CN103913571B - A kind of immunoassay device of array fracture electrode and application - Google Patents
A kind of immunoassay device of array fracture electrode and application Download PDFInfo
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- CN103913571B CN103913571B CN201410164371.3A CN201410164371A CN103913571B CN 103913571 B CN103913571 B CN 103913571B CN 201410164371 A CN201410164371 A CN 201410164371A CN 103913571 B CN103913571 B CN 103913571B
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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
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Abstract
The invention discloses immunoassay device and the method for a kind of array fracture electrode, these apparatus and method are that analysis platform ruptures electrode for detecting electrode with array with chip, what be marked with noble metal nano particles forms binary or ternary immune complex for the antibody analyzing thing at the gap location of detecting electrode, silver enhancement solution forms deposition of silver under the catalytic action of noble metal nano particles, formation has the electrode of different impedance to form different photo-signals, and then carries out quantitative measurment to determinand.Apparatus of the present invention signal stabilization, comparability are strong, highly sensitive, excitation wavelength and photoelectric activity material range of choices large, can realize flux analysis.
Description
Technical field
The present invention relates to photoelectricity immunoassay technology, is a kind of device utilizing the immune detection of photoelectric technology and human lymph node effect.
Background technology
High flux, microminiaturization, portability are one of study hotspots of immunoassay always.Photic electrochemical process refers to that photoelectric activity material makes electric charge produce transfer because of absorb photons, thus forms photocurrent.
Be for the device of photic electrochemical analysis up to now and build voluntarily, there is 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 realize on one chip integrated.At present, there are no report chip technology being applied to photic Electrochemical Detection.
Utilizing photoelectric technology to carry out immune detection is all directly be fixed on optoelectronic pole by antigen-antibody, and exciting light direct irradiation on the analyte.This way produces sex change after immunoassay being caused to be irradiated by illumination on the one hand, and causes the amount that photosignal in fact can not reflect analysis thing truly; On the other hand, easily affect its surface topography when immunoassay directly fixes photoelectric material at the electrode surface, and the comparability caused before and after photosignal reduces.
Summary of the invention
The object of the invention is the immunoassay device of openly a kind of array fracture electrode, this device by overcoming prior art jitter, operate loaded down with trivial details, can not high flux and system complexity problem and set up a kind of New Photoinduced electrochemical immunoassay method that can realize high-sensitivity detection, the method background signal is low, 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 immunoassay device of array fracture electrode, it is the gap location utilizing silver ion to be deposited on fracture electrode (gapelectrode or splitelectrode) under the catalysis of the noble metal nano particles of variable concentrations, form the electrode with different impedance, be expressed as a kind of immunoassay device of not identical photocurrent, it is constructed as follows:
1, a photic electrochemical analyser, photic electrochemical analyser is a kind of photic electrochemical photocurrent detection device, it has a darkroom casing, light path LED lamp is upwards had at the bottom device in darkroom, the LED light intensity control module that what this LED had wire to connect be located at outside darkroom, the light path of LED has LED light focusing unit, this light focusing unit can regulate the size of the optically focused hot spot of LED, a photoelectrochemistrpool pool support is had in the light path front of light focusing unit, on support stably, be placed with photoelectrochemistrpool pool freely, photoelectrochemistrpool pool is that quartzy material is made, its sensitive surface aims at the center of the exciting light that LED sends, working electrode is equipped with in photoelectrochemistrpool pool, contrast electrode and the three-electrode system to electrode composition, this three-electrode system is connected with the electrochemical workstation be arranged on outside darkroom, electrochemical workstation provides the constant voltage that can preset to three-electrode system, and can record the electrochemical source of current (photocurrent) of corresponding generation, electrochemical workstation is connected with computing machine, and computing machine is used for the setting of photocurrent experiment parameter and data acquisition (see patented claim " a kind of photic galvanochemistry optoelectronic pole pick-up unit " (application number: 201310473553.4)),
