CN108982630B - Preparation method and application of sandwich type electrochemical immunosensor for detecting prostate specific antigen - Google Patents
Preparation method and application of sandwich type electrochemical immunosensor for detecting prostate specific antigen Download PDFInfo
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Abstract
The invention belongs to the technical field of novel nano materials, immunoassay and biosensing, and provides a preparation method and application of a sandwich type electrochemical immunosensor for detecting prostate specific antigen, in particular to a sandwich type immunosensor which is constructed by adopting a nano composite material of molybdenum disulfide nanoflowers loaded with hollow core-shell gold-silver-platinum nanocubes as a mimic enzyme to mark a secondary antibody and adopting triangular gold nanoplates as a substrate material, so that the prostate specific antigen can be quickly and sensitively detected.
Description
Technical Field
The invention belongs to the technical field of novel nano materials, immunoassay and biosensing, and provides a preparation method and application of a sandwich type electrochemical immunosensor for detecting prostate specific antigen, in particular to a sandwich type immunosensor which is constructed by adopting a nano composite material of molybdenum disulfide nanoflowers loaded with hollow core-shell gold-silver-platinum nanocubes as a mimic enzyme to mark a secondary antibody and adopting triangular gold nanoplates as a substrate material, so that the prostate specific antigen can be quickly and sensitively detected.
Background
Prostate cancer (PCa) is the second most common non-skin cancer (second only to lung cancer) in men, accounting for 14% of all new cancer cases in men worldwide. The detection of the concentration of the prostate specific antigen can be used for diagnosing prostate cancer, and can also be used for judging the nature and the state of the prostate cancer by detecting the content and the change of the amount of the prostate specific antigen, so that the prostate specific antigen is quantitatively detected in serum in medicine, plays an important role in the aspects of diagnosis, lesion range assessment, curative effect prediction assessment, relapse monitoring and the like of the prostate cancer, early diagnosis is carried out by specific and sensitive biomarkers and analysis tools, the incidence rate of the prostate cancer can be reduced, and the survival rate can be effectively improved.
The electrochemical immunosensor is an analysis method based on the specific combination of antigen and antibody, and has the advantages of rapid detection, low detection limit, high sensitivity, simple operation and low preparation cost. In recent years, electrochemical immunosensors have attracted attention and are widely used for detecting tumor markers. The invention utilizes a self-assembly technology, utilizes a novel molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocube nanocomposite as a mimic enzyme to mark a secondary antibody as a signal amplifier, and utilizes triangular gold nanoplates as a substrate material to prepare the sandwich type electrochemical immunosensor for detecting prostate specific antigen. The novel molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocubes nanocomposite material has a unique structure, has a large number of catalytic active sites, and is H-shaped2O2The reduction of the nano composite material shows excellent catalytic performance, the nano composite material not only can fully exert the excellent catalytic performance and the large specific surface area of the molybdenum disulfide nanoflower, but also can effectively utilize the unique catalytic performance, the excellent conductive performance and the good biocompatibility of the hollow core-shell gold-silver-platinum nanocube, and by exerting the synergistic effect of the two novel nano materials, the nano composite material of the molybdenum disulfide nanoflower loaded with the hollow core-shell gold-silver-platinum nanocube is used as a mimic enzyme to mark a second antibody as a signal amplifier, and the triangular gold nanoplate is used as the excellent conductive performance exhibited by a substrate material, so that the sensitivity of the immunosensor can be effectively improved. Based on the advantages, the sandwich type immunosensor is constructedThe quantitative detection of the prostate specific antigen is realized, and the kit has the advantages of wide detection range, low detection lower limit, high sensitivity, simple operation, high detection speed and the like, has good reproducibility, stability and selectivity, and provides a reliable detection means for early diagnosis of prostate cancer.
Disclosure of Invention
The invention provides a preparation method and application of a sandwich type electrochemical immunosensor for detecting prostate specific antigen, wherein the electrochemical immunosensor comprises: the device comprises a working electrode, a counter electrode and a reference electrode, wherein the working electrode is a glassy carbon electrode, the surface of the working electrode is sequentially modified with triangular gold nanoplates, a prostate specific antibody, bovine serum albumin, a prostate specific antigen and a molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocube nanocomposite material for hatching a secondary antibody marker of the prostate specific antibody, the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode.
The invention aims to provide a preparation method of a sandwich type electrochemical immunosensor for detecting prostate specific antigens.
The other purpose of the invention is to use the prepared sandwich type electrochemical immunosensor for quantitative detection of prostate specific antigen.
The technical scheme of the invention comprises the following steps:
(1) preparing triangular gold nanoplates;
(2) preparing a nano composite material of the molybdenum disulfide nanoflower loaded hollow core-shell gold, silver and platinum nanocubes;
(3) preparing a second antibody marker of the prostate specific antibody incubated by the nano composite material of the molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocubes;
(4) preparing a working electrode of a sandwich type electrochemical immunosensor for detecting prostate specific antigens;
(5) preparing a working curve of the sandwich type electrochemical immunosensor for detecting the prostate specific antigen.
Wherein the step (1) of preparing the triangular gold nanoplates comprises the following steps:
① preparation of gold seed solution
Adding 25.0-30.0 muL and 50.0 mmol/L chloroauric acid solution into 4.0-5.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 200.0-300.0 muL and 10.0mmol/L sodium borohydride into the solution under continuous stirring, and reacting for 2h at room temperature to obtain gold seed solution;
② preparation of triangular gold nanoplates
Firstly, preparing a solution and a solution b respectively: the solution a is prepared by adding 40.0 muL and 50.0 mmol/L chloroauric acid solution and 15.0 muL and 10.0mmol/L sodium iodide solution into 8.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution; the solution b is prepared by adding 500.0 muL, 50.0 mmol/L chloroauric acid solution and 300.0 muL, 10.0mmol/L sodium iodide solution into 40.0 mL, 0.1mol/L hexadecyl trimethyl ammonium chloride solution; respectively adding 40.0-60.0 muL, 400.0-600.0 muL and 0.1mol/L ascorbic acid solution into the solution a and the solution b, then adding 100.0 muL-200.0 muL of 10-fold diluted gold seed solution into the solution a, reacting for 1 s, rapidly adding 3.0-4.0 mL of the solution a into the solution b, stirring for ten seconds, then standing for 1-3 h at room temperature, and finally centrifuging and cleaning for 3 times to obtain the triangular gold nanoplatelets.
