CN1549744A

CN1549744A - Treatment of substrates

Info

Publication number: CN1549744A
Application number: CNA02817030XA
Authority: CN
Inventors: ��ġ��ա�ɳɭ; 爱德文·麦勒·沙森; 赖安·爱戈兰德
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford Gene Technology IP Ltd
Priority date: 2001-08-31
Filing date: 2002-09-02
Publication date: 2004-11-24
Anticipated expiration: 2022-09-02
Also published as: GB0121155D0; EA005836B1; WO2003020415A2; WO2003020415A3; JP4261350B2; EA200400379A1; CN100441283C; AU2002321587A1; JP2005502032A; EP1420879A2; US20040238369A1

Abstract

A method of treating a substrate, which method comprises providing an electrolyte in contact with the substrate and an array of electrodes adjacent the surface and in contact with the electrolyte, and altering the potential of at least one electrode so as to generate an active redox product which modifies the substrate adjacent the at least one electrode, characterised in that the electrolyte is chosen such that the active redox product is quenchable by a second redox product. The method is particularly suitable for the step-wise chemical synthesis of oligomers such as oligonucleotides bound to a surface.

Description

Substrate processing method

[ technical field]to

The present invention relates to a method for treating a substrate using an electrochemical technique, and more particularly, to a method for modifying a substrate using an electrochemical technique.

[ background of the invention]

Many devices require a specific pattern of a material to be formed on their surface, semiconductor chips being an obvious example. Recently, DNA chips have also been provided with an array of oligonucleotides attached to a solid surface [ G.Lamy (G.Ramsay), "Nature Biotechnology", 1998, Vol.16, 40-44].

The performance of such devices depends on the pattern and properties of the surface material. Furthermore, improvements to existing devices are on the one hand due to the need for miniaturization and on the other hand the need for new surfaces that integrate chemical, physical properties. Therefore, there is a real need for new methods to fabricate devices with desired patterns on their surfaces.

There are several methods for treating specific areas of a surface. One method is photolithography. Specific areas of the surface are covered by a photolithographic mask and the exposed areas are trimmed by ultraviolet radiation. This method has been widely used in semiconductor manufacturing, by which holes can be formed in the surface of a semiconductor wafer coated with a photoresist.

Another photolithography technique is also applied to the manufacture of DNA chips. In this method, oligonucleotides with photolabile protecting groups are formed on a solid surface, a photolithographic mask is used to cover certain areas of the surface, and the exposed areas of the surface are then irradiated with UV light. Finally, the oligonucleotides covering the exposed regions are removed [ G.Ramsay (Nature Biotechnology 1998, Vol.16, 40-44)].

WO93/22480 discloses a method for treating a surface by electrochemical techniques. In this method, the electrolyte floats on the surface and the electrode array is proximate to the surface. By varying the potential of one or more electrodes of the electrode array, the surface proximate to the one or more electrodes is modified. The electrolyte is an acetonitrile solution of triethylamine and sulfuric acid.

U.S. patent No. 6,093,302 discloses an electrochemical method for placing materials at specific locations on a substrate. The material is produced at an electrode and then reacted with a substance in close proximity to the electrode. Also, this patent uses a buffering or scavenging solution (a buffering or scavenging solution). The effect of this buffer or decontamination solution is to improve the resolution of the substrate being treated by reaction with the reagents flowing from the adjacent electrodes. A large volume of solution containing a buffer or purification material will inhibit not only the reagent distributed to a particular electrode, but also the reagent used to react with the substrate adjacent to the particular electrode.

Another method for improving resolution using surface electrochemical treatment is disclosed in science 2000, 289, 98-101 by Schuster et al. Schoude utilizes complex current pulses to limit diffusion time.

[ summary of the invention]

It is an object of the present invention to provide an improved method for electrochemically modifying a substrate, in particular to provide a method for modifying a substrate having a higher resolution.

To achieve this object: the present invention provides a method of controlling diffusion of a first redox species generated at a first electrode, comprising: a second redox species is generated at a second electrode adjacent to the first electrode, the first and second electrodes being in contact with an electrolyte, wherein the electrolyte is such that the first redox species is inhibited by the second redox species.

The invention is further improved in that: the second electrode is an auxiliary electrode.

The invention is further improved in that: the first redox species is an active redox species that can be used to condition a substrate adjacent to an electrode.

In addition, the present invention provides a method of treating a substrate comprising providing an electrolyte in contact with the substrate, and one or more electrodes adjacent to the substrate and in contact with the electrolyte; modifying at least one electrode to produce an active redox species that can modify the substrate proximate the at least one electrode; the electrolyte may cause the active redox species to be inhibited by the second redox species.

By inhibited it is meant that the second redox species can react with the first redox species and change its manner of reaction such that the first redox species cannot react as it is. When the first redox species is an active redox species, the reaction between the active redox species and the second redox species will prevent the active redox species from modifying the substrate. For example, when the active redox species is an acid, the second redox species will likely be a base. The reaction between the acid and base inhibits the acid and prevents it from modifying the substrate.

