JP4261350B2 - Substrate processing - Google Patents

Description

Detailed Description of the Invention

The present invention relates to substrate processing using electrochemical methods. More particularly, the present invention relates to a method for chemically modifying a substrate using an electrochemical method.
Many devices require a pattern of specific material on the surface. A semiconductor chip is a well-known example of the device. A very recent example of the device is a DNA chip, which contains a sequence of oligonucleotides bound to a solid surface (G. Ramsay, Nature Biotechnology , 1998, vol. 16, 40-44).

  The nature of the device depends on the nature and pattern of the material on the surface. Furthermore, improvements to existing devices are accelerated on the one hand by miniaturization requirements and on the other hand by the desire for new types of surfaces that combine chemical and physical characteristics. Accordingly, there is a continuing need for new methods of processing devices that require patterns on the surface.

There are several ways to treat a specific area of the surface. One method uses photolithography technology. A specific area of the surface is covered with a photolithographic mask and the exposed area is modified by exposure to ultraviolet light. This method is widely used in the manufacture of semiconductors, and an opening is formed on the surface of a semiconductor wafer coated with a photosensitive compound.

The photolithographic method is also used in the production of DNA chips. In this method, an oligonucleotide having a photolabile protecting group is attached to a solid surface. The surface area is covered with a photolithographic mask, and the exposed area of the surface is irradiated with ultraviolet rays. Thus, photolabile protecting groups can be removed from exposed areas of the surface (G. Ramsay, Nature Biotechnology , 1998, vol. 16, 40-44).

WO 93/22480 describes a surface treatment method using an electrochemical method. In this method, an array of electrolyte covering the surface and electrodes near the surface is provided. The surface near one or more electrodes can be modified by changing the potential of one or more electrodes in the array. The electrolyte used is an acetonitrile solution of triethylamine and sulfuric acid.

  U.S. Pat. No. 6,093,302 describes an electrochemical method of placing material at a specific location on a substrate. This material is generated at the electrode and usually reacts with the material in the vicinity of the electrode. The use of a buffer or capture solution is described. The buffer or capture solution is for improving the solubility of the substrate to be treated by reaction with a reagent leaving the immediate vicinity of the electrode. However, a bulk solution containing a buffer or capture substance has the disadvantage that it captures not only the reagent that diffuses away from the specific electrode, but also the reagent to be reacted on the substrate in the vicinity of the specific electrode.

Schuster et al., Science , 2000, 289 , 98-101 describes another method for improving the solubility of electrochemical methods for treating surfaces. Schuster uses a complex sequence of current pulses to limit the diffusion time.

It is an object of the present invention to provide an improved method of modifying a substrate using electrochemical means. In particular, an object of the present invention is to provide a method for modifying a substrate with excellent solubility.
Accordingly, the present invention is a method for controlling the diffusion of a first redox product produced by a first electrode, wherein a second redox product is produced by a second electrode in the vicinity of the first electrode, A method is provided wherein the second electrode is in contact with an electrolyte, wherein the electrolyte is such that the first redox product can be extinguished by the second redox product.

Preferably, the second electrode is a counter electrode.
Preferably, the first redox product is an active redox product and can be used to modify the substrate in the vicinity of the electrode.

Accordingly, in another aspect, the present invention is a method of processing a substrate, the method comprising an electrolyte in contact with the substrate and one or more electrodes in proximity to the substrate and in contact with the electrolyte, and at least one Changing the potential of at least one electrode to produce an active redox product that modifies a substrate in the vicinity of one electrode, such that the electrolyte can be extinguished by the second redox product. Provided is a method characterized by

Expirable means that the second redox product can react with the first redox product to modify its reactivity, so that the first redox product does not react in the same way as the original shape. Means. When the first redox product is an active redox product, the reaction of the active redox product with the second redox product prevents the active redox product from modifying the substrate. For example, if the active redox product is an acid, the second redox product can be a base. The reaction between the acid and base destroys the acid and prevents it from modifying the substrate.

In preferred embodiments, the quenching reaction regenerates one or more substances in the electrolyte.
By active redox product is meant an oxidation or reduction product that can modify the substrate. The active redox product can be produced directly by oxidation or reduction of substances in the electrolyte. Alternatively, the active redox product can be generated indirectly by one or more subsequent reactions with other substances in the electrolyte following oxidation or reduction of the substance in the electrolyte.

Usually, an active redox product is formed on the electrode surface. The active redox product can then modify the nearby substrate. Acid is a preferred example of an active redox product. The acid can participate in many types of reactions on the substrate, such as elimination, substitution, rearrangement and chemical etching. Preferably, when the active redox product is an acid, the acid is used to remove the acid labile protecting group from the substrate.

