This is well known form any text in electrochemistry
or even physical chemistry. The threshold potential is the formal
half cell potential for the reaction driven. When the reaction is
reversible, then the potential is the thermodynamic potential, -
G/(nF), where G is the change in the Gibbs free energy, n is the
number of electrons gained or lost by the reduced or oxidized
species and F is Faraday"s constant. For example, the threshold
potential for electrolysis of water is 1.23 V (measured relative
the standard hydrogen electrode). A Faradaic current resulting in
water electrolysis flows only when this potential is exceeded.
[0032] Acid is produced by electrolysis of water at a half cell
potential depending on the pH and temperature. The higher the pH,
the more reducing (negative) this potential is, shifting at
25.sup.oC by 59 mV for each pH unit (when the partial pressure of
hydrogen is fixed).Reversible potentials are listed in most
handbooks of chemistry, such as the Handbook of Chemistry and
Physics.
[0033] The Faradaic reaction need not be reversible. For example,
when ascorbic acid is electrooxidized the reaction is irreversible.
The composition of the solution within the cell changes in the
reaction. The compound is not only redistributed but converted.
[0034] The local concentration of ions, particularly of protons,
and thus the local pH can be controlled through controlling the
current density passing through a particular electrode, which can
be in turn, controlled by the applied potential. Upon
electrooxidation, protons are usually released and upon
electroreduction protons are usually consumed.
[0035] The reactant in the faradaic process can be oxygen, the
solvent itself, e.g. water; a readily electrooxidizable organic
compound such as ascorbic acid, or a readily electroreducible
organic compound, such as benzoquinone. The reactant can also be an
inorganic or metal-organic ion, such as a complex of iron, cobalt,
ruthenium, osmium or copper. For example, the reactant can be a
bipyridine complex of ruthenium or osmium or cobalt; a cyanide
complex of iron; or a metallocene derivative, such as a ferrocene
derivative.
[0036] The metal of the electrode or a metal ion may be reduced or
oxidized to an oxidation state which is suitable for the modulation
of the chemical reaction at the surface of the electrode. The
reactant may also be an organic molecule which can be
electroreduced or electrooxidized to a species which chelates with
a dissolved metal ion. The electrochemically formed chelator
reduces the local free metal ion concentration at the electrode
surface.
[0037] Control through application of a potential without the
occurrence of a substantial faradaic (electroreduction or
electrooxidation) reaction:As taught in Heller et.al., U.S. Patent
No. 5,605,662, the local concentration of ions at a particular
microelectrode or group of microelectrodes may be controlled by a
capacitive process, such as a process of attracting cations,
repelling cations, attracting anions or repelling anions. Examples
of the anions attracted or repelled are hydrated protons (H.sup.+),
hydroxide anions (OH.sup.-) and Zn.sup.2+, Mg.sup.2+, Ca.sup.2+ or
Cu.sup.2+ ions.
[0038] The potential applied to the electrode for eletrophoretic
transport and concentration of a charged species is mirrored by the
ions in the solution. Thus a positive applied potential (relative
to the potential of zero charge (PZC) of the particular metal in
the solution) will draw anions from the bulk solution, causing a
local build up of anions at the electrode interface relative to the
bulk solution. The attracted anions will replace cations at the
surface. The pH or ion concentration in the proximity of an
electrode can be controlled through the potential applied, and/or
the buffer added to the solution and/or the concentration of the
relevant ion in the solution.
[0039] The number of electrodes in the array is greater than ten
thousand and the dimension of each microelectrode is 10 m or
smaller. The use of microelectrodes ensures that mass transport of
species to and away from the electrode surface is efficient.
Concentration polarization, which is the change in concentration of
a reactant near an operating electrode, and is important in
defining the necessary spacing of the electrodes, is also reduced
when microelectrodes are used. Thus denser electrode arrays can be
made.
