CN1549744A - A base treatment method - Google Patents

Abstract

A method for treating a substrate includes: providing an electrolyte in contact with the substrate, and an array of one or more electrodes adjacent to the substrate and in contact with the electrolyte; altering at least one electrode to generate an active redox substance that modifies the substrate adjacent to the at least one electrode; and the electrolyte being capable of inhibiting the active redox substance by a second redox substance. The method of the present invention is particularly suitable for the stepwise chemical synthesis of oligomers, such as low nucleotides.

Description

A base treatment method

【Technical field】

The invention relates to a method for treating a substrate with an electrochemical technique, in particular to a method for modifying a substrate with an electrochemical technique.

【Background technique】

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

The performance of such devices depends on the pattern and nature of the surface material. In addition, the improvement of existing devices is due to the need for miniaturization on the one hand, and the need for new surfaces with integrated chemical and physical properties on the other hand. Therefore, new methods are needed to fabricate devices with desired patterns on their surfaces.

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

Another photolithographic technique has also been applied to the fabrication of DNA chips. In this method, oligonucleotides with photolabile protecting groups are formed on a solid surface, a photolithographic mask covers certain areas of the surface, and the exposed areas of the surface are then irradiated with ultraviolet light. Finally, the oligonucleotides covering the exposed areas are removed [G. Ramsay, Nature Biotechnology, 1998, Vol. 16, 40-44].

WO93/22480 discloses a method for surface treatment using electrochemical techniques. In this method, the electrolyte floats on the surface and the electrode array is attached 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 a solution of triethylamine and sulfuric acid in acetonitrile.

US 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 reacts with substances near the electrode. At the same time, the patent applies a buffering or scavenging solution. The purpose of this buffer or purge solution is to improve the resolution of the substrate being treated by reacting with reagents flowing past adjacent electrodes. A solution containing a large amount of buffering or purifying substance will not only inhibit the distribution of reagents to a particular electrode, but also inhibit the reagents used to react with the substrate adjacent to a particular electrode.

In Science, 2000, 289, 98-101, Schuster et al. disclosed another method for improving resolution by using surface electrochemical treatment technology. Skoder used complex electric current pulses to limit the diffusion time.

【Content of invention】

The object of the present invention is to provide an improved method for trimming a substrate by electrochemical means, especially to provide a method for trimming a substrate with higher resolution.

To achieve this purpose: the present invention provides a method for controlling the diffusion of a first redox species generated at a first electrode, which includes: generating a second redox species at a second electrode close to the first electrode, the first and second The electrodes are in contact with an electrolyte that allows the first redox species to be inhibited by the second redox species.

A further improvement of the present invention is that: the second electrode is an auxiliary electrode.

A further improvement of the present invention is that the first redox species is an active redox species that can be used to modify the substrate adjacent to the electrode.

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

Inhibiting means that the second redox species can react with the first redox species and change its reaction mode so that the first redox species cannot react in the original way. 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 conditioning 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 the base inhibits the acid and prevents it from conditioning the substrate.

Compared with the prior art, the electrolyte solution of the present invention can make the active redox species be suppressed by at least one other redox species, so that the resulting substrate has a high resolution.

【Description of drawings】

The present invention will be described in detail below in conjunction with accompanying drawing:

Fig. 1 shows the equipment used for realizing the method of the present invention;

Figure 2 shows how selected areas of the substrate are trimmed;

Figure 3 shows the effect of changing the electrolysis time;

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

【Detailed ways】

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

By activating redox species, any oxidation or reduction products will modify the substrate. The active redox species can be produced directly by oxidizing or reducing species in the electrolyte. Or the active redox species is obtained by oxidizing or reducing substances in the electrolyte, and then reacting with other substances in the electrolyte in one or more ways.

Usually, the redox species are generated on the electrode surface. Redox species modify their adjacent substrates. Acids are preferred reactive redox species that undergo a variety of reactions on substrates, such as eliminations, displacements, reforming, and chemical etching. When the active redox species is an acid, it is preferably used to remove acid labile protecting groups on the substrate.