  Acid labile protecting groups are well known to those skilled in the art and include, for example, acetals (eg, methoxymethyl, methylthiomethyl, (2-methoxyethoxy) methyl, benzoyloxymethyl, β- (trimethylsilyl) ethoxymethyl, tetrahydropyranyl, Benzylidene, isopropylidene, cyclohexylidene, cyclopentylidene), esters (eg, benzoyl, benzoyloxycarbonyl, tertiary butoxycarbonyl), ethers (eg, trityl, dimethoxytrityl, tertiary butyl) and silyl ethers ( For example, tertiary butyldimethylsilyl, trimethylsilyl, triethylsilyl). Preferably, the acid labile protecting group is trityl or dimethoxytrityl (DMT) ether, which are the protecting groups commonly used in the synthesis of oligonucleotides.

  Similarly, the active redox product can be a base. The base can participate in various reactions on the substrate. For example, a base can be used to remove a base labile protecting group.

Base labile protecting groups are well known to those skilled in the art and include, for example, 9-fluorenylmethoxycarbonyl (Fmoc) and cyanoethyl groups.
A radical is another example of an active redox product. Radicals can be used to initiate radical reactions on the substrate. Electrochemical methods for generating radicals are well known to those skilled in the art. One commonly used method for generating radicals electrochemically is the oxidation of carboxylate anions.

  Halogen is another example of an active redox product. Halogen can be used, for example, for the oxidation reaction or addition reaction on the substrate. Halogen can be generated electrochemically by oxidation of the corresponding halide ion.

These and other examples of active redox products will be readily apparent to those skilled in the art.
The method of the present invention is used to process a substrate. As used herein, the term substrate refers to any material that can be modified by an active redox product in proximity to an electrode. The substrate can be separated from the electrode, in which case the substrate is placed in the vicinity of the electrode and then separated from the vicinity of the electrode after the redox reaction. Alternatively, the substrate can be bonded to the electrode itself or to the same surface as the electrode. If necessary, after the redox reaction is performed, the substrate can be peeled off from the electrode or the same surface.

Thus, in one aspect, the substrate is a surface of a material that is separated from and proximate to the electrode. Thus, the substrate can be the surface of glass, plastic, solid fiber matrix, metal, semiconductor or gel material. The surface of this material can be modified directly by a redox reaction. Further, in this embodiment, the surface of the substance can be bound with the substance. For example, the organic compound can be bound (and optionally peeled off) to the material surface by known methods. Thus, the substance bound to the surface of the substance can be modified by a redox reaction.

  In other embodiments, the substrate is a substance bound to the same surface as the electrode or to the electrode itself by a linker group. U.S. Pat. No. 6,093,302 describes the latter of this assembly, i.e., the substrate is attached to the electrode by a linker group.

  The method of the present invention is similar to the method described in WO 93/22480. However, the method of the present invention differs in the choice of electrolyte. WO 93/2240 uses an electrolyte which is an acetonitrile solution of trimethylamine and sulfuric acid. The present invention uses an electrolyte in which the active redox product can be extinguished by at least one other redox product. The advantage of this electrolyte is that the active redox product can be accurately confined to the region that rapidly surrounds the electrode that produced the electrolyte.

  In the method described in WO93 / 22480, acid confinement in a specific region is controlled by variation in electrode potential. However, the present inventors have found that when the electrolyte is an acetonitrile solution of triethylamine and sulfuric acid, the acid is not confined after long-time electrolysis. Insufficient acid confinement results in inadequate solubility of the substrate being processed. For example, protons diffusing away from the immediate vicinity of the anode can react on the substrate in the zone between the electrodes. Such accidental reaction of diffused protons is undesirable from the standpoint of obtaining a high resolution patterned substrate. By choosing an electrolyte according to the present invention, the problems of prior art electrolytes can be avoided. It is an important feature of the present invention to select the electrolyte so that the active redox product can be extinguished by at least one other redox product.

Those skilled in the art are aware of many examples of electrolytes that produce active redox products that can be extinguished by other redox products.
An example of such an electrolyte is a combination of I ⁻ and S ₄ O ₆ ²⁻ . The oxidation of iodide at the anode produces iodine (an active redox product), while the reduction of S ₄ O ₆ ^{2− at} the cathode produces S ₂ O ₃ ²⁻ , which eliminates the iodine produced at the anode. Can do. The reaction in the electrolyte can be expressed as:
anode:

Cathode:

The disappearance of iodine by the reaction:

Preferably, the active redox product is an acid and the annihilation redox product is an anion, preferably an organic radical anion. Usually, the acid is generated at the anode by oxidation of an alcohol (which can be any aliphatic or aromatic alcohol). In the electrolyte, the disappearing anion is usually generated at the cathode by reduction of a suitable substance. Many materials are reduced at the cathode to produce anions, which can extinguish the acid produced at the anode. For example, dissolved molecular oxygen can be reduced at the cathode, thereby producing O ₂ ⁻ and / or O ₂ ²⁻ .

  An example of an electrolyte that produces a suitable redox product is a combination of a ketone and the corresponding alcohol. Alcohol oxidation at the anode produces protons (active redox products), while reduction of the ketone at the cathode produces radical anions, which can extinguish the protons produced at the anode.

