CN104897758A - Method for manufacturing and modifying electrochemiluminescence electrode array - Google Patents
Method for manufacturing and modifying electrochemiluminescence electrode array Download PDFInfo
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- CN104897758A CN104897758A CN201510360195.5A CN201510360195A CN104897758A CN 104897758 A CN104897758 A CN 104897758A CN 201510360195 A CN201510360195 A CN 201510360195A CN 104897758 A CN104897758 A CN 104897758A
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Abstract
The invention provides a method for manufacturing an electrochemiluminescence electrode array which is simple and efficient and an electrode surface modifying method. A square electrode structure with a circular-arc top angle is adopted to maximize electrode surface area and reduce corner effect, a synchronous method is adopted to use a ligand or a grafting molecule to modify the surface of each electrode in the electrode array, and a stamp method is used to load and capture a probe molecule on the surface of each electrode to enable the electrode array to be more uniform. The method has the advantages of simplicity in manufacturing and using, low consumable cost, convenience in operation and high in detection result repeatability.
Description
Technical field
The present invention relates to electrode preparation and the surface modification method of a kind of electrochemiluminescence micro-array chip used in technical field of biological.
Background technology
Electrochemiluminescence (electrochemiluminescence) be in electrochemical reaction and chemiluminescence phenomenon in conjunction with product.In chemical reaction process, molecule produces what is called " energy jump " due to the energy absorbing chemical reaction and discharge, and is converted into unstable excited state by ground state.Discharge photon when molecule decays into ground state again from excited state and produce chemiluminescence phenomenon.Electrochemiluminescence is otherwise known as electricity to chemiluminescence, with general chemistry luminous unlike, electrochemiluminescence relies on the redox reaction that exciting electrode surface produces.In tripropyl amine (TPA) the most frequently used at present-pyridine ruthenium electrochemical luminescence system, chemiluminescence agent tris (bipyridine) ruthenium [Ru (bpy)
3]
2+[Ru (bpy) is oxidized to respectively at anode surface with electron donor tripropyl amine (TPA) (TPA)
3]
3+and TPA
+TPA
+very unstable, be easy to lose a proton (H
+) and form strong reductant TPA.As [Ru (bpy)
3]
3+when there is redox reaction with TPA, [Ru (bpy)
3]
3+divalence [the Ru (bpy) of excited state is formed by TPA reduction
3]
2+, TPA is oxidized to di-n-propylamine and propionic aldehyde.Then [the Ru (bpy) of excited state
3]
2+decay into [the Ru (bpy) of ground state
3]
2+, launch a wavelength is the photon of 614nm simultaneously.In tripropyl amine (TPA)-pyridine ruthenium electrochemical luminescence system, light emitting molecule tris (bipyridine) ruthenium carries out the energy trasfer of ground-excited state-ground state again and again at electrode surface, produce a large amount of photon.
Reaction electrode is one of major control factors participating in electrochemiluminescence reaction.Electrochemiluminescence detecting electrode the most frequently used at present mainly adopts the conductive material such as gold, silver, carbon, electro-conductive glass, carry out intensifier electrode to the catalytic capability of chemical reaction occurring in its near surface by carrying out complicated finishing to electrode, thus improve light signal strength.
Compared with general chemistry luminescence, electrochemiluminescence has the advantages such as reagent stability is good, reaction easily controls, test findings is reproducible, but electrochemiluminescence reacts more complicated comparatively speaking, especially for exciting electrode making and modify for detection numerical stability abnormal important.For improving detection sensitivity and reducing the coefficient of variation (Coefficient of Variation), usually need to do many modifications to improve the parameters such as capture probe loading density, uniformity coefficient and degree of freedom to electrode surface.
Capture probe loading method the most frequently used at present in micro-array chip field pin type point sample method and liquid pearl specking method, but, for electrochemiluminescence microarray, said method usually causes loaded reagent at electrode surface skewness, and these methods all need to purchase expensive special microarray point sample equipment.This causes current electrochemiluminescence microarray detection processes cannot be widely used like that to the luminous microarray detection processes of general chemistry.
Summary of the invention
Technical matters to be solved by this invention is the electrod-array preparation and the method for modifying that provide a kind of simple and efficient in the preparation of electrochemiluminescence tiny array electrode and modification field.It has equipment making and uses the advantages such as simple, consumables cost is low, easy to operate, testing result is reproducible.