Acid-labile protecting groups are well known to those skilled in the art, and include acetals (such as methoxymethyl, dimethyl sulfide, (2-methoxyethoxy) methyl, methyl benzoate ester, β(trimethylsilyl)ethoxymethyl, tetrahydropyranyl, benzylidene, isopropylidene, cyclohexylene and cyclopentylene), esters (such as benzoyl, benzoic acid carbonyl and tert-butoxycarbonyl), ethers (such as trityl, dimethoxytrityl, and tert-butyl), and silyl ethers (such as tert-butyldimethylsilyl, trimethyl base silicon and triethyl silicon). The acid labile protecting group is preferably trityl ether or dimethoxytrityl (DMT) ether, which are commonly used in the synthesis of oligonucleotides.

Likewise, the active redox species can be a base that undergoes a variety of reactions on the substrate. For example, for 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 (Radicals) are another active redox species. Free radicals are used to initiate free 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 the oxidation of carboxylate anions.

Halogen is another active redox species, eg, used to carry out oxidation reactions or addition reactions on substrates. Halogen can be obtained by oxidation of the corresponding halide ion.

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

The methods of the present invention can be used to treat substrates. The substrate of the present invention can employ any material or substance that is attached to the electrodes and that can be modified by active redox species. The substrate is placed close to the electrodes and can be removed from the electrodes after the redox reaction is complete. Alternatively, the substrate may be attached to the electrode, or be attached thereon as the surface of the electrode. If desired, the substrate can be removed from the electrode or the aforementioned surface after completion of the redox reaction.

Thus, in one embodiment, the substrate is the surface of a material that is separate from, but adjacent to, the electrodes. The substrate may be the surface of glass, plastic, solid fibers, metal, semiconductor or gel material. The surfaces of these materials can be directly modified through redox reactions. In addition, in this embodiment, the surface of these materials may be attached with other substances. For example, the attachment of organic compounds to the surface of these materials is a known method. Substances attached to the surface of these materials can be modified through redox reactions.

In another embodiment, the substrate may be a substance attached to the surface as an electrode. Alternatively the substrate is a substance that is attached to the electrode itself via linking groups. US Patent No. 6,093,302 discloses the latter method, in which the substrate is attached to the electrode via a linking group.

The method of the present invention is similar to the method disclosed in WO93/22480. However, the electrolyte solution selected for the method of the present invention is different therefrom. The electrolyte used in WO93/2240 is an acetonitrile solution of triethylamine and sulfuric acid. Active redox species may be inhibited by at least one other redox species in the electrolyte of the invention. The electrolyte can precisely confine the active redox species to the surrounding area of the electrode where the active redox species is generated.

In the method described in WO 93/22480, the confinement of the acid in a specific area is controlled by a change in the electrode potential. However, the inventors of the present invention have found that after long-time electrolysis, when the electrolyte is an acetonitrile solution of triethylamine and sulfuric acid, the acid loses its confinement. Failure to effectively confine the acid will result in reduced resolution of the treated substrate. For example, protons diffusing from the vicinity of the anode will react with the substrate region between the electrodes. Incidental reactions of diffusing protons by this means are undesirable in order to obtain substrates with high-resolution patterns. According to the selected electrolyte of the present invention, the problems of the prior art electrolyte can be avoided. It is an important feature of the present invention that the electrolyte of the present invention allows active redox species to be inhibited by at least one other redox species.

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

For example, an electrolyte that is a combination of ^I- and S ₄ O ₆ ^2- . Oxidation of iodide at the anode produces iodine (an active redox species), while reduction of S ₄ O ₆ ^2- at the cathode produces S ₂ O ₃ ^2- , which inhibits iodine production at the anode. The reaction in the electrolyte can be expressed as follows:

anode:

cathode:

Iodine is inhibited by the following reactions:

The active redox substance is preferably an acid, and the redox-inhibiting substance is an anion, preferably an organic anion (Radical anion). Typically, the acid is produced by anodic oxidation of an alcohol, which can be an aliphatic alcohol, or an aromatic alcohol. In such electrolytes, the suppressed anions are usually produced by reduction of suitable species at the cathode. Many species can be reduced at the cathode to produce anions and inhibit acid formation at the anode. For example, dissolved oxygen molecules can be reduced at the cathode to produce O ₂ ⁻ and/or O ₂ ²⁻ .

For example, an electrolyte that produces the appropriate redox species, which is a combination of a ketone and the corresponding alcohol. Alcohols are oxidized at the anode to produce protons (an active redox species), while ketones are reduced at the cathode to produce negative ions, which inhibit proton production at the anode.