The preparation method of the molybdenum disulfide nanoflower-loaded hollow core-shell gold-silver-platinum nanocube nanocomposite material in the step (2) comprises the following steps:
① preparation of hollow core-shell gold, silver and platinum nanocubes
Adding 10.0 mu L and 25.0 mmol/L chloroauric acid into 10.0 mL and 75.0 mmol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 0.6 mL and 10.0mmol/L sodium borohydride solution, and reacting for 2h at room temperature to obtain gold seed solution. Then adding 0.1-0.3 mL, 10.0mmol/L chloroauric acid solution and 2.0-4.0 mL, 0.2 mol/L hexadecyltrimethylammonium chloride solution into 25.0 mL deionized water, adding 1.5-2.5 mL, 0.1mol/L ascorbic acid into the above solutions for reaction for more than ten seconds, then adding 0.3-0.5 mL of gold seed solution diluted by 100 times, standing for 8h at room temperature, adding 0.5-1.0 mL, 10.0mmol/L silver nitrate, 2.0-4.0 mL, 0.1mol/L ascorbic acid solution into the above solutions, and standing for 12 h in a 50 ℃ water bath kettle to obtain the gold and silver nano cubic colloidal solution. Taking 4.0-6.0 mL of gold and silver nano cubic colloidal solution, continuously stirring and heating in an oil bath pot, refluxing to 100 ℃, dropwise adding 200.0-400.0 muL and 5.0 mmol/L of chloroplatinic acid solution into the solution after 15 min, continuously stirring for reaction for 15 min, cooling, centrifuging and cleaning for 3 times to obtain a hollow core-shell gold and silver platinum nano cube;
② preparation of molybdenum disulfide nanoflower
Adding 1.2-1.6 g of ammonium molybdate into 15.0 mL of deionized water, then adding 0.8-1.0 g of ethylenediamine into the solution, after ultrasonic dissolution, adding a proper amount of 1.0 mol/L hydrochloric acid to adjust the pH value to 4.0-6.0, reacting for 2h at 50 ℃, centrifugally cleaning for 3 times, and carrying out vacuum drying at 50 ℃ to obtain a complex of ammonium molybdate and ethylenediamine; adding 0.1-0.2 g of ammonium molybdate and ethylenediamine complex into 15.0 mL of deionized water, then adding 0.2-0.4 g of L-cysteine, ultrasonically dispersing, placing into a 25.0 mL high-pressure reaction kettle with a polytetrafluoroethylene lining, heating to 200 ℃, preserving heat for 14h, finally centrifugally cleaning for 3 times, and vacuum drying to obtain molybdenum disulfide nanoflowers;
③ preparation of nanometer composite material of molybdenum disulfide nanometer flower load hollow core-shell gold, silver and platinum nanometer cube
Ultrasonically dispersing 0.1-0.2 g of molybdenum disulfide nanoflower into 20.0 mL of absolute ethyl alcohol, heating to 100 ℃ in an oil bath kettle in a refluxing manner, then adding 3-aminopropyltriethoxysilane of 20.0-30.0 mu L, reacting for 2h, cooling, centrifuging, cleaning for 3 times, and freeze-drying to obtain an aminated molybdenum disulfide nanoflower; adding 2.0-4.0 mL and 2.0 mg/mL hollow core-shell gold and silver platinum nanocubes into 2.0 mL and 2.0 mg/mL aminated molybdenum disulfide nanoflowers, ultrasonically dispersing for 1h, oscillating for 6h at room temperature, and centrifugally cleaning for 2 times to obtain the molybdenum disulfide nanoflower-loaded hollow core-shell gold and silver platinum nanocube nanocomposite.
Wherein the second antibody marker of the hatching prostate specific antibody of the nanocomposite material of the molybdenum disulfide nanoflower-loaded hollow core-shell gold and silver platinum nanocubes prepared in the step (3) comprises the following steps:
① adding 2.0-4.0 mL and 10.0 mug/mL prostate specific antibodies into the 2.0 mL and 2.0 mg/mL molybdenum disulfide nanoflower loaded hollow core-shell gold and silver platinum nanocube nanocomposite, oscillating for 12 h at 4 ℃, and centrifugally cleaning and dispersing in a phosphoric acid buffer solution.
Wherein the working electrode of the electrochemical immunosensor for detecting the prostate specific antigen in the sandwich type prepared in the step (4) comprises:
① Al for glassy carbon electrode with diameter of 3.0-5.0 mm2O3Polishing the polishing powder into a mirror surface, and respectively and sequentially ultrasonically cleaning in absolute ethyl alcohol and ultrapure water;
② dripping 6.0 muL and 0.5-3.0 mg/mL triangular gold nanoplate dispersion liquid on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying at room temperature;
③ continuously dripping 6.0 muL and 4.0-8.0 mug/mL prostate specific antibodies on the surface of the electrode, and drying in a refrigerator at 4 ℃;
④ dropwise adding a bovine serum albumin solution of 3.0 muL and 0.7-1.4 wt% to the surface of the electrode to seal the nonspecific active sites, washing the surface of the electrode with a phosphate buffer solution of which the pH is 7.0, and drying in a refrigerator at 4 ℃;
⑤, dripping a series of prostate specific antigen solutions with different concentrations, namely 6.0 muL and 0.00005-100.0 ng/mL, washing the surface of the electrode by using a phosphate buffer solution with the pH value of 7.0, and airing in a refrigerator at 4 ℃;
⑥ dropping a second antibody marker of the prostate specific antibody incubated by the nanocomposite material with the hollow core-shell gold and silver platinum nanocubes loaded by the molybdenum disulfide nanoflowers in the concentration of 6.0 muL and 2.0-4.0 mg/mL on the surface of the electrode, washing the surface of the electrode by using a phosphate buffer solution with the pH value of 7.0, and drying the electrode in a refrigerator at 4 ℃ to prepare the working electrode of the sandwich type electrochemical immunosensor for detecting the prostate specific antigen.
