CN105606673A - Chip applicable to electrochemical real-time PCR and preparation method of chip - Google Patents
Chip applicable to electrochemical real-time PCR and preparation method of chip Download PDFInfo
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- CN105606673A CN105606673A CN201610074948.0A CN201610074948A CN105606673A CN 105606673 A CN105606673 A CN 105606673A CN 201610074948 A CN201610074948 A CN 201610074948A CN 105606673 A CN105606673 A CN 105606673A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
Abstract
The invention discloses a chip applicable to electrochemical real-time PCR and a preparation method of the chip. The preparation method comprises the following steps: growing an insulating layer on a substrate, and then preparing three electrodes including a working electrode, an auxiliary electrode and a reference electrode; then growing an insulating film on the surface of the working electrode, and transferring a mask pattern from a microarray electrode to the insulating film by virtue of a photoetching process and an etching process; and etching a region of the insulating film on the working electrode, so as to generate the microarray electrodes. Each microarray electrode is an independent reaction unit and is applied to electrochemical real-time PCR. The chips can be combined in series or in parallel for use and can be used for simultaneously carrying out multiple PCRs, so that the detection throughput is greatly increased; the chip has the advantages of being high in precision and throughput and capable of realizing low-concentration quantitative detection.
Description
Technical field
The present invention relates to biochip research field, particularly a kind of chip and making side thereof that can be used for electrochemistry PCR in real timeMethod.
Background technology
PCR (PolymeraseChainReaction is called for short PCR) is the synthetic specific DNA sheet of external enzymaticA kind of method of section, by reaction composition one-periods such as high-temperature denatured, process annealing (renaturation) and thermophilic extensions, loops,Make target DNA be able to rapid amplification, there is high specificity, highly sensitive, easy and simple to handle, the feature such as save time. It not only canFor basic research such as Gene Isolation, clone and nucleic acid sequence analysises, also can be used for pathogen detection, pre-natal diagnosis, tumour baseBecause of medical treatment, food, health fields such as detection, curative effect evaluation, food safety detection, the detections of food transgenosis.
The DNA copy number producing in PCR course of reaction is exponential manner to be increased, along with the increase of reaction cycle number,Whole PCR reaction no longer generates template with exponential manner, thus the plateau of entering. In traditional PCR, conventional gel electrophoresisThe final amplified production that separates and detect with fluorescent staining PCR reaction, therefore quantitatively exists PCR product with this end-point methodUnreliable part. And the gel electrophoresis detection of need to uncapping, very easily cause the pollution of PCR product.
Real-time fluorescence quantitative PCR (QuantitativeReal-timePCR), in pcr amplification process, by adding fluorescenceMatter, detects in real time to PCR process, has effectively solved traditional limitation that quantitatively can only end point determination, has realized each and has taken turnsCirculation all detects the intensity of first order fluorescence signal, and is recorded among computer software, by the calculating to each sample Ct value, rootObtain quantitative result according to calibration curve. Quantitative real time PCR Instrument is by basic PC R part, fluoroscopic examination part and host computerThe compositions such as part, wherein fluoroscopic examination part comprises the parts such as exciting light source, photomultiplier, signals collecting and processing, thereforeDesign is complicated, body is huge, cost is higher, is difficult to basic unit and promotes the use of.
Electrochemical chip provides a kind of effective ways for PCR detects, and without using specific light source, needn't carry out photoelectric conversion doublyThe steps such as increasing, therefore simple, be easy to realize instrument miniaturization and portability. Electrochemical chip research is carried out for many years, butCurrent most of electrochemical chip is all after pcr amplification completes, and amplified production is joined in chip, according to yin and yang attribute sampleOriginally the different electrical signals providing judges, complex operation, can not realize Real-Time Monitoring on the one hand, on the other hand PCR productThe operation of uncapping has increased the possibility of polluting greatly. Although therefore electrochemical chip possesses series of advantages, not yet realize large ruleMould uses.
Electrochemistry PCR in real time has been reported in recent years in the literature, and the utilizations such as Thibaut add hybridization instruction in reaction systemAgent, the square wave volt-ampere curve that circulates in Real-Time Monitoring pcr amplification process, in the situation that having template to exist, along with pcr amplificationCarrying out, the peak point current of circulation square wave volt-ampere curve reduces (ThibautDef é veretal.Real-Time graduallyElectrochemicalPCRwithaDNAIntercalatingRedoxProbe.Anal.Chem.,2011,83(5),pp1815 – 1821). The method does not relate to the modification of primer/probe, and design and processing to reaction electrode do not have particular/special requirement.
In order to increase the specificity of electrochemistry PCR in real time, can consider by introduce electro-chemical activity functional group on primer/probe,The amplification situation of Real-Time Monitoring PCR system. But, in prior art, also there is no to carry out the chip of electrochemistry PCR in real time.