The reaction in this electrolyte can be expressed as follows:

anode:

cathode:

Among them, R ¹ and R ² can be substituted C ₁ to C ₁₅ hydrocarbon groups respectively, wherein more than three carbon atoms can be optionally substituted by N, O and/or S atoms; or R ¹ and R ² together form a substituted C ₁ to C ₁₅ cyclohydrocarbylene (cyclohydrocarbylene), wherein more than three carbon atoms can be optionally substituted by N, O and/or S atoms.

Preferably, R ¹ and R ² can each be substituted C _1-8 alkyl, C _3-8 cycloalkyl or phenyl.

"Hydrocarbyl" in the present invention refers to a monovalent group which contains carbon and hydrogen. Hydrocarbyl groups thus include alkyl, alkenyl and alkynyl groups (in straight and branched chain structures), cycloalkyl groups (including polycycloalkyl groups), cycloalkene and aryl groups, and combinations of the above, such as alkylcycloalkanes radical, alkylpolycycloalkyl, alkylaryl, alkenylaryl, alkynylaryl, cycloalkylaryl and cycloalkenylaryl.

"Hydrocarbylene" in the present invention refers to a divalent group, which contains carbon and hydrogen. Cycloalkylene groups thus include cycloalkylene or cycloalkylene groups, cycloalkenylene groups and arylene or arylene groups.

"Aryl" in the present invention refers to an aromatic group, for example, phenyl, naphthyl or anthracenyl. Or when the aryl group has carbon atoms substituted by O, N and/or S, the aryl group refers to an aromatic heterocyclic group, for example, pyridyl, pyrrolyl, thienyl, furyl, imidazolyl, triazolyl, Quinolinyl, isoquinolyl, oxazolyl or isoxazolyl.

The substituents of the present invention can be selected from C ₁ to C ₆ alkyl, C ₁ to C ₆ alkoxy, thio, C ₁ to C ₆ thioalkyl, carboxyl, carboxyl (C ₁ to C ₆ ) Alkyl, formate, C ₁ to C ₆ alkylcarbonyl, C ₁ to C ₆ alkylcarbonylalkoxy, nitro, trihalomethane, hydroxy, C ₁ to C ₆ hydroxyalkyl, hydroxy (C ₁ to C ₆ ) alkyl, amino, C1 to C6 alkylamino, di(C ₁ to C ₆ ) amino, aminocarboxyl, C ₁ to C ₆ alkylaminocarboxy (alkylaminocarboxy), di(C ₁ to C ₆ alkyl) diaminocarboxy, aminocarboxy (C ₁ to C ₆ ) alkyl, C ₁ to C ₆ alkylaminocarboxy (C ₁ to C ₆ ) alkyl, di(C ₁ to C ₆ Alkyl) aminocarboxy (C ₁ to C ₆ ) alkyl, C ₁ to C ₆ alkylcarbonylamine, C ₁ to C ₆ cycloalkyl, C ₁ to C ₆ cycloalkyl (C ₁ to C ₆ ) alkyl, C ₁ to C ₆ alkylcarbonyl (C ₁ to C ₆ alkyl)amino, halogen-containing, C ₁ to C ₆ haloalkyl, sulfamoyl, tetrazolyl and cyano.

"Halogen-containing" or "halogen" in the present invention refers to iodine, bromine, chlorine or fluorine.

The properties of R1 and R2 determine the redox characteristics of the electrolyte. For example, substitution reduction on ^R1 and ^R2 can change the potential by oxidation or reduction.

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

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

和

and

Wherein R3, R4, R5 and R6 can be selected respectively: hydrogen, halogen, nitro group, hydroxyl group, thio group, amino group, substituted C ₁ to C ₁₅ hydrocarbon group, wherein more than three carbon atoms can be formed by N, O and / or S atom substitution. It can also be R3 and R4, and/or R5 and R6 combined to form a substituted _C1 to _C15 cycloalkylene group, wherein more than three carbon atoms can be replaced by N, O and/or S atoms.

R3, R4, R5 and R6 are preferably hydrogen, C _1-8 alkyl or R3/R4 and R5/R6 combined to form a substituted C _5-12 arylene, such as phenylene.

The properties of R3, R4, R5 and R6 can determine the redox properties of the electrolyte, for example, oxidation or reduction can change the precise potential. The electrolyte solution of the benzoquinone/hydroquinone derivative is preferably an organic solution of anthraquinone/anthradiol and tetramethyl-p-benzoquinone/tetramethylhydroquinone.

