CN103913571A - Immunodetection method of array breakage electrode - Google Patents
Immunodetection method of array breakage electrode Download PDFInfo
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- CN103913571A CN103913571A CN201410164371.3A CN201410164371A CN103913571A CN 103913571 A CN103913571 A CN 103913571A CN 201410164371 A CN201410164371 A CN 201410164371A CN 103913571 A CN103913571 A CN 103913571A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
Abstract
The invention discloses an immunodetection method of an array breakage electrode. The method comprises the following steps: taking a chip as an analytic platform and the array breakage electrode as a detection electrode, forming a binary or ternary immunocomplex at a gap of the detection electrode by a noble metal nanoparticle-marked antibody for an analyte, forming silver deposition under the catalytic action of noble metal nanoparticles by a silver-enhanced solution, forming different light current signals by forming electrodes with different impedance, and quantitatively measuring a material to be detected. According to the method, the signal is stable, the comparability is good, the sensitivity is high, the selectable range of excitation light wavelengths and photoelectric activity materials is wide, and the quantitative analysis can be realized.
Description
Technical field
The present invention relates to photoelectricity immunoassay technology, is a kind of immunologic detection method that utilizes photoelectric technology and silver-colored enhancement effect.
Background technology
High flux, microminiaturization, portability are one of study hotspots of immunoassay always.Photic electrochemical process refers to that photoelectric activity material shifts charge generation because absorbing photon, thereby forms photocurrent.
Be voluntarily and build for the device of photic electrochemical analysis up to now, have the shortcomings such as bulky, complex structure.Be difficult to realize high flux, microminiaturization, portability.
The advantages such as it is few that chip apparatus has reagent consumption, convenient and swift, in addition, several functions can be realized and being integrated on chip piece.At present, there are no the report that chip technology is applied to photic Electrochemical Detection.
Utilizing photoelectric technology to carry out immune detection is all that antigen-antibody is directly fixed on optoelectronic pole, and exciting light direct irradiation is on analyte.This way produces sex change after can causing on the one hand immunoassay to be irradiated by illumination, and causes photosignal in fact and can not reflect truly the amount of analyte; On the other hand, when immunoassay is directly fixed on the photoelectric material on electrode surface, easily affect its surface topography, and the comparability causing before and after photosignal reduces.
Summary of the invention
The object of the invention is the openly immunologic detection method of a kind of array fracture electrode, by overcoming prior art jitter, operate loaded down with trivial details, can not high flux and the problem of system complexity.Set up a kind of New Photoinduced electrochemical immunoassay method that can realize high-sensitivity detection, the method background signal is low, and signal to noise ratio (S/N ratio) is high, can meet the testing requirement of trace materials.
For achieving the above object, technical solution of the present invention is:
A kind of immunologic detection method of array fracture electrode, it utilizes silver ion under the catalysis of the noble metal nano particles of variable concentrations, to be deposited on the gap location of fracture electrode (gap electrode or split electrode), formation has the electrode of different impedances, is expressed as not identical photocurrent; Comprise the following steps:
Step 1. is prepared a photic electrochemical analyser;
Step 2. is produced required optoelectronic pole and array fracture electrode pattern to be coated with conductive material glass as chip substrates, its figure is that optoelectronic pole is connected with one end of array fracture electrode, the described photoelectricity circle that very diameter is 5 ± 1mm, described array fracture electrode width is 8 ± 2mm, and described fracture electrode is to have to be less than the wide fracture electrode that forms the electric gap of opening circuit of 1mm; (seeing Fig. 1)
Step 3. is by dimethyl silicone polymer (PDMS) thin slice with photic electrochemical cell circular hole and array detection pond aperture and chip bonding and photic electrochemical cell circular hole is alignd with optoelectronic pole form photic electrochemical cell, the detection cell aperture formation detection cell that aligns with the detection position of chip, described detection position is at every display fracture electrode on the fracture electrode with respect to the fracture gap other end of photic electrochemical cell;
Step 4. is prepared noble metal nano particles and this particle is marked on a kind of antibody for determinand;
Step 5. is fixed for another antibody molecule of determinand or is directly fixed determinand molecule in the bottom of detection cell;
Step 6. adds in determinand molecule and step 4 antibody molecule that is marked with noble metal of preparation to form in detection cell: be marked with the antibody molecule-testing molecule-step 5 of noble metal another antibody molecule composition ternary sandwich immune complex or only add the antibody molecule that is marked with noble metal of preparation in step 4 to form the antibody molecule and the molecular binary immune complex of determinand that are marked with noble metal;
Step 7. adds silver enhancement solution in detection cell, removes liquid in detection cell and dry up detection cell with nitrogen after 15-20 minute;
Step 8. is modified photoelectric activity material on the electrode of photoelectrochemistrpool pool bottom;
Step 9. is by chip and photocurrent measuring instrument connects and chip is placed on photic galvanochemistry platform, and starting light source can measuring-signal.