The reaction in the electrolyte can be expressed as:
anode:

Cathode:

Wherein R ¹ and R ² are independently optionally substituted C ₁ to C ₁₅ hydrocarbyl (wherein 3
Up to C atoms can be substituted with N, O and / or S atoms); or R ¹ and R ² together, optionally substituted C ₁ to C ₁₅ cyclohydrocarbi To form a ren, where up to 3 C atoms can optionally be replaced by N, O and / or S atoms.

Preferably R ¹ and R ² are independently selected from optionally substituted C _1-8 alkyl, C _3-8 cycloalkyl or phenyl groups.
The term “hydrocarbyl” is used herein to refer to a monovalent group consisting of carbon and hydrogen. Thus, hydrocarbyl groups include alkyl, alkenyl and alkynyl groups (both linear and branched), cycloalkyl (including polycycloalkyl), cycloalkenyl and aryl groups, and alkylcycloalkyl, alkylpolycycloalkyl, alkylaryl, Such conjugations as alkenyl aryl, alkynyl aryl, cycloalkyl reel and cycloalkenyl aryl groups are included.

  The term “hydrocarbylene” is used herein to refer to a divalent group consisting of carbon and hydrogen. Cyclohydrocarbylene groups therefore include cycloalkylene, cycloalkenylene and arylene groups.

  The term “aryl” is used herein to refer to an aromatic group such as phenyl, naphthyl or anthracyl. Alternatively, when the aryl group has a carbon atom substituted with O, N and / or S, the term aryl is as pyridyl, pyrrolyl, thienyl, furanyl, imidazolyl, triazolyl, quinolinyl, isoquinolinyl, oxazolyl or isoxazolyl. A heterocyclic aromatic group.

When referring to any substituents herein, the substituents are preferably C ₁ to C ₆ alkyl, C ₁ to C ₆ alkoxy, thio, C ₁ to C ₆ alkylthio, carboxy, carboxy (C ₁ to C ₆₎ alkyl, formyl, C ₁ to C ₆ alkylcarbonyl, to no C ₁ C ₆ alkylcarbonyl alkoxycarbonyl, nitro, trihalomethyl, hydroxy, C ₁ to C ₆ alkyl hydroxy, to hydroxy (C ₁ no C ₆₎ alkyl, Amino, C ₁ to C ₆ alkylamino, di (C ₁ to C ₆ alkyl) amino, aminocarboxy, C ₁ to C ₆ alkylaminocarboxy, di (C ₁ to C ₆ alkyl) aminocarboxy, aminocarboxy (C ₁ To 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 alkylcarbonylamino, C ₅ to C ₈ cycloalkyl, C ₅ to C ₈ cycloalkyl (C ₁ to C ₆ ) alkyl, C ₁ -C ₆ alkylcarbonyl (C ₁ -C ₆ alkyl) amino, halo, C ₁ -C ₆ alkylhalo, sulfamoyl, tetrazoyl and cyano.

As used herein, “halo” or “halogen” refers to iodine, bromine, chlorine or fluorine.
In order to change the redox properties of the electrolyte, the properties of R ¹ and R ² can be varied. For example, the introduction of substituents at R ¹ and R ² can change the potential at which oxidation or reduction occurs.

Preferred examples of ketone / alcohol electrolytes are 2-propanone / isopropanol and benzophenone / benzhydrol in suitable organic solvents.
Other examples of suitable electrolytes are benzoquinone / hydroquinone and their derivatives. The electrolyte can be a combination of:

Wherein R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from:
Hydrogen, halo, nitro, hydroxyl, thio, nitro, amino, optionally substituted C ₁ to C ₁₅ hydrocarbyl (where up to 3 C atoms can optionally be replaced by N, O and / or S atoms) Or C ₁ to C ₁₅ cyclohydrocarbylene in which R ³ and R ⁴ and / or R ⁵ and R ⁶ are optionally substituted together (wherein up to 3 C atoms are N, O and / or Or can be substituted with S atoms).

Preferably, R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from hydrogen, optionally substituted C _1-8 alkyl, or R ³ / R ⁴ and R ⁵ / R ⁶ are combined. To form an optionally substituted C ₅ -C ₁₂ arylene group such as phenylene.

The properties of R ³ , R ⁴ , R ⁵ and R ⁶ can be changed to change the redox properties of the electrolyte, for example to change the exact potential at which oxidation or reduction occurs. Preferred examples of electrolytes based on benzoquinone / hydroquinone derivatives are anthraquinone / anthraquinol and duroquinone / duroquinol in a suitable solvent.

  In a preferred embodiment, the electrolyte comprises a mixture of benzoquinone and hydroquinone in acetonitrile. This mixture yields an active redox product that is hydrogen ions. Hydrogen ions (protons) can be extinguished by the benzoquinone radical anion.

  Specifically, hydroquinone is oxidized at the anode to produce benzoquinone and protons:

Most of the protons liberated by the oxidation of hydroquinone are localized at the anode, and the substrate in the vicinity thereof can be modified . For example, protons can deprotect substrates having acid labile protecting groups.