Compared with the prior art, the electrolyte of the invention can inhibit the active redox substance by at least one other redox substance, so that the obtained substrate has high resolution.

[ description of the drawings]

The invention will be described in detail below with reference to the following drawings:

FIG.1 shows an apparatus for carrying out the method of the invention;

FIG.2 shows how selected areas of a substrate are modified;

FIG.3 shows the effect of varying electrolysis time;

fig.4 shows the effect of removing a cathode from the electrode array.

[ detailed description]embodiments

In a preferred embodiment of the invention, the inhibition reaction regenerates one or more species in the electrolyte.

Any oxidation or reduction product will modify the substrate by the active redox species. The active redox species may be generated directly by oxidation or reduction of species in the electrolyte. Or the active redox species may be obtained by oxidation or reduction of species in the electrolyte and then one or more reactions with other species in the electrolyte.

Usually, the redox species is generated on the surface of the electrode. The redox species will modify its adjacent substrate. Acids are the preferred active redox species that undergo a number of reactions on the substrate, such as eliminations (eliminations), displacement, reformation, and chemical etching. When the active redox species is an acid, it is preferred that it is used to remove an acid labile protecting group (acid protecting group) on the substrate.

Acid labile protecting groups are known to those skilled in the art and include acetals (such as methoxymethyl, dimethyl sulfide, (2-methoxyethoxy) methyl, methyl benzoate, β (trimethylsilyl) ethoxymethyl, tetrahydropyranyl, benzylidene, isopropylidene, cyclohexylidene, and cyclopentylidene), esters (such as benzoyl, benzoicacid carbonyl, and t-butoxycarbonyl), ethers (such as trityl, dimethoxytrityl, and t-butyl), and silyl ethers (such as t-butyldimethylsilyl, trimethylsilyl, and triethylsilyl).

Likewise, the active redox species can be a base that undergoes a number of reactions on the substrate. For example, for the removal of base labile protecting groups.

Base labile protecting groups are well known to those skilled in the art and include 9-fluorenylmethoxycarbonyl (Fmoc) and Cyanoethyl (Cyanoethyl).

Free Radicals (radials) are another active redox species. The radicals are used to initiate radical reactions at the substrate. Electrochemical methods for generating free radicals are known to those skilled in the art. A commonly used electrochemical method for generating free radicals is carboxylate anion oxidation.

Halogen is another active redox species, which is used, for example, to perform oxidation or addition reactions on a substrate. Halogens are obtained by oxidation of the corresponding halide ions.

These and other active redox species will be apparent to those skilled in the art.

The method of the present invention can be used to treat a substrate. The substrate of the present invention may be employed with any material or substance that is associated with an electrode and that can be conditioned by an active redox species. The substrate is positioned adjacent to the electrode and is removed from the electrode after the redox reaction is completed. Alternatively, the substrate may be attached to the electrodes or attached to the surface thereof as the electrodes. If desired, after the redox reaction is complete, the substrate may be removed from the electrode or the surface.

Thus, in one embodiment, the substrate is a surface of a material that is separate from but proximate to the electrodes. The substrate may be a surface of glass, plastic, solid fiber, metal, semiconductor, or gel material. The surface of these materials can be directly conditioned by redox reactions. In addition, in this embodiment, other substances may be attached to the surfaces of these materials. For example, it is known to attach organic compounds to the surface of these materials. Substances attached to the surface of these materials can be conditioned by redox reactions.

In another embodiment, the substrate may be a substance that is attached to a surface that acts as an electrode. Or the substrate is a substance that is attached to the electrode itself via a linking group. U.S. Pat. No. 6,093,302 discloses the latter method, in which a substrate is attached to an electrode via a linker.

The process of the present invention is similar to that disclosed in WO 93/22480. However, the method of the present invention selects an electrolyte different therefrom. The electrolyte used in WO93/2240 is a solution of triethylamine in sulfuric acid in acetonitrile. The active redox species may be inhibited in the electrolyte of the present invention by at least one other redox species. The electrolyte may accurately confine the active redox species to the surrounding area of the electrode where the active redox species is generated.

In the method described in WO93/22480, the confinement of the acid at a specific region is controlled by variation of the electrode potential. However, the inventors of the present invention found that after a long time of electrolysis, when the electrolyte was a solution of triethylamine and sulfuric acid in acetonitrile, the acid was out of limits. Failure of the acid to be effectively confined will result in reduced resolution of the treated substrate. For example, protons diffusing from the vicinity of the anode may react with the substrate region between the electrodes. To obtain a substrate with a high resolution pattern, it is undesirable for the diffusing protons to react accidentally in this way. The problems of the prior art electrolytes can be avoided according to the selected electrolyte of the present invention. The electrolyte of the present invention allows the active redox species to be inhibited by at least one other redox species, which is an important feature of the present invention.

Those skilled in the art are aware of many electrolytes that can produce an active redox species that is inhibited by another redox species.

