CN101655473B - Preparation method of nanogold immunoelectrode - Google Patents
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- 239000000243 solution Substances 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 20
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000002484 cyclic voltammetry Methods 0.000 claims abstract description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 13
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims abstract description 6
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000008151 electrolyte solution Substances 0.000 claims description 7
- 239000001117 sulphuric acid Substances 0.000 claims description 7
- 235000011149 sulphuric acid Nutrition 0.000 claims description 7
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- 244000309466 calf Species 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 3
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- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 abstract 1
- 238000004832 voltammetry Methods 0.000 abstract 1
- 239000000427 antigen Substances 0.000 description 18
- 102000036639 antigens Human genes 0.000 description 18
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- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
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- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 2
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
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Abstract
The invention discloses a preparation method of a nanogold immunoelectrode, which comprises the following steps: polishing a gold disk electrode with aluminum oxide powder; soaking the gold disk electrode in a mixed solution of concentrated sulfuric acid and hydrogen peroxide; cleaning the gold disk electrode with ultrasonic waves; polishing the gold disk electrode in a 0.5M sulfuric acid solution by cyclic voltammetry; using a chlorogold acid electrolyte as a base solution, and depositing nanogold particles on the gold disk electrode by linear cyclic sweep voltammetry; connecting a specific antibody; and sealing. The invention combines the electrodeposition technology and the nanogold technology; by directly using an electrodeposition method, gold ions in a gold sodium chloride solution are directly oxidized under the control of the electrochemical technology to form the nanogold particles; and finally the nanogold particles are assembled on the surface of the gold electrode. A sensor formed by the prepared immunoelectrode, a reference electrode and an auxiliary electrode not only keeps the advantages of high sensitivity, strong specificity, good repeatability and strong stability of the sensor, but also greatly shortens the preparation time of the working electrode.
Description
Technical field
The invention belongs to biological chemistry and technical field of electrochemistry, particularly a kind of method that adopts electro-deposition method at quick assemble nanometer gold particle of gold electrode surfaces and connection specific antibody.
Background technology
The immunosensor detection technique is that immunoassay technology is combined with the sensing detection technology and one type of novel detection technique forming; Be field of biosensors development one of technology the most rapidly, have highly sensitive, high specificity, detection is quick, easy to use, low-cost and realizes advantage perhaps such as online detection easily.The current mode immunosensor is a type of immunosensor; Usually form by conversion element (supporting electrode), biosensor (working electrode that antibody or antigen are modified), electrolytic cell and data Collection & Processing System, realize qualitative/detection by quantitative through change in current before and after detecting antigen-antibody reaction.Over year, the current mode immunosensor has obtained extensive studies in medical science, animal medicine, environmental analysis and food safety detection etc. are multi-field surplus in the of nearly ten, and wherein the package technique of biomolecule is the key content of this research field always on the working electrode.
At present; Nanogold particle adsorbs and the good carrier of support biomolecule and being applied in the research of immunosensor because of having that specific surface area is big, the surfactivity site is many, absorption affinity is strong, photoelectric characteristic is good, bioaffinity is strong and can keep advantage such as protein active to a certain extent, being used as.The common method of having reported that nm of gold is assembled into the immunosensor working electrode surface is: at first with some with active function groups (as-COOH ,-SH ,-S-S-,-OH etc.) molecule (like mercaptoethylmaine; The L-halfcystine; Cysteamine) modifies working electrode, utilize the covalent bond of these small molecule active functional groups and nm of gold again and nano Au particle is assembled into working electrode surface.Nano Au particle through covalent bond effect absorption can stably be combined in gold electrode surfaces, and still, the method for this assemble nanometer gold particle is consuming time longer, not only prolongs the test period, and has increased the uncertain factor of test, influences accuracy of experimental results.Such as; In the immunosensor manufacturing process that with the gold electrode is working electrode; Usually need gold electrode be soaked in halfcystine solution and just can make cysteine modified in 12 hours to electrode, after this need in the nm of gold colloidal solution of preparation in advance, place again and just can make nano Au particle be attached to electrode surface in 6-12 hour.And halfcystine is prone to be oxidised with air to cystine, directly has influence on the stability of immunosensor.
Summary of the invention
In order to overcome the above-mentioned defective of prior art, the invention provides a kind of immunosensor working electrode surface assemble nanometer gold grain be connected the method that specific antibody prepares nanogold immunoelectrode.