Wherein the working curve of the electrochemical immunosensor for detecting the prostate specific antigen prepared in the step (5) comprises the following steps:
① testing with an electrochemical workstation by using a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, taking the prepared sensor as a working electrode, and testing in 10.0 mL of 50.0 mmol/L phosphate buffer solution with pH of 5.3-8.0;
② detecting the analyte by time-current method, with input voltage of-0.4V, sampling interval of 0.1 s, and running time of 200 s;
③ when the background current tends to be stable, injecting 10.0-15.0 muL and 5.0 mol/L hydrogen peroxide solution into 10.0 mL and 50.0 mmol/L phosphate buffer solution with pH of 7.4, recording current values corresponding to prostate specific antigens under different concentrations, and drawing a working curve of the electrochemical immunosensor for detecting the prostate specific antigens;
④ the concentration of prostate specific antigen in the sample to be tested is obtained by using the working curve method.
The raw materials used in the present invention are all available from chemical or biopharmaceutical companies.
Advantageous results of the invention
(1) According to the invention, the novel molybdenum disulfide nanoflower-loaded hollow core-shell gold and silver platinum nanocubes nanocomposite is used as a mimic enzyme to mark the second antibody as a signal amplifier, and the molybdenum disulfide nanoflower and the hollow core-shell gold and silver platinum nanocubes have unique structures, possess a large number of catalytic active sites, and can be used for H2O2The prepared novel molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocube nanocomposite can fully exert synergistic effect, show excellent catalytic performance, conductivity and biocompatibility, and the triangular gold nanosheet as a substrate material has excellent conductivity, so that the sensitivity of the sensor can be effectively improved, the constructed sandwich-type immunosensor realizes quantitative detection of prostate specific antigen, has the advantages of wide detection range, low detection lower limit, high sensitivity, simplicity in operation, high detection speed and the like, has good reproducibility, stability and selectivity, and provides a reliable detection means for early diagnosis;
(2) the sandwich type electrochemical immunosensor constructed by the invention realizes the purpose of accurately and quantitatively detecting the prostate specific antigen, the linear detection range is 0.00005 ng/mL-100 ng/mL, and the lowest detection lower limit is 16.6 fg/mL;
(3) the electrochemical immunosensor constructed by the method is simple to operate and rapid to detect, and can be used for rapid detection of actual samples.
Detailed Description
The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.
Example 1 preparation of triangular gold nanoplates includes:
① preparation of gold seed solution
Adding 25.0 muL and 50.0 mmol/L chloroauric acid solution into 4.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 200.0 muL and 10.0mmol/L sodium borohydride into the solution under continuous stirring, and reacting for 2h at room temperature to obtain gold seed solution;
② preparation of triangular gold nanoplates
Firstly, preparing a solution and a solution b respectively: the solution a is prepared by adding 40.0 muL and 50.0 mmol/L chloroauric acid solution and 15.0 muL and 10.0mmol/L sodium iodide solution into 8.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution; the solution b is prepared by adding 500.0 muL, 50.0 mmol/L chloroauric acid solution and 300.0 muL, 10.0mmol/L sodium iodide solution into 40.0 mL, 0.1mol/L hexadecyl trimethyl ammonium chloride solution; respectively adding 40.0 muL, 400.0 muL and 0.1mol/L ascorbic acid solutions into the solution a and the solution b, then adding a gold seed solution diluted by 10 times by 100.0 muL into the solution a, reacting for 1 s, rapidly adding 3.0 mL of the solution a into the solution b, stirring for ten seconds, then standing for 1h at room temperature, and finally centrifuging and cleaning for 3 times to obtain the triangular gold nanoplatelets.
Example 2 preparation of triangular gold nanoplates includes:
① preparation of gold seed solution
Adding a chloroauric acid solution of 27.5 muL and 50.0 mmol/L into a hexadecyltrimethylammonium chloride solution of 4.5 mL and 0.1mol/L, then dropwise adding sodium borohydride of 250.0 muL and 10.0mmol/L into the solution under continuous stirring, and reacting for 2h at room temperature to obtain a gold seed solution;
② preparation of triangular gold nanoplates
Firstly, preparing a solution and a solution b respectively: the solution a is prepared by adding 40.0 muL and 50.0 mmol/L chloroauric acid solution and 15.0 muL and 10.0mmol/L sodium iodide solution into 8.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution; the solution b is prepared by adding 500.0 muL, 50.0 mmol/L chloroauric acid solution and 300.0 muL, 10.0mmol/L sodium iodide solution into 40.0 mL, 0.1mol/L hexadecyl trimethyl ammonium chloride solution; respectively adding 50.0 muL, 500.0 muL and 0.1mol/L ascorbic acid solutions into the solution a and the solution b, then adding a gold seed solution diluted by 10 times by 250.0 muL into the solution a, reacting for 1 s, rapidly adding 3.5 mL of the solution a into the solution b, stirring for ten seconds, then standing for 2h at room temperature, and finally centrifuging and cleaning for 3 times to obtain the triangular gold nanoplatelets.
Example 3 preparation of triangular gold nanoplates includes:
① preparation of gold seed solution
Adding 30.0 muL and 50.0 mmol/L chloroauric acid solution into 5.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 300.0 muL and 10.0mmol/L sodium borohydride into the solution under continuous stirring, and reacting for 2h at room temperature to obtain gold seed solution;
② preparation of triangular gold nanoplates
Firstly, preparing a solution and a solution b respectively: the solution a is prepared by adding 40.0 muL and 50.0 mmol/L chloroauric acid solution and 15.0 muL and 10.0mmol/L sodium iodide solution into 8.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution; the solution b is prepared by adding 500.0 muL, 50.0 mmol/L chloroauric acid solution and 300.0 muL, 10.0mmol/L sodium iodide solution into 40.0 mL, 0.1mol/L hexadecyl trimethyl ammonium chloride solution; respectively adding 60.0 muL, 600.0 muL and 0.1mol/L ascorbic acid solutions into the solution a and the solution b, then adding 200.0 muL of gold seed solution diluted by 10 times into the solution a for reaction for 1 s, rapidly adding 4.0 mL of the solution a into the solution b, stirring for ten seconds, then standing for 3h at room temperature, and finally centrifuging and cleaning for 3 times to obtain triangular gold nanoplates.