For example, an electrolyte, which is I^-And S₄O₆ ^2-A conjugate of (1). Iodide is oxidized at the anode to produce iodine, an active redox species, and at S₄O₆ ^2-Reduced at the cathode to produce S₂O₃ ^2-It can inhibit iodine generation at the anode. The reaction in the electrolyte can be represented as follows:

anode:

cathode:

iodine is inhibited by the following reaction:

the active redox species is preferably an acid, and the redox-inhibiting species is an anion, preferably an organic anion (radius). Typically, the acid is generated by anodic oxidation of an alcohol, which may be an aliphatic alcohol, or an aromatic alcohol. In such electrolytes, the inhibiting anions are generally generated by reduction of a suitable substance at the cathode. Many substances can be reduced at the cathode to produce anions and can inhibit acid formation at the anode. For example, dissolved oxygen molecules may be reduced at the cathode, thereby producing O₂ ^-And/or O₂ ^2-。

For example, an electrolyte that produces a suitable redox species that is a combination of a ketone and the corresponding alcohol. The alcohol oxidizes at the anode to produce protons (an active redox species), while the ketone reduces at the cathode to produce negative ions, which inhibit proton production at the anode.

The reaction in the electrolyte can be represented as follows:

anode:

cathode:

wherein R is¹And R²Respectively optionally substituted C₁To C₁₅A hydrocarbon group in which three or more carbon atoms may be optionally substituted with N, O and/or S atoms; or R¹And R²Together form a substituted C₁To C₁₅Cycloalkylene (cyclohydrocarbylene) in which three or more carbon atoms may optionally be substituted with N, O and/or S atoms.

Preferably, R¹And R²Respectively optionally substituted C_1-8Alkyl radical, C_3-8Cycloalkyl or phenyl.

"hydrocarbyl" in the context of the present invention refers to a monovalent group containing carbon and hydrogen. Hydrocarbyl thus includes alkyl, alkenyl and alkynyl groups (in both straight and branched chain configurations), cycloalkyl (including polycycloalkyl), cycloalkene and aryl groups, and combinations of the above, such as alkylcycloalkyl, alkylpolycycloalkyl, alkylaryl, alkenylaryl, alkynylaryl, cycloalkylaryl and cycloalkenylaryl groups.

The term "hydrocarbylene" as used herein refers to a divalent group containing carbon and hydrogen. Cycloalkylene groups here include cycloalkylene or cycloalkylene groups, cycloalkylene groups (cycloalkylenene) and arylene or arylene groups.

The "aryl group" of the present invention means an aromatic group, for example, a phenyl group, a naphthyl group or an anthracenyl group. Or when the aryl group has a carbon atom substituted with O, N and/or S, the aryl group refers to an aromatic heterocyclic group such as pyridyl, pyrrolyl, thienyl, furyl, imidazolyl, triazolyl, quinolyl, isoquinolyl, oxazolyl or isoxazolyl.

The substituents of the present invention may be selected from C₁To C₆Alkyl of (C)₁To C₆Alkoxy, thio, C₁To C₆Thioalkyl, carboxyl (C)₁To C₆) Alkyl, formate, C₁To C₆Alkylcarbonyl of C₁To C₆Alkylcarbonylalkoxy, nitro, trihalomethane, hydroxy, C₁To C₆Hydroxyalkyl, hydroxy (C)₁To C₆) Alkyl, amino, alkylamino of C1 to C6, di (C)₁To C₆) Amino, aminocarboxy, C₁To C₆Alkylamino carboxyl group of (1), di (C)₁To C₆Alkyl) diamino carboxyl, aminocarboxy (C)₁To C₆) Alkyl radical, C₁To C₆Alkylamino carboxyl group (C) of₁To C₆) Alkyl, di (C)₁To C₆Alkyl) aminocarboxy (C)₁To C₆) Alkyl radical, C₁To C₆Alkyl carbonylamines of (C)₁To C₆Cycloalkyl of, C₁To C₆Cycloalkyl (C)₁To C₆) Alkyl radical, C₁To C₆Alkylcarbonyl (C)₁To C₆Alkyl) amino, halogen-containing, C₁To C₆Haloalkyl, sulfamoyl, tetrazolyl and cyano.

"halogen-containing" or "halogen" in the context of the present invention means iodine, bromine, chlorine or fluorine.

The nature of R1 and R2 determines the redox characteristics of the electrolyte. For example, R¹And R²OnSubstitutional reduction may change potential through oxidation or reduction.

The ketone/alcohol electrolyte is preferably an organic solution of 2-propanone/isopropanol and benzophenone/benzhydrol.

Another electrolyte is benzoquinone/hydroquinone and derivatives thereof. Such an electrolyte may be a mixture of:

and

wherein R3, R4,R5 and R6 are respectively selectable: hydrogen, halogen, nitro, hydroxyl, thio, amino, substituted C₁To C₁₅A hydrocarbon group in which three or more carbon atoms may be replaced by N, O and/or an S atom. Or R3 and R4, and/or R5 and R6 combine to form a substituted C₁To C₁₅A cycloalkylene group in which three or more carbon atoms may be replaced by N, O and/or an S atom.