[0040] The extent of concentration polarization, meaning the
distance to which a concentration change extends from a particular
electrode depends on the diffusion coefficient of the reactant, the
dimension of the microelectrode and the applied potential. The
occurrence or prevention of a reaction at a particular electrode is
controlled by applying a potential to, or passing a current
through, the electrode at which the occurrence of the reaction is
desired or in which the occurrence of the reaction is to be
prevented. The application of a potential induces a faradaic or
non-faradaic process, depending upon the specific potential applied
and the species present in the vicinity of the electrode, which
changes the concentration of at least one ion or molecule relative
to the bulk solution.
[0041] The concentration of an ion near a particular microelectrode
can be controlled through a faradaic process, such as an
electroreduction reaction or an electrooxidation reaction. It is
well known that the potential where an electrooxidation or
electroreduction reaction takes place depends on the presence of a
particular electrooxidizable or electroreducible species in the
solution. In general the local pH at and near the electrode surface
is increased when an electroreduction reaction is taking place and
is decreased when an electrooxidation reaction is taking place.
Usually the higher the current density the greater the change in
local pH. The magnitude of the change also depends on the
concentration of buffering agents, decreasing when the buffer
concentration is raised. For example, the local pH may be increased
at a microelectrode by the electroreduction of oxygen, a reaction
where protons are consumed.
[0042] The pH can be decreased locally by the electrooxidation of
water to hydrogen peroxide or to oxygen, or by the electrooxidation
of a solute, such as ascorbate ion, that takes place at a less
oxidizing potential than the potential for electrooxidation of
water. In these reactions protons are released. The extent of pH
change may be controlled by the potential applied or by the
buffering capacity of the solution. In summary the local
concentration of a particular species near a particular electrode
is controlled through the potential applied or the current passed,
or by the concentration and nature of an added buffer.
[0043] There are different ways through which the local
concentrations of ions at a microelectrode surface may control the
occurrence or non-occurrence of a reaction. For example, a reactive
species such as an O-acylisourea or an N-hydroxysuccinimide ester
does not react with an amine in an acid environment, where the
amine is protonated. However, at neutral or slightly basic pH the
reaction with the amine does take place and an amide is formed.
Through controlling the local concentration of protons, or of other
ions, or of a molecule such as ascorbate which is required for a
reaction to take place at a defined potential, it is also possible
to modulate the activity of enzymes that catalyze either the
formation or the breakage of chemical bonds, for example bonds
formed in condensation or hydrolysis reactions. There are numerous
well documented examples, found in textbooks of biochemistry, where
an enzyme is active only in a well defined pH range. Also some
enzyme-catalyzed reactions require the presence of a particular
ion, such as Zn.sup.2+ or Mg.sup.2+, or Ca.sup.2+; others can be
reversibly or irreversibly inhibited by the presence of other ions,
e.g. Cu.sup.2+.
[0044] Examples of non-faradaic processes whereby the local
concentration of an ion is changed include local increase of the
concentration of a cation when a negative potential is applied to
an electrode; increase in the local concentration of an anion when
a positive potential is applied; local decrease in concentration of
a cation when a positive potential is applied; and local decrease
in the concentration of an anion when a negative potential is
applied. The above four processes do not require the occurrence of
an electrochemical reaction.
[0045] Examples of relevant reactions known to take place in a
particular pH domain: Examples of such reactions include the
syntheses of amides such as those of oligopeptides including
peptides, proteins and in peptide nucleic acids via a carbodiimide
involving reaction; syntheses involving N-hydroxysuccinimide
esters; syntheses involving imidates; and synthesis involving
polyphosphates. Figure 2 shows an example of a flow diagram of a
synthesis utilizing a carbodiimide and Nfor the formation of a
peptide bond on an electrode surface.