Summary of the invention
The object of the present invention is to provide a kind of chip of reasonable in design, processing precise, detect for electrochemistry PCR in real time, andA kind of preparation method of this chip is provided, requires preparation simple, with low cost, can mass production.
Technical scheme of the present invention is as follows:
For a chip for electrochemistry PCR in real time, comprise the insulating barrier of substrate, substrate front surface, and be positioned on insulating barrierThree electrodes, described three electrodes are working electrode (WE), auxiliary electrode (CE) and reference electrode (RE), it is characterized in that,Described working electrode surface is coated with insulation film, is placed with tiny array electrode in insulation film, below each tiny array electrodeConnect working electrode, and side is spaced by insulation film.
From the surface of said chip, the array arrangement of tiny array electrode can be parallel arrangement, cross arrangement, circular arrangement etc.;The shape of described tiny array electrode upper surface can be the forms such as circle, rectangle, annular, regular polygon.
Preferably, the upper surface of described tiny array electrode have at least one dimension size within the scope of 1~100 μ m (for circular,Described one dimension size refers to its diameter); From upper surface, the one dimension size of described tiny array electrode and adjacent tiny array electrodeThe ratio of spacing be 1:1~1:20.
Described substrate can be the substrate of the materials such as silicon, glass, polymer, is preferably silicon chip.
The insulating layer material of described substrate front surface can be silica (SiO2), alundum (Al2O3) (Al2O3), zirconium dioxide (ZrO2)Deng.
The material of three electrodes and tiny array electrode can be gold, silver, platinum, chromium, carbon etc. Three electrode patterns can be plane formula alsoCan be column type, the shape of described plane formula can be circle, annular, regular polygon etc.
Chip of the present invention, can pass through the coupling of serial or parallel connection mode, carries out several PCR reactions simultaneously, greatly improves and detects flux.
The present invention also provides the preparation method of the above-mentioned chip for electrochemistry PCR in real time, comprises the steps:
1) clean substrate dry, the insulating barrier of then growing in substrate;
2) on insulating barrier, make three electrodes, contact conductor and lead-in wire interface, described three electrodes comprise working electrode (WE), auxiliaryHelp electrode (CE) and reference electrode (RE);
3) at working electrode surface growth insulation film;
4) by photoetching process and etching process, the mask plate patterns of tiny array electrode is transferred to step 3) growth insulation filmOn;
5) on working electrode, erode insulation film region generate tiny array electrode.
Above-mentioned steps 1) substrate can be the substrate of the materials such as silicon, glass, polymer, is preferably silicon chip. Can adopt organicSolvent and water carry out ultrasonic cleaning to substrate, remove the organic impurities of substrate surface; Can use the mixing of inorganic acid and hydrogen peroxideSubstrate is boiled in liquid heating, then uses deionized water rinsing, removes the inorganic impurity of substrate surface. Substrate after cleaning is blown with nitrogenDry, in vacuum drying chamber, dry afterwards.
Step 1) insulating layer material of growing in substrate and growth thickness be according to insulating properties and cost and determine. Insulating layer materialCan be selected from silica (SiO2), alundum (Al2O3) (Al2O3), zirconium dioxide (ZrO2) etc.; The method bag of growth insulating barrierDraw together physical vaporous deposition, chemical vapour deposition technique, thermal oxidation method etc., preferably thermal oxidation method.
Step 2) by photoetching process and evaporation process, on insulating barrier, make electrode, contact conductor and lead-in wire interface. FirstFirst on insulating barrier, make the figure needing by lithography by photoetching process, photoresist, as mask, can use positive glue, also can makeWith negative glue; Then by evaporation process, at graph area evaporation metal, form electrode, finally peel off mask. For silicon chip,Because the adhesiveness of metal electrode and silica is bad, can first evaporation one deck titanium or the metal buffer layer of zirconium, play adhere to slowPunching effect, the metal evaporation of afterwards electrode pair being answered is to metal buffer layer.
Electrode material can be gold, silver, platinum, chromium, carbon etc., electrode material can with contact conductor and lead-in wire interface materialUnanimously, also can be different. Three electrode patterns can be plane formulas, can be also column types, and wherein the shape of plane formula can beCircle, annular, arc, regular polygon etc., as shown in Figure 2, wherein working electrode (WE) is circular, auxiliary electrode (CE)With reference electrode (RE) is arc, be arranged on the periphery of circular working electrode.
Step 3) insulation at the insulation film of working electrode surface growth in order to tiny array electrode. The grow side of this insulation filmMethod is chemical vapour deposition technique, the less and even thickness of film particles that adopts the method to form. The material of this insulation film is optionalFrom SiO2、Al2O3、ZrO2Deng.
