CN100441283C - Treatment of substrates - Google Patents

Treatment of substrates Download PDF

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Publication number
CN100441283C
CN100441283C CNB02817030XA CN02817030A CN100441283C CN 100441283 C CN100441283 C CN 100441283C CN B02817030X A CNB02817030X A CN B02817030XA CN 02817030 A CN02817030 A CN 02817030A CN 100441283 C CN100441283 C CN 100441283C
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electrolyte
electrode
substrate
array
protecting group
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CN1549744A (en
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爱德文·麦勒·沙森
赖安·爱戈兰德
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Oxford Gene Technology IP Ltd
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    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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Abstract

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

Description

A kind of substrate treating method
[affiliated technical field]
The present invention relates to a kind of method of substrate being handled with electrochemical techniques, particularly a kind of method of substrate being repaired (modify) with electrochemical techniques.
[background technology]
Many devices need form the specific pattern of certain material on its surface, and semiconductor chip is exactly an obvious example.The DNA chip that occurs also has an oligonucleotide array recently, and this array is attached to a surface of solids [G. Ramsay (G.Ramsay), " Nature Biotechnol ", 1998, volume .16,40～44].
The performance of this type of device depends on the pattern and the character of surfacing.In addition, the improvement conventional device is the demand for miniaturization on the one hand, is the needs of the novel surface of pair set chemistry, physical property on the other hand.Therefore, making the device that its surface has required pattern with new method is necessary in fact.
The method of at present surperficial specific region being handled has several.A kind of method is a photolithographic techniques.The specific region on surface is covered by the lithoprinting mask, and the zone of exposing is repaired by the ultraviolet ray irradiation.This method has been widely used in the semiconductor manufacturing, and the semiconductor wafer surface that utilizes the method to be coated with the photoetching agent can form the hole.
Another kind of photolithographic techniques also is applied to the manufacturing of DNA chip.In the method, the oligonucleotide with light unstability protecting group is formed on the surface of solids, and some zone of photolithography mask covering surfaces utilizes ultraviolet ray that the zone of exposing on surface is shone then.At last, be covered in the oligonucleotide that exposes the zone and be removed [G. Ramsay (G.Ramsay), " Nature Biotechnol ", 1998, volume .16,40～44].
WO93/22480 has disclosed a kind of method of handling with electrochemical techniques his-and-hers watches face.In the method, electrolyte floats on the surface, and electrod-array is pressed close to this surface.The electromotive force of the one or more electrodes by changing electrod-array is trimmed near the surface of these one or more electrodes.This electrolyte is the acetonitrile solution of triethylamine and sulfuric acid.
United States Patent (USP) the 6th, 093 has disclosed a kind of electrochemical method that material is placed the substrate ad-hoc location No. 302.This material production is at an electrode, and the material with close electrode reacts then.Simultaneously, this patent has been used a kind of buffering or purified solution (a buffering or scavengingsolution).The effect of sort buffer or purified solution is to improve the discrimination rate of substrate, and this substrate is handled by reacting with the reagent of crossing from contiguous electrode stream.Include the buffering or the solution of cleaning of substances in a large number and not only can suppress to be distributed in the reagent of special electrodes, and can suppress to be used for reagent with the substrate reaction of adjacent specific electrode.
This Goethe people such as (Schuster) is in " science ", and 2000,289,98～101 have disclosed another kind utilizes the surface electrochemistry treatment technology to improve the method for discrimination rate.This Goethe has utilized complicated current impulse to limit diffusion time.
[summary of the invention]
The object of the present invention is to provide a kind of with electrochemical means to improving one's methods that substrate is repaired, a kind of method of repairing out the substrate with higher discrimination rate particularly is provided.
For realizing this purpose: the method that the invention provides the first redox material diffusion of a kind of control first electrode generation, it comprises: produce the second redox material at second electrode near above-mentioned first electrode, first and second electrodes contact with electrolyte, and wherein above-mentioned electrolyte can make the first redox material be suppressed by the second redox thing.
Further improvement of the present invention is: second electrode is an auxiliary electrode.
Further improvement of the present invention is: this first redox material is the active oxidation reducing substances, and it can be used for repairing the substrate of adjacent electrode.
In addition, the invention provides a kind of method of handling substrate, it comprise provide an electrolyte that contacts with substrate and one or more near substrate and with the electrolyte electrodes in contact; Change at least one electrode to produce the active oxidation reducing substances, it can repair the substrate near this at least one electrode; This electrolyte can make this active oxidation reducing substances be suppressed by the second redox material.
The meaning that suppresses is that the second redox material can and change its reactive mode with the first redox substance reaction, and this first redox material can not be reacted in original mode.When the first redox material is the active oxidation reducing substances, the reaction between this active oxidation reducing substances and the second redox material will prevent active oxidation reducing substances finishing substrate.For example, when the active oxidation reducing substances is acid, the second redox material may be an alkali.Reaction between this acid and the alkali suppresses acid and prevents its finishing substrate.
Compared to prior art, electrolyte of the present invention can make the active oxidation reducing substances be suppressed by at least a other redox materials, makes the substrate of gained have high discrimination rate.
[description of drawings]
Below with reference to accompanying drawing the present invention is elaborated:
The equipment that Fig. 1 Display Realization method of the present invention is used;
Fig. 2 shows how the selection area of substrate is trimmed;
The effect of Fig. 3 display change electrolysis time;
Fig. 4 shows the effect that removes a negative electrode from electrod-array.
[specific embodiment]
In the preferred embodiment of invention, inhibitory reaction can regenerate one or more materials in electrolyte.