The preparation method of nanogold immunoelectrode is characterized in that may further comprise the steps:
A. the disk gold electrode being used diameter successively is that the alumina powder of 1 μ m, 0.3 μ m and 0.05 μ m is polished to light;
B. the electrode after the polishing was with the concentrated sulphuric acid and hydrogen peroxide mixed liquid dipping 10 minutes;
C. take out electrode, successively in absolute ethyl alcohol and distilled water with ultrasonic cleaning 15 minutes;
D. the electrode after will cleaning is put into the 0.5M sulfuric acid solution, polishes 30 minutes under-0.3～1.5V voltage with cyclic voltammetry, dries subsequent use;
E. be end liquid with gold chloride electrolytic solution, adopt linear cyclic voltammetry scan method, depositing nano gold grain on the disk gold electrode gets the nm of gold electrode;
F. the nm of gold electrode is immersed in the antibody-solutions of 0.5 μ g/ml, hatches under the room temperature after 20 minutes and to take out, with 0.25% calf serum sealing 15 minutes, dry after pure water is cleaned subsequent use, must nanogold immunoelectrode.
The present invention is the working electrode of immunosensor with the gold electrode of three-electrode system; Earlier to pre-service such as working electrode surface polish; Be electrolyte solution again with the chlorauric acid solution; Make gold ion produce golden simple substance in electrode surface generation reduction reaction with cyclic voltammetry, chien shih gold simple substance is deposited on electrode surface with the nano particle form during through gated sweep voltage and surface sweeping, and the gold particle diameter of acquisition is about 20nm; Adsorb specific antibody, seal nonspecific binding site through nano Au particle at last, make nanogold immunoelectrode with calf serum.
Nanogold immunoelectrode of the present invention; In the nm of gold assembling process, do not use intermediary's connection materials such as halfcystine; Do not need to prepare in advance nm of gold colloidal solution yet; Be assembled in gold electrode surfaces but directly use electro-deposition method to use electrochemical process control direct oxidation to form nanogold particle the gold ion in the sodium chloraurate solution; The sensor that the immunoelectrode of preparing forms with contrast electrode and auxiliary electrode has not only kept highly sensitive, the high specificity, good reproducibility of sensor, the advantage that stability is strong, and has shortened the preparation time of working electrode to a great extent.
Further, the said concentrated sulphuric acid of step b and hydrogen peroxide mixed liquor are 98% concentrated sulphuric acid and the preparation of 3: 1 by volume ratio of 30% superoxol.
Further, the starting potential of the said linear cyclic voltammetry scan method of step e is 1.1V, and final voltage is 0V, surface sweeping speed 0.1V/s, and the scanning number of turns is 1 circle.
The prepared nanogold immunoelectrode of the present invention can be used for detection specificity antigen.The current-responsive that adopts cyclic voltammetry to detect antibody and seized antigen-reactive front and back sensor changes, thereby confirms the content of seized antigen, and concrete detection method is:
The antigenic solution of normal concentration is carried out serial dilution according to the requirement of drawing standard curve, carries out cyclic voltammetry scan with the above-mentioned antigenic solution of electrode pair of the present invention respectively and detect, obtain a series of oxidation peak currents (i) decline number percent data k:
k＝(i
1-i
2)/i
1×100％
I in the formula
1Be the oxidation peak current before the immune response, i
2Be the oxidation peak current after the immune response.
Be ordinate and horizontal ordinate with peak current electric current decline number percent K and antigen concentration corresponding respectively with it; With Excel software processes drawing standard curve map; Obtain its corresponding regression equation, and sensitivity, specificity and stability that the immunosensor that this method is assembled detects are analyzed.
When the antigenic content in the test sample; Carry out cyclic voltammetry scan with electrode pair of the present invention solution to be measured and detect, calculate oxidation peak current (i) decline number percent k, according to the k value; Make the regression equation of curve with standard, can go out the content of antigen in the testing sample.
The method that the present invention prepares nanogold immunoelectrode has merged electro-deposition techniques and nm of gold technology, has simply, advantage fast and effectively, and can significantly shorten the test period, reduce experimentation cost, and the stability and the sensitivity that improve immunosensor.Utilize the working electrode of this method assembling highly sensitive, high specificity, good stability, cost of manufacture is low, can significantly improve immunosensor make efficiency and quality.