R3, R4, R5 and R6 are preferably hydrogen, C_1-8Or R3/R4 and R5/R6 combine to form a substituted C_5-12Arylene groups (arylene), such as phenylene.

The nature of R3, R4, R5 and R6 may determine the redox properties of the electrolyte, for example, oxidation or reduction may occur to alter the precise potential. The electrolyte of the benzoquinone/hydroquinone derivative is preferably an organic solution of anthraquinone/anthracene-diol and tetramethyl p-benzoquinone/tetramethyl hydroquinone.

In a preferred embodiment, the electrolyte comprises an acetonitrile mixture of benzoquinone and hydroquinone. The mixture provides an active redox species, which is a hydrogen ion. The hydrogen ion (proton) can inhibit benzoquinone anion.

Hydroquinone is oxidized at the anode to produce benzoquinone and protons.

The protons released by oxidation of the hydroquinone stay mostly at the anode, which can modify its adjacent substrate. For example, the proton may deprotect (deprotect) a substrate bearing an acid labile protecting group.

Benzoquinone is reduced at the cathode to produce benzoquinone anions:

the benzoquinone anion is stable in solution, for example acetonitrile. The negative ions suppress any protons escaping from the vicinity of the anode, and the reaction can be expressed as follows:

thus, by allowing the active redox species generated by the electrode to reside in a region on the substrate where, for example, protons are generated at the anode, the resolution of that region can be improved.

The electrolyte of the present invention includes any suitable solvent, such as water, Tetrahydrofuran (THF), methanol, ethanol, Dimethylformamide (DMF), dichloromethane (dichloromethane), diethyl ether, Dimethylsulfoxide (DMSO), or acetonitrile. One skilled in the art will recognize that the choice of solvent can affect the equilibrium or kinetics of the redox and/or inhibition reactions at the electrode. The solvent may affect the activity of certain structures in solution, for example, blending structures, hydrogen bonding, dipole-dipole, or "delocalization" of charge. The solvent is preferably an aprotic solvent (aproticsolvent) which stabilizesthe negative ions. The aprotic solvents are: dichloromethane, dimethylformamide, dimethylsulfoxide, acetonitrile and tetrahydrofuran. Acetonitrile is a more preferred solvent.

In a preferred embodiment, the electrolyte may additionally include a conductivity enhancer (conductivity) for improving the conductivity of the electrolyte. The electrolysis voltage required to add the conductive reinforcement is lower than without the conductive reinforcement. Any electrolyte-soluble ion can achieve this. For example, when the electrolyte includes an organic solvent, such as acetonitrile, the conductivity enhancer is tetra (C)_1-8Alkyl) ammonium salts, for example ammonium hexafluorophosphate (tetrabutylammonium hexafluorophosphate).

The skilled person is aware that salts have their effect in the electrolyte, rather than merely increasing the conductivity of the electrolyte. The salt may affect the equilibrium or kinetics of the inhibition reaction and/or the redox reaction at the electrode. Salts can affect electrostatic interaction forces of charged species in solution and correspondingly can also affect reaction kinetics. For example, when the electrolyte is hydroquinone/benzoquinone in acetonitrile, the addition of ammonium hexafluorophosphate can alter the degree of inhibition of the reaction and also increase the conductivity.

The method of the invention is implemented by the apparatus disclosed in WO 93/22480. The device disclosed in WO93/22480 comprises an array of electrodes spaced apart on an insulating surface. The electrodes are plated with platinum for providing electrical connections (electrical connections) for changing their electrical potential.

However, the electrode used in the method of the present invention is preferably an iridium electrode. The invention provides an electrode array comprising a block of insulating materialhaving a surface on which are formed spaced apart deposits of iridium, each deposit being provided as electrical connection means for varying its potential.

Iridium has the advantage of being highly conductive and chemically inert. Furthermore, iridium does not degrade at the high potentials of the method of the present invention. Platinum has been widely used as an electrode material, however platinum does not adhere well to some materials, such as silicon wafers, especially at high electrode potentials. The internal inhibition requires longer use of high electrode potentials without a decrease in resolution of the substrate being treated, which requires changes to existing electrode designs.

Many metals were tested as suitable electrodes, including aluminum, silver, and gold. However, the inventors of the present invention have surprisingly found that iridium is a good material for electrodes. Iridium does not degrade in the electrolyte and adheres well to materials such as oxidized silicon wafer materials.

One piece of material on the electrode array can be an insoluble polymer, a ceramic oxide (such as alumina) or a silicon oxide wafer. Preferably an oxidized silicon wafer.