By the active oxidation reducing substances, any oxidation or go back original product and can repair substrate.This active oxidation reducing substances can directly produce by the material in oxidation or the reduction electrolyte.Or this active oxidation reducing substances by oxidation or reduction the material in the electrolyte, and then one or more reaction take place and obtain with other materials in the electrolyte.
Usually, this redox thing is created on electrode surface.The redox material can be repaired its contiguous substrate.Acid is preferred active oxidation reducing substances, and many kind reactions can take place in substrate for it, for example, eliminate (eliminations), displacement, reformation and chemical etching.When the active oxidation reducing substances was acid, preferably it was used to remove suprabasil sour unstability protecting group (acidlabile protecting group).
Acid unstability protecting group has been known by those skilled in the art to be known; it includes acetal (as methoxyl methyl, dimethyl sulphide, (2-methoxy ethoxy) methyl; methyl benzoate, β (trimethyl silicane) ethoxymethyl, THP trtrahydropyranyl, benzylidene, isopropylidene, cyclohexylene and ring pentylidene); ester (as benzoyl, benzoic acid carbonyl and tert-butoxy carbonyl); ether (as trityl, dimethoxytrityl and tertiary butyl), and silyl ether (silyl ethers) (as tertiary butyl dimethyl-silicon, trimethyl silicane and triethyl group silicon).Acid unstability protecting group is preferably trityl ether or dimethoxytrityl (DMT) ether, and they are normally used for the synthetic of oligonucleotide.
Similarly, this active oxidation reducing substances can be a kind of alkali, and many kind reactions can take place in substrate this alkali.For example, be used to remove alkali unstability protecting group.
Alkali unstability protecting group is that those skilled in the art know and know that it comprises 9-fluorenes methoxy carbonyl acyl (Fmoc) and cyanoethyl (Cyanoethyl).
Free radical (Radicals) is another kind of active oxidation reducing substances.Free radical is used for causing radical reaction in substrate.The electrochemical method that is used to produce free radical is that those skilled in the art know and know.Usually the electrochemical method of used generation free radical is carboxylate anion's oxidation.
Halogen is another kind of active oxidation reducing substances, and for example, it is used to carry out oxidation reaction or addition reaction in substrate.Halogen can obtain by the corresponding halide ion of oxidation.
These or other active oxidation reducing substances all is apparent and easy to know to those skilled in the art.
Method of the present invention can be used for substrate is handled.Substrate of the present invention can be adopted any material or the material that links to each other with electrode and can be repaired by the active oxidation reducing substances.This substrate places near the electrode place, it can be moved apart electrode after redox reaction is finished.In addition, substrate also is attachable to electrode, perhaps pastes thereon the surface as electrode.When needing, after redox reaction is finished, substrate can be taken off from electrode or above-mentioned surface.
Therefore, in one embodiment, substrate is the surface of a material, itself and electrode separation but near electrode.This substrate can be the surface of glass, plastics, solid fiber, metal, semiconductor or gel rubber material.The surface of these materials can directly be repaired by redox reaction.In addition, in this embodiment, other materials can be enclosed in the surface of these materials.For example, to enclose organic compound be exactly an existing method on the surface of these materials.The material that is attached to these material surfaces can be repaired by redox reaction.
In another embodiment, substrate can be a kind of material that invests as the surface of electrode.Perhaps this substrate is a kind of material that is attached to electrode itself by linking group.United States Patent (USP) the 6th, 093 has disclosed back a kind of method No. 302, and its substrate invests electrode by linking group.
Method of the present invention is similar to the method that WO93/22480 discloses.Yet the selected electrolyte of method of the present invention is different with it.The used electrolyte of WO93/2240 is the acetonitrile solution of triethylamine and sulfuric acid.The active oxidation reducing substances can be suppressed by at least a other redox materials in electrolyte of the present invention.This electrolyte accurately restricted activity redox material to the peripheral region of the electrode that produces this active oxidation reducing substances.
In the described method of WO93/22480, be to control by the variation of electrode potential in the restriction of the acid of specific region.After yet the present inventor found long-time electrolysis, when electrolyte was the acetonitrile solution of triethylamine and sulfuric acid, acid lost restriction.Acid can not will be caused the discrimination rate of processed substrate to reduce by effective restriction.For example, the proton that goes out from the contiguous diffusion of anode can with react at interelectrode basal region.Obtain to have the substrate of high discrimination rate pattern, it is undesirable that the proton of diffusion by this mode accidental reaction takes place.According to the selected electrolyte of the present invention, the problem of prior art electrolyte can be avoided.Electrolyte of the present invention can make the active oxidation reducing substances be suppressed by at least a other redox materials, and this is a key character of the present invention.
Those skilled in the art will know that many electrolyte, it can produce the active oxidation reducing substances that is suppressed by another redox material.
For example, a kind of electrolyte, it is I -And S 4O 6 2-Bond.Iodide produce iodine (a kind of active oxidation reducing substances) in the oxidized meeting of anode, and at S 4O 6 2-Being reduced at negative electrode can generation S 2O 3 2-, it can suppress iodine and produce at anode.Reaction at electrolyte can be expressed as follows:
Anode: 2I --2e -→ I 2
Negative electrode: S 4O 6 2-+ 2e -→ 2S 2O 3 2-
Iodine is suppressed by following reaction: 2S 2O 3 2-+ I 2→ S 4O 6 2-+ 2I -
This active oxidation reducing substances is preferably acid, and this suppresses redox material is anion, is preferably organic anion (Radical anion).Usually, acid is that it is aliphatic alcohol, or aromatic alcohols by the pure oxidation generation of anode.In this electrolyte, the anion of this inhibition produces in cathodic reduction by suitable material usually.Many materials can be reduced and produce anion at negative electrode, and can suppress acid and form at anode.For example, the oxygen molecule of dissolving can be in cathodic reduction, thereby produce O 2 -And/or O 2 2-
For example, a kind of electrolyte, it can produce suitable redox material, and this redox material is the bond of ketone and corresponding alcohol.Alcohol can oxidation produces proton (a kind of active oxidation reducing substances) at anode, and ketone can reduce the generation anion at negative electrode, and it can suppress proton and produce at anode.