Example 4 preparation of a nanocomposite material of molybdenum disulfide nanoflowers loaded with hollow core-shell gold, silver and platinum nanocubes includes:
① preparation of hollow core-shell gold, silver and platinum nanocubes
Adding 10.0 mu L and 25.0 mmol/L chloroauric acid into 10.0 mL and 75.0 mmol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 0.6 mL and 10.0mmol/L sodium borohydride solution, and reacting for 2h at room temperature to obtain gold seed solution. Then adding 0.1 mL, 10.0mmol/L chloroauric acid solution and 2.0 mL, 0.2 mol/L hexadecyltrimethylammonium chloride solution into 25.0 mL deionized water, adding 1.5 mL, 0.1mol/L ascorbic acid into the above solution for reaction for more than ten seconds, then adding 0.3 mL of gold seed solution diluted by 100 times, standing for 8h at room temperature, adding 0.5 mL, 10 mmol/L silver nitrate and 2.0 mL, 0.1mol/L ascorbic acid solution into the above solution, and standing in a 50 ℃ water bath for 12 h to obtain the gold and silver nano cubic colloidal solution. Taking 4.0 mL of gold and silver nano cubic colloidal solution, continuously stirring and heating in an oil bath pot, refluxing to 100 ℃, dropwise adding 200.0 muL and 5.0 mmol/L chloroplatinic acid solution into the solution after 15 min, continuously stirring for reaction for 15 min, cooling, centrifuging and cleaning for 3 times to obtain a hollow core-shell gold and silver platinum nano cube;
② preparation of molybdenum disulfide nanoflower
Adding 1.2 g of ammonium molybdate into 15.0 mL of deionized water, then adding 0.8 g of ethylenediamine into the solution, after ultrasonic dissolution, adding a proper amount of 1.0 mol/L hydrochloric acid to adjust the pH value to 4.0, reacting for 2h at 50 ℃, centrifugally cleaning for 3 times, and drying in vacuum at 50 ℃ to obtain a complex of ammonium molybdate and ethylenediamine;
adding 0.1 g of a complex of ammonium molybdate and ethylenediamine into 15.0 mL of deionized water, then adding 0.2 g of L-cysteine, ultrasonically dispersing, putting into a 25.0 mL high-pressure reaction kettle with a polytetrafluoroethylene lining, heating to 200 ℃, preserving heat for 14h, finally centrifugally cleaning for 3 times, and vacuum drying to obtain molybdenum disulfide nanoflowers;
③ preparation of nanometer composite material of molybdenum disulfide nanometer flower load hollow core-shell gold, silver and platinum nanometer cube
Ultrasonically dispersing 0.1 g of molybdenum disulfide nanoflowers into 20.0 mL of absolute ethyl alcohol, heating the mixture to 100 ℃ in an oil bath kettle in a refluxing manner, then adding 20.0 muL of 3-aminopropyltriethoxysilane, reacting for 2 hours, cooling, centrifuging, cleaning for 3 times, and freeze-drying to obtain aminated molybdenum disulfide nanoflowers;
adding 2.0 mL and 2.0 mg/mL hollow core-shell gold and platinum nanocubes into 2.0 mL and 2.0 mg/mL aminated molybdenum disulfide nanoflowers, ultrasonically dispersing for 1h, shaking for 6h at room temperature, and centrifugally cleaning for 2 times to obtain the molybdenum disulfide nanoflower-loaded hollow core-shell gold and platinum nanocube nanocomposite.
Example 5 preparation of a nanocomposite material of molybdenum disulfide nanoflowers loaded with hollow core-shell gold, silver and platinum nanocubes includes:
① preparation of hollow core-shell gold, silver and platinum nanocubes
Adding 10.0 mu L and 25.0 mmol/L chloroauric acid into 10.0 mL and 75.0 mmol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 0.6 mL and 10.0mmol/L sodium borohydride solution, and reacting for 2h at room temperature to obtain gold seed solution. Then adding 0.2 mL, 10.0mmol/L chloroauric acid solution and 3.0 mL, 0.2 mol/L hexadecyltrimethylammonium chloride solution into 25.0 mL deionized water, adding 2.0 mL, 0.1mol/L ascorbic acid into the above solution for reaction for more than ten seconds, then adding 0.4 mL of gold seed solution diluted by 100 times, standing for 8h at room temperature, adding 0.75 mL, 10.0mmol/L silver nitrate and 3.0 mL, 0.1mol/L ascorbic acid solution into the above solution, and standing for 12 h in a 50 ℃ water bath kettle to obtain the gold and silver nano cubic colloidal solution. Taking 5.0 mL of gold and silver nano cubic colloidal solution, continuously stirring and heating in an oil bath pot, refluxing to 100 ℃, dropwise adding 300.0 muL and 5.0 mmol/L chloroplatinic acid solution into the solution after 15 min, continuously stirring for reaction for 15 min, cooling, centrifuging and cleaning for 3 times to obtain a hollow core-shell gold and silver platinum nano cube;
② preparation of molybdenum disulfide nanoflower
Adding 1.4 g of ammonium molybdate into 15.0 mL of deionized water, then adding 0.9 g of ethylenediamine into the solution, after ultrasonic dissolution, adding a proper amount of 1.0 mol/L hydrochloric acid to adjust the pH value to 5.0, reacting for 2h at 50 ℃, centrifugally cleaning for 3 times, and drying in vacuum at 50 ℃ to obtain a complex of ammonium molybdate and ethylenediamine; adding 0.15 g of a complex of ammonium molybdate and ethylenediamine into 15.0 mL of deionized water, then adding 0.3 g of L-cysteine, ultrasonically dispersing, putting into a 25.0 mL high-pressure reaction kettle with a polytetrafluoroethylene lining, heating to 200 ℃, preserving heat for 14h, finally centrifugally cleaning for 3 times, and vacuum drying to obtain molybdenum disulfide nanoflowers;
③ preparation of nanometer composite material of molybdenum disulfide nanometer flower load hollow core-shell gold, silver and platinum nanometer cube
Ultrasonically dispersing 0.15 g of molybdenum disulfide nanoflowers into 20.0 mL of absolute ethyl alcohol, heating the mixture to 100 ℃ in an oil bath kettle in a refluxing manner, then adding 25.0 muL of 3-aminopropyltriethoxysilane, reacting for 2 hours, cooling, centrifuging, cleaning for 3 times, and freeze-drying to obtain aminated molybdenum disulfide nanoflowers; adding 3.0 mL and 2.0 mg/mL hollow core-shell gold and platinum nanocubes into 2.0 mL and 2.0 mg/mL aminated molybdenum disulfide nanoflowers, ultrasonically dispersing for 1h, shaking for 6h at room temperature, and centrifugally cleaning for 2 times to obtain the molybdenum disulfide nanoflower-loaded hollow core-shell gold and platinum nanocube nanocomposite.