  Benzoquinone is reduced at the cathode to produce a benzoquinone radical anion:

  The benzoquinone radical anion is a relatively stable species in solvents such as acetonitrile. This radical anion extinguishes incidental protons that desorb from the immediate vicinity of the anode by the following reaction:

  In this way, it is possible to improve the solubility of the substrate region to be processed by localizing active redox products generated at the electrode, for example protons generated at the anode.

  The electrolyte used in the present invention may contain a suitable solvent such as water, THF (tetrahydrofuran), methanol, ethanol, DMF (dimethylformamide), dichloromethane, diethyl ether, DMSO (dimethylsulfoxide) or acetonitrile. One skilled in the art will appreciate that the choice of solvent can affect the kinetics or equilibrium of the redox and / or quenching reactions at the electrode. Solvents can affect the reactivity of dissolved species, such as complex formation, hydrogen bonding, dipole interactions, or charge delocalization. Preferably, the solvent is an aprotic solvent capable of stabilizing radical anions. Examples of aprotic solvents are dichloromethane, DMF, DMSO, acetonitrile and THF. More preferably, the solvent is acetonitrile.

In a preferred embodiment, the electrolyte further includes a conductivity improver. The conductivity improver is a substance that increases the conductivity of the electrolyte. It is desirable to increase the conductivity of the electrolyte so that electrolysis can be carried out at a lower voltage than when no conductivity improver is present. An ionic substance that is soluble in the electrolyte is suitable for this purpose. For example, when the electrolyte includes an organic solvent such as acetonitrile, a suitable conductivity improver can be a tetra (C _1-8 alkyl) ammonium salt such as tetrabutylammonium hexafluorophosphate.

Those skilled in the art will appreciate that salts in the electrolyte can have effects other than merely increasing the conductivity of the electrolyte. The salt can affect the kinetics or equilibrium of the annihilation and / or redox reaction at the electrode. It is known that the presence of salt affects the electrostatic interaction between charged species in solution. Conversely, this can affect reactivity. For example, when the electrolyte is a hydroquinone / benzoquinone acetonitrile solution, the addition of tetrabutylammonium hexafluorophosphate was found to not only modify the extent of the quenching reaction but also increase the conductivity.

The process according to the invention can be carried out using an apparatus as described in WO 93/22480. The device described in WO 93/22480 includes an array of electrodes spaced on an insulating surface. The electrode is a platinum deposit with electrical connection means to change the potential.

However, it has been found that the method of the present invention is preferably performed using an iridium electrode. Accordingly, the present invention comprises a deposit of iridium are arranged spaced blocks of electrically insulating material, and a surface having a surface, the deposits are provided with electrical connection means for varying the potential, the An array of electrodes suitable for use in the method described in the specification is provided.

  The advantage of using iridium is that it is highly conductive and chemically inert. Furthermore, iridium does not degrade at the high potentials that can be performed using the method of the present invention. Conventionally, platinum has been used as an electrode material. However, it has been observed that platinum does not adhere well to materials such as silicon wafers, especially at high electrode potentials. The internal annihilation reaction described herein allows long-term use of a high electrode potential without significant loss of solubility in the substrate being processed. The use of a high electrode potential required electrode design changes.

  Many metals including aluminum, silver and gold were tested for suitability as electrodes. Surprisingly, however, the inventors have recognized that iridium is the best material excellent as an electrode. Iridium was found to adhere well to materials such as silicon oxide wafers without degradation during electrolysis.

  A block of material can be made from an appropriate material such as an insoluble polymer, ceramic oxide (eg, alumina) or silicon oxide wafer to form an array of electrodes. Preferably, the material is a silicon oxide wafer.

An array of iridium electrodes can be made using any suitable method. In a preferred embodiment, the electrode array is made by a method comprising the following steps:
(I) providing a silicon wafer having a silicon dioxide layer on the surface;
(Ii) deposit spaced apart iridium on the silicon dioxide surface; and (iii) anneal the iridium in air at a temperature in the range of 200-500 ° C.

In a typical method, a positive organic photoresist is applied to a silicon dioxide layer on a silicon wafer. The photoresist is exposed to UV light through a suitable photomask to expose the silicon dioxide region. Iridium metal is deposited on the surface of the material using an electron beam gun. The electrode array is then exposed except for the photoresist layer. Finally, the iridium electrode is annealed in air to improve adhesion to the wafer surface. Typically, iridium is annealed at about 350 ° C. for 15 minutes to 3 hours, preferably about 1 hour.

The annealing process is important for the adhesion of iridium to silicon dioxide. Assuming that iridium has a melting point of 2545 ° C., the effect of annealing at a temperature of about 350 ° C. is surprising. It has been observed that even in the case of a 50 nm iridium layer, the annealed electrode can withstand scratching by a steel scrampel. Furthermore, the iridium electrode can withstand the harsh chemical environment, high potential and high current that can be used in the method of the present invention.

  The electrodes are preferably an array of parallel lines spaced less than 0.5 mm. The electrodes are preferably spaced 0.1-200 microns, more preferably 1-100 microns, and even more preferably 10-60 microns.