Reaction in this electrolyte can be expressed as follows:
Anode: R 1CH (OH) R 2→ R 1C (O) R 2+ 2H ++ 2e -
Negative electrode: R 1C (O) R 2+ e -→ [R 1C (O) R 2] -
Wherein, R 1And R 2Can select the C that replaces respectively 1To C 15Alkyl, wherein three above carbon atoms can be replaced by N, O and/or S atom arbitrarily; Perhaps R 1And R 2Form the C that replaces together 1To C 15Cyclic alkylene (cyclohydrocarbylene), wherein three above carbon atoms can be replaced by N, O and/or S atom arbitrarily.
Preferably, R 1And R 2Can select the C that replaces respectively 1-8Alkyl, C 3-8Cycloalkyl or phenyl.
" alkyl " of the present invention is meant monoradical, and it contains carbon and hydrogen.Therefore alkyl comprises alkyl, alkylene and alkynyl (in straight chain and branch chain structure), cycloalkyl (comprising poly-cycloalkyl), cycloolefin and aryl, with the bond of above group, alkyl-cycloalkyl for example, alkyl gathers cycloalkyl, alkylaryl, alkylene aryl, alkynyl aryl, cycloalkyl aryl and cycloalkenyl group aryl.
" alkylene (hydrocarbylene) " of the present invention is meant divalent group, and it contains carbon and hydrogen.This comprises cycloalkanes support or ring alkylidene, ring alkenylene (cycloalkenylene) and arylene or arlydene cyclic alkylene.
" aryl " of the present invention is meant aromatic group, for example, and phenyl, naphthyl or anthryl.Or when aryl had carbon atom by O, N and/or S replacement, this aryl was meant fragrant heterocyclic radical, for example, and pyridine radicals, pyrrole radicals, thienyl, furyl, imidazole radicals, triazolyl, quinolyl, isoquinolyl, oxazolyl or different azoles base.
Substituting group of the present invention can be selected C 1To C 6Alkyl, C 1To C 6Alkoxyl, sulfo-, C 1To C 6Alkylthio, carboxyl, carboxyl (C 1To C 6) alkyl, formyl, C 1To C 6Alkyl-carbonyl, C 1To C 6The alkyl-carbonyl alkoxyl, nitro, three halides, hydroxyl, C 1To C 6Hydroxyalkyl, hydroxyl (C 1To C 6) alkyl, amino, C 1To C 6Alkyl amino, two (C 1To C 6) amino, amino carboxyl, C 1To C 6Alkyl amino carboxyl (alkylaminocarboxy), two (C 1To C 6Alkyl) diaminourea carboxyl, amino carboxyl (C 1To C 6) alkyl, C 1To C 6Alkyl amino carboxyl (C 1To C 6) alkyl, two (C 1To C 6Alkyl) amino carboxyl (C 1To C 6) alkyl, C 1To C 6Alkyl-carbonyl amine, C 1To C 6Cycloalkyl, C 1To C 6Cycloalkyl (C 1To C 6) alkyl, C 1To C 6Alkyl-carbonyl (C 1To C 6Alkyl) amino, halogen-containing, C 1To C 6Haloalkyl, ammonia sulphur anilide, tetrazole radical and cyano group.
" halogen-containing " of the present invention or " halogen " are meant iodine, bromine, chlorine or fluorine.
R 1And R 2The redox characteristic of character decision electrolyte.For example, R 1And R 2On replacement reduction can be by oxidation or reduction change electromotive force.
Ketone/pure electrolyte is preferably the organic solution of 2-acetone/isopropanol and Benzophenone/benzohydrol.
Another electrolyte is benzoquinones/hydroquinones and its derivative.This electrolyte can be the mixture of following material:
Figure C0281703000151
Wherein R3, R4, R5 and R6 can select respectively: hydrogen, halogen, nitro, hydroxy, thio group, amino, the C of replacement 1To C 15Alkyl, wherein three above carbon atoms can be substituted by N, O and/or S atom.Also R3 and R4, and/or R5 and R6 are in conjunction with forming the C that replaces 1To C 15Cyclic alkylene, wherein three above carbon atoms can be substituted by N, O and/or S atom.
R3, R4, R5 and R6 are preferably hydrogen, C 1-8Alkyl or R3/R4 and R5/R6 in conjunction with forming the C that replaces 5-12Arylene (arylene), for example phenylene.
The character of R3, R4, R5 and R6 can determine the redox property of electrolyte, for example, oxidation takes place or reduce to change accurate electromotive force.The electrolyte of the derivative of benzoquinones/hydroquinones is preferably the organic solution of anthraquinone/oxanthranol and duroquinone/duroquinol.
In a preferred embodiment, electrolyte comprises the acetonitrile mixed liquor of benzoquinones and hydroquinones.This mixed liquor provides an active oxidation reducing substances, and it is a hydrogen ion.This hydrogen ion (proton) can suppress the benzoquinones anion.