2, detection chip: be coated with conductive material glass for chip substrates produce required optoelectronic pole and array fracture electrode pattern, its figure is that optoelectronic pole is connected with one end of array fracture electrode, described photoelectricity very diameter is the circle of 5 ± 1mm, described array fracture electrode width is 8 ± 2mm, and described fracture electrode is have the fracture electrode (see Fig. 1) being less than the electric open circuit gap of the wide formation of 1mm;
3, dimethyl silicone polymer (PDMS) thin slice with photic electrochemical cell circular hole and array detection pond aperture is made photic electrochemical cell circular hole align with optoelectronic pole to form photic electrochemical cell with chip bonding, the detection aligned in position of detection cell aperture and chip forms detection cell, and described detection position is ruptured on the fracture electrode of electrode relative to the fracture gap other end of photic electrochemical cell in every bar display;
4, noble metal nano particles is prepared and by this particle label on a kind of antibody of determinand;
5. another antibody molecule for determinand or direct fixing determinand molecule is fixed in the bottom of detection cell;
6, the antibody molecule being marked with noble metal added in detection cell in determinand molecule and 4 is formed: the ternary sandwich immune complex being marked with another antibody molecule composition in the antibody molecule-testing molecule-5 of noble metal or the antibody molecule being marked with noble metal only added in 4 are marked with antibody molecule and the molecular binary immuno compound of determinand of noble metal with formation;
7, in detection cell, add silver enhancement solution, remove the liquid in detection cell after 15-20 minute and dry up detection cell with nitrogen;
8, the electrode bottom photoelectrochemistrpool pool is modified photoelectric activity material;
9, the chip obtained in 3 and photic electrochemical analyser connected and chip be placed on photic galvanochemistry platform.
The immunoassay device of above-mentioned array fracture electrode, the gap of described fracture electrode is that nanometer is to micron order.
The immunoassay device of above-mentioned array fracture electrode, the photoelectric activity material that described electrode is modified is CdS quantum dot.
A kind of immunologic detection method adopting the immunoassay device of above-mentioned array fracture electrode, it is the gap location utilizing silver ion to be deposited on fracture electrode (gapelectrode or splitelectrode) under the catalysis of the noble metal nano particles of variable concentrations, form the electrode with different impedance, be expressed as not identical photocurrent; Comprise the following steps:
Step 1. prepares a photic electrochemical analyser;
Step 2. be coated with conductive material glass for chip substrates produce required optoelectronic pole and array fracture electrode pattern, its figure is that optoelectronic pole is connected with one end of array fracture electrode, described photoelectricity very diameter is the circle of 5 ± 1mm, described array fracture electrode width is 8 ± 2mm, and described fracture electrode is have the fracture electrode being less than the electric open circuit gap of the wide formation of 1mm; (see Fig. 1)
Dimethyl silicone polymer (PDMS) thin slice with photic electrochemical cell circular hole and array detection pond aperture makes photic electrochemical cell circular hole align with optoelectronic pole to form photic electrochemical cell with chip bonding by step 3., the detection aligned in position of detection cell aperture and chip forms detection cell, and described detection position is ruptured on the fracture electrode of electrode relative to the fracture gap other end of photic electrochemical cell in every bar display;
Step 4. prepares noble metal nano particles and by this particle label on a kind of antibody of determinand;
Step 5. fixes another antibody molecule for determinand or direct fixing determinand molecule in the bottom of detection cell;
Step 6. adds the antibody molecule being marked with noble metal of preparation in determinand molecule and step 4 and is formed in detection cell: the ternary sandwich immune complex being marked with another antibody molecule composition of the antibody molecule-testing molecule-step 5 of noble metal or the antibody molecule being marked with noble metal only adding preparation in step 4 are to form the antibody molecule and the molecular binary immuno compound of determinand that are marked with noble metal;
Step 7. adds silver enhancement solution in detection cell, removes the liquid in detection cell and dry up detection cell with nitrogen after 15-20 minute;
The electrode of step 8. bottom photoelectrochemistrpool pool modifies photoelectric activity material;
Chip and photocurrent measuring instrument connect and are placed in by chip on photic galvanochemistry platform by step 9., and starting light source namely can measuring-signal.