Example 6 preparation of a molybdenum disulfide nanoflower loaded hollow core-shell gold, silver and platinum nanocube nanocomposite material includes:
① preparation of hollow core-shell gold, silver and platinum nanocubes
Adding 10.0 mu L and 25.0 mmol/L chloroauric acid into 10.0 mL and 75.0 mmol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 0.6 mL and 10.0mmol/L sodium borohydride solution, and reacting for 2h at room temperature to obtain gold seed solution. Then adding 0.3 mL, 10.0mmol/L chloroauric acid solution and 4.0 mL, 0.2 mol/L hexadecyltrimethylammonium chloride solution into 25.0 mL deionized water, adding 2.5 mL, 0.1mol/L ascorbic acid into the above solution for reaction for more than ten seconds, then adding 0.5 mL of gold seed solution diluted by 100 times, standing for 8h at room temperature, adding 1.0 mL, 10.0mmol/L silver nitrate and 4.0 mL, 0.1mol/L ascorbic acid solution into the above solution, and standing in a 50 ℃ water bath for 12 h to obtain the gold and silver nano cubic colloidal solution. Taking 6.0mL of gold and silver nano cubic colloidal solution, continuously stirring and heating in an oil bath pot, refluxing to 100 ℃, dropwise adding 400.0 muL and 5.0 mmol/L chloroplatinic acid solution into the solution after 15 min, continuously stirring for reaction for 15 min, cooling, centrifuging and cleaning for three times to obtain a hollow core-shell gold and silver platinum nano cube;
② preparation of molybdenum disulfide nanoflower
Adding 1.6 g of ammonium molybdate into 15.0 mL of deionized water, then adding 1.0 g of ethylenediamine into the solution, after ultrasonic dissolution, adding a proper amount of 1.0 mol/L hydrochloric acid to adjust the pH value to 6.0, reacting for 2h at 50 ℃, centrifugally cleaning for 3 times, and drying in vacuum at 50 ℃ to obtain a complex of ammonium molybdate and ethylenediamine; adding 0.2 g of a complex of ammonium molybdate and ethylenediamine into 15.0 mL of deionized water, then adding 0.4 g of L-cysteine, ultrasonically dispersing, putting into a 25.0 mL high-pressure reaction kettle with a polytetrafluoroethylene lining, heating to 200 ℃, preserving heat for 14h, finally centrifugally cleaning for 3 times, and vacuum drying to obtain molybdenum disulfide nanoflowers;
③ preparation of nanometer composite material of molybdenum disulfide nanometer flower load hollow core-shell gold, silver and platinum nanometer cube
Ultrasonically dispersing 0.2 g of molybdenum disulfide nanoflowers into 20.0 mL of absolute ethyl alcohol, heating the mixture to 100 ℃ in an oil bath kettle in a refluxing manner, then adding 30.0 mu L3-aminopropyltriethoxysilane, reacting for 2 hours, cooling, centrifuging, cleaning for 3 times, and freeze-drying to obtain aminated molybdenum disulfide nanoflowers; adding 4.0 mL and 2.0 mg/mL hollow core-shell gold and platinum nanocubes into 2.0 mL and 2.0 mg/mL aminated molybdenum disulfide nanoflowers, ultrasonically dispersing for 1h, shaking for 6h at room temperature, and centrifugally cleaning for 2 times to obtain the molybdenum disulfide nanoflower-loaded hollow core-shell gold and platinum nanocube nanocomposite.
Example 7 preparation of a second antibody marker of a prostate specific antibody incubated by a nanocomposite material in which molybdenum disulfide nanoflowers supported hollow core-shell gold and silver platinum nanocubes were:
① adding 2.0 mL and 10.0 mug/mL prostate specific antibodies into 2.0 mL and 2.0 mg/mL molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocubes, shaking for 12 h at 4 ℃, and centrifugally cleaning and dispersing in phosphate buffer solution.
Example 8 the preparation of the second antibody marker of the hatching prostate specific antibody of the molybdenum disulfide nanoflower-loaded hollow core-shell gold, silver and platinum nanocube nanocomposite comprises:
① adding 3.0 mL and 10.0 mug/mL prostate specific antibodies into 2.0 mL and 2.0 mg/mL molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocubes, shaking for 12 h at 4 ℃, and centrifugally cleaning and dispersing in phosphate buffer solution.
Example 9 preparation of a second antibody marker of a prostate specific antibody incubated by a nanocomposite material in which molybdenum disulfide nanoflowers supported hollow core-shell gold, silver and platinum nanocubes were prepared, the second antibody marker comprising:
① adding 4.0 mL and 10.0 mug/mL prostate specific antibodies into 2.0 mL and 2.0 mg/mL molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocubes, shaking for 12 h at 4 ℃, and centrifugally cleaning and dispersing in phosphate buffer solution.
Example 10 preparation of a sandwich-type working electrode for an electrochemical immunosensor for the detection of prostate-specific antigens, comprising:
① use of Al for glassy carbon electrode with diameter of 3.0 mm2O3Polishing the polishing powder into a mirror surface, and respectively and sequentially ultrasonically cleaning in absolute ethyl alcohol and ultrapure water;
② dripping 6.0 muL and 0.5 mg/mL triangular gold nanoplate dispersion liquid on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying at room temperature;
③ continuously dripping 6.0 mu L and 4.0 mu g/mL prostate specific antibodies on the surface of the electrode, and drying in a refrigerator at 4 ℃;
④ dropping a bovine serum albumin solution of 3.0 muL and 0.7 wt% on the surface of the electrode to seal the nonspecific active sites, washing the surface of the electrode with a phosphate buffer solution with pH of 7.0, and drying in a refrigerator at 4 ℃;
⑤, dripping a series of prostate specific antigen solutions with different concentrations, namely 6.0 muL and 0.00005-100.0 ng/mL, washing the surface of the electrode by using a phosphate buffer solution with the pH value of 7.0, and airing in a refrigerator at 4 ℃;
⑥ adding the second antibody marker of the prostate specific antibody incubated by the nanocomposite material of the molybdenum disulfide nanoflower-loaded hollow core-shell gold and silver platinum nanocubes with the concentration of 6.0 muL and 2.0 mg/mL to the surface of the electrode, washing the surface of the electrode by phosphate buffer with the pH value of 7.0, and drying the electrode in a refrigerator at 4 ℃ to prepare the working electrode of the sandwich type electrochemical immunosensor for detecting the prostate specific antigen.