[0046] When the objective is not to make amides but other
compounds, other active reactants may be used. For example an
epoxide may be used for a reaction with an amine; or an alkyl
halide, particularly an alkyl iodide or an alkyl bromide, for a
reaction of an amine. The reaction takes place in neutral or basic
solutions, but not in acid ones. Control can be either by adjusting
the local pH through a faradaic or a non-faradaic process such that
the reaction proceeds, or through inhibiting the reaction through
adjusting the local pH such that the reaction is prevented.
[0047] For the electrically controlled synthesis of
oligonucleotides, the well-known phosphoramidite method can be
applied. Figure 3 shows the flow diagram of a current or a
potential controllable synthetic route, based on the
phosphoramidite method and involving a dimethoxytrityl (DMT)
protecting group on the 5" terminus of the reacting base. When
protons are generated at the microelectrode, the protecting group
on the 5" is removed. This removal or de-protection allows the
extension of the oligonucleotide once the cycle is repeated.
Because the coupling step is also pH dependent, the coupling can
also be modulated by an applied potential or a current passed
through the electrode. Also the iodine required for the oxidation
step may be electrochemically generated when the solution comprises
iodide ions. Any unreacted oligonucleotide is capped by acylation
with acetic anhydride to avoid extension of any undesired
sequences. This capping is not shown in Figure 3.
[0048] Enzyme Catalyzed Reactions: There are families of enzymes
known to catalyze the hydrolysis or formation of amides in
peptides, proteins, and protein nucleic acids and of phosphate
ester links in oligonucleotides or DNA and of glycosidic linkages
in oligosaccharides. The families of these enzymes include, for
example, kinases, peptidases, proteolytic enzymes and hydrolases,
transferases, ligases. The enzymes" activity can be controlled by
the local adjustment of the pH. Also the activity of some enzymes
may be enhanced by the local electrochemical reaction or process
(meaning application of potential or passage of current) increase
in the concentration of ions such as Mg.sup.2+, Ca.sup.2+ , or
Zn.sup.2+, or may be decreased by electrochemically increasing the
local concentration of ions such as Cu.sup.2+. The enzyme may cause
the addition, through formation of a covalent bond, of a dissolved
species in the solution to which the array is exposed; or it may be
such that it cleaves a terminal function on a molecule already on
the electrode so as to enable a reaction at that particular
electrode; or it may cause hydrolysis of a functional group of a
molecule on an electrode.
[0049] A schematic diagram of the steps involved in the enzyme
catalyzed reaction is shown in figure 4. The method described is
the step by step synthesis of a peptide nucleic acid modulated by a
non-specific amidase, particularly acrylamide amidohydrolase from
Pseudomonas aeruginosa. Enrichment of the zone near the
microelectrode in protons prevents the enzymatic cleavage of the
terminal amide and therefore the subsequent carbodiimide or
N-hydroxysuccinimide ester (NHS) utilizing condensation reaction
whereby an amino acid is added to the peptide, peptide nucleic acid
or protein on the electrode.Automated system: The system may be
automated by integrating a computer to control the flow of liquid
containing reactants by a series of valves and also the
potentiostat as shown in figure 5.
[0050] For example, a series of different oligonucleotides can be
formed on an array of electrodes using the apparatus illustrated in
figure 5 and the process illustrated in figure 3. An array of
carbon or metallic electrodes is formed. Each of the electrodes is
coated with a coupling species. The coupling species includes a
reactive functionality that is initially capped with a protective
group. The array is placed within a flow device that is coupled to
a valve, which is, in turn, coupled to four reservoirs of molecular
subunits, corresponding to the four different bases of DNA;
adenine, guanine, thymine, and cytosine. Each of the molecular
subunits includes a first reactive functionality for coupling to a
deprotected reactive functionality of the coupling species or a
previously deposited molecular subunit. Each of the molecular
subunits also includes a second reactive functionality that is
initially capped by a protecting group.