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

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

Most of the protons released by the oxidation of hydroquinone stay at the anode, which modifies its adjacent substrate. For example, the proton can deprotect a substrate bearing an acid labile protecting group.

Benzoquinone is reduced at the cathode to produce benzoquinone negative ions:

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

In this way, the resolution of an area on the substrate can be improved by localizing the active redox species produced by the electrodes, eg protons are generated at the anode.

The electrolyte solution of the present invention includes any suitable solvent, such as water, tetrahydrofuran (THF), methanol, ethanol, dimethylformamide (DMF), dichloromethane (dichloromethane), ether, dimethylsulfoxide (DMSO) or acetonitrile. Those skilled in the art will know that the choice of solvent can affect the balance or kinetics of redox reactions and/or inhibit reactions at the electrodes. The solvent can affect the activity of certain structures in solution, eg, blended structures, hydrogen bonding, dipole-dipole or "delocalisation" of charge. The solvent is preferably an aprotic solvent, which stabilizes the negative ions. Aprotic solvents are: dichloromethane, dimethylformamide, dimethylsulfoxide, acetonitrile and tetrahydrofuran. Acetonitrile is a more preferred solvent.

In a preferred embodiment, the electrolyte may further include a conductivity enhancer for improving the conductivity of the electrolyte. The electrolytic voltage required to add conductivity enhancers is lower than that without conductivity enhancers. Any ion that is soluble in the electrolyte will serve this purpose. For example, when the electrolyte solution includes an organic solvent such as acetonitrile, the conductivity enhancer is a tetra(C _1-8 alkyl)ammonium salt, such as tetrabutylammonium hexafluorophosphate.

Those skilled in the art know that salts have a role to play in the electrolyte, and not just to increase the conductivity of the electrolyte. Salts can affect the balance or kinetics of inhibitory reactions and/or redox reactions at the electrodes. Salt can affect the electrostatic interaction force of charged substances in solution, and correspondingly, it can also affect the reaction kinetics. For example, when the electrolyte is a solution of hydroquinone/benzoquinone in acetonitrile, additional ammonium hexafluorophosphate can change the degree of inhibition of the reaction and can also increase the conductivity.

The method of the present invention is realized by the device disclosed in WO93/22480. The device disclosed in WO 93/22480 comprises an array of electrodes spaced apart on an insulating surface. The electrodes are plated with platinum to provide electrical connecting means for changing their potential.

However, the electrodes used in the process of the invention are preferably iridium electrodes. The present invention provides an electrode array comprising a piece of insulating material having a surface on which spaced apart arrays of iridium deposits are formed, each deposit being provided as an electrical connection means for changing its potential.

The advantages of iridium are its high conductivity and chemical inertness. 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 attach well to some materials, such as silicon wafers, especially at high electrode potentials. Inhibition reactions internally require higher electrode potentials to be used for longer periods of time without loss of resolution of the substrate being processed, requiring changes to existing electrode designs.

Many metals were tested for suitability as 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 electrolytes and adheres well to materials such as oxidized silicon wafer material.

A piece of material on the electrode array can be an insoluble polymer, a ceramic oxide (such as alumina) or a silicon oxide wafer. Oxidized silicon wafers are preferred.

Iridium electrode arrays can be fabricated by any number of suitable methods. In a preferred embodiment, the electrode array can be obtained by:

(i) providing a silicon wafer with a silicon dioxide layer on its surface;

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

(iii) Annealing iridium in air at a temperature of 200-500°C.

In a typical process, a positive organic photoresist is applied to the silicon dioxide layer of a silicon wafer. Covering with a photomask followed by exposure to UV light removes the exposed photoresist, exposing the silicon dioxide regions where the photoresist was removed. Iridium metal is deposited on the exposed silicon dioxide regions using an electron beam gun. Areas of the photoresist layer are removed to form an electrode array. Finally, the iridium electrodes are annealed in air to improve adhesion to the wafer surface. Typically, iridium is annealed at 350°C for 15 minutes to 3 hours, preferably about 1 hour.

For iridium to adhere to silica, the annealing step is important. The annealing temperature is about 350°C, and iridium can have a melting temperature of 2545°C. It was even found that an annealed electrode with a 50 nm iridium layer was resistant to scratching by a steel scalpel. In addition, iridium electrodes are resistant to harsh chemical environments, and the high potentials and high currents of the method of the present invention.