Example 11 preparation of a sandwich-type working electrode for an electrochemical immunosensor for the detection of prostate-specific antigens, comprising:
① use of Al for glassy carbon electrode with diameter of 4.0 mm2O3Polishing the polishing powder into a mirror surface, and respectively and sequentially ultrasonically cleaning in absolute ethyl alcohol and ultrapure water;
② dripping 6.0 muL and 2.0 mg/mL triangular gold nanoplate dispersion liquid on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying at room temperature;
③ continuously dripping 6.0 mu L and 6.0 mu g/mL prostate specific antibodies on the surface of the electrode, and drying in a refrigerator at 4 ℃;
④ dropping a bovine serum albumin solution of 3.0 muL and 1.0 wt% on the surface of the electrode to seal the nonspecific active sites, washing the surface of the electrode with a phosphate buffer solution of which the pH value is 7.0, and drying in a refrigerator at 4 ℃;
⑤, dripping a series of prostate specific antigen solutions with different concentrations, namely 6.0 muL and 0.00005-100.0 ng/mL, washing the surface of the electrode by using a phosphate buffer solution with the pH value of 7.0, and airing in a refrigerator at 4 ℃;
⑥ adding a second antibody marker of the prostate specific antibody incubated by the molybdenum disulfide nanoflower loaded hollow core-shell gold and silver platinum nanocubes nanocomposite material with the volume of 6.0 muL and the volume of 3.0 mg/mL to the surface of the electrode, washing the surface of the electrode by phosphate buffer with the pH value of 7.0, and drying the electrode in a refrigerator at 4 ℃ to prepare the working electrode of the sandwich type electrochemical immunosensor for detecting the prostate specific antigen.
Example 12 preparation of a sandwich-type working electrode for an electrochemical immunosensor for the detection of prostate-specific antigens, comprising:
① use of 5.0 mm diameter Al for glassy carbon electrode2O3Polishing the polishing powder into a mirror surface, and respectively and sequentially ultrasonically cleaning in absolute ethyl alcohol and ultrapure water;
② dripping 6.0 muL and 3.0 mg/mL triangular gold nanoplate dispersion liquid on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying at room temperature;
③ continuously dripping 6.0 mu L and 8.0 mu g/mL prostate specific antibodies on the surface of the electrode, and drying in a refrigerator at 4 ℃;
④ dropping a bovine serum albumin solution of 3.0 muL and 1.4 wt% on the surface of the electrode to seal the nonspecific active sites, washing the surface of the electrode with a phosphate buffer solution with pH of 7.0, and drying in a refrigerator at 4 ℃;
⑤, dripping a series of prostate specific antigen solutions with different concentrations, namely 6.0 muL and 0.00005-100.0 ng/mL, washing the surface of the electrode by using a phosphate buffer solution with the pH value of 7.0, and airing in a refrigerator at 4 ℃;
⑥ adding a second antibody marker of the prostate specific antibody incubated by the molybdenum disulfide nanoflower loaded hollow core-shell gold and silver platinum nanocubes nanocomposite material with the volume of 6.0 muL and the volume of 4.0 mg/mL to the surface of the electrode, washing the surface of the electrode by phosphate buffer with the pH value of 7.0, and drying the electrode in a refrigerator at 4 ℃ to prepare the working electrode of the sandwich type electrochemical immunosensor for detecting the prostate specific antigen.
Example 13 preparation of a sandwich-type electrochemical immunosensor for the detection of prostate specific antigens, the working curve includes:
① testing with electrochemical workstation in three-electrode system, saturated calomel electrode as reference electrode, platinum wire electrode as counter electrode, and prepared sensor as working electrode, in 10.0 mL, 50.0 mmol/L phosphate buffer solution with pH of 5.3;
② detecting the analyte by time-current method, with input voltage of-0.4V, sampling interval of 0.1 s, and running time of 200 s;
③ when the background current tends to be stable, 10.0 muL and 5.0 mol/L hydrogen peroxide solution is injected into 10.0 mL and 50.0 mmol/L phosphate buffer solution with pH of 5.3, current values corresponding to prostate specific antigens under different concentrations are recorded, and a working curve of the electrochemical immunosensor for detecting the prostate specific antigens is drawn;
④ the concentration of prostate specific antigen in the sample to be tested is obtained by using the working curve method.
Example 14 working curves for preparing a sandwich-type electrochemical immunosensor for the detection of prostate specific antigens include:
① testing with electrochemical workstation in three-electrode system, saturated calomel electrode as reference electrode, platinum wire electrode as counter electrode, and prepared sensor as working electrode, in 10.0 mL, 50.0 mmol/L phosphate buffer solution with pH of 7.4;
② detecting the analyte by time-current method, with input voltage of-0.4V, sampling interval of 0.1 s, and running time of 200 s;
③ when the background current tends to be stable, injecting 13.0 muL and 5.0 mol/L hydrogen peroxide solution into 10.0 mL and 50.0 mmol/L phosphate buffer solution with pH of 6.8, recording current values corresponding to prostate specific antigens under different concentrations, and drawing a working curve of the electrochemical immunosensor for detecting the prostate specific antigens;
④ the concentration of prostate specific antigen in the sample to be tested is obtained by using the working curve method.
Example 15 the working curve for preparing a sandwich-type electrochemical immunosensor for the detection of prostate specific antigens includes:
① testing with electrochemical workstation in three-electrode system, saturated calomel electrode as reference electrode, platinum wire electrode as counter electrode, and prepared sensor as working electrode, in 10.0 mL, 50.0 mmol/L phosphate buffer solution with pH of 8.0;
② detecting the analyte by time-current method, with input voltage of-0.4V, sampling interval of 0.1 s, and running time of 200 s;
③ when the background current tends to be stable, injecting 15.0 muL and 5.0 mol/L hydrogen peroxide solution into 10.0 mL and 50.0 mmol/L phosphate buffer solution with pH of 7.4, recording current values corresponding to prostate specific antigens under different concentrations, and drawing a working curve of the electrochemical immunosensor for detecting the prostate specific antigens;
④ the concentration of prostate specific antigen in the sample to be tested is obtained by using the working curve method.