Hydroquinones is oxidized and produce benzoquinones and proton at anode.
Figure C0281703000161
Hydroquinone oxidation and the proton major part that discharges rests on anode, it can repair its contiguous substrate.For example, this proton can be separated the substrate of protection (deprotect) with sour unstability protecting group.
Benzoquinones is reduced at negative electrode and produces the benzoquinones anion:
Figure C0281703000162
This benzoquinones anion is stable in solution, for example acetonitrile solution.Anion can suppress near any proton from fleeing from out the anode, and this reaction can be expressed as follows:
Figure C0281703000163
Like this, rest on suprabasil certain zone by the active oxidation reducing substances that electrode is produced, discrimination rate that then should the zone can improve, and for example, proton is created in anode.
Electrolyte of the present invention comprises any suitable solvent, as water, oxolane (THF), methyl alcohol, ethanol, dimethyl formamide (DMF), carrene (dichloromethane), ether, methyl-sulfoxide (DMSO) or acetonitrile.Those skilled in the art should know that choice of Solvent can influence at the redox reaction of electrode and/or the balance or the power of inhibitory reaction.This solvent can influence the activity of some structure in the solution, for example, " the non-localityization " of blending structure, hydrogen bond, dipole-dipole or electric charge (delocalisation).Solvent is preferably non-protonic solvent (aproticsolvent), and it can stablize anion.Non-protonic solvent has: carrene, dimethyl formamide, methyl-sulfoxide, acetonitrile and oxolane.Acetonitrile is more preferably solvent.
In a preferred embodiment, electrolyte can comprise conductibility stiffener (conductivityenhancer) in addition, is used to improve the electric conductivity of electrolyte.It is lower than the voltage of inconductivity stiffener to add the required decomposition voltage of conductibility stiffener.Any ion that dissolves in electrolyte all can reach this purpose.For example, when electrolyte comprises organic solvent, acetonitrile for example, then the conductibility stiffener is four (C 1-8Alkyl) ammonium salt, for example, ammonium hexafluorophosphate (tetrabutylammoniumhexafluorophosphate).
Those skilled in the art will know that salt has its effect in electrolyte, rather than only increase the electric conductivity of electrolyte.Salt can influence the inhibitory reaction on electrode and/or the balance or the power of redox reaction.Salt can influence the electrostatic interaction power of charged thing in the solution, correspondingly also can influence reaction power.For example, when electrolyte was the acetonitrile solution of hydroquinones/benzoquinones, the ammonium hexafluorophosphate that other adds can change the degree of inhibitory reaction, also can increase electric conductivity.
Method of the present invention is to realize by the disclosed equipment of WO93/22480.The disclosed equipment of WO93/22480 comprises electrod-array, and its compartment of terrain is distributed in insulating surface.This electrode plates platinum, is used to provide the arrangements of electric connection (electricalconnecting means) that changes its electromotive force.
Yet method electrode used therein of the present invention is preferably iridium electrode.Electrod-array provided by the invention, it comprises an insulating materials with a surface, forms the iridium deposit of array on this surface with being separated by, each deposit is provided as the arrangements of electric connection that changes its electromotive force.
The advantage of iridium is its high conductivity and chemical inertness.In addition, iridium can not degenerated under the high potential of method of the present invention.Platinum has been widely used as electrode material, however platinum can not be attached to well on some materials, as silicon wafer, particularly under the high electrode electromotive force.Under the situation that the discrimination rate of processed substrate does not reduce, inner inhibitory reaction needs to use for more time the high electrode electromotive force, and this just need the existing electrode design of change.
Many metals are tested to be fit to do electrode, comprises aluminium, silver and golden.Yet it is the good material of making electrode that the present inventor is surprised to find iridium.Iridium can not be degenerated in electrolyte and can be attached to well on the material, as the silicon wafer material of oxidation.
Block of material on electrod-array can be undissolved polymer, ceramic oxide (as aluminium oxide) or silicon wafer.Be preferably the silicon wafer of oxidation.
The iridium electrode array can be by many suitable method systems.In a preferred embodiment, electrod-array can be obtained by following method:
(i) provide a silicon wafer, its surface has a silicon dioxide layer;
(ii) iridium on compartment of terrain deposition on the silicon dioxide layer makes it form array;
(iii) under 200～500 ℃ of temperature, iridium is annealed in air.
In common processing procedure, just organic photoetching agent is applied to the silicon dioxide layer of silicon wafer.Cover photomask, be exposed to ultraviolet light then, the photoetching agent that exposes is removed, the silicon dioxide zone at the photoetching agent place of removing is exposed.Iridium metals is deposited on the silicon dioxide zone of this exposure with electron beam gun.The zone that removes photoetching agent layer can form electrod-array.At last, iridium electrode is annealed in air to improve the adhesion in wafer surface.Usually, iridium is to anneal 15 minutes to 3 hours down at 350 ℃, is preferably about 1 hour.
Be adhered to silica for iridium, annealing steps is important.Annealing temperature is approximately 350 ℃, and iridium can have 2545 ℃ fusion temperature.Even find to have the scratching of annealing electrode ability steel scalpel of 50nm iridium layer.In addition, the abominable chemical environment of iridium electrode ability and the high potential and the high electric current of method of the present invention.
Preferably, electrode is the array of parallel lines of each interval, and it is less than 0.5 millimeter at interval.Preferably, electrode gap is 0.1～200 micron, more preferably 1～100 micron, is more preferably 10～60 microns.