The technical solution used in the present invention is: (1), in electrod-array, adopts the square-shaped electrode structure of band circular arc drift angle electrode surface area is maximized and reduces sharp angle effect; (2) adopt each electrode surface in Synchronos method part or grafting molecules modified electrode array to obtain homogeneous electrode surface; (3) use imprint method to load capture probe molecules at electrode surface, when loading capture probe impression, Loading Method or loaded in parallel method can be adopted successively; (4) adopt larger electrode surface and relatively little similar figures die, make each electrode surface all can obtain a complete capture probe impression, to improve the homogeneity of reaction.(5) sufficient Seal treatment is carried out to reduce non-specific adsorption to electrode.(electrod-array and capture probe impression pattern see
fig. 1,
fig. 2).
When the probe being fixed on electrode surface catches target molecule to be checked-electrochemiluminescence marker complex, electrochemiluminescence reaction system is carried out electrochemiluminescence reaction and is produced light signal on reaction electrode surface, detect the light signal produced with photomultiplier (PMT).
Described electrod-array refers to: by the square-shaped electrode electrode group pattern that formed of proper alignment in length and breadth of multiple band circular arc drift angle, wherein the surface area of each electrode is 0.25 ~ 25 mm
2, the spacing between each electrode is 0.2 ~ 2 mm.
Described Synchronos method electrode face finish refers to: electrod-array same batch of reagent is carried out moditied processing simultaneously.
Described part comprises: biotin, Avidin, Streptavidin, antiantibody and molecularly imprinted polymer (Molecular Imprinted Polymer, MIP).
Described grafting molecules component comprises: maleic anhydride (Maleic anhydride), 2-acrylamide-2-methylpro panesulfonic acid (2 Acrylamido-2-Methyl Propane Sulfonic Acid), cellulose, polyvinyl alcohol (PVA) (Polyvinyl alcohol), polyglycol diallyl ether (Polyethylene Glycol Diallyl Ether), polyvinylpyrrolidone-vinyl acetate co-polymer (Polyvinyl pyrrolidone-vinyl acetate copolymer), polyacrylic acid (Polyacrylic acid), poly (glycidyl methacrylate) (Glycidyl methacrylate).
Described capture probe molecules comprises: biotin, Avidin, capture antibody, peptide nucleic acid (Peptide Nucleic Acid) probe or nucleic acid probe.Wherein nucleic acid probe comprises genome DNA probe, cDNA probe, rna probe, cRNA probe and oligonucleotide probe.
Described successively Loading Method or loaded in parallel method are: load capture probe impression successively one by one at each electrode surface of electrod-array or adopt that the die corresponding with whole electrod-array is disposable loads capture probe impression on each electrode simultaneously.
Described electrochemiluminescence label is luminol and derivant thereof or terpyridyl ruthenium derivative.
Described electrochemiluminescence reaction system comprises: Luminol, pyridine ruthenium system.In Luminol, electrochemiluminescence label is luminol and derivant thereof, and buffer solution contains: 1.0 mM3.0 mM H
2o
2, 0.05 M Na
2cO
3-NaHCO
3, pH 811.In pyridine ruthenium system, electrochemiluminescence label is terpyridyl ruthenium derivative, and buffer solution contains: 0.03 mM2.0 mM dibutylethanolamine (DBAE), 0.05 M phosphate buffer, pH 69.
Principle of the present invention is as follows:
In electrochemiluminescence testing process, electrochemiluminescence reaction system produces electricity to oxidation-reduction reaction at electrode surface, and then produces electrochemiluminescence by " transition-decay " process.When other condition is identical, luminous intensity and electrode surface useful area and the being proportionate property of capture probe molecules density loaded.So the change of electrode surface useful area size and capture probe molecules density will cause the change of luminous value, thus cause testing result unstable.
No matter now conventional microarray dot matrix method, be pin type point sample method or liquid-spraying type point sample method, all cannot ensure the homogeneity of electrode surface useful area.Pin type point sample method produces rigid contact due to point sample face and electrode surface, and than being easier to the impact being subject to electrode surface flatness and surface roughness, pin type point sample method is not suitable for the larger electrode of area simultaneously.Liquid-spraying type point sample method is subject to the impact of epidemic disaster, meanwhile, because the mobility at electrode surface is comparatively large, easily by the impact of electrode surface wettability, thus causes reagent at electrode surface skewness.