Step 4) on the insulation film of working electrode surface, apply one deck photoresist, make the figure needing by lithography by photoetching processShape, then transfers to mask pattern on insulation film by wet etching, and wet etching ends to electrode.
Step 5) by evaporation or sputtering technology, on working electrode, wet etching falls the region generation microarray electricity of insulation filmThe utmost point, the each micro-boss in microarray is a microelectrode, becomes an independent reaction unit, anti-for electrochemistry PCR in real timeShould.
Beneficial effect of the present invention:
The present invention, for the chip of electrochemistry PCR in real time, has high steady-state current density, high s/n ratio, Minimum-time constant,Low solution potential degradation advantage. Tiny array electrode time constant is little, reaches at short notice stable state, and electrode can be realized quick soundShould. In addition, the electrode size of tiny array electrode of the present invention, shape, density all have very large adjustable range, are applicable to multiple applicationDemand. By chip of the present invention for PCR in real time not only possess regular-PCR high specificity, highly sensitive, easy fastEtc. feature, also possesses the quantitatively advantage of detection of high accuracy, high flux, low concentration.
Brief description of the drawings
Fig. 1 is the flow chart of the present invention for the preparation of the chip of electrochemistry PCR in real time;
Fig. 2 is that the embodiment of the present invention is for the preparation of the step schematic diagram of the chip of electrochemistry PCR in real time from depression angle;
Fig. 3 is for from vertical section, and the embodiment of the present invention is for the preparation of the step schematic diagram of the chip of electrochemistry PCR in real time, wherein,101-substrate, 102-insulating barrier, 103-auxiliary electrode, 104-working electrode, 105-reference electrode, 106-insulation film, 107-Tiny array electrode.
Detailed description of the invention
Below in conjunction with accompanying drawing, further set forth the present invention by embodiment.
Refer to Fig. 1~Fig. 3, the preparation method of electrochemistry PCR in real time chip of the present invention, comprises the steps:
Step 1: get a substrate 101, clean the organic and inorganic impurity of removing substrate 101 surfaces, and carry out vacuum drying.
Described substrate is silicon chip, first uses ultrasonic wave to clean in analysis pure acetone, deionized water, removes the organic of silicon chip surfaceImpurity, then uses the mixed liquor heating of the concentrated sulfuric acid and hydrogen peroxide to boil silicon chip, and uses deionized water rinsing, removes silicon chip tableThe inorganic impurity of face, packs in culture dish after the silicon chip after cleaning is dried up with nitrogen, puts into afterwards 120 DEG C of vacuum drying chambersDry, this operation object is to remove the subsidiary steam of base silicon chip itself.
Step 2: at the positive growth insulating barrier 102 of substrate 101.
Described insulating layer material and growth thickness are according to insulating properties and cost and determine. Insulating layer material comprises silica, three oxygenChange two aluminium, zirconium dioxide etc. The method of growth insulating barrier comprises physical vaporous deposition, chemical vapour deposition technique, thermal oxidation methodDeng, the present embodiment uses thermal oxidation method.
Step 3: make electrode, contact conductor and lead-in wire interface by photoetching process and evaporation process on insulating barrier. DescribedPhotoetching process comprises the steps such as even glue, front baking, exposure, development, makes the figure needing by photoetching process on insulating barrier by lithography,Photoresist, as mask, can use positive glue, also can use negative glue. By evaporation process, the first evaporation layer of gold in graph areaBelong to cushion, metal buffer layer can be titanium, can be also zirconium. Because the adhesiveness of metal electrode and silica is bad, titaniumOr zirconium plays the cushion effect that adheres to. The metal evaporation of afterwards electrode pair being answered, to cushion, finally peels off mask. ElectricityThe utmost point is divided into three electrodes, working electrode 104, auxiliary electrode 103 and reference electrode 105, electrode material can be gold, silver, platinum,Chromium, carbon etc., electrode material can be consistent with the material of contact conductor and lead-in wire interface, also can be different. As shown in Figure 2,The working electrode 104 of the present embodiment is circular, and irradiation electrode 103 and reference electrode 105 are around this circular arc section.
Step 4: at working electrode 104 superficial growth insulation films 106, in order to the insulation of tiny array electrode.
The method of growth insulation film 106 is chemical vapour deposition technique, and the film particles that adopts the method to form is less and thickness is equalEven.
Step 5: mask plate patterns is transferred on insulating layer of thin-film by photoetching process and etching process.
On insulation film 106, apply one deck photoresist, on insulation film 106, make the figure needing by photoetching process by lithography,By wet etching, mask pattern to be transferred on insulation film 106, wet etching is to electrode cutoff layer.