One or more electrodes are as auxiliary electrode (counter electrode).Preferably, with respect to the method for existing electrochemical treatments substrate, desire of the present invention is handled substrate can not form an electrode or an auxiliary electrode.Method of the present invention is similar to the described method of WO93/22480.And the substrate that this desire is handled can be insulating surface.
In preferred enforcement, the invention provides a kind of method that realizes several treatment steps in regular turn.The electrode of array is coupled together,, can finish a treatment step so that change the electromotive force of selected one or more electrodes of array.
In the method for the invention, the substrate of this desire processing comprises that one invests the material of the surface of solids.This surface of solids can be pressed close to electrode.Preferably, this surface of solids is the surface inequality of adjacent electrode.The redox material can be used for repairing the material that invests the surface of solids.Those skilled in the art can find out many redox materials and corresponding chemical finishing.In a preferred embodiment, the substrate of desire processing comprises the material with sour unstability protecting group.In the preferred embodiment, by at least one electrode in the array is connected power supply and with it as anode, can realize treatment step, this anode is used for producing acid at electrolyte.The acid of this generation can remove the sour unstability protecting group that is attached to surface and close anode region.
The active oxidation reducing substances participates in the number of chemical reaction of substrate and is approved.A kind of potential application be Si Gede people such as (Schuster) in " science ", 2000,289,98～101 electrochemistry micro mechanical technologies that disclose.The disclosed instrument of this Goethe can be used in the electrolyte of the present invention, centers on probe pinpoint in case the diffusion of oxidation reducing substances with the auxiliary electrical polar ring.The present invention will use existing nanoscale pattern technology.For example, acid just can be used for etching or nanometer processing procedure, and it can remove material in a small amount from a surface.
In addition, acid can participate in many organic or inorganic reactions.Those skilled in the art know many potential reactions of the present invention that can be used for.For example, organic reaction comprises open loop of epoxy compound, is connected to multikey, rearrange (rearrangement), replace (as, the unimolecular nucleophilic substitution (S of the tertiary alcohol N1)), the generation of elimination, enol and acylate is simply protonated.
When the active oxidation reducing substances is a halogen, it can participate in mild oxidation, bleaching substrate or halogenation.The active oxidation reducing substances is halide ion also, and it can be used for substitution reaction.
Method of the present invention also can be used for being attached to synthetic [the seeing Si Geleibo (schreiber), " science ", 2000,287,1964～1969] of the low organic compound on a surface.Low organic compound is important in the drug invention field.The applied reaction range of the present invention means and is suitable for the synthetic of such material in theory.
Method of the present invention can be used for the progressively synthetic of oligomer, as, oligonucleotide, polysaccharide and protein.Preferably, method of the present invention can be used for the synthetic of nucleotides.
A kind of method of a synthetic cover oligomer, it may further comprise the steps:
(a) provide a substrate, this substrate is with an array with material of protecting group, and an electrolyte contacts with substrate with the close substrate of an electrod-array and with electrolyte and contacts;
(b) optionally change the electromotive force of one or more electrodes, to produce the active oxidation reducing substances, this active oxidation reduzate removes above-mentioned protecting group from selected material;
(c) a protection monomer is attached to form in the step (b) separate the protection material;
(d) repeating step (b) and (c) changes one or more electrodes selected in the step (b), with a synthetic cover oligomer;
It is characterized in that electrolyte can make the active oxidation reducing substances be suppressed by at least one other redox material.
When said method was used to low nuclear former times acid synthetic, the active oxidation reducing substances was preferably proton, and protecting group is preferably sour unstability protecting group, and as triphenylmethyl or dimethoxytrityl, it can protect furans hydroxyl (furanyl hydroxyl).Those skilled in the art will admit this method, and to be particularly suitable for the combination of DNA chip synthetic, as described in WO93/22480.
Said method can be used for the synthetic of peptide equally.For example, the proton that produces with anode removes Boc (t-butyloxycarbonyl) protecting group continuously from nitrogen-atoms and can synthesize peptide.The synthetic of other oligomer is to show and easily know to those skilled in the art.
See also Fig. 1, electrod-array is positioned at the high resistivity silicon wafer 1 of oxidation, and its upper surface deposits an iridium metals layer.At interval 2 be formed at iridium metals layer on the silicon wafer, so that a parallel pole array forms by the photolithography technology.The width of each electrode and each about 40 microns every 2 width.Another silicon wafer 4 is arranged on the electrod-array.The surface of this silicon wafer 4 is trimmed out dimethoxytrityl protection nucleosides.
See also 2, the part of this figure show electrode array and substrate thereof.Middle electrode is an anode, and all the other two electrodes are negative electrode.The electrolyte that includes the acetonitrile solution of benzoquinones and hydroquinones contacts with the substrate that electrode and desire are handled.At anode, hydroquinones is oxidized, thereby produces benzoquinones and proton.Most protons are limited in the basal region near anode.Confined proton (nucleotide moiety) on the shielded nucleosides fragment that is attached to substrate removes dimethoxytrityl.Yet some protons can diffuse into the zone between anode and the negative electrode.
On negative electrode, the benzoquinones reduction produces the benzoquinones anion.The benzoquinones anion is relatively stable, can diffuse into the zone between anode and the negative electrode.The benzoquinones anion suppresses to diffuse into this regional proton, thereby produces hydroquinones and benzoquinones.Therefore, the proton in the basal region diffusion that keeps clear of anode can be prevented from reaction.By preventing the accidental reaction in the zone of proton between electrode, can improve the discrimination rate of substrate.