One or more electrodes are preferably used as the counter electrode. In contrast to the usual methods of electrochemically treating a substrate, it is preferred that the substrate to be modified does not form an electrode or counter electrode. Therefore, the method of the present invention can be similar to the method described in WO93 / 22480. Furthermore, the substrate to be processed can be an electrically insulating surface.

  In a preferred embodiment, the present invention provides a method for performing several processes sequentially. Accordingly, the electrodes of the array are preferably connected to perform each treatment by changing a selected set of potentials of one or more electrodes in the array.

Preferably, in the method of the invention, the substrate to be treated comprises a substance bound to a solid surface. The solid surface can be a surface with electrodes. Preferably, the solid surface is a different surface near the electrode. The active redox product can be used to cause chemical modification of the material bound to the solid. One skilled in the art can envision many active redox products and corresponding chemical modifications . In a preferred embodiment, the substrate to be treated comprises a material having an acid labile protecting group. In this preferred embodiment, the treatment is performed by connecting at least one electrode in the array as an anode at a potential that produces acid in the electrolyte. The acid thus generated then removes the acid labile protecting group from the material bound to the surface in the region near the anode.

However, it will be understood that the active redox product can participate in various chemical reactions on the substrate. One potential application is in the electrochemical microfabrication technique described by Schuster et al., Science , 2000, 289 , 96-101. The tool described by Schuster can be adapted using the electrolyte according to the present invention and surrounding the probe tip with a ring of counter electrodes to prevent diffusion of the redox product. Thus, the present invention can be applied to existing nanoscale patterning technology. For example, acids can be used for etching or nanofabrication that requires the removal of a small amount of material from the surface.

Alternatively, the acid can be involved in an organic or inorganic reaction promoted by the acid. Those skilled in the art will be aware of the numerous potential reactions that may be suitable for use in the present invention. Epoxide opening Examples of organic reactions, addition to multiple bonds, rearrangement, substitution (e.g., S _N I-substituted tertiary alcohols), elimination, enol product with subsequent reaction of the enol, and a simple organic acid salts There is a protonation.

  Similarly, if the active redox product is a halogen, the halogen can be involved in mild oxidation, substrate bleaching or halogenation. The active redox product can also be a halide ion, which can be used in the substitution reaction.

The methods of the invention can also be used to synthesize libraries of small organic compounds bound to a surface (see, for example, Schreiber, Science 2000, 287 , 1964-1969). Small organic compound libraries are important in the field of drug discovery. The range of reactions to which this method can be applied means that it is optimal for the synthesis of the library.

  Preferably, the method of the invention is used for the stepwise chemical synthesis of oligomers such as oligonucleotides, polysaccharides and proteins. More preferably, the method of the invention is used for the synthesis of oligonucleotides.

A method for synthesizing a set of oligomers is as follows:
(A) providing a substrate to which an array of substances having protective groups, an electrolyte in contact with the substrate, and an array of electrodes in the vicinity of the substrate and in contact with the electrolyte are combined;
(B) selectively altering the potential of one or more electrodes to produce an active redox product that removes the protecting group from the selected material;
(C) couple the protective monomer with the deprotection material made in step (b); and (d) one or more electrodes selected in step (b) to synthesize a set of oligomers. While changing, repeat steps (b) and (c)
A step characterized by selecting an electrolyte such that the active redox product can be extinguished by at least one other redox product.

  When using the above method in the synthesis of the oligonucleotide, the active redox product is preferably a proton and the acid labile, such as a trityl or dimethoxytrityl (DMT) group, preferably protecting the furanyl hydroxyl group Protecting group. One skilled in the art will appreciate that this method is particularly suitable for ligated synthesis of DNA chips, as described in WO 93/22480.

  The above methods can also be used for peptide synthesis. For example, peptides can be synthesized by sequential removal of tertiary butyloxycarbonyl (Boc) protecting groups from nitrogen atoms using protons generated at the anode. The synthesis of other oligomers will be readily apparent to those skilled in the art.

The invention will be described in more detail with reference to the drawings.
Referring to FIG. 1, an electrode array is arranged on a silicon oxide wafer (1) having a high resistivity, and an iridium layer is attached to the upper surface thereof. A gap (2) is formed in the iridium layer on the silicon wafer using a positive resist photolithographic method to obtain an array (3) of parallel electrodes. The width of each electrode and each gap is about 40 microns. A silicon wafer (4) is placed on the electrode array. The silicon wafer is modified to make DMT protected nucleosides present on the surface.

  Referring to FIG. 2, the arrangement of electrodes and a portion of the substrate are shown. The central 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 electrode and the substrate to be treated. At the anode, hydroquinone is oxidized to produce benzoquinone and protons. Most protons are confined in the substrate region near the anode. Thus, the trapped proton removes the DMT group from the protected nucleotide moiety bound to the substrate. However, some of the protons can diffuse into the zone between the anode and cathode.