Step 6: generate tiny array electrode 107.
By evaporation or sputtering technology, the region that wet etching falls on working electrode generates tiny array electrode 107, in microarrayEach micro-boss be a microelectrode, become an independent reaction unit, for electrochemistry real-time PCR reactions.
Tiny array electrode described in Fig. 2 and Fig. 3 is cylindric, and upper surface is circular, arranges as regular hexagon array.
It should be noted that, the present invention illustrates process of the present invention by above-described embodiment, not the present invention is done anyPro forma restriction, everyly replaces any equivalence made for any of the above embodiments according to the technology of the present invention essence, simple modification, etc.With changing and modifying, the selection of concrete mode etc., still belong within the scope of technical solution of the present invention, therefore protection scope of the present inventionWhen being as the criterion with claims.
Claims (10)
1. for a chip for electrochemistry PCR in real time, comprise the insulating barrier of substrate, substrate front surface, and be positioned on insulating barrierThree electrodes, described three electrodes are working electrode, auxiliary electrode and reference electrode, it is characterized in that described working electrode surfaceBe coated with insulation film, in insulation film, be placed with tiny array electrode, connect working electrode below each tiny array electrode,And side is spaced by insulation film.
2. chip as claimed in claim 1, is characterized in that, described tiny array electrode is parallel arrangement, cross arrangement or circular rowCloth; Described tiny array electrode upper surface is shaped as circle, annular or regular polygon.
3. chip as claimed in claim 1, is characterized in that, the upper surface of described tiny array electrode has at least one dimension size to existWithin the scope of 1~100 μ m; From upper surface, the ratio of the one dimension size of described tiny array electrode and the spacing of adjacent tiny array electrodeFor 1:1~1:20.
4. chip as claimed in claim 1, is characterized in that, described base material is silicon, glass or polymer; Described insulating barrierMaterial is silica, alundum (Al2O3) or zirconium dioxide; The material of described three electrodes and tiny array electrode be gold, silver, platinum,Chromium or carbon.
5. for a preparation method for the chip of electrochemistry PCR in real time, comprise the following steps:
1) clean substrate dry, the insulating barrier of then growing in substrate;
2) on insulating barrier, make three electrodes, contact conductor and lead-in wire interface, described three electrodes comprise working electrode, auxiliary electrodeAnd reference electrode;
3) at working electrode surface growth insulation film;
4) by photoetching process and etching process, the mask plate patterns of tiny array electrode is transferred to step 3) growth insulation filmOn;
5) on working electrode, erode insulation film region generate tiny array electrode.
6. preparation method as claimed in claim 5, is characterized in that step 1) described substrate is silicon chip, sheet glass or polymerSubstrate, cleans the organic and inorganic impurity of removing substrate surface, then dry; Described insulating layer material is silica, threeAl 2 O or zirconium dioxide, the method for growth insulating barrier is physical vaporous deposition, chemical vapour deposition technique or thermal oxidation method.
7. preparation method as claimed in claim 5, is characterized in that step 2) by photoetching process and evaporation process, in insulationOn layer, make electrode, contact conductor and lead-in wire interface, described electrode material is gold, silver, platinum, chromium or carbon.
8. preparation method as claimed in claim 5, is characterized in that step 2) described in working electrode be circular, described auxiliaryElectrode and reference electrode are arc, are arranged on the periphery of working electrode.
9. preparation method as claimed in claim 1, is characterized in that step 3) at working electrode surface by chemical vapour deposition (CVD)Method growth insulation film.
10. chip as claimed in claim 5, is characterized in that step 4) on the insulation film of working electrode surface, apply oneLayer photoetching glue, makes the figure needing by lithography by photoetching process, then by wet etching, mask pattern is transferred to insulation thinOn film, wet etching ends to electrode; Step 5) by evaporation or sputtering technology, fall the district of insulation film at wet etchingTerritory generates tiny array electrode.
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CN108627553A (en) * | 2018-05-15 | 2018-10-09 | 青岛理工大学 | Platinum electrode Multifunction Sensor |
CN109865539A (en) * | 2017-12-04 | 2019-06-11 | 上海新微技术研发中心有限公司 | Micro-fluidic chip of integrated electrochemical sensor and manufacturing method and application thereof |
CN113109408A (en) * | 2021-04-08 | 2021-07-13 | 海南师范大学 | Portable palm electrochemical sensor for detecting enzyme concentration based on chip electrode and preparation method and detection method thereof |
CN113533787A (en) * | 2021-06-23 | 2021-10-22 | 浙江大学 | Electrochemical reaction process in-situ monitoring device based on atomic force microscope and monitoring method thereof |
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Application publication date: 20160525 |