Fig. 2 has also shown follow-up base treatment.This free hydroxyl is coupled with acetyl group under standard conditions, the residue of dimethoxytrityl is removed.The free hydroxyl of gained is handled with fluorescent dye (Cy5 phosphoramidite), and this fluorescent dye makes can be by the imaging of confocal microscope method observation substrate.Therefore, the discrimination rate of initial detritylation (detritylation) step can conveniently be observed.Obviously, can be with above-mentioned technology at the selection zone of substrate synthesis of oligonucleotides thing.
See also Fig. 3, shown the influence that under the 1.33V fixed potential, changes electrolysis time with the confocal microscope method.In the figure, bright area is the basal region of fluorescence, can not be removed in electrolytic process in this zone dimethoxytrityl.Remaining subsequently dimethoxytrityl is replaced by the Cy5 dyestuff of fluorescence.This bright area is usually near negative electrode.At black region, dimethoxytrityl is eliminated in electrolytic process.The free hydroxyl that produces is in conjunction with the acetyl group of last non-fluorescence.This black region is usually near anode.
After Fig. 3 is presented at 2.0 seconds, removed fully in dimethoxytrityl near anode region.In addition, the discrimination rate of substrate can not change after 80 seconds.There is confined striped in corresponding zone near anode and negative electrode.The proton that these proofs produce in electrolytic process strictly is limited in the zone near anode, even also is like this after prolonging electrolysis time.
Fig. 4 (a) and (b) shown the effect of knowing that from electrod-array, removes negative electrode.What black region showed is the zone that dimethoxytrityl is removed, this electrode potential is arranged on 1.33V, when the negative electrode of centre is removed, the proton that produces at anode can freely diffuse into zone line, this material that has proved that clearly negative electrode produces has restriction, can be suppressed at the proton that anode produces.
Experimental section
The electrode combination
Existing photolithography technology has been widely used in generating iridium metals (50 nanometer thickness) electrode on oxidation high conductivity silicon wafer.This silicon wafer is coated on positive photoetching agent, is exposed to ultraviolet ray by photomask then.Wash this wafer with deionization, under 100 ℃, cured 20 minutes, by the ion etching descum.This photomask can be made the array of 96 parallel poles, about 7500 microns long and 40 microns wide.The spacing of adjacent electrode is approximately 40 microns.
Iridium is to be deposited on this wafer by beam methods.Iridium metals can be placed in the crucible of cold boiler, and two or three wafers are placed in about 20 centimeters of crucible near cold boiler.The chamber of cold boiler is evacuated to 3 * 10 -6Torr uses the electron beam gun of 300mA, 5kV to heat iridium metals about 3 minutes, can be coated with the iridium metals that is covered with 50nm on wafer.
Wafer is positioned in the ultrasonic wave acetone photoetching agent can be removed in 30 minutes, electrod-array can manifest.Electrode is placed air, annealed 1 hour down for 350 ℃,, clean by ion etching then with the adhesion strength of raising with wafer substrates.
Behind annealing and cleaning, each electrode all is connected with ultrasonic gold thread separately, and this gold thread and printed circuit board (PCB) link, with the circuit integrated chosen electrode of " logic switch " (analog switch) activation, so that the release step to be provided.Electric current is employed the amplifier control voltage source as a plurality of independent operations.Parallel each electrode of low noise amplifier feedback circuit continuous measurement receive an ampere level electric current.
The solid support combination
Silica wafers after the polishing can be used as substrate support.Before forming pattern,,, organic reagent connects [Gray D.E., Keyes Green S.C., Fil T.S. on the molecule so that being attached to connecting molecule with this wafer surface functionalization, Dao Bosen P.J. and Sa Sen E.M. (Gray, D.E., CaseGreen, S.C., Fell, T.S., Dobson, P.J.﹠amp; Southern E.M.), is fixed on the ellipsometry of the dna probe on the combination array and the characteristics of interferometry, Languir13,2833～2842 (1997)].Wafer is placed in the chamber of vacuum drying oven, and the capacity of this chamber is 19.1 liters, also comprises the ampoule (ampoule) that contains 5ml GPTS (glycidoxypropyltrimethoxysilane).After stove was heated to 185 ℃, this ampoule was heated to 205 ℃ and chamber and is pumped to 25～30mBar.After approximately the silane of 2.5ml is evaporated, in vacuum (10 -3Torr) lower chambers can be cooled off.By immerse have GPTS wafer in containing the polyglycol solution of a small amount of sulfuric acid, " connection molecule " can be enclosed.By existing oligonucleotide synthetic technology, the dimethoxytrityl that will contain phosphoramidite (phosphoramidite) is attached to [the triumphant outstanding S.L. of Bill and Ai Ya R.P. (Beaucage, S.L.﹠amp on the hydroxyl of polyethylene glycol in the mode of covalency; Iyer, R.P.), by the improvement of the synthetic oligonucleotide of phosphoramidite method, Tetrahedron 48,2223～2311 (1992)].This wafer substrates is cut into 1 centimetre x1 centimetre then, in order to using when forming pattern.
Embodiment one
Above prepared electrode array is positioned over apart from 20 microns places of solid support.This solid support is prepared by above-mentioned step, and thymidine phosphoramidite (thymidinephosphoramidite) is attached to polyethylene glycol and connects molecule.The thymidine phosphoramidite has 5 '-hydroxyl, and it is protected by dimethoxytrityl.
One electrolytic solution (anhydrous acetonitrile of 25mM hydroquinones/25mM quinone/25mM ammonium hexafluorophosphate) is injected into the cavity (cavity) between electrod-array and the solid support.Chosen anode is set in 1.33V and voltage is held 0.2 to 0.8 second (as shown in Figure 3).