Iridium electrode arrays may be made by a number of suitable methods. In a preferred embodiment, the electrode array is obtainable by:

(i) providing a silicon wafer, the surface of which has a silicon dioxide layer;

(ii) depositing iridium on the silicon dioxide layer at intervals to form an array;

(iii) annealing the iridium in the air at the temperature of 200-500 ℃.

In a typical process, a positive organic photoresist is applied to a silicon dioxide layer of a silicon wafer. The photomask is covered and then exposed to ultraviolet light to remove the exposed photoresist, exposing the silicon dioxide regions where the photoresist was removed. Iridium metal is deposited on the exposed silicon dioxide region with an electron beam gun. Removing the regions of the photoresist layer may form an array of electrodes. Finally, the iridium electrode is annealed in air to improve adhesion at the wafer surface. Typically, the iridium is annealed at 350 ℃ for 15 minutes to 3 hours, preferably about 1 hour.

The annealing step is important for iridium to adhere to silicon dioxide. The annealing temperature is about 350 c and the iridium may have a melting temperature of 2545 c. Even annealed electrodes with a 50nm iridium layer were found to be scratch resistant to steel scalpels. Furthermore, iridium electrodes are resistant to harsh chemical environments, and the high potentials and high currents of the methods of the present invention.

Preferably, the electrodes are an array of parallel lines spaced from one another by less than 0.5 mm. Preferably, the electrode spacing is 0.1 to 200 microns, more preferably 1 to 100 microns, and even more preferably 10 to 60 microns.

One or more of the electrodes serve as an auxiliary electrode (counter electrode). Preferably, the substrate to be treated of the present invention does not form an electrode or an auxiliary electrode, as opposed to existing methods of electrochemically treating substrates. The process of the invention is similar to that described in WO 93/22480. Furthermore, the substrate to be treated may be an insulating surface.

In a preferred embodiment, the invention provides a method of carrying out several process steps in sequence. One processing step may be accomplished by connecting the electrodes of the array such that the potential of a selected electrode or electrodes of the array is changed.

In the method of the present invention, the substrate to be treated comprises a substance attached to a solid surface. The solid surface may be proximate to an electrode. Preferably, the solid surfaces are non-identical surfaces adjacent to the electrodes. Redox species can be used to modify species attached to a solid surface. Many redox species and corresponding chemical modifications may be envisaged by the person skilled in the art. In a preferred embodiment, the substrate to be treated comprises a material having an acid labile protecting group. In the preferred embodiment, the treatment step is accomplished by connecting at least one electrode in the array to a power source and using it as an anode for generating acid in the electrolyte. The generated acid can remove acid labile protecting groups attached to the surface and near the anode region.

Active redox species have been recognized to participate in a variety of chemical reactions at a substrate. One potential application is the electrochemical micromechanical technology disclosed in science, 2000, 289, 98-101, by Schuster et al. The scold tool may be used in the electrolyte of the present invention with an auxiliary electrode ring surrounding the probe tip to prevent diffusion of the redox species. The present invention will use existing nano-scale patterning techniques. For example, acids may be used for etching or nano-fabrication processes, which may remove small amounts of material from a surface.

In addition, the acid may participate in a number of organic or inorganic reactions. One skilled in the art will recognize many potential reactions that may be used in the present invention. For example, organic reactions include ring opening of epoxy compounds, attachment to multiple bonds, rearrangement (rearrangement), substitution (e.g., unimolecular nucleophilic substitution of tertiary alcohols) (S)_N1) Elimination, production of enols and simple protonation of organic acid salts.

When the active redox species is a halogen, it may participate in mild oxidation, bleaching of the substrate or halogenation. The active redox species may also be a halide ion, which may be used for the substitution reaction.

The method of the present invention can also be used for the synthesis of low organic compounds attached to a surface [ see schreiber (science, 2000, 287, 1964-1969)]. Low organic compounds are important in the field of pharmaceutical invention. The reaction range used in the present invention is meant to be theoretically suitable for the synthesis of such substances.

The methods of the invention are useful for the stepwise synthesis of oligomers, such as oligonucleotides, polysaccharides, and proteins. Preferably, the method of the invention is useful for the synthesis of nucleotides.

A method of synthesizing a set of oligomers comprising the steps of:

(a) providing a substrate with an array of materials having protecting groups, an electrolyte in contact with the substrate and an electrode array adjacent to the substrate and in contact with the electrolyte;

(b) selectively altering the potential of one or more of the electrodes to produce an active redox species which removes the protecting group from the selected species;

(c) incorporating a protecting monomer into the deprotecting species formed in step (b);

(d) repeating steps (b) and (c) and altering the one or more electrodes selected in step (b) to synthesize a set of oligomers;

characterised in that the electrolyte is such that the active redox species is inhibited by at least one other redox species.

When the above method is used for oligonucleotide synthesis, the active redox species is preferably a proton and the protecting group is preferably an acid labile protecting group such as trityl or dimethoxytrityl, which protects the furanhydroxy group. The skilled person will appreciate that the method is particularly suitable for combinatorial synthesis of DNA chips, as described in WO 93/22480.