Example 16 working curves for preparing a sandwich-type electrochemical immunosensor for the detection of prostate specific antigens include:
① testing with electrochemical workstation in three-electrode system, saturated calomel electrode as reference electrode, platinum wire electrode as counter electrode, and prepared sensor as working electrode, in 10.0 mL, 50.0 mmol/L phosphate buffer solution with pH of 8.0;
② detecting the analyte by time-current method, with input voltage of-0.4V, sampling interval of 0.1 s, and running time of 200 s;
③ when the background current tends to be stable, injecting 15.0 muL and 5.0 mol/L hydrogen peroxide solution into 10.0 mL and 50.0 mmol/L phosphate buffer solution with pH of 8.0, recording current values corresponding to prostate specific antigens under different concentrations, and drawing a working curve of the electrochemical immunosensor for detecting the prostate specific antigens;
④ the concentration of prostate specific antigen in the sample to be tested is obtained by using the working curve method.
Claims (3)
1. A method for preparing a sandwich-type electrochemical immunosensor for detecting prostate-specific antigens, the method comprising:
(1) preparing triangular gold nanoplates;
(2) preparing a nano composite material of the molybdenum disulfide nanoflower loaded hollow core-shell gold, silver and platinum nanocubes;
(3) preparing a second antibody marker of the prostate specific antibody incubated by the nano composite material of the molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocubes;
(4) preparing a working electrode of a sandwich type electrochemical immunosensor for detecting prostate specific antigens;
the step (1) of preparing the triangular gold nanoplates comprises:
① preparation of gold seed solution
Adding 25.0-30.0 muL and 50.0 mmol/L chloroauric acid solution into 4.0-5.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 200.0-300.0 muL and 10.0mmol/L sodium borohydride into the solution under continuous stirring, and reacting for 2h at room temperature to obtain gold seed solution;
② preparation of triangular gold nanoplates
Firstly, preparing a solution and a solution b respectively: the solution a is prepared by adding 40.0 muL and 50.0 mmol/L chloroauric acid solution and 15.0 muL and 10.0mmol/L sodium iodide solution into 8.0 mL and 0.1mol/L hexadecyl trimethyl ammonium chloride solution; the solution b is prepared by adding 500.0 muL, 50.0 mmol/L chloroauric acid solution and 300.0 muL, 10.0mmol/L sodium iodide solution into 40.0 mL, 0.1mol/L hexadecyl trimethyl ammonium chloride solution; respectively adding 40.0-60.0 muL, 400.0-600.0 muL and 0.1mol/L ascorbic acid solution into the solution a and the solution b, then adding 100.0 muL-200.0 muL gold seed solution diluted by 10 times into the solution a, reacting for 1 s, rapidly adding 3.0-4.0 mL of the solution a into the solution b, stirring for ten seconds, then standing for 1-3 h at room temperature, and finally centrifuging and cleaning for 3 times to obtain triangular gold nanoplates;
the preparation of the nano composite material of the molybdenum disulfide nanoflower loaded hollow core-shell gold-silver-platinum nanocubes in the step (2) comprises the following steps:
① preparation of hollow core-shell gold, silver and platinum nanocubes
Adding 10.0 muL and 25.0 mmol/L chloroauric acid into 10.0 mL and 75.0 mmol/L hexadecyl trimethyl ammonium chloride solution, then dropwise adding 0.6 mL and 10.0mmol/L sodium borohydride solution, and reacting for 2h at room temperature to obtain gold seed solution; then adding 0.1-0.3 mL, 10.0mmol/L chloroauric acid solution and 2.0-4.0 mL, 0.2 mol/L hexadecyltrimethylammonium chloride solution into 25.0 mL deionized water, adding 1.5-2.5 mL, 0.1mol/L ascorbic acid into the above solutions for reaction for more than ten seconds, then adding 0.3-0.5 mL of gold seed solution diluted by 100 times, standing for 8 hours at room temperature, adding 0.5-1.0 mL, 10.0mmol/L silver nitrate, 2.0-4.0 mL, 0.1mol/L ascorbic acid solution into the above solutions, and standing for 12 hours in a 50 ℃ water bath kettle to obtain a gold and silver nanocube colloidal solution; taking 4.0-6.0 mL of gold and silver nanocube colloidal solution, continuously stirring, refluxing and heating to 100 ℃ in an oil bath pot, dripping 200.0-400.0 mu L and 5.0 mmol/L of chloroplatinic acid solution into the solution after 15 min, continuously stirring and reacting for 15 min, cooling, centrifuging and cleaning for 3 times to obtain a hollow core-shell gold and silver platinum nanocube;
② preparation of molybdenum disulfide nanoflower
Adding 1.2-1.6 g of ammonium molybdate into 15.0 mL of deionized water, then adding 0.8-1.0 g of ethylenediamine into the solution, after ultrasonic dissolution, adding a proper amount of 1.0 mol/L hydrochloric acid to adjust the pH value to 4.0-6.0, reacting for 2h at 50 ℃, centrifugally cleaning for 3 times, and carrying out vacuum drying at 50 ℃ to obtain a complex of ammonium molybdate and ethylenediamine; adding 0.1-0.2 g of ammonium molybdate and ethylenediamine complex into 15.0 mL of deionized water, then adding 0.2-0.4 g of L-cysteine, ultrasonically dispersing, placing into a 25.0 mL high-pressure reaction kettle with a polytetrafluoroethylene lining, heating to 200 ℃, preserving heat for 14h, finally centrifugally cleaning for 3 times, and vacuum drying to obtain molybdenum disulfide nanoflowers;
③ preparation of nanometer composite material of molybdenum disulfide nanometer flower load hollow core-shell gold, silver and platinum nanometer cube
Ultrasonically dispersing 0.1-0.2 g of molybdenum disulfide nanoflowers into 20.0 mL of absolute ethyl alcohol, heating to 100 ℃ in an oil bath pot in a refluxing manner, then adding 20.0-30.0 muL 3-aminopropyltriethoxysilane, reacting for 2 hours, cooling, centrifuging, cleaning for 3 times, and freeze-drying to obtain aminated molybdenum disulfide nanoflowers; adding 2.0-4.0 mL and 2.0 mg/mL hollow core-shell gold and silver platinum nanocubes into 2.0 mL and 2.0 mg/mL aminated molybdenum disulfide nanoflowers, ultrasonically dispersing for 1h, oscillating for 6h at room temperature, and centrifugally cleaning for 2 times to obtain the molybdenum disulfide nanoflower-loaded hollow core-shell gold and silver platinum nanocube nanocomposite;
the second antibody marker of the hatching prostate specific antibody of the nanocomposite material of the molybdenum disulfide nanoflower-loaded hollow core-shell gold and silver platinum nanocubes prepared in the step (3)
Adding 2.0-4.0 mL and 10.0 mug/mL prostate specific antibodies into a 2.0 mL and 2.0 mg/mL molybdenum disulfide nanoflower loaded hollow core-shell gold and silver platinum nanocube nanocomposite, oscillating for 12 h at 4 ℃, and centrifugally cleaning and dispersing in a phosphate buffer solution.