  At the cathode, benzoquinone radical anions are generated by reduction of benzoquinone. The benzoquinone radical anion is a relatively stable species and can diffuse into the zone between the anode and the cathode. The benzoquinone radical anion extinguishes the protons diffusing in this zone and produces hydroquinone and benzoquinone as shown. Thus, the diffused protons prevent reaction in the substrate region that is not near the anode. By preventing accidental reaction of protons in the region between the electrodes, the resolution of the patterned substrate is improved.

  FIG. 2 also shows the next processing of the substrate following said detritylation from a specific area of the substrate. The free hydroxyl group is acetylated under standard conditions to remove the remainder of the DMT group. The resulting free hydroxyl group is treated with a fluorescent dye (Cy5 phosphoramidite) to allow imaging of the substrate by confocal microscopy. Therefore, the resolution of the initial detritylation process can be suitably analyzed. However, it will be readily apparent that oligomers can be synthesized in selected regions of the substrate using the methodology described above.

  Referring to FIG. 3, the effect of electrolysis time variation at a constant potential of 1.33 V is shown using confocal microscopy. In this figure, the bright area shows the fluorescence of the substrate area where the DMT group was not removed during electrolysis. Thereafter, the residual DMT group is substituted with the fluorescent dye Cy5. The bright areas are usually adjacent cathodes. The dark area is the area where the DMT group has been removed during electrolysis. The resulting free hydroxyl group is acetylated with a non-fluorescent acetyl group. The dark areas are usually adjacent anodes.

  FIG. 3 shows that after 2.0 s, the DMT group is completely removed in the anode adjacent region. Furthermore, the resolution of the patterned substrate remains unchanged after 80s. There are sharply shown streaks that correspond to the areas near the anode and cathode. This indicates that even after prolonged electrolysis, protons generated during electrolysis are completely confined in the region near the anode.

  Figures 4 (a) and (b) show the significant effect of removing the cathode from the array of electrodes. The dark area indicates the area where the DMT group has been removed. The electrode potential is fixed at 1.33V. Once the central cathode is removed, protons generated at the anode are free to diffuse into the central region. This clearly shows the confinement effect of having a cathode that produces a species that can annihilate protons generated in the ano.

The method of the present invention is described in further detail in the following examples.
(Example)
(Experimental part)
(Electrode assembly)
An iridium metal (thickness 50 nm) electrode was formed on a high resistivity silicon oxide wafer using a normal positive resist photolithography method. A silicon oxide wafer was coated with a positive photoresist layer and exposed to UV light through a photomask. The wafer was washed with deionized water, baked at 100 ° C. for 20 minutes, and then “de-scummed” by reactive ion etching. The photomask was chosen to obtain an array of 96 parallel electrodes approximately 7500 microns long and 40 microns wide. The gap between adjacent electrodes was about 40 microns.

Iridium was deposited on the wafer by the electron beam method. Iridium metal was placed in the crucible in the vacuum evaporator and a few wafers were placed about 20 cm from the crucible in the vacuum evaporator. The chamber was evacuated to 3 × 10 ⁻⁶ Torr and the wafer was coated with 500 nm iridium by heating the metal with an electron beam gun set at 5 kV and 300 mA for about 3 minutes.

  The wafer was then placed in an ultrasonic acetone bath for 30 minutes to remove the photoresist layer, thus exposing the array of electrodes. The electrode was annealed in air at 350 ° C. for about 1 hour to increase the adhesion with the wafer substrate and was cleaned by reactive ion etching.

After heating annealing and cleaning process, each electrode is connected to printed circuit board by ultrasonic gold wire bonding, where digitally controlled “analog switch” integrated circuit activates selected electrode for certain deblocking process Make it. Current was applied as a multiple independent operability amplifier control voltage source. The parallel low noise instrumentation amplifier feedback circuit constantly measured the nanoamp precision current at each electrode. A computer specially programmed for this operation controlled all voltage, timing, and electrode switching and collected current measurement data.

(Solid support assembly)
A polished silicon dioxide wafer was used as a patterned substrate support. Prior to electrochemical patterning, the wafer surface was functionalized with a linker molecule to bind organic reagents (Gray, DE, CaseGreen, SC, Fell, TS, Dobson, P J. & Southern, EM Ellipsometric and interferometric char- acterization of DNA probes on a combinatorial array. Langmuir 13 , 1933-42). The wafer was placed in a vacuum furnace chamber with a volume of 19.1 l along with 5 ml glycidoxypropyltrimethoxysilane containing ampoule. After heating the furnace to 185 ° C, the ampoule was heated to 205 ° C and the chamber was evacuated to 25-30 mBar. After evaporation of about 2.5 ml of silane, the chamber was cooled under reduced pressure (10 ⁻³ Torr). “Linker molecules” were attached by immersing the glycidoxypropyltrimethoxysilane-derived wafer in a 100% solution of polyethylene glycol in traces of sulfuric acid. Followed by conventional oligonucleotide synthesis methods, and the DMT-containing phosphoramidite is covalently bonded to the free hydroxyl of polyethylene glycol (Beaucage, S.L. & Iyer, R.P.Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach. Tetrahedron 48 , 2223-2311 (1992)). The wafer substrate thus prepared was cut into 1 cm square for use in patterning.