The above method is also applicable to the synthesis of peptides. For example, peptides can be synthesized by successively removing a Boc (t-butyloxycarbonyl) protecting group from a nitrogen atom with an anode-generated proton. The synthesis of other oligomers will be apparent to those skilled in the art.

Referring to FIG.1, the electrode array is located on an oxidized high resistivity silicon wafer 1 having a layer of iridium metal deposited on its upper surface. The spacers 2 are formed by photolithographic techniques on an iridium metal layer on a silicon wafer so that an array of parallel electrodes is formed. The width of each electrode and the width of each space 2 are about 40 microns. Another silicon wafer 4 is arranged above the electrode array. The surface of the silicon wafer 4 was trimmed to remove dimethoxytrityl protected nucleosides.

Referring to FIG.2, an electrode array and a portion of a substrate thereof are shown. The middle electrode is the anode and the other two electrodes are the cathodes. An electrolyte comprising a solution of benzoquinone and hydroquinone in acetonitrile is contacted with the electrode and the substrate to be treated. At the anode, hydroquinone is oxidized to produce benzoquinone and protons. Most of the protons are confined to the region of the substrate near the anode. The restricted proton removes the dimethoxytrityl group from the protected nucleoside fragment (nucleotide moiety) attached to the substrate. However, some protons may diffuse into the region between the anode and the cathode.

On the cathode, the benzoquinone is reduced to produce benzoquinone anions. The benzoquinone negative ions are relatively stable and can diffuse into the region between the anode and the cathode. The benzoquinone anion suppresses the proton diffusion into this region, thereby producing hydroquinone and benzoquinone. Thus, protons diffusing in the substrate area not close to the anode can be prevented from reacting. By preventing the proton from accidentally reacting in the region between the electrodes, the resolution of the substrate can be improved.

Figure 2 also shows the subsequent substrate processing. The free hydroxyl group is acetyl added under standard conditions and the remainder of the dimethoxytrityl group is removed. The resulting free hydroxyl groups were treated with a fluorescent dye (Cy5 phosphoramidite) which allowed the imaging of the substrate to be observed by confocal microscopy. Thus, the resolution of the initial detritylation step can be easily observed. It is clear that oligomers can be synthesized in selected regions of the substrate using the techniques described above.

Referring to FIG.3, the effect of varying electrolysis time at a fixed potential of 1.33V is shown using confocal microscopy. In this figure, the bright area is the fluorescent substrate area where the dimethoxytrityl group is not removed during electrolysis. The remaining dimethoxytrityl group was then replaced by fluorescent Cy5 dye. The bright area is typically near the cathode. In the black region, the dimethoxytrityl group has been removed during the electrolysis. The free hydroxyl groups generated were bound to non-fluorescent acetyl groups. The black region is typically near the anode.

Figure 3 shows that after 2.0 seconds the dimethoxytrityl group was completely removed in the area close to the anode. Furthermore, the resolution of the substrate did not change after 80 seconds. There are limited striations corresponding to the areas near the anode and cathode. These demonstrate that the protons produced during electrolysis are strictly confined to the region close to the anode, even after prolonged electrolysis times.

Fig.4(a) and (b) show the clear effect of removing the cathode from the electrode array. The black area shows the area where the dimethoxytrityl group was removed, the electrode potential was set at 1.33V, and when the intermediate cathode was removed, protons generated at the anode could freely diffuse into the intermediate area, clearly demonstrating that the species generated at the cathode had a limiting effect, and could suppress protons generated at the anode.

Experimental part

Electrode assembly

Current photolithographic techniques have been widely used to produce iridium metal (50 nm thick) electrodes on oxidized high conductivity silicon wafers. The oxidized silicon wafer is coated with a positive photoresist and then exposed to ultraviolet light through a photomask. The wafer was washed with deionized water, baked at 100 ℃ for 20 minutes, and scum was removed by ion etching. The photomask produced an array of 96 parallel electrodes, approximately 7500 microns long and 40 microns wide. The spacing between adjacent electrodes is about 40 microns.

Iridium is deposited on the wafer by electron beam methods. Iridium metal may be placed in the crucible of the vacuum evaporator with two or three wafers placed approximately 20 centimeters near the crucible of the vacuum evaporator. The chamber of the vacuum evaporator is pumped to 3X 10^-6Torr, 50nm of iridium metal was coated on the wafer by heating the iridium metal for about 3 minutes with a 300mA, 5kV electron beam gun.

The photoresist can be removed and the electrode array visualized by placing the wafer in ultrasonic acetone for 30 minutes. The electrode was placed in air, annealed at 350 ℃ for 1 hour to improve adhesion to the wafer substrate, and then cleaned by ion etching.

After the annealing and cleaning steps, each electrode is individually connected to an ultrasonic gold wire, which is connected to a printed circuit board, and a "logic switch" (analog switch) integrated with the circuit activates the selected electrode to provide the unlatching step. The current is applied as a plurality of individually operating amplifier control voltage sources. A parallel low noise amplifier feedback circuit continuously measures the nanoamp level current at each electrode.