[0051] In operation, the valve is directed to open and allow one of
the four solutions of molecular subunits to flow into contact with
the array. Each electrode of the array is individually coupled to a
potentiostat, typically, under computer-control. A potential is
selectively applied to (or, alternatively, a current is passed
through) those electrodes at which the particular molecular subunit
is to be deposited. The electrical potential (or current) causes a
change in the concentration of an anionic or cationic species
(e.g., a change in pH) that leads to the removal of the protecting
group on the coupling species and results in a reaction of the
reactive subunit on the coupling species and the first reactive
functionality of the molecular subunit.
[0052] The solution containing the first molecular subunit is then
removed and the valve is directed to open and allow a second
solution with a different molecular subunit to flow into contact
with the array. Again, a potential is selectively applied to those
electrodes at which this particular molecular subunit is to be
attached, including those electrode at which this second molecular
subunit is to be attached to the first molecular subunit. The
application of the potential results in the removal of the
protecting group from the reactive group of the coupling species or
the second reactive group of the previously coupled molecular
subunit. This procedure is repeated until the desired
oligonucleotide sequences are all formed on the array of
electrodes.
[0053] As an example, a four electrode array can be formed with the
following oligonucleotides on the electrodes:
[0054] Electrode 1 - AGTC
[0055] Electrode 2 - ATGC
[0056] Electrode 3 - GTGC
[0057] Electrode 4 - TGCA
[0058] One exemplary process includes the steps in the following
table:
[0059] Other sequences of steps could also be used to obtain the
same array of electrodes. In addition, the same principle can be
used to form other molecules, such as peptides or proteins, on
electrodes that utilize a small set of subunits, such as amino
acids.
[0060] Combinatorial Synthesis of Inorganic MaterialsIn the
combinatorial syntheses of inorganic compounds or organic compounds
having inorganic backbones a potential is applied to a
microelectrode of the array or a current is passed through an
electrode of the array, such that the local pH is changed or the
local concentration of another ion is changed. When the reactive
material on the microelectrode is a metal or metal oxide, then
typically a reduction in pH, the application of a positive
potential, or the occurrence of a local electrooxidation reaction
can accelerate the dissolution of the oxide or the metal. For
example, metals, such as zinc or aluminum, or an oxide of such
metals are more rapidly dissolved by a local rise in pH. The rate
of removal is adjusted through local control of the pH, the
potential, or the current. Thus by varying in a gradual manner the
potential or the current density the amount of residual material
residing on an electrode after partial stripping of a layer can be
increased or decreased. Similarly by driving an electroreduction
reaction, whereupon the pH is increased, the amount of residual
material may be increased when the film is in an etching solution,
such as an acid solution.
[0061] The removal and/or deposition of inorganic material can be
useful in a variety of circumstances, including, for example, the
combinatorial formation of non-stoichiometric materials. These
materials are often tested for various properties, including, for
example, fluorescence wavelength, fluorescence quantum yield,
magnetic properties, and dielectric constant. It is often useful to
test a range of different non-stoichiometric combinations. A
material is "non-stoichiometric" if the composition can not be
expressed as a chemical formula using numbers or integers of
smaller than five.
[0062] One method for forming a range of non-stoichiometric
combinations includes forming an array of metal electrodes 102 on a
substrate 114, as shown in Figure 1. By selectively applying
different potentials or by varying the duration of the applied
potential to the electrodes of the array, different amounts of the
electrode can be removed.
[0063] The rate of removal is determined, at least in part, by the
local concentration of other anionic or cationic species around the
electrode. This local concentration is modified by the potential
applied to the electrode. For example, the pH can be altered by
applying a potential, which can then cause the dissolution of the a
portion of the metal electrode. The amount of the metal electrode
that is removed depends, at least in part, on the potential, the
current density through the electrode, and the period of time the
potential is applied or the current is passed. By varying any of
these parameters across the array, the amount of material removed
varies.