(Example 1)
The array of electrodes (prepared as described above) was placed at a distance of 20 microns from the solid support. A solid support was prepared as described above to attach thymidine phosphoramidite to a polyethylene glycol linker molecule. Thymidine phosphoramidite protected the 5'-hydroxyl group with a DMT group.

  An electrolyte solution (25 mM hydroquinone / 25 mM quinone / 25 mM tetrabutylammonium hexafluorophosphate in anhydrous acetonitrile) was introduced into the cavity between the electrode array and the solid support. The selected anode was then set to 1.33 V with respect to the cathode, and the voltage was maintained from 2 to 80 s, as shown in FIG.

  After electrolysis, the silicon wafer was washed with acetonitrile and acetylated with acetic anhydride using standard methods. In this step, only the DMT deprotected portion of the silicon wafer having an exposed hydroxy group was acetylated.

  Then, the entire substrate was treated with a dichloromethane solution of dichloroacetic acid to remove DMT groups that were not removed by the electrochemical step. By electrochemically producing the acid, the hydroxyl group thus exposed is coupled to the fluorescent dye Cy5 using standard phosphoramidate coupling methods, so that the fluorescence of Cy5 is observed by confocal microscopy. The resulting pattern was revealed. This process sequence is shown in FIG.

FIG. 3 shows the effect of increasing electrolysis time at 1.33V. Once the maximum bandwidth is reached after about 2.0 s, the resolution of the substrate is maintained even after 80.0 s of electrolysis.
(Example 2)
Example 1 was repeated exactly as described above, except that the selected anode maintained a voltage of 1.33 V for 16 s. As shown in FIGS. 4 (a) and (b), the significant effect of cathode removal was investigated. When the central cathode is removed, there is no control of diffused protons. The diffused protons can sneak into the central region and do not localize around the anode. This is demonstrated by the central dark region that does not contain the fluorescent group of FIG.

(Example 3)
In the synthesis of 17-mer oligomers on a solid support, the general method described in Example 1 was used along with the 16 steps of electrochemically controlled DMT deprotection. The method of use was the same as in Example 1, except that the electrode array was placed at a distance of 40 microns from the substrate surface.

  A uniform coating of dimethoxytrityl (DMT) protected deoxyadenosine (dA) residues was coupled to a polyethylene glycol linker group on a solid support using standard phosphoramidite coupling chemistry.

  After thorough cleaning with acetonitrile, the electrolyte used in Example 1 was introduced into the cavity between the electrode array and the solid support. A potential of 1.33 V was applied to the selected anode for 9 s to remove the DMT group in the vicinity thereof. Two anodes surrounded the side of the anode.

Following further washing with acetonitrile, DMT protected nucleotide residues were coupled to exposed hydroxyl groups using standard phosphoraramid coupling methods. The obtained trivalent phosphorus bond was oxidized with iodine to obtain a pentavalent phosphorus bond, and the entire silicon wafer was washed with acetonitrile followed by dichloromethane. Standard coupling and oxidation steps used to synthesize oligonucleotides on solid supports are well known in the art (see, eg, ABI Synthesizer Manual , Section 2: “Chemistry for Automated DNA Synthesis”).

  This method was repeated, changing the DMT protected nucleotide residues introduced into the phosphoramidite coupling step. In this way, oligonucleotides were synthesized on the solid support.

  Two 17-mer oligonucleotides: This method with an appropriately programmed computer-controlled automated device in the synthesis of wild type “A” human hemoglobin mRNA and the corresponding “S” sickle erythrocyte mRNA. It was used. The 17-mer formed in excellent yield on a constant strip of solid support. One skilled in the art will readily appreciate that this method can be used for ligated synthesis of DNA chips, as described in WO 93/22480.

  Of course, it will be understood that the present invention has been described by way of example only and that variations in detail fall within the scope of the invention.

FIG. 1 shows an apparatus suitable for carrying out the method of the present invention. FIG. 2 is a diagram illustrating a method by which selected regions of a substrate can be modified . FIG. 3 shows the effect of varying the electrolysis time. FIG. 4 shows the effect of removing the cathode from the electrode array.

Claims (32)

  A method for treating a substrate, comprising: an electrolyte in contact with the substrate; and an electrode in the vicinity of the substrate and in contact with the electrolyte, wherein the potential of at least one first electrode is set in the vicinity of the first electrode. Producing an active redox product that modifies the substrate of the second electrode and generating a second redox product with a second electrode in the vicinity of the first electrode, wherein the electrolyte is activated by the second redox product. That the redox product is quenchable and that the substrate to be modified does not form either the first electrode or the second electrode and is separated from the first and second electrodes; A method characterized by being.   The method of claim 1, wherein the electrolyte comprises a solvent, and neither the active redox product nor the second redox product is formed from the solvent.   The method of claim 1 or 2, wherein the electrolyte comprises an organic solvent.   4. A process according to claim 3, wherein the solvent is selected from tetrahydrofuran (THF), methanol, ethanol, dimethylformamide (DMF), dichloromethane, diethyl ether, dimethyl sulfoxide (DMSO), or acetonitrile.   The method according to claim 1, wherein an extinction reaction regenerates the electrolyte.   6. The method according to any one of claims 1 to 5, wherein the active redox product is a proton.   The method according to any one of claims 1 to 6, wherein the second redox product is an organic radical anion.   The method according to any one of claims 1 to 7, wherein the electrolyte is a solution of hydroquinone and benzoquinone, or a derivative thereof. Electrolyte