Carry out washing this silicon wafer with acetonitrile after the electrolysis, and, make silicon wafer in conjunction with last acetyl group with acetic anhydride with standard method.In this step, only the dimethoxytrityl of silicon wafer is separated the protection zone and is added acetyl group.
The dimethoxytrityl that does not remove in electrochemical step is removed when handling whole substrate with the dichloromethane solution of dichloroacetic acid.The hydroxyl that will expose with the phosphoramidite associated methods of standard is in conjunction with last Cy5 (fluorescent dye), taken place and the pattern that generates manifests by the electrochemistry of acid, and it can be seen by the fluorescence of confocal microscopy sem observation Cy5.Sequence of steps as shown in Figure 2.
Fig. 3 has shown the effect that prolongs electrolysis time under 1.33V.It is wide to arrive maximum band after about 2.0 seconds, and the discrimination rate of substrate is stable, even 80 seconds discrimination rates of electrolysis afterwards can not change yet.
Embodiment two
Embodiment two is basic identical with embodiment one, and difference is that selected anode kept 16 seconds under the voltage of 1.33V.As Fig. 4 (a) with (b), remove the Considerable effect of negative electrode and examine.Negative electrode when the center is removed, and the proton of diffusion is out of hand.The proton of this diffusion can flow into the central area, and can not rest on around the anode.In Fig. 4 (b), middle black region clearly, fluorophor is no longer contained in this zone.
Embodiment three
Embodiment one described method is used on solid support the oligomer of synthetic 17-mer, and what this method comprised 16 dimethoxytrityl separates the protection step.The used method of present embodiment is basic identical with embodiment one, and part inequality is that electrod-array is positioned over apart from 40 microns places of substrate surface.
The phosphoramidite associated methods of use standard protects the uniform coating of desoxyadenossine remnants (residue) to be attached to polyethylene glycol linking group on the solid support dimethoxytrityl.
After cleaning on a large scale with acetonitrile, the electrolyte among the embodiment one is injected into the cavity between electrod-array and the solid support.Selected anode adds 9 seconds 1.33V voltage, to remove near the dimethoxytrityl the selected anode.This anode is between two negative electrodes.
After the further cleaning of acetonitrile, protect nucleosides remnants to connect the hydroxyl that exposes dimethoxytrityl with the phosphoramidite associated methods of standard.With the phosphorus key (phosphorus linkage) of iodine oxidation trivalent, thus the phosphorus key of generation pentavalent.Clean whole silicon wafer with acetonitrile, and then use carrene.The used binding of method of synthetic oligonucleotide and oxidation step are existing technology (see, " AbI synthesizes the handbook second portion, is used for the chemical method of automatic synthetic DNA) on solid support.
Repeat this processing procedure, change the dimethoxytrityl protection nucleosides remnants that in the phosphoramidite integrating step, introduce.Thereby oligonucleotide is synthesized on solid support.
This processing procedure can be finished by automation equipment, this automation equipment is by computer control, comprises in the oligonucleotide of two 17-mer synthetic: wild type (wild type) " A " human hemochrome mRNA (ribonucleic acid) and " S " type sickle cell mutant mRNA accordingly.17-mer is created on the qualification bar (strip) of solid support and can obtains high production.The combination synthetic method of this DNA chip is that those skilled in the art know and know, discloses as WO93/22480.
Although the present invention describes with reference to specific embodiment, this description and not meaning that is construed as limiting the present invention.With reference to description of the invention, other distortion of the disclosed embodiments all can be expected for those skilled in the art.Therefore, such distortion can not break away from affiliated claim restricted portion and spirit.

Claims (40)

1. method of handling substrate, it comprises provides an electrolyte that contacts with substrate, with near substrate and with electrolyte electrodes in contact array, change at least one first electrode in the above-mentioned electrod-array to produce the active oxidation reducing substances, this active oxidation reducing substances finishing is near the substrate of this first electrode, produce the second redox material at one or more auxiliary electrodes near above-mentioned first electrode in the electrod-array, it is characterized in that above-mentioned electrolyte can make the first redox material be suppressed and be limited to the peripheral region of first electrode by the second redox thing, do not form first electrode or auxiliary electrode with the substrate of this desire finishing, and be separated with first electrode and auxiliary electrode.
2. method according to claim 1 is characterized in that: this electrolyte comprises organic solvent.
3. method according to claim 2 is characterized in that: this electrolyte can be selected: oxolane (THF), methyl alcohol, ethanol, dimethyl formamide (DMF), carrene (dichloromethane), ether, methyl-sulfoxide (DMSO) or acetonitrile.
4. according to the arbitrary described method of claim 1 to 3, it is characterized in that: this inhibitory reaction liquid that produces electrolysis again.
5. according to the arbitrary described method of claim 1 to 3, it is characterized in that: this active oxidation reducing substances is a proton.
6. method according to claim 4 is characterized in that: this active oxidation reducing substances is a proton.
7. according to the arbitrary described method of claim 1-3, it is characterized in that: the second redox material is organic anion (radical anion).
8. method according to claim 4 is characterized in that: the second redox material is organic anion (radical anion).
9. method according to claim 5 is characterized in that: the second redox material is organic anion (radical anion).
10. method according to claim 6 is characterized in that: the second redox material is organic anion (radical anion).
11. method according to claim 1 is characterized in that: this electrolyte is the solution of hydroquinones and benzoquinones, or derivatives thereof.