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 that described electrode is modified 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 present invention has the following advantages compared with other method existing:
1) current photic electrochemical immunoassay method, be all be fixed on by immune molecule on photoelectric activity material, before and after the signal of this detection method, comparability, reappearance are not good.Detecting electrode is separated with optoelectronic pole by the present invention, and photoelectric material surface is injury-free, substantially increases comparability and the reappearance of data.
2) in photic electro-chemistry immunity popular at present detects, all chip is not combined with it, be difficult to experiment high throughput analysis.Chip combines with photic galvanochemistry by the present invention, achieves flux immune detection.
3) in existing photic electrochemical immunoanalytical, in testing process, analyze the thing illumination that is all excited to penetrate, some are analyzed the easily illuminated and sex change of things and affect detection signal, therefore this method limits the light of some wavelength and the application of some photographone active substances.Present invention achieves effectively being separated of detecting electrode and optoelectronic pole, effectively prevent analyze thing by illumination, improve the stability of signal, extend 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.
The layout structure figure of Fig. 2 apparatus 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.
The fracture electrode conduction schematic diagram of Fig. 3 apparatus of the present invention when detecting, 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 the array fracture electrode of Fig. 4 apparatus of the present invention.
Fig. 5 detection signal figure of the present invention, wherein a does not add curve when analyzing thing; The signal that b is analyte concentration when being 10ng/ml; The signal that c is analyte concentration when being 100ng/ml.
Embodiment
Embodiment 1:
The reagent adopting apparatus of the present invention to use in the implementation process of immunologic detection method comprises following several composition: the antibody being marked with the nano particle of noble metal, antibody and determined antigen, the electron donor solution needed for photic electrochemical reaction and silver enhancement solution.The device used comprises photic electroanalysis instrument, multi-channel electrochemical workstation.
For golden nanometer particle, prostate cancer antigen and antibody, CdS quantum dot, the concrete implementing procedure of apparatus of the present invention in immune detection is described:
The making of chip:
Fracture electrode is one section of discontinuous conductors for detecting, gap place betwixt, when analyzing reaction that thing participates in and produce conducting to a certain degree to fracture electrode, can measure and carry out the amount of metric analysis thing (see Chia-HsienYeh by the electric current of this conductor; Wei-TingChen; Hong-PingLin; Tsung-ChainChang; Yu-ChengLin, Developmentofanimmunoassaybasedonimpedancemeasurementsut ilizinganantibody-nanosilverprobe, silverenhancement, andelectro-microchip.SensorsandActuatorsB-Chemical2009,139 (2), 387-393.).
(1) preparation of chip substrates electrode and the bonding of dimethyl siloxane (PDMS) thin slice
(1) graphic plotting
The figure of optoelectronic pole is finished 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 for being less than 0-1mm.
(2) print
By the Graphic transitions that makes in (1) on silk screen, 63mm ╳ 63mm tin indium oxide (ITO) electro-conductive glass is fixed on below the silk screen with electrode pattern, on silk screen, brush ink transfers on ITO electro-conductive glass by electrode pattern, at the moon place airing ink.
(3) corrode
The above-mentioned ito glass with ink is immersed 37 DEG C of ITO corrosive liquids (15% hydrochloric acid solution) 15 minutes, takes out ito glass and rinse well with water.
(4) removal ink
Ito glass in (3) to be immersed in 10% sodium hydroxide solution about 15 minutes, and the glass-chip of ITO electrode is rinsed and obtained to taking-up water well.
(5) dimethyl siloxane (PDMS) thin slice makes
Get PDMS15g, hardening agent 1.5g, mixes, and vacuum outgas is poured in double dish, and heating 1 hour on the warm table being flatly placed in 80 DEG C, peels off PDMS and be cut into about 50mm ╳ 63mm size with scalpel after its cooling from double dish.With card punch, the circular port that diameter is 6mm is being opened to the position being applied to optoelectronic pole, opening the hole of 3mm ╳ 6mm corresponding to detection position.
(6) bonding
The glass substrate of the ITO electrode of cleaning and the porose PDMS thin slice of band are carried out reversibility bonding and forms photoelectric cell and detection cell, obtain chip.