Preferably, the electrodes are an array of parallel wires spaced apart from each other by less than 0.5mm. Preferably, the distance between the electrodes is 0.1-200 microns, more preferably 1-100 microns, even more preferably 10-60 microns.

One or more electrodes are used as counter electrodes. Preferably, compared to the existing methods for electrochemically treating substrates, the substrate to be treated in the present invention does not form an electrode or an auxiliary electrode. The method of the present invention is similar to that described in WO93/22480. Furthermore, the substrate to be treated may be an insulating surface.

In a preferred implementation, the present invention provides a method for implementing several processing steps in sequence. Connecting the electrodes of the array such that changing the potential of selected one or more electrodes of the array completes a processing step.

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 in close proximity to the electrodes. Preferably, the solid surface is a different surface adjacent to the electrode. Redox species can be used to modify species attached to solid surfaces. Many redox species and corresponding chemical modifications can be devised by those skilled in the art. In preferred embodiments, the substrate to be treated comprises a material having an acid labile protecting group. In this preferred embodiment, the processing step is accomplished by connecting at least one electrode in the array to a power source and serving as an anode for generating acid in the electrolyte. This generated acid removes acid labile protecting groups attached to the surface and near the anode region.

Active redox species are recognized to participate in a variety of chemical reactions on substrates. One potential application is the electrochemical micromechanical technology disclosed by Schuster et al. in Science, 2000, 289, 98-101. The tool disclosed by Skoder can be used in the electrolyte of the present invention, with an auxiliary electrode ring surrounding the probe tip to prevent diffusion of redox species. The present invention will use existing nanoscale patterning technology. For example, acids can be used in etching or nanofabrication, which remove small amounts of material from a surface.

Additionally, acids can participate in many organic or inorganic reactions. Those skilled in the art will know of many potential reactions that can be used in the present invention. For example, organic reactions include ring opening of epoxides, attachment to multiple bonds, rearrangement, substitution (e.g., unimolecular nucleophilic substitution of tertiary alcohols ( _SN 1)), elimination, formation of enols, and organic Simple protonation of acid salts.

When the active redox species is a halogen, it can participate in mild oxidation, bleaching of the substrate or halogenation. Active redox species can also be halide ions, which can be used in substitution reactions.

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 drug discovery. The range of reactions to which the present invention applies is meant to be theoretically suitable for the synthesis of such substances.

The method of the invention can be used for the stepwise synthesis of oligomers, eg, oligonucleotides, polysaccharides and proteins. Preferably, the method of the invention is used for the synthesis of nucleotides.

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

(a) providing a substrate with an array of substances having protecting groups attached thereto, an electrolyte in contact with the substrate and an array of electrodes proximate to the substrate and in contact with the electrolyte;

(b) selectively altering the potential of one or more electrodes to generate an active redox species that removes the aforementioned protecting groups from selected species;

(c) incorporation of a protected monomer into the deprotected species formed in step (b);

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

The electrolyte is characterized in that the active redox species is inhibited by at least one other redox species.

When the above method is used for the synthesis of oligonucleotides, the active redox species is preferably a proton, and the protecting group is preferably an acid-labile protecting group, such as a trityl group or a dimethoxytrityl group, which can be Protect furanyl hydroxyl. Those skilled in the art will recognize that this method is particularly suitable for combinatorial synthesis of DNA chips, as described in WO93/22480.

The above method can also be used for the synthesis of peptides. For example, peptides can be synthesized by sequentially removing the Boc (t-butyloxycarbonyl) protecting group from the nitrogen atom using anode-generated protons. 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 with an iridium metal layer deposited on its upper surface. The spacer 2 is formed by photolithography on the iridium metal layer on the silicon wafer, so that a parallel electrode array is formed. The width of each electrode and the width of each space 2 is approximately 40 microns. Another silicon wafer 4 is disposed on the electrode array. The surface of the silicon wafer 4 is trimmed to form dimethoxytrityl protected nucleosides.

See 2, which shows the electrode array and part of its substrate. The middle electrode is the anode, and the other two electrodes are the cathode. An electrolyte comprising benzoquinone and hydroquinone in acetonitrile is in contact with the electrodes and the substrate to be treated. At the anode, hydroquinone is oxidized, producing benzoquinone and protons. Most protons are confined to the substrate region close to the anode. The confined proton removes the dimethoxytrityl group from a protected nucleotide moiety attached to the substrate. However, some protons can diffuse into the region between the anode and cathode.