Description of drawings
Fig. 1 is the working curve of nanogold immunoelectrode of the present invention in hoptoglobin detects.
Embodiment
Below in conjunction with specific embodiment content of the present invention is further described.
Embodiment 1
1. the configuration of required solution
Piranha solution: 98% concentrated sulphuric acid and 30% superoxol are 3: 1 ratio preparation by volume.
The gold chloride electrolyte solution: the 1g gold chloride is dissolved into is mixed with 1% chlorauric acid solution in the 100ml pure water, the chlorauric acid solution of getting 1ml 1% again joins in the 30ml 0.5M dilution heat of sulfuric acid, mixes.
Electrolytic solution cell: take by weighing the 0.0832g potassium ferricyanide, the 0.1056g potassium ferrocyanide, 0.3728g potassium chloride is dissolved in the 50ml pure water.
2. preparation nanogold immunoelectrode
It is that the alumina powder of 1 μ m, 0.3 μ m and 0.05 μ m is polished to light that the disk gold electrode is used diameter successively; With the above-mentioned Piranha solution soaking for preparing 10 minutes; Be soaked in absolute ethyl alcohol and the distilled water ultrasonic cleaning more successively 15 minutes; Electrode after cleaning is put into the 0.5M sulfuric acid solution, under-0.3～1.5V voltage, polished 30 minutes, dry subsequent use with cyclic voltammetry.
Gold chloride electrolytic solution with above-mentioned preparation is end liquid, adopts linear cyclic voltammetry scan method, and it is 1.1V that starting potential is set, and final voltage is 0V, surface sweeping speed 0.1V/s, and the scanning number of turns is 1 circle, depositing nano gold grain on the disk gold electrode gets the nm of gold electrode.
The nm of gold electrode is immersed in the ox hoptoglobin antibody-solutions of 0.5 μ g/ml, hatches under the room temperature after 20 minutes and to take out, with 0.25% calf serum sealing 15 minutes, dry after pure water is cleaned subsequent use, must nanogold immunoelectrode.
In the preparation process of nanogold immunoelectrode, adopt cyclic voltammetry (voltage-0.2V-0.6V, sweep velocity 0.1V/s, electrolytic solution cell are the 5mM potassium ferricyanide, 5mM potassium ferrocyanide, 0.1M Klorvess Liquid) that each step is handled and characterize.
Immunoelectrode and contrast electrode, auxiliary electrode assembling are formed the nm of gold immunosensor.
3, detect
1) drawing curve:
Ox hoptoglobin standard antigen (concentration is 0.77mg/ml) is diluted to 1540ng/ml, 770ng/ml, 385ng/ml, 154ng/ml, 77ng/ml, 38.5ng/ml, 7.7ng/ml, 3.85ng/ml, 1.54ng/ml and 0.77ng/ml respectively.
Use the nm of gold immunosensor that makes in the standard antigen solution of above-mentioned concentration, to soak after 30 minutes respectively; Adopt cyclic voltammetry that ox hoptoglobin standard antigen is carried out detection by quantitative, the number percent K that descends with oxidation peak currents (i) before and after the immune response is as quantitative basis.
k＝(i
1-i
2)/i
1×100％，
I in the formula
1Be the oxidation peak current before the immune response, i
2Be the oxidation peak current after the immune response.
To each concentration standard Detection of antigen three times, the mean value of getting oxidation peak is as the pairing peak point current of each standard antigen concentration.Logarithm value with standard antigen concentration is horizontal ordinate (X), and the k value is ordinate (Y), with Excel software processes and drawing standard curve, promptly obtains working curve diagram (as shown in Figure 1) equation of linear regression relevant with it of standard antigen solution:
Y＝0.2672X-0.0567
X=log wherein
10C, C are antigen concentration, and Y is an oxidation peak decline number percent), linearly dependent coefficient R
2=0.9959, satisfy the requirement of quantitative analysis method to precision.
100000)～(V=1: in the time of 2000), the percentage k that immunosensor oxidation peak current (i) descends and the denary logarithm of antigen concentration are good linear relationship to 350ng/ml the dilute concentration of ox hoptoglobin antigen (V=V standard antigen: V water) at 7ng/ml (V=1:.