(2) mean diameter is the synthesis of the golden nanometer particle of 16nm:
By the mass percentage concentration of 100ml be 0.01% aqueous solution of chloraurate be heated with stirring to slight boiling condition, add rapidly the trisodium citrate aqueous solution that 5ml mass percentage concentration is 1%, solution can become claret, continue stir and naturally cooling, 4 DEG C save backup.
(3) mark of prostate cancer second antibody:
Getting concentration is that the anti-100 μ l of prostate cancer two of 100 μ g/ml add in the above-mentioned golden nanometer particle of 2ml, 20min is placed in 25 DEG C after shake well, add the bovine serum albumin (BSA) that 150 μ l concentration are 1mg/ml again, shake well, place 20min for 25 DEG C.Then be the centrifugal 10min of 14000rpm by it at rotating speed, abandoning supernatant, is scattered in the pure water of 500 μ l again by sediment, 4 DEG C 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 DEG C, dry up with nitrogen with ethanol purge secondary.Add 5% glutaraldehyde (GA) (with 50mM phosphate buffer, pH7.6 is solvent), place 2 hours for 37 DEG C, with water cleaning twice, nitrogen dries up.Add the prostate cancer first antibody that concentration is 20 μ g/ml again, hatch 4 hours for 37 DEG C, with phosphate buffer cleaning twice, nitrogen dries up, different concentration known (1ng is added in five detection cells, 10ng, 100ng, 1000ng, 10000ng, prostate cancer antigen (determinand) 100000ng), the prostate cancer antigen (determinand) of unknown concentration is added in a detection cell, in 37 DEG C of hatchings 2 hours, with phosphate buffer cleaning twice, nitrogen dries up, inject the second antibody being marked with golden nanometer particle of (3), hatch 2 hours for 37 DEG C, with phosphate buffer cleaning twice, nitrogen dries up, finally add silver enhancement solution.With water cleaning twice, nitrogen dries up.
(5) synthesis of CdS quantum dot:
Getting 50ml concentration is 1.0 х 10
?2caddy (the CdCl of mol/L
2) aqueous solution, put in 100ml three-necked bottle, stir and pass into nitrogen, to reinject 250 μ l mercaptoacetic acid, after stirring, regulate above-mentioned pH value of solution to 11.0 with NaOH (NaOH) solution of 1.0mol/L, after nitrogen continues 30min, add the sodium sulfide solution that 5.0mL concentration is 0.1mol/L, reflux 4 hours, then remove thermal source and naturally cool and get final product.4 DEG C of storages are for subsequent use.
(6) modification of optoelectronic pole:
2% poly-(diallyldimethylammonium chloride) (PDDA) solution 50 μ l is added in photic electrochemical cell, after 10 minutes, carefully clean three times with water, nitrogen dries up, add the CdS quantum dot of preparation in (5), after 10 minutes, carefully clean three times with water, after nitrogen dries up, add 2% (PDDA) solution 50 μ l, 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 handled well is placed on photic electrochemical analysis platform, 1030a type electrochemical workstation and each wire is connected by Fig. 2, the aqueous ascorbic acid of 0.1mol/L is injected in photic electrochemical cell, open light source and electrochemical workstation measurement 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 is by the reaction of specific biocompatible and noble metal nano particles to the catalytic deposition effect of silver ion, and formation has the electrode of different impedance, is expressed as different photo-signals, and then carries out quantitative measurment to determinand.Compare with method with existing photic electrochemical analysis device, the range of choice that the apparatus and method of this invention have exciting light and photoelectric activity material is wide, signal stabilization, comparability are strong also can carry out realizing the advantages such as high flux detection, may be used for the highly sensitive detection of environmental chemistry pollution, medicine, hormone, antibody, protein, nucleic acid.