Comparatively speaking, the present invention is owing to adopting similar to electrode surface shape and being less than the flexible point sample face of electrode surface, so have the following advantages: (1) point sample face contacts with electrode surface closely, affects little by electrode surface shape; (2) size of the size determination electrode useful area in point sample face, thus guarantee the consistance of electrode useful area; (3) because point sample face is similar to electrode surface shape and be less than electrode surface, therefore for the impression position in deposition process, there is good tolerance; (4) point sample face and electrode surface can have longer duration of contact, effectively can reduce the mobility of test solution at electrode surface, improve the uniformity coefficient that reagent distributes at electrode surface.In addition, each electrode surface adopting Synchronos method part or grafting molecules to modify in microarray not only can obtain homogeneous electrode surface proterties, also can improve the loading density of capture probe at electrode surface, thus improves detection sensitivity.
The method applied in the present invention not only can improve stability and the sensitivity of electrochemiluminescence testing result, and does not need to buy expensive equipment, and operation is simple, and use cost reduces greatly.
Accompanying drawing explanation
fig. 1it is the electrod-array pattern signal before loading capture probe impression
figure.
fig. 2it is the electrod-array pattern signal after loading capture probe impression
figure.
Embodiment
Specific embodiment one
The preparation of electrod-array: telegraph circuit adopts the preparation of single or multiple lift printed circuit board (PCB), electrode can use gold paste, silver slurry or carbon pulp material, adopts low temperature method for printing screen to prepare electrod-array.Starching low temperature silk screen print method with ultra-fine silver slurry low-temperature silver is below example, the preparation method of statement electrod-array:
(1) preparation of substrate: adopt the preparation of single or multiple lift printed circuit board technology according to the wiring of design;
(2) preparation of super fine silver powder: dissolve silver nitrate with ionized water, makes silver nitrate concentration be 100 g/L; Add sodium carbonate liquor and be settled out Ag
2cO
3; Add formaldehyde reduction about adjust ph to 8.5, under agitation under room temperature, obtain super fine silver powder; Silver powder is clean by washed with de-ionized water, add spreading agent, dry at low temperatures, sieving is for subsequent use;
(3) surface treatment of silver powder: take polyvalent alcohol as spreading agent, be equipped with the steel ball of respective numbers in mass ratio, with horizontal ball mill ball milling 72 ~ 96 hours, obtain flake silver powder, flake silver powder is clean by washed with de-ionized water, add spreading agent, dry at low temperatures, sieving is for subsequent use;
(4) low-temperature silver slurry preparation: take polyurethane as cured resin, polyurethane is dissolved in appropriate solvent, be mixed with the resin solution that concentration is 25%; Silver powder is mixed with the ratio (weight ratio) of resin solution according to 6:1; Add solvent and auxiliary agent, wherein solvent comprises: the molten acid anhydride acetic acid esters of butyl, butyl acetate, DGDE acetate, isophorone, and auxiliary agent is common antioxidant and stabilizing agent; On three-roller mixing to slurry fineness be 2 ~ 4 μm;
(5) low-temperature silver slurry serigraphy: adopt 300 ~ 420 order polyester nets or stainless steel cloth web plate, web plate emulsion thickness 10 ~ 12 μm, glue is scraped hardness 60 to 70 and is spent; Adopt hot setting, 110 DEG C are toasted 30 ~ 60 minutes;
(6) electrod-array cutting: prepare electrod-array according to design size, each electrode in electrod-array all sized by, the square or rectangular of band circular arc drift angle that shape is identical, the surface area of single electrode is 0.25 ~ 25 mm
2, the spacing between adjacent electrode is 0.2 ~ 2 mm; The electrod-array mould precise cutting produced is for subsequent use.