2) recovery research:
After getting 3 parts of ox hoptoglobin antigenic solutions and diluting,, measure result such as following table by its content of above step measurements:
Its average recovery rate is 93.01%, and the standard deviation (RSD) of the corresponding recovery is 0.09%, meets the precision requirement of quantitative analysis method.
3) stability study:
After getting 3 parts of ox hoptoglobin standard antigen solution and diluting, by its content of above step measurements; Again prepare nanogold immunoelectrode, put in 4 ℃ of refrigerators preserve 48h after, redeterminate 3 parts of same concentration standard antigenic solutions, measure result such as following table:
Mean deviation is 6.07%, explains that electrode stability is good.
4) repetitive research:
Prepare 2 nanogold immunoelectrodes, be labeled as 1# respectively, the 2# electrode.After getting 3 parts of ox hoptoglobin standard antigen solution and diluting, use 1# respectively, the 2# electrode detection, measurement result such as following table:
Detect three groups of antigenic solutions together with 2 electrodes, detect deviation between the same concentration bottom electrode and be respectively 8.39%, 11.27% and 3.68%.Mean deviation is 7.79%, meets the requirement of immunosensor repeatability.
Claims (1)
1. the preparation method of nanogold immunoelectrode is characterized in that may further comprise the steps:
A. the disk gold electrode being used diameter successively is that the alumina powder of 1 μ m, 0.3 μ m and 0.05 μ m is polished to light;
B. the electrode after the polishing was with the concentrated sulphuric acid and hydrogen peroxide mixed liquid dipping 10 minutes; The said concentrated sulphuric acid and hydrogen peroxide mixed liquor are 98% concentrated sulphuric acid and the preparation of 3: 1 by volume ratio of 30% superoxol;
C. take out electrode, successively in absolute ethyl alcohol and distilled water with ultrasonic cleaning 15 minutes;
D. the electrode after will cleaning is put into the 0.5M sulfuric acid solution, polishes 30 minutes under-0.3 ~ 1.5V voltage with cyclic voltammetry, dries subsequent use;
E. be end liquid with gold chloride electrolytic solution, adopt linear cyclic voltammetry scan method, depositing nano gold grain on the disk gold electrode gets the nm of gold electrode; The starting potential of said linear cyclic voltammetry scan method is 1.1V, and final voltage is 0V, surface sweeping speed 0.1V/s, and the scanning number of turns is 1 circle;
F. the nm of gold electrode is immersed in the antibody-solutions of 0.5 μ g/ml, hatches under the room temperature after 20 minutes and to take out, with 0.25% calf serum sealing 15 minutes, dry after pure water is cleaned subsequent use, must nanogold immunoelectrode.
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| CN101966594A (en) * | 2010-10-18 | 2011-02-09 | 上海市七宝中学 | Electro-deposition gold nanoparticles, preparation method and application thereof |
| CN103207218B (en) * | 2012-01-11 | 2015-07-22 | 西南大学 | Electrochemical immunosensor making method and Streptococcus suis detection method using electrochemical immunosensor |
| CN102879582A (en) * | 2012-09-11 | 2013-01-16 | 宁波大学 | In-vitro sandwich immunoassay process with magnetic separation for detecting alpha fetoprotein |
| US20150053555A1 (en) * | 2013-08-22 | 2015-02-26 | King Fahd University Of Petroleum And Minerals | Pencil graphite electrode modified with gold nanoparticles |
| 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 |
| CN109632903A (en) * | 2018-12-05 | 2019-04-16 | 泰山学院 | A method of in glassy carbon electrode surface fabricated in situ single layer uniform gold nanoparticle |
| CN112986553A (en) | 2019-12-14 | 2021-06-18 | 南京岚煜生物科技有限公司 | Preparation method of immunity electrode |
| CN113049671B (en) * | 2021-03-18 | 2023-05-12 | 宁波江丰电子材料股份有限公司 | Method for detecting content of impurity element in cobalt |
| CN114994155A (en) * | 2022-05-05 | 2022-09-02 | 河南农业大学 | Electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts as well as preparation method and application of electrochemical immunosensor |
| CN118050500A (en) * | 2024-02-23 | 2024-05-17 | 中国人民解放军军事科学院军事医学研究院 | Clostridium perfringens alpha toxin electrochemical immunosensor and preparation and application thereof |
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