Claims (4)
1. the immunoassay device of an array fracture electrode, it is characterized in that: it is the gap location utilizing silver ion to be deposited on fracture electrode under the catalysis of the noble metal nano particles of variable concentrations, form the electrode with different impedance, be expressed as a kind of immunoassay device of not identical photocurrent, it is constructed as follows:
1, a photic electrochemical analyser, photic electrochemical analyser is a kind of photic electrochemical photocurrent detection device, it has a darkroom casing, light path LED lamp is upwards had at the bottom device in darkroom, the LED light intensity control module that what this LED had wire to connect be located at outside darkroom, the light path of LED has LED light focusing unit, this light focusing unit can regulate the size of the optically focused hot spot of LED, a photoelectrochemistrpool pool support is had in the light path front of light focusing unit, on support stably, be placed with photoelectrochemistrpool pool freely, photoelectrochemistrpool pool is that quartzy material is made, its sensitive surface aims at the center of the exciting light that LED sends, working electrode is equipped with in photoelectrochemistrpool pool, contrast electrode and the three-electrode system to electrode composition, this three-electrode system is connected with the electrochemical workstation be arranged on outside darkroom, electrochemical workstation provides the constant voltage that can preset to three-electrode system, and can record the electrochemical source of current of corresponding generation, electrochemical workstation is connected with computing machine, and computing machine is used for setting and the data acquisition of photocurrent experiment parameter,
2, detection chip: be coated with conductive material glass for chip substrates produce required optoelectronic pole and array fracture electrode pattern, its figure is that optoelectronic pole is connected with one end of array fracture electrode, described photoelectricity very diameter is the circle of 5 ± 1mm, described array fracture electrode width is 8 ± 2mm, and described array fracture electrode is have the array fracture electrode being less than the electric open circuit gap of the wide formation of 1mm;
3, dimethyl silicone polymer (PDMS) thin slice with photic electrochemical cell circular hole and array detection pond aperture is made photic electrochemical cell circular hole align with optoelectronic pole to form photic electrochemical cell with detection chip bonding, the detection aligned in position of detection cell aperture and detection chip forms detection cell, and described detection position is ruptured on the fracture electrode of electrode relative to the fracture gap other end of photic electrochemical cell at every strip array;
4, noble metal nano particles is prepared and by this particle label on a kind of antibody of determinand;
5, another antibody molecule for determinand or direct fixing determinand molecule is fixed in the bottom of detection cell;
6, the antibody molecule being marked with noble metal added in detection cell in determinand molecule and 4 is formed: the ternary sandwich immune complex being marked with another antibody molecule composition in the antibody molecule-testing molecule-5 of noble metal or the antibody molecule being marked with noble metal only added in 4 are marked with antibody molecule and the molecular binary immuno compound of determinand of noble metal with formation;
7, in detection cell, add silver enhancement solution, remove the liquid in detection cell after 15-20 minute and dry up detection cell with nitrogen;
8, the optoelectronic pole bottom photic electrochemical cell is modified photoelectric activity material;
9, the chip obtained in 3 and photic electrochemical analyser connected and chip be placed on photic galvanochemistry platform.
2. the immunoassay device of array fracture electrode according to claim 1, is characterized in that: the gap of described array fracture electrode is that nanometer is to micron order.
3. the immunoassay device of array fracture electrode according to claim 1, is characterized in that: the photoelectric activity material that described optoelectronic pole is modified is CdS quantum dot.
4. pick-up unit according to claim 1, the application in the highly sensitive detection of environmental chemistry pollution, medicine, hormone, antibody, protein, nucleic acid.
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| 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 |
| WO2018138105A1 (en) * | 2017-01-27 | 2018-08-02 | Merck Patent Gmbh | Method for detecting breakage of a substrate of a switchable optical element and switchable optical device |
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| CN101858918A (en) * | 2010-06-09 | 2010-10-13 | 长沙市食品质量安全监督检测中心 | Microgap array electrode-based electrochemical immunosensor and method for detecting ractopamine in animal-derived food thereof |
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| Electrochemiluminescence on bipolar electrodes for visual bioanalysis;Mei-Sheng Wu 等;《Chemical Science》;20121221;第4卷;第1182-1188页 * |
| 纳米间隙电极的制备及应用;董晓东 等;《显微、测量、微细加工技术与设备》;20100131;第47卷(第1期);第50-55、63页 * |
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