Specific embodiment two
Prepared by die:
With dimethyl silicone polymer (Polydimethylsiloxane, PDMS) the soft silica gel material of surface graft modification, according to Design Mode (pattern) and size, prepare die by soft lithography.Die shape is similar with corresponding tiny array electrode, and size is less than corresponding electrode, its mid point and corresponding electrode mid point inregister; It is 0.20 ~ 20 mm that stamp surfaces is amassed
2;
(1) die preparation flow: draw mask plate
figure→ with laser printer (12000 dpi) thoroughly
gelatinsheet prints mask blank → by the mixing of high precision photoetching making SU-8 mould → carry out PDMS performed polymer and hardening agent, cast, solidification, bonding → surface graft modification;
(2) SU-8 2100 lithography step: cleaning substrate; Substrate is dried 30 minutes at 200 DEG C; At SU-8 2100 glue of substrate surface spin coating 1 ~ 5 mm; With hot plate, front baking process is carried out to SU-8 glue, then Slow cooling on hot plate; Ultraviolet photolithographic machine carries out contact exposure; After SU-8 2100 glue of exposure-processed carries out on hot plate, warm process, because crosslinked SU-8 glue internal stress is comparatively large, so heating must slowly, cooling should with hot plate cool to room temperature on hot plate; Crosslinked SU-8 2100 plastic structure is carried out ultrasonic development, obtains photoresist
figureshape; SU-8 2100 plastic structure is cured at 150 DEG C ~ 200 DEG C on hot plate;
(3) PDMS die preparation: by PDMS performed polymer and hardening agent with 10: 1 ratio fully mix, vacuumize degasification; The PDMS performed polymer glue of mixing is cast on SU-8 2100 mould; Put heating in 85 DEG C of hot plates or baking oven to be cured for 60 minutes; Taken off from mould by the PDMS of solidification, put into plasma cleaner and carry out plasma treatment (treating that graft modification is towards upper), process parameter is: vacuumize 5 minutes, 45 seconds processing times; PDMS chip after plasma treatment is placed in water at once or polar organic solvent environment preserves the water wettability that can keep PDMS chip surface for a long time;
(4) PDMS die hydrophilic surface graft modification: the process of PDMS die using plasma is in silastic surface forming reactions functional group; PDMS die is treated grafted surface immerse polyglycol graft crosslinking agent polyglycol diallyl ether (Polyethylene Glycol Diallyl Ether), and put into 85 DEG C of hot plates or baking oven heating 30 ~ 60 minutes; Take out PDMS die, thermal treatment 5 ~ 30 minutes in 140 DEG C of baking ovens; Totally for subsequent use by washed with de-ionized water.
Specific embodiment three
Electrochemical luminescence electrode array Avidin and Streptavidin method of modifying: the electrochemical luminescence electrode array prepared by employing, in the basic conditions Avidin or Streptavidin are fixed on tiny array electrode surface by physisorption, concrete steps are as follows: (1) weighs Na respectively with balance
2cO
31.5 grams, NaHCO
32.93 grams, with distilled water diluting to 1000 milliliter, be configured to 0.05 mol/L pH9.6 carbonic acid buffer, 4 DEG C, preserve.(2) with 0.05 mol/L pH 9.6 carbonic acid buffer dilution Avidin or Streptavidin, be 1 ~ 10 microgram/microlitre to final concentration.(3) in microarray reaction chamber, add the Avidin after 200 ~ 400 microlitres dilutions or solution of streptavidin, 37 DEG C of incubations 1 hour, 4 DEG C of refrigerators are placed 8 ~ 12 hours.(4) repeatedly clean microarray reaction chamber three times with pure water, finally blot residual liquid with thieving paper, 4 DEG C for subsequent use.
Specific embodiment four
Electrochemiluminescence microarray oligonucleotide probe method of modifying: adopt and UV-crosslinked oligonucleotide probe is fixed on tiny array electrode surface, concrete steps are as follows: (1) probe dilution: the oligonucleotide probe of 5' end band poly thymine structure is dissolved in 50% dimethyl trident maple damping fluid (oligonucleotide probe final concentration is 10 ~ 20 micromoles).(2) according to Design Mode, with PDMS die, the single stranded oligonucleotide DNA probe impression after dilution is loaded into corresponding electrode surface, 50 degrees Celsius of oven dry.(3) microarray of oven dry is put into 254 nano wave length UV-crosslinked equipment reaction, 3 ~ 5min to carry out probe and fix.(4) fill it up with reacting hole with pure water, quietly put three minutes, three times repeatedly, finally reaction plate is upside down on thieving paper and blots residual liquid, 50 degrees Celsius dry after for subsequent use.
Specific embodiment five
Biotin method electrochemiluminescence antigen detects: antigen to be checked 0.05 mol/L pH 7.6 phosphate buffer is pressed 1:1 dilution; Get the antigen to be checked of 100 microlitres dilutions respectively, two anti-, the biotin labeled primary antibodie of 50 microlitre of 50 microlitre bipyridyl rutheniums marks add in the electrochemical luminescence electrode array reaction chamber modified in advance with Avidin or Streptavidin, 37 DEG C of incubations 2 hours; Clean three times with pure water, finally blot residual liquid with thieving paper; In microarray reaction chamber, add 200 ~ 400 microlitre pyridine ruthenium electrochemical luminescence-producing reaction systems buffering molten (0.03 mM2.0 mM dibutylethanolamine, 0.05 M phosphate buffer, pH 69), put into electrochemiluminescence checkout equipment and detect electrochemiluminescence value.