[0064] Next, a second metal can be deposited on the electrodes and
the process of applying a potential is repeated. This can continue
for any number of metal deposition steps. Alternatively, the metal
may be selectively deposited by applying a potential that causes
electroreduction of metal cations from a solution. The amount of
metal that is deposited depends on the potential, the current
density through the electrode, and the period of time the potential
is applied. In yet another embodiment, porous films of metal
compounds, such as, for example, metal oxides, can be formed on the
electrode and a portion of the metal compounds can be selectively
removed by applying a potential.
[0065] After all of the metal and/or metal compound depositions are
performed, and an array of electrode structures with different
combinations of metals and metal compounds are formed, the metal
and/or metal compounds can be converted into a desired alloy or
compound by, for example, heating, oxidation, sulfidation, other
chemical reactions, or consolidation of the various layers.
[0066] This method can be useful for the formation of an array of
different non-stoichiometric combinations of materials, each
combination being determined, at least in part, by the particular
potentials and durations applied to the electrode during each
step.
[0067] Yet another means of control involves increasing the local
concentration of an anion with which the metal of the electrode may
react to form a soluble complex. For example, gold is known to
react at mildly oxidizing potentials with dissolved chloride ions.
If the local concentration of chloride ions is increased by
applying a positive potential, then the rate of the dissolution of
the gold will also increase, even if the local pH is unchanged.
[0068] In the hydrolytic reaction and precipitation of solution
phase inorganic or mixed organic-inorganic compounds, such as
halides or alkoxides of Si, Ti, Zr or Al, the nature and reactivity
of the product may be controlled at an electrode of the array
through the local change in pH. The formation of the solution phase
inorganic or mixed organic-inorganic compounds, such as a polymer,
can be controlled by application or non-application of a potential
to each electrode in the array. In addition, the structure of the
compound may, at least in some cases, be dependent on the potential
applied at the electrode. For example, the polymer formed upon
hydrolysis of a silicone precursor, such as methyl trimethoxysilane
depends on the local pH. Ladder-type silsesquioxanes are often
formed at higher pH.
[0069] Exemplary array and usePVP-Os-NH.sub.2: A redox polymer
comprising a poly (4-vinyl pyridinie) backbone here about 0.10 of
the pyridines are complexed with [Os(bpy).sub.2Cl].sup.+/2+; and
about 20% are reacted with 2-bromoethylamine and are thereby
quarternized ( bpy is 2,2'-bipyridine; PEGDGE is poly(ethylene
glycol)diglycidyl ether , molecular weight 400-600; SCE is standard
calomel electrode potential.
[0070] An array of four gold electrodes is produced on a quartz
plate, each electrode spaced a distance of 100 micrometers from the
other, and insulated from eachother. Each gold electrode (2
micrometers in diameter) is connected to a contact pad. A solution
of PVP-OS-NH.sub.2 (10 mg/ml) is incubated with PEGDGE (1 mg/ml)
for two hours at 37.sup.oC, pH7.
[0071] The electrodes are placed into contact with the
PVP-OS-NH.sub.2 / PEGDGE solution, and a potential of -0.6 Volts
(SCE) is applied for 10 minutes to electrophoretically deposit and
crosslink the redox polymer. The solution is replaced with a
solution of oligonucleotides (DNA or RNA) (10 mg/ml), and the
potential is reversed to +0.6 Volts (SCE) for ten minutes. The
array is then placed into a solution of ascorbic acid and sodium
ascorbate (pH 7.5), total concentration 0.5mM. A potential of 0.4
volts (SCE) is then applied to two of the electrodes of the
array.
[0072] A drop of micrococcal nuclease (300,000 U/ml) from
Staphlococcus aureus is added to the array. DNA or RNA is
hydrolized only at those electrodes to which no potential is
applied, and is not hydrolyzed at those electrodes at those
electrodes to which a potential is applied.
[0073] The foregoing description contains numerous references to
publications and patents, each of which is hereby incorporated by
reference, for all purposes, as if fully set forth.
* * * * *