{Wherein R ³ , R ⁴ , R ⁵ and R ⁶ are:
Hydrogen, halo, hydroxyl, thio, nitro, amino, optionally substituted C ₁ -C ₁₅ hydrocarbyl (where up to 3 C atoms can optionally be replaced by N, O and / or S atoms) Or R ³ and R ⁴ and / or R ⁵ and R ⁶ together are optionally substituted C ₁ -C ₁₅ cyclohydrocarbylene (wherein up to 3 C atoms Is optionally formed by N, O and / or S atoms).   The method according to any one of claims 1 to 9, wherein the electrolyte is an acetonitrile solution of hydroquinone and benzoquinone.   The method according to claim 1, wherein the electrolyte further comprises a conductivity improver. The method of claim 11, wherein the conductivity enhancer is a tetra (C _1-8 alkyl) ammonium salt.   The method of claim 12, wherein the conductivity enhancer is tetrabutylammonium hexafluorophosphate.   14. A column according to any of claims 1 to 13, wherein the electrodes of the column are connected to perform each treatment by changing the potential of a selected set of one or more electrodes of the column to perform several processes sequentially. The method according to claim 1.   15. A method according to any one of the preceding claims, wherein the substrate comprises an array of materials bound to the surface.   The method of claim 15, wherein the surface is a surface of a silicon oxide wafer.   The method of claim 15, wherein the material to be treated comprises an acid labile protecting group.   18. The method of claim 17, wherein each treatment is performed by connecting at least one electrode in a row as an anode at a potential that removes acid labile protecting groups from the material on the surface.   19. A method according to any one of claims 14 to 18, wherein the or each treatment is performed during the stepwise chemical synthesis of the oligomer. (A) providing a substrate bonded to a row of substances having a protecting group, an electrolyte in contact with the substrate, and a row of electrodes in the vicinity of the substrate and in contact with the electrolyte;
(B) (i) an active redox product that removes the protecting group from the selected material, and the potential of one or more of the electrodes is selectively selected to yield a second redox product. Change;
(C) coupling the protected monomer with the deprotected material produced in step (b); and (d) one or more selected in step (b) to synthesize a set of oligomers. The step of repeating steps (b) and (c) while changing the electrode of the method, wherein the electrolyte is selected so that the active redox product can be extinguished by the second redox product. A method of synthesizing a set of oligomers wherein the substrate does not form an electrode and is separated from the row of electrodes.   21. The method of claim 20, wherein the electrolyte comprises a solvent and neither the active redox product nor the second redox product is formed from the solvent. The method according to claim 20 or 21, wherein the electrolyte comprises an organic solvent.   23. A process according to claim 22, wherein the solvent is selected from tetrahydrofuran (THF), methanol, ethanol, dimethylformamide (DMF), dichloromethane, diethyl ether, dimethyl sulfoxide (DMSO) or acetonitrile.   24. A method according to any one of claims 20 to 23, wherein the extinction reaction regenerates the electrolyte.   25. A method according to any one of claims 20 to 24, wherein an array of substances is bound to a surface and the oligomer is synthesized on the surface.   26. The method according to any one of claims 20 to 25, wherein the oligomer is an oligonucleotide.   27. A method according to any one of claims 20 to 26, wherein the active redox product is a proton and the protecting group is an acid labile protecting group.   28. The method according to any one of claims 20 to 27, wherein the electrolyte is a solution of hydroquinone and benzoquinone, or derivatives thereof. Electrolyte

{Wherein R ³ , R ⁴ , R ⁵ and R ⁶ are:
Hydrogen, halo, hydroxyl, thio, nitro, amino, optionally substituted C ₁ -C ₁₅ hydrocarbyl (where up to 3 C atoms can optionally be replaced by N, O and / or S atoms) Or R ³ and R ⁴ and / or R ⁵ and R ⁶ together are optionally substituted C ₁ -C ₁₅ cyclohydrocarbylene (wherein up to 3 C atoms 29. The method according to any one of claims 20 to 28, wherein can be optionally substituted with N, O and / or S atoms.   30. The method according to any one of claims 20 to 29, wherein the electrolyte is an acetonitrile solution of hydroquinone and benzoquinone.   31. A method according to any one of claims 20 to 30, wherein the electrolyte further comprises a conductivity improver. 32. The method of claim 31, wherein the conductivity enhancer is a tetra ( _C1-8 alkyl) ammonium salt.

Images