The immunologic detection method of above-mentioned array fracture electrode, the gap of described fracture electrode is that nanometer is to micron order.
The immunologic detection method of above-mentioned array fracture electrode, the diameter of described noble metal nano particles is nanoscale.
The immunologic detection method of above-mentioned array fracture electrode, described noble metal nano particles is golden nanometer particle or Nano silver grain.
The immunologic detection method of above-mentioned array fracture electrode, the photoelectric activity material of modifying on described electrode is CdS quantum dot.
The immunologic detection method of above-mentioned array fracture electrode, described determinand is organic compound, nucleotide, RNA (ribonucleic acid), DNA (deoxyribonucleic acid), monose, polysaccharide, amino acid, polypeptide or protein.
The immunologic detection method of above-mentioned array fracture electrode, described photic electrochemical analyser is a kind of photic galvanochemistry photocurrent pick-up unit, it has a darkroom casing, there is light path LED lamp upwards at the bottom device in darkroom, this LED lamp has the strong control module of LED light outside darkroom that is located at of wire connection, in the light path of LED lamp, there is LED lamp light focusing unit, this light focusing unit can regulate the size of the optically focused hot spot of LED lamp, there is a photoelectrochemistrpool pool support in the light path front of light focusing unit, on support stably, be placed with freely photoelectrochemistrpool pool, photoelectrochemistrpool pool is that quartzy material is made, the center of the exciting light that its sensitive surface aligning LED lamp sends, in photoelectrochemistrpool pool, be equipped with working electrode, contrast electrode and the three-electrode system to electrode composition, this three-electrode system is connected with the electrochemical workstation being arranged on outside darkroom, electrochemical workstation provides energy default constant voltage to three-electrode system, and can record the electrochemical source of current (photocurrent) of corresponding generation, electrochemical workstation is connected with computing machine, computing machine for the setting of photocurrent experiment parameter and data acquisition (referring to patented claim " a kind of photic galvanochemistry optoelectronic pole pick-up unit " (application number: 201310473553.4) identical.
The present invention has the following advantages compared with existing other method:
1) current photic electrochemical immunoassay method, is all that immune molecule is fixed on photoelectric activity material, and comparability before and after the signal of this detection method, reappearance are not good.The present invention separates detecting electrode with optoelectronic pole, photoelectric material surface is injury-free, has greatly improved comparability and the reappearance of data.
2) at present popular photic electro-chemistry immunity detects, all chip is not combined with it, be difficult to experiment high throughput analysis.The present invention combines chip with photic galvanochemistry, realized flux immune detection.
3) in existing photic electrochemical immunoanalytical, in testing process, the analyte irradiation that is all excited, the easily illuminated and sex change and affect detection signal of some analytes, therefore this method has limited the application of light He some photographone active substances of some wavelength.The present invention realized detecting electrode and separated with the effective of optoelectronic pole, effectively prevented analyte by illumination, improved signal stability, expanded the range of choice of photoelectric activity material.
Accompanying drawing explanation
Optoelectronic pole and display fracture electrode schematic diagram on Fig. 1 base material of the present invention.1 is optoelectronic pole; 2 is fracture electrode (6), and 3 is fracture electrode gap.
Fig. 2 layout structure figure of the present invention, wherein 4 is chip; 5 is photic electrochemical analyser; 6 is photic electrochemical cell; 7 is detection cell; 8 is contrast electrode; 9 is to electrode; 10 electrochemical workstations.