Solid support assembly

The polished silicon dioxide wafer can be used asAnd (4) supporting the substrate. Prior to patterning, the wafer surface is functionalized with linker molecules to attach organic agents to the linker molecules [ Gray d.e., ksaglin s.c., fel t.s., dobeson p.j. and samson E.M. (Gray, d.e., CaseGreen, s.c., Fell, t.s., Dobson, P.J).&Southern, E.M.), features of ellipsometry and interferometry for DNA probes immobilized on combinatorial arrays, Languir13, 2833-2842 (1997)]. The wafer was placed in a chamber of a vacuum furnace having a capacity of 19.1 liters and further comprising an ampoule (ampoule) containing 5ml of GPTS (glycidoxypropyl method oxysiline). After the furnace is heated to 185 ℃, the ampoule is heated to 205 ℃ and the chamber is pumped to 25-30 mBar. After about 2.5ml of silane has been evaporated, in vacuo (10)^-3Torr) the lower chamber can be cooled. The "linker" can be attached by immersing the wafer with GPTS in a solution of polyethylene glycol containing a small amount of sulfuric acid. Dimethoxytrityl groups containing phosphoramidite (phosphoramidite) were covalently attached to the hydroxyl group of polyethylene glycol by existing oligonucleotide synthesis techniques [ birkejie s.l. and eiya R.P. (Beaucage, s.l.).&Iyer R.P), improvement in oligonucleotide synthesis by the phosphoramidite method, Tetrahedron 48, 2223-2311 (1992)]. The wafer substrate was then cut to 1 cm x1 cm for use in patterning.

Example two

The electrode array prepared above was placed 20 microns from the solid support. The solid support is prepared by the above procedure, with thymidine phosphoramidite (thymidylphosphamide) attached to a polyethylene glycol linker molecule. Thymidine phosphoramidite has a 5' -hydroxyl group, which is protected by a dimethoxytrityl group.

An electrolytic solution (25mM hydroquinone/25 mM quinone/25 mM ammonium hexafluorophosphate in dry acetonitrile) was injected into the cavity between the electrode array and the solid support. The selected anode was set at 1.33V and the voltage was held for 0.2 to 0.8 seconds (as shown in fig. 3).

After electrolysis, the silicon wafer was washed with acetonitrile and bound with acetyl groups using acetic anhydride in a standard manner. In this step, only the dimethoxytrityl deprotection area of the silicon wafer is added with acetyl groups.

Dimethoxytrityl groups not removed in the electrochemical step were removed when the entire substrate was treated with a dichloromethane solution of dichloroacetic acid. The exposed hydroxyl group was bound to Cy5 (fluorescent dye) by a standard phosphoramidite binding method, and a pattern generated by electrochemical generation of an acid was revealed, which was observed by observing fluorescence of Cy5 with a confocal microscope. The sequence of steps is shown in figure 2.

FIG.3 shows the effect of extending the electrolysis time at 1.33V. The maximum bandwidth is reached after about 2.0 seconds, the resolution of the substrate is stable, and thereafter the resolution does not change even after 80 seconds of electrolysis.

Example two

Example two is essentially the same as example one except that the selected anode was held at a voltage of 1.33V for 16 seconds. As shown in fig.4(a) and (b), the effect of removing the cathode was observed. When the central cathode is removed, the diffusing protons lose control. The diffused protons may flow into the central region without staying around the anode. In FIG.4(b), the middle black region is evident, which no longer contains fluorophores.

EXAMPLE III

Example one of the methods described was used to synthesize 17-mer oligomers on a solid support, which included 16 dimethoxytrityl deprotection steps. The method used in this example is essentially the same as in the first example, except that the electrode array is placed 40 microns from the substrate surface.

A uniform coating of dimethoxytrityl protected deoxyadenosine residue (residual) was attached to polyethylene glycol linker groups on the solid support using standard phosphoramidite binding methods.

After extensive washing with acetonitrile, the electrolyte of example one was injected into the cavity between the electrode array and the solid support. A voltage of 1.33V was applied to the selected anode for 9 seconds to remove the dimethoxytrityl group near the selected anode. The anode is positioned between two cathodes.

After further washing with acetonitrile, the dimethoxytrityl protected nucleoside residue was grafted with an exposed hydroxyl group using standard phosphoramidite conjugation methods. Trivalent phosphorus bonds (phosphorus bonds) are oxidized with iodine, thereby generating pentavalent phosphorus bonds. The entire silicon wafer was washed with acetonitrile and then with dichloromethane. The ligation and oxidation steps used in the synthesis of oligonucleotides on solid supports are prior art (see, part II of the AbI synthesis handbook, chemical methods for automated synthesis of DNA).

The process was repeated to change the dimethoxytrityl protected nucleoside residue introduced during the phosphoramidite conjugation step. Thus, the oligonucleotides are synthesized on a solid support.