At the cathode, benzoquinone is reduced to produce benzoquinone anion. Benzoquinone anions are relatively stable and can diffuse into the region between the anode and cathode. The benzoquinone negative ion suppresses the diffusion of protons into this region, resulting in hydroquinone and benzoquinone. Thus, protons diffusing in regions of the substrate that are not close to the anode can be prevented from reacting. Substrate resolution is improved by preventing accidental proton reactions in the region between the electrodes.

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

Please refer to Fig. 3, the effect of varying the electrolysis time at a fixed potential of 1.33V is shown by confocal microscopy. In this figure, the bright region is the fluorescent basal region where the dimethoxytrityl group is not removed during electrolysis. The remaining dimethoxytrityl groups are then replaced by the fluorescent Cy5 dye. This bright area is usually close to the cathode. In the black area, the dimethoxytrityl group has been removed during electrolysis. The resulting free hydroxyl groups are bound to non-fluorescent acetyl groups. This black area is usually close to the anode.

Figure 3 shows that after 2.0 seconds, the dimethoxytrityl group was completely removed in the region near the anode. Also, the resolution of the substrate does not change after 80 seconds. Confined fringes exist in regions corresponding to the proximity of the anode and cathode. These demonstrate that protons produced during electrolysis are strictly confined to the region close to the anode, even after prolonged electrolysis.

Figure 4(a) and (b) show the clear effect of removing the cathode from the electrode array. The black region shows the region where the dimethoxytrityl group was removed. The electrode potential was set at 1.33 V. When the cathode in the middle was removed, the protons generated at the anode were free to diffuse into the middle region, which It is clearly demonstrated that species produced at the cathode have a confinement effect that suppresses protons produced at the anode.

Experimental part

electrode combination

Existing photolithography techniques have been widely used to generate iridium metal (50 nm thick) electrodes on oxidized high-conductivity silicon wafers. The silicon oxide 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° C. 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 was deposited on the wafer by electron beam method. The iridium metal can be placed in the crucible of the vacuum evaporator, and two or three wafers are placed approximately 20 cm close to the crucible of the vacuum evaporator. Pump the chamber of the vacuum evaporator to 3×10 ^-6 Torr, heat the iridium metal with a 300mA, 5kV electron beam gun for about 3 minutes, and coat the wafer with 50nm iridium metal.

Placing the wafer in ultrasonic acetone for 30 minutes removes the photoresist and reveals the electrode array. The electrodes were placed in air, annealed at 350°C 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 the ultrasonic gold wire, which is connected to the printed circuit board, and an "analog switch" integrated with the circuit activates the selected electrode to provide the unlocking step . The current is applied as a control voltage source for multiple individually operated amplifiers. A parallel LNA feedback circuit continuously measures the nanoampere current at each electrode.

solid support combination

Polished silica wafers can be used as substrate supports. Prior to patterning, the wafer surface is functionalized with linker molecules to allow the attachment of organic reagents to the linker molecules [Gray DE, Keith Green SC, Fell TS, Dobson PJ and Sassen EM (Gray, DE, CaseGreen, SC, Fell, TS, Dobson, PJ & Southern, EM), Characterization of ellipsometry and interferometry of DNA probes immobilized on combinatorial arrays, Languir 13, 2833-2842 (1997)]. The wafer was placed in a chamber of a vacuum furnace, which had a capacity of 19.1 liters and included an ampoule containing 5 ml of GPTS (glycidoxypropyltrimethoxysilane). After the furnace was heated to 185°C, the ampoule was heated to 205°C and the chamber was pumped to 25-30 mBar. After about 2.5 ml of silane has evaporated, the chamber can be cooled under vacuum (10 ⁻³ Torr). The "linker molecules" can be attached by immersing the wafer with GPTS in a polyethylene glycol solution containing a small amount of sulfuric acid. Through the existing oligonucleotide synthesis technology, the dimethoxytrityl group containing phosphoramidite (phosphoramidite) is covalently attached to the hydroxyl group of polyethylene glycol [Bill Kaijie SL and Aiya RP (Beaucage, SL & Iyer, RP), Improved synthesis of oligonucleotides by the phosphoramidite method, Tetrahedron 48, 2223-2311 (1992)]. The wafer substrate was then cut to 1 cm x 1 cm for use in patterning.