Fig. 3 fracture electrode conduction of the present invention schematic diagram, wherein 11 is glass; 12 is prostate cancer first antibody; 13 is prostate cancer antigen; 14 is prostate cancer second antibody; 15 is conductor part; 16 is noble metal nano particles; 17 is depositing silver.
The immunologic detection method process flow diagram of Fig. 4 array fracture of the present invention electrode.
Fig. 5 detection signal figure of the present invention, wherein a is the curve while not adding analyte; B is the signal of analyte concentration while being 10ng/ml; C analyses the signal of substrate concentration while being 100ng/ml.
Embodiment
Embodiment 1:
The reagent that the inventive method is used in implementation process comprises following several composition: be marked with the antibody of the nano particle of noble metal, antibody and determined antigen, electron donor solution and silver enhancement solution that photic electrochemical reaction is required.The device using comprises photic electroanalysis instrument, multi-channel electrochemical workstation.
Take golden nanometer particle, prostate cancer antigen and antibody, CdS quantum dot as example, the concrete implementing procedure of the inventive method is described:
The making of chip
Fracture electrode be one section for detection of discontinuous conductors, gap place betwixt, the reaction participating in when analyte is to fracture electrode while producing conducting to a certain degree, can measure the amount of carrying out metric analysis thing by the electric current of this conductor (referring to Chia-Hsien Yeh; Wei-Ting Chen; Hong-Ping Lin; Tsung-Chain Chang; Yu-Cheng Lin, Development of an immunoassay based on impedance measurements utilizing an antibody-nanosilver probe, silver enhancement, and electro-microchip.Sensors and Actuators B-Chemical2009,139 (2), 387-393.).
(1) bonding of the preparation of chip substrates electrode and dimethyl siloxane (PDMS) thin slice
(1) graphic plotting
Finish the figure of optoelectronic pole with Coredraw mapping software, a diameter 5mm optoelectronic pole and 6 width are that 8mm detecting electrode is distributed in both sides, one end of optoelectronic pole and 6 detecting electrodes is all connected, and there is a gap centre of detecting electrode, and its spacing size is 0-1mm.
(2) printing
The figure making in (1) is transferred on silk screen, 63mm ╳ 63mm tin indium oxide (ITO) electro-conductive glass is fixed on below the silk screen of electrode pattern, on silk screen, brush ink and transfer on ITO electro-conductive glass by electrode pattern, at the moon place airing ink.
(3) corrosion
The above-mentioned ito glass with ink is immersed to 37 ℃ of ITO corrosive liquids (15% hydrochloric acid solution) 15 minutes, take out ito glass water and rinse well.
(4) removal ink
Ito glass in (3) is immersed in 10% sodium hydroxide solution to approximately 15 minutes, takes out water and rinse well and obtain the glass-chip of ITO electrode.
(5) dimethyl siloxane (PDMS) thin slice is made
Get PDMS15g, hardening agent 1.5g, mixes, and vacuum outgas is poured in double dish, is flatly placed on the warm table of 80 ℃ and heats 1 hour, after it is cooling, PDMS is peeled off from double dish and is cut into about 50mm ╳ 63mm size with scalpel.With card punch the position that is applied to optoelectronic pole being opened to the circular port that diameter is 6mm, in the hole of opening 3mm ╳ 6mm corresponding to detection position.
(6) bonding
Carry out reversibility bonding formation photoelectric cell and detection cell by the glass substrate of clean ITO electrode with porose PDMS thin slice, obtain chip.
(2) synthesizing of the golden nanometer particle that mean diameter is 16nm:
The aqueous solution of chloraurate that is 0.01% by the mass percentage concentration of 100ml is heated with stirring to slight boiling condition, and adding rapidly 5ml mass percentage concentration is 1% trisodium citrate aqueous solution, and solution can become claret, continues to stir and naturally cooling, and 4 ℃ save backup.
(3) mark of prostate cancer second antibody:
The anti-100 μ l of prostate cancer two that get concentration and be 100 μ g/ml add in the above-mentioned golden nanometer particle of 2ml, after shake well, place 20min in 25 ℃, add the bovine serum albumin (BSA) that 150 μ l concentration are 1mg/ml, shake well, places 20min for 25 ℃ again.Then be the centrifugal 10min of 14000rpm by it at rotating speed, abandoning supernatant, is scattered in sediment in the pure water of 500 μ l again, and 4 ℃ of storages are for subsequent use.