2. The method for preparing an electrochemical immunosensor for sandwich type detection of prostate specific antigen according to claim 1, wherein the step (4) of preparing the working electrode of the electrochemical immunosensor for sandwich type detection of prostate specific antigen comprises:
① Al for glassy carbon electrode with diameter of 3.0-5.0 mm2O3Polishing the polishing powder into a mirror surface, and respectively and sequentially ultrasonically cleaning in absolute ethyl alcohol and ultrapure water;
② dripping 6.0 muL and 0.5-3.0 mg/mL triangular gold nanoplate dispersion liquid on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying at room temperature;
③ continuously dripping 6.0 muL and 4.0-8.0 mug/mL prostate specific antibodies on the surface of the electrode, and drying in a refrigerator at 4 ℃;
④ dropwise adding a bovine serum albumin solution of 3.0 muL and 0.7-1.4 wt% to the surface of the electrode to seal the nonspecific active sites, washing the surface of the electrode with a phosphate buffer solution of which the pH is 7.0, and drying in a refrigerator at 4 ℃;
⑤, dripping a series of prostate specific antigen solutions with different concentrations, namely 6.0 muL and 0.00005-100.0 ng/mL, washing the surface of the electrode by using a phosphate buffer solution with the pH value of 7.0, and airing in a refrigerator at 4 ℃;
⑥ dropping a second antibody marker of the prostate specific antibody incubated by the nanocomposite material with the hollow core-shell gold and silver platinum nanocubes loaded by the molybdenum disulfide nanoflowers in the concentration of 6.0 muL and 2.0-4.0 mg/mL on the surface of the electrode, washing the surface of the electrode by using a phosphate buffer solution with the pH value of 7.0, and drying the electrode in a refrigerator at 4 ℃ to prepare the working electrode of the sandwich type electrochemical immunosensor for detecting the prostate specific antigen.
3. The method for preparing an electrochemical immunosensor for sandwich type detection of prostate specific antigen according to claim 1, wherein the step (4) is to prepare a working electrode of the electrochemical immunosensor for sandwich type detection of prostate specific antigen for detection of prostate specific antigen, and the detection steps are as follows:
① testing with an electrochemical workstation by using a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, taking the prepared sensor as a working electrode, and testing in 10.0 mL of 50.0 mmol/L phosphate buffer solution with pH of 5.3-8.0;
② detecting the analyte by time-current method, with input voltage of-0.4V, sampling interval of 0.1 s, and running time of 200 s;
③ when the background current tends to be stable, injecting 10.0-15.0 muL and 5.0 mol/L hydrogen peroxide solution into 10.0 mL and 50.0 mmol/L phosphate buffer solution, recording current values corresponding to prostate specific antigens under different concentrations, and drawing a working curve of the electrochemical immunosensor for detecting the prostate specific antigens;
④ the concentration of prostate specific antigen in the sample to be tested is obtained by using the working curve method.
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| CN109738641A (en) * | 2019-01-19 | 2019-05-10 | 山东理工大学 | A kind of preparation method and application without enzyme electrochemical immunosensor based on platinum palladium functionalization molybdenum disulfide |
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| CN110045121B (en) * | 2019-04-30 | 2022-02-11 | 山东理工大学 | Preparation method and application of hollow cube-shaped trimetal nanocomposite immunosensor |
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| CN111721820B (en) * | 2020-07-13 | 2023-05-09 | 江西科技师范大学 | Non-labeled electrochemical immunosensor for detecting prostate specific antigen |
| CN112858420A (en) * | 2021-03-30 | 2021-05-28 | 济南大学 | Preparation method and application of sandwich type electrochemical sensor constructed based on vanadium selenide/gold nanoparticles |
| CN113075202B (en) * | 2021-03-31 | 2022-10-14 | 山东理工大学 | Preparation of immunosensor based on MIL-101@ SnS2 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297480A (en) * | 2014-09-26 | 2015-01-21 | 济南大学 | Preparation method and application of sandwich type immunosensor for prostate specific antigens |
| CN105699464A (en) * | 2016-03-05 | 2016-06-22 | 济南大学 | Preparation method and application of immunity sensor based on cuprous oxide doped palladium nanoparticle marks |
| CN107505466A (en) * | 2017-10-20 | 2017-12-22 | 山东理工大学 | A kind of preparation method and application for the Amperometric Immunosensor for detecting hepatitis B surface antibody |
| CN108132287A (en) * | 2017-12-04 | 2018-06-08 | 山东理工大学 | A kind of preparation method and application of the Amperometric Immunosensor based on polypyrrole nanosheet composite material |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105683745A (en) * | 2013-08-29 | 2016-06-15 | 阿波赛尔公司 | Method and apparatus for isolation, capture and molecular analysis of target particles |
| KR101751500B1 (en) * | 2015-02-09 | 2017-06-27 | 한국과학기술원 | Thiolated ligand conjugated MoS2 Chemiresistor gas sensor and fabrication method thereof |
-
2018
- 2018-07-20 CN CN201810802209.8A patent/CN108982630B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297480A (en) * | 2014-09-26 | 2015-01-21 | 济南大学 | Preparation method and application of sandwich type immunosensor for prostate specific antigens |
| CN105699464A (en) * | 2016-03-05 | 2016-06-22 | 济南大学 | Preparation method and application of immunity sensor based on cuprous oxide doped palladium nanoparticle marks |
| CN107505466A (en) * | 2017-10-20 | 2017-12-22 | 山东理工大学 | A kind of preparation method and application for the Amperometric Immunosensor for detecting hepatitis B surface antibody |
| CN108132287A (en) * | 2017-12-04 | 2018-06-08 | 山东理工大学 | A