12. method according to claim 1 is characterized in that: this electrolyte is the solution of following compound:
Figure C028170300003C1
Wherein R3, R4, R5 and R6 can select respectively: hydrogen, halogen, nitro, hydroxy, thio group, amino, the C of replacement 1To C 15Alkyl, wherein three above carbon atoms can be substituted by N, O and/or S atom; Perhaps R3 and R4, and/or R5 and R6 are in conjunction with forming the C that replaces 1To C 15Cyclic alkylene (Cyclohydrocarbylene), wherein three above carbon atoms can be substituted by N, O and/or S atom.
13. require 1 described method according to aforesaid right, it is characterized in that: electrolyte is the acetonitrile solution of hydroquinones and benzoquinones.
14. according to claim 1-3, the arbitrary described method of 11-13 is characterized in that: electrolyte further comprises conductibility stiffener (conductivity enhancer).
15. method according to claim 14 is characterized in that: this conductibility stiffener is four (C 1-8Alkyl) ammonium salt.
16. method according to claim 15 is characterized in that: this conductibility stiffener is ammonium hexafluorophosphate (tetrabutylammonium hexafluorophosphate).
17. method according to claim 1 is characterized in that: be to realize several treatment steps in regular turn, the electrode of array is coupled together, make the electromotive force of selected one or more electrodes of changing array, can finish a treatment step.
18. according to claim 1-3,11-13,17 arbitrary described methods is characterized in that: this substrate comprises that one is attached to the array of a material on its surface.
19. method according to claim 18 is characterized in that: this surface is the silicon wafer surface of oxidation.
20. method according to claim 18 is characterized in that: the material that this desire is handled comprises sour unstability protecting group (acid labile protecting group).
21. method according to claim 20 is characterized in that: by at least one electrode in the array is added electromotive force and as anode to remove sour indefinite peace protecting group at the material on surface, can realize single treatment.
22. method according to claim 17 is characterized in that: in the progressively chemical synthesis process of oligomer, processing is implemented.
23. the method for a synthetic cover oligomer, it may further comprise the steps:
(a) provide a substrate, this substrate is with an array with material of protecting group, and an electrolyte contacts with substrate, contacts with the close substrate of an electrod-array and with electrolyte;
(b) optionally change the electromotive force of one or more electrodes in the above-mentioned electrod-array, to produce the active oxidation reducing substances, this active oxidation reduzate removes above-mentioned protecting group to revise the substrate near this first electrode from selected material;
(c) a protection monomer is attached to form in the step (b) separate the protection material;
(d) repeating step (b) and (c) changes one or more electrodes selected in the step (b), with a synthetic cover oligomer;
It is characterized in that electrolyte can make this active oxidation reducing substances be suppressed and be limited to the peripheral region of above-mentioned one or more electrodes by at least one other redox material, wherein electrode is not formed in the substrate of desire finishing, and it separates with electrod-array.
24. according to the described method of claim 23, it is characterized in that: this electrolyte comprises a kind of solvent.
25. method according to claim 24 is characterized in that: this electrolyte can be selected: oxolane (THF), methyl alcohol, ethanol, dimethyl formamide (DMF), carrene (dichloromethane), ether, methyl-sulfoxide (DMSO) or acetonitrile.
26., it is characterized in that: this inhibitory reaction liquid that produces electrolysis again according to the arbitrary described method of claim 23 to 25.
27. according to the arbitrary described method of claim 23 to 25, it is characterized in that: the array of material is attached to a surface, this oligomer is synthesized on above-mentioned surface.
28. method according to claim 26 is characterized in that: the array of material is attached to a surface, and this oligomer is synthesized on above-mentioned surface.
29. according to the arbitrary described method of claim 23 to 25, it is characterized in that: this oligomer is an oligonucleotide.
30. method according to claim 26 is characterized in that: this oligomer is an oligonucleotide.
31. according to the arbitrary described method of claim 23-25, it is characterized in that: this active oxidation reducing substances is a proton, this protecting group is sour unstability protecting group.
32. method according to claim 26 is characterized in that: this active oxidation reducing substances is a proton, and this protecting group is sour unstability protecting group.
33. method according to claim 27 is characterized in that: this active oxidation reducing substances is a proton, and this protecting group is sour unstability protecting group.
34. method according to claim 28 is characterized in that: this active oxidation reducing substances is a proton, and this protecting group is sour unstability protecting group.
35. method according to claim 29 is characterized in that: this active oxidation reducing substances is a proton, and this protecting group is sour unstability protecting group.
36. method according to claim 23 is characterized in that: this electrolyte is the solution of hydroquinones and benzoquinones for this electrolyte, or derivatives thereof.
37. method according to claim 23 is characterized in that: this electrolyte is the solution of following compound:
Figure C028170300006C1
Wherein R3, R4, R5 and R6 can select respectively: hydrogen, halogen, nitro, hydroxy, thio group, amino, the C of replacement 1To C 15Alkyl, wherein three above carbon atoms can be substituted by N, O and/or S atom; Perhaps R3 and R4, and/or R5 and R6 are in conjunction with forming the C that replaces 1To C 15Cyclic alkylene (Cyclohydrocarbylene), wherein three above carbon atoms can be substituted by N, O and/or S atom.
38. method according to claim 23 is characterized in that: this electrolyte is the acetonitrile solution of hydroquinones and benzoquinones.
39. method according to claim 23 is characterized in that: electrolyte further comprises conductibility stiffener (conductivity enhancer).
40. method according to claim 23 is characterized in that: this conductibility stiffener is four (C 1-8Alkyl) ammonium salt.
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AU2002321587A1 (en) 2003-03-18
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EA200400379A1 (en) 2004-08-26
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US20040238369A1 (en) 2004-12-02

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