(4) modification of chip and the preparation of immune complex:
In the detection cell of chip, add 5% (3-aminopropyl) triethoxysilane (APTES) ethanolic solution, place 2 hours for 37 ℃, clean secondary and dry up with nitrogen with ethanol.Add 5% glutaraldehyde (GA) (with 50mM phosphate buffer, pH7.6 is solvent), place 2 hours for 37 ℃, water cleans twice, and nitrogen dries up.Adding concentration is the prostate cancer first antibody of 20 μ g/ml again, hatch 4 hours for 37 ℃, with twice of phosphate buffer cleaning, nitrogen dries up, in five detection cells, add different concentration known (1ng, 10ng, 100ng, 1000ng, 10000ng, prostate cancer antigen (determinand) 100000ng), in a detection cell, add the prostate cancer antigen (determinand) of unknown concentration, in 37 ℃ of hatchings 2 hours, with twice of phosphate buffer cleaning, nitrogen dries up, inject the second antibody that is marked with golden nanometer particle of (3), hatch 2 hours for 37 ℃, with twice of phosphate buffer cleaning, nitrogen dries up, finally add silver enhancement solution.Water cleans twice, and nitrogen dries up.
(5) CdS quantum dot is synthetic:
Getting 50ml concentration is 1.0 х 10
-2caddy (CdCl2) aqueous solution of mol/L, put in 100ml three-necked bottle, stir and pass into nitrogen, the 250 μ l mercaptoacetic acid of reinjecting, after stirring, regulate above-mentioned pH value of solution to 11.0 with NaOH (NaOH) solution of 1.0mol/L, nitrogen continues after 30min, adding 5.0mL concentration is the sodium sulfide solution of 0.1mol/L, refluxes 4 hours, then removes thermal source naturally cooling and get final product.4 ℃ of storages are for subsequent use.
(6) modification of optoelectronic pole:
In photic electrochemical cell, add 2% poly-(diallyldimethylammonium chloride) (PDDA) solution 50 μ l, after 10 minutes, careful water cleans three times, and nitrogen dries up, add the CdS quantum dot of preparation in (5), after 10 minutes, careful water cleans three times, adds 2% (PDDA) solution, 50 μ l after nitrogen dries up, after 10 minutes, careful water cleans three times, and nitrogen dries up, three times and get final product so repeatedly.
(7) measurement of photocurrent:
The chip of handling well is placed on photic electrochemical analysis platform, connect 1030a type electrochemical workstation and each wire by Fig. 1, in photic electrochemical cell, inject the aqueous ascorbic acid of 0.1mol/L, open light source and electrochemical workstation and measure photo-signal, according to the current signal production standard curve of the prostate cancer antigen of five concentration known, record the concentration of prostate cancer antigen to be measured according to the current signal of the prostate cancer antigen of unknown concentration.
The present invention, by specific biocompatible reaction and the catalytic deposition effect of noble metal nano particles to silver ion, forms the electrode with different impedances, is expressed as different photo-signals, and then determinand is carried out to quantitative measurment.Compared with existing photic electrochemical analysis method, it is wide that the method has the range of choice of exciting light and photoelectric activity material, signal stabilization, comparability also can realize the advantages such as high flux detection by force, can be for the highly sensitive detection of all kinds of materials such as environmental chemistry pollution, medicine, hormone, antibody, protein, nucleic acid.