This process can be carried out by an automated apparatus, controlled by a computer, which comprises, in the synthesis of the oligonucleotides of the two 17-mers: wild type (wild type) "A" human hemoglobin mRNA (ribonucleic acid) and the corresponding "S" type sickle cell mutant mRNA. High yields can be obtained by growing the 17-mer on a solid supported confining strip (strip). The combinatorial synthesis of such DNA chips is well known to those skilled in the art, for example, as disclosed in WO 93/22480.

While the invention has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Other variations of the disclosed embodiments may be contemplated by those skilled in the art in view of the description of the present invention. Accordingly, such modifications do not depart from the scope and spirit as defined by the appended claims.

Claims

1. A method of controlling diffusion of a first redox species generated at a first electrode, the method comprising: the method comprises the following steps: a second redox species is generated at a second electrode adjacent the first electrode, the first and second electrodes being in contact with an electrolyte, wherein the electrolyte is such that the first redox species is inhibited by the second redox species.

2. The method of claim 1, wherein: the second electrode is an auxiliary electrode.

3. The method according to claim 1 or 2, characterized in that: the first redox species is an active redox species.

4. The method of claim 3, wherein: the active redox species is used to condition the substrate near the electrode.

5. A method of treating a substrate, the method comprising: providing an electrolyte in contact with the substrate, and one or more electrodes adjacent to the substrate and in contact with the electrolyte; modifying at least one electrode to produce an active redox species that tailors a substrate proximate the at least one electrode; characterised in that the electrolyte is such that the active redox species is inhibited by a second redox species.

6. The method of claim 3, 4 or 5, wherein: the active redox species is a proton.

7. The method of any preceding claim, wherein: the second redox species is an organic anion (chemical anion).

8. The method of any preceding claim, wherein: the electrolyte is a solution of hydroquinone and benzoquinone, or a derivative thereof.

9. The method of any preceding claim, wherein: the electrolyte is a solution of the following compounds;

and

whereinR3, R4, R5 and R6 are respectively selectable: hydrogen, halogen, nitro, hydroxyl, thio, amino, substituted C₁To C₁₅A hydrocarbyl group in which three or more carbon atoms may be replaced by N, O and/or S atoms; or R3 and R4, and/or R5 and R6 combine to form substituted C₁To C₁₅Cycloalkylene (Cyclohydrocarbylene) in which more than three carbon atoms may be replaced by N, O and/or S atoms.

10. The method of any preceding claim, wherein: the electrolyte is acetonitrile solution of hydroquinone and benzoquinone.

11. The method of any preceding claim, wherein: the electrolyte further includes a conductivity enhancer.

12. The method of claim 7, wherein: the conductivity enhancer is four (C)_1-8Alkyl) ammonium salts.

13. The method according to any one of claims 5 to 12, wherein: one or more of the electrodes are used as auxiliary electrodes, and the substrate to be finished does not constitute an electrode or an auxiliary electrode.

14. The method of any preceding claim, wherein: to achieve several process steps in sequence, the electrodes of the array are connected such that varying the potential of a selected electrode or electrodes of the array completes a process step.

15. The method of any preceding claim, wherein: the substrate includes an array of substances attached to a surface thereof.

16. The method of claim 15, wherein: the surface is an oxidized silicon wafer surface.

17. The method of claim 15, wherein: the substance to be treated comprises an acid labile protecting group (acid labile protecting group).

18. The method of claim 17, wherein: one treatment can be achieved by applying an electrical potential to at least one electrode in the array and acting as an anode to remove acid labile protecting groups of material at the surface.

19. The method according to any one of claims 14 to 18, wherein: during the stepwise chemical synthesis of the oligomer, the treatment is effected.

20. A method of synthesizing a set of oligomers comprising the steps of:

(a) providing a substrate with an array of materials having protecting groups, an electrolyte in contact with the substrate, and an electrode array adjacent to the substrate and in contact with the electrolyte;

21. The method of claim 20, wherein: an array of substances is attached to a surface where the oligomers are synthesized.

22. The method according to claim 20 or 21, characterized in that: the oligomer is an oligonucleotide.

23. The method of any one of claims 20 to 22, wherein: the active redox species is a proton and the protecting group is an acid labile protecting group.

24. The method of any one of claims 20 to 22, wherein: the electrolyte is as defined in claims 8 to 12.

25. The method of any one of claims 20 to 24, wherein: one or more of the electrodes of the array are auxiliary electrodes.

26. An electrode array comprising a block of insulating material having a surface on which are formed spaced apart deposits of iridium, each deposit being provided as electrical connection means to vary its potential.

27. The array of claim 26, wherein: the block of insulating material is an oxidized silicon wafer.

28. An array according to claim 26 or 27, wherein: the deposits of iridium are in the form of parallel lines spaced apart from one another.

29. A method of manufacturing an array according to any one of claims 26 to 28, comprising the steps of:

(iii)annealing the iridium in the air at the temperature of 200-500 ℃.