Specific embodiment six
The electrochemiluminescence detecting step of PCR primer:
(1) PCR amplification system:
Following ingredients is added to final volume 50 μ L in 0.2mL PCR reaction tube:
10 μ L template DNAs
5 μ L 10 × PCR damping fluids
1.5μL 50mM MgCl2
1 μ L 10mM dNTP potpourri
1 μ L marks the combined upstream amplimer (10 μMs) of bipyridyl ruthenium
1 μ L marks the mixed downstream amplimer (10 μMs) of bipyridyl ruthenium
0.5 μ l Taq archaeal dna polymerase (5U/ μ L)
Add sterile purified water to cumulative volume 50 μ L;
(2) PCR reaction conditions:
94 DEG C, 2 minutes
94 DEG C, 25 seconds
60 DEG C, 25 seconds
30 circulations
72 DEG C, 40 seconds
72 DEG C, 5 minutes;
(3) system is hybridized:
Hybridization buffer: 20 × SSC (3.0M NaCl, 0.3 M sodium citrate, pH 7.08.0);
0.1 micromole's pcr amplification product 55 μ L
20×SSC 25μL
1% SDS 10μL
Total reaction volume 100 μ L
(4) hybridization procedures:
A. the electrochemical luminescence electrode array that oligonucleotide probe of learning from else's experience is modified, adds above-mentioned hybridization system reactant liquor 200400 μ L in the reactor chamber, and 100 DEG C are heated 5 minutes, are then quickly cooled to room temperature.58 DEG C are incubated 1 hour;
B. add 200400 μ L 0.2 × SSC and 0.1%SDS in the reactor chamber, put 42 DEG C of cleanings in 5 minutes 2 times;
C. to the greatest extent solution in plate hole, fills it up with reacting hole with pure water, quietly puts three minutes, three times repeatedly, is finally upside down on thieving paper by reaction plate and blots residual liquid;
D. every hole adds 200 microlitre pyridine ruthenium electrochemical luminescence-producing reaction systems buffering molten (0.03 mM2.0 mM dibutylethanolamine, 0.05 M phosphate buffer, pH 69), puts into and adopts the electrochemiluminescence checkout equipment of floated electrode to detect electrochemiluminescence value.
Claims (6)
1. the present invention relates to electrode preparation and the method for modifying of a kind of electrochemiluminescence micro-array chip used in technical field of biological, it is characterized in that: each electrode in electrod-array all sized by, the square or rectangular of band circular arc drift angle that shape is identical, the surface area of single electrode is 0.25 ~ 25 mm
2, the spacing between adjacent electrode is 0.2 ~ 2 mm.
2. electrochemical luminous detection method according to claim 1, it is characterized in that: adopt each electrode surface in Synchronos method part or graft crosslinking molecular modification microarray to obtain simple, fast and efficient processing process and homogeneous electrode surface---described Synchronos method refers to and electrod-array same batch of reagent is carried out moditied processing simultaneously, described part comprises biotin, Avidin, Streptavidin, antiantibody and molecularly imprinted polymer (Molecular Imprinted Polymer, MIP).
3. the electrochemical luminous detection method according to claim 1,2, is characterized in that: use imprint method to load capture probe molecules at electrode surface, when loading impression, can adopt successively Loading Method or loaded in parallel method.
4. the capture probe molecules described in comprises: biotin, Avidin, capture antibody, acceptor, molecularly imprinted polymer, peptide nucleic acid (Peptide Nucleic Acid) probe or nucleic acid probe.
5. wherein nucleic acid probe comprises genome DNA probe, cDNA probe, rna probe, cRNA probe and oligonucleotide probe.
6. the electrochemical luminous detection method according to claim 1,2, it is characterized in that: use the dimethyl silicone polymer (Polydimethylsiloxane through surface graft modification, PDMS) die, capture probe impression is loaded at the electrode surface with imprint method, die shape is similar with corresponding tiny array electrode, size is less than corresponding electrode, and its mid point overlaps with corresponding electrode mid point, makes each electrode surface all can obtain a complete capture probe impression.
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