Claims (6)
1. the immunologic detection method of an array fracture electrode, it is characterized in that utilizing silver ion under the catalysis of the noble metal nano particles of variable concentrations, to be deposited on the gap location of fracture electrode (gap electrode or split electrode), formation has the electrode of different impedances, is expressed as not identical photocurrent; It specifically comprises the following steps:
Step 1. is prepared a photic electrochemical analyser;
Step 2. is produced required optoelectronic pole and array fracture electrode pattern to be coated with conductive material glass as chip substrates, its figure is that optoelectronic pole is connected with one end of array fracture electrode, the described photoelectricity circle that very diameter is 5 ± 1mm, described array fracture electrode width is 8 ± 2mm, and described fracture electrode is to have to be less than the wide fracture electrode that forms the electric gap of opening circuit of 1mm; (seeing Fig. 1)
Step 3. is by dimethyl silicone polymer (PDMS) thin slice with photic electrochemical cell circular hole and array detection pond aperture and chip bonding and photic electrochemical cell circular hole is alignd with optoelectronic pole form photic electrochemical cell, the detection cell aperture formation detection cell that aligns with the detection position of chip, described detection position is at every display fracture electrode on the fracture electrode with respect to the fracture gap other end of photic electrochemical cell;
Step 4. is prepared noble metal nano particles and this particle is marked on a kind of antibody for determinand;
Step 5. is fixed for another antibody molecule of determinand or is directly fixed determinand molecule in the bottom of detection cell;
Step 6. adds in determinand molecule and step 4 antibody molecule that is marked with noble metal of preparation to form in detection cell: be marked with the antibody molecule-testing molecule-step 5 of noble metal another antibody molecule composition ternary sandwich immune complex or only add the antibody molecule that is marked with noble metal of preparation in step 4 to form the antibody molecule and the molecular binary immune complex of determinand that are marked with noble metal;
Step 7. adds silver enhancement solution in detection cell, removes liquid in detection cell and dry up detection cell with nitrogen after 15-20 minute;
Step 8. is modified photoelectric activity material on the electrode of photoelectrochemistrpool pool bottom;
Step 9. is by chip and photocurrent measuring instrument connects and chip is placed on photic galvanochemistry platform, and starting light source can measuring-signal.
2. the immunologic detection method of array fracture electrode according to claim 1, is characterized in that: the gap of described fracture electrode is that nanometer is to micron order.
3. the immunologic detection method of array fracture electrode according to claim 1, is characterized in that: the diameter of described noble metal nano particles is nanoscale.
4. the immunologic detection method of array fracture electrode according to claim 1, is characterized in that: described noble metal nano particles is golden nanometer particle or Nano silver grain.
5. the immunologic detection method of array fracture electrode according to claim 1, is characterized in that: the photoelectric activity material of modifying on described electrode is CdS quantum dot.
6. the immunologic detection method of array fracture electrode according to claim 1, is characterized in that: described determinand is organic compound, nucleotide, RNA (ribonucleic acid), DNA (deoxyribonucleic acid), monose, polysaccharide, amino acid, polypeptide or protein.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105021575A (en) * | 2015-07-21 | 2015-11-04 | 青岛大学 | Photoelectric sensor for detection of kinase activity on the basis of local area surface plasma resonance |
| CN105628931A (en) * | 2016-01-19 | 2016-06-01 | 济南大学 | Preparation and application of alpha fetoprotein electrochemical immunosensor based on silver deposition |
| CN105806908A (en) * | 2016-03-07 | 2016-07-27 | 济南大学 | Preparation method and application of label-free type electrochemical immunosensor based on MoS2/Au composite material |
| CN110178075A (en) * | 2017-01-27 | 2019-08-27 | 默克专利股份有限公司 | For detecting the method and switchable optical device of the substrate breakage of switchable optical elements |
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| WO2003071259A1 (en) * | 2002-02-15 | 2003-08-28 | Ut-Battelle, Llc | Photoelectrochemical molecular comb |
| US20050069905A1 (en) * | 2003-09-30 | 2005-03-31 | Carl Myerholtz | Detection of molecular binding events |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105021575A (en) * | 2015-07-21 | 2015-11-04 | 青岛大学 | Photoelectric sensor for detection of kinase activity on the basis of local area surface plasma resonance |
| CN105628931A (en) * | 2016-01-19 | 2016-06-01 | 济南大学 | Preparation and application of alpha fetoprotein electrochemical immunosensor based on silver deposition |
| CN105806908A (en) * | 2016-03-07 | 2016-07-27 | 济南大学 | Preparation method and application of label-free type electrochemical immunosensor based on MoS2/Au composite material |
| CN110178075A (en) * | 2017-01-27 | 2019-08-27 | 默克专利股份有限公司 | For detecting the method and switchable optical device of the substrate breakage of switchable optical elements |
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