Embodiment two

The electrode array prepared above was placed 20 microns away from the solid support. The solid support was prepared by the above steps, thymidine phosphoramidite (thymidinephosphoramidite) was attached to polyethylene glycol linker molecules. Thymidine phosphoramidites have a 5'-hydroxyl group, which is protected by a dimethoxytrityl group.

An electrolytic solution (25 mM hydroquinone/25 mM quinone/25 mM ammonium hexafluorophosphate in anhydrous 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 Figure 3).

After electrolysis, the silicon wafers were washed with acetonitrile and acetyl groups were incorporated with acetic anhydride in a standard manner. In this step, only the dimethoxytrityl deprotected regions of the silicon wafer add acetyl groups.

Dimethoxytrityl groups not removed during the electrochemical step were removed upon treatment of the entire substrate with dichloroacetic acid in dichloromethane. The exposed hydroxyl groups were bound to Cy5 (a fluorescent dye) using standard phosphoramidite conjugation methods, and the pattern generated by the electrochemical generation of the acid was revealed, which could be visualized by confocal microscope observation of Cy5 fluorescence. The sequence of steps is shown in Figure 2.

Figure 3 shows the effect of prolonged electrolysis time at 1.33V. After about 2.0 seconds, the maximum frequency bandwidth is reached, and the resolution of the substrate is stable, and the resolution will not change even after electrolysis for 80 seconds.

Embodiment two

Embodiment 2 is basically the same as Embodiment 1, except that the selected anode is kept at a voltage of 1.33V for 16 seconds. As shown in Figure 4(a) and (b), the effect of removing the cathode can be observed. When the central cathode is removed, the diffusion of protons gets out of hand. The diffused protons can flow into the central region instead of staying around the anode. In Fig. 4(b), the black area in the middle is evident, which no longer contains the fluorophore.

Embodiment Three

The method described in Example 1 was used to synthesize a 17-mer oligomer on a solid support, and the method included 16 deprotection steps of dimethoxytrityl. The method used in this embodiment is basically the same as that in Embodiment 1, except that the electrode array is placed at a distance of 40 microns from the surface of the substrate.

A uniform coating of dimethoxytrityl-protected deoxyadenosine residue was attached to a polyethylene glycol linker on the solid support using standard phosphoramidite conjugation methods.

After extensive cleaning with acetonitrile, the electrolyte in Example 1 was injected into the cavity between the electrode array and the solid support. A voltage of 1.33 V was applied to the selected anode for 9 seconds to remove the dimethoxytrityl group near the selected anode. The anode is located between the two cathodes.

After further washing with acetonitrile, the dimethoxytrityl-protected nucleoside residues were attached to the exposed hydroxyl groups using standard phosphoramidite conjugation methods. A trivalent phosphorus linkage is oxidized with iodine to generate a pentavalent phosphorus linkage. Rinse the entire silicon wafer with acetonitrile followed by dichloromethane. The ligation and oxidation steps used in methods for the synthesis of oligonucleotides on solid supports are known in the art (see, "AbI Synthesis Handbook", Part II, Chemical Methods for Automated DNA Synthesis).

This procedure was repeated, varying the dimethoxytrityl-protected nucleoside residues introduced in the phosphoramidite conjugation step. Thus, oligonucleotides are synthesized on solid supports.

The process can be accomplished by an automated device controlled by a computer, including in the synthesis of two 17-mer oligonucleotides: wild type (wild type) "A" human hemoglobin mRNA (ribonucleic acid) and the corresponding " S" sickle cell mutant xenogeneic mRNA. High yields can be achieved by generating 17-mers on solid-supported defined strips. The combinatorial synthesis method of the DNA chip is known to those skilled in the art, as disclosed in WO93/22480.

Although the invention has been described with reference to specific embodiments, such description is not meant to limit the invention. With reference to the description of the present invention, other variations of the disclosed embodiments are expected by those skilled in the art. Therefore, such modifications do not depart from the scope and spirit defined by the appended claims.

Claims (29)

1. A method for controlling the diffusion of a first redox substance produced by a first electrode, characterized in that: the method comprises: generating a second redox substance at a second electrode close to the above-mentioned first electrode, the first and second electrodes In contact with an electrolyte solution, wherein the electrolyte solution allows the first redox species to be inhibited by the second redox species. 2. The method according to 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. A method according to claim 3, characterized in that the active redox species is used to condition the substrate close to 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 proximate to the substrate and in contact with the electrolyte; altering at least one electrode to produce an active redox species, the active A redox species modifies the substrate adjacent to the at least one electrode; characterized in that the electrolyte enables the active redox species to be inhibited by a second redox species. 6. The method according to claim 3, 4 or 5, characterized in that the active redox species is a proton. 7. The method according to any one of the preceding claims, characterized in that: the second redox substance is an organic negative ion (radical anion). 8. The method according to any one of the preceding claims, wherein the electrolyte is a solution of hydroquinone and benzoquinone, or a derivative thereof. 9. The method according to any one of the preceding claims, characterized in that: the electrolyte is a solution of the following compounds;

and

Wherein R3, R4, R5 and R6 can be selected respectively: hydrogen, halogen, nitro group, hydroxyl group, thio group, amino group, substituted C ₁ to C ₁₅ hydrocarbon group, wherein more than three carbon atoms can be formed by N, O and / or S atom replacement; or R3 and R4, and/or R5 and R6 combine to form a substituted C ₁ to C ₁₅ cyclohydrocarbylene (Cyclohydrocarbylene), wherein more than three carbon atoms can be replaced by N, O and/or S atoms. 10. The method according to any one of the preceding claims, characterized in that: the electrolyte is an acetonitrile solution of hydroquinone and benzoquinone. 11. A method according to any preceding claim, wherein the electrolyte further comprises a conductivity enhancer. 12. The method according to claim 7, wherein the conductivity enhancer is tetrakis(C _1-8 alkyl)ammonium salt. 13. A method according to any one of claims 5 to 12, characterized in that one or more electrodes are used as auxiliary electrodes, and the substrate to be trimmed does not constitute an electrode or an auxiliary electrode. 14. A method according to any one of the preceding claims, characterized in that, in order to realize several processing steps in sequence, the electrodes of the array are connected such that the potential of selected one or more electrodes of the array is changed, One processing step can be done. 15. A method according to any preceding claim, wherein the substrate comprises an array of substances attached to its surface. 16. The method of claim 15, wherein the surface is an oxidized silicon wafer surface. 17. The method according to claim 15, characterized in that: the substance to be treated comprises an acid labile protecting group. 18. The method according to claim 17, characterized in that a single treatment is 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 species on the surface. 19. A method according to any one of claims 14 to 18, characterized in that the processing is carried out during the stepwise chemical synthesis of the oligomers. 20. A method of synthesizing a set of oligomers comprising the steps of: (a) providing a substrate with an array of substances having protecting groups attached to the substrate, an electrolyte in contact with the substrate, and an array of electrodes proximate the substrate and in contact with the electrolyte; (b) selectively altering the potential of one or more electrodes to generate an active redox species that removes the aforementioned protecting groups from selected species; (c) incorporation of a protected monomer into the deprotected species formed in step (b); (d) repeating steps (b) and (c), changing one or more electrodes selected in step (b), to synthesize a set of oligomers; It is characterized in that the electrolyte enables the active redox species to be inhibited by at least one other redox species. 21. The method of claim 20, wherein the array of substances is attached to a surface on which the oligomers are synthesized. 22. The method according to claim 20 or 21, wherein the oligomer is an oligonucleotide. 23. The method according to any one of claims 20-22, characterized in that: the active redox species is a proton, and the protecting group is an acid labile protecting group. 24. The method according to any one of claims 20-22, wherein the electrolyte is the electrolyte defined in claims 8-12. 25. A method according to any one of claims 20 to 24, wherein one or more electrodes of the array are auxiliary electrodes. 26. An electrode array comprising a piece of insulating material having a surface on which spaced apart arrays of iridium deposits are formed, each deposit being provided as an electrical connection means for changing its potential. 27. The array of claim 26, wherein the mass of insulating material is an oxidized silicon wafer. 28. An array as claimed in claim 26 or 27, characterized in that the deposits of iridium are in the form of parallel lines spaced apart from each other. 29. A method of manufacturing the array of any one of claims 26 to 28, comprising the steps of: (i) providing a silicon wafer with a silicon dioxide layer on its surface; (ii) depositing iridium at intervals on the silicon dioxide layer to form an array; (iii) Annealing iridium in air at a temperature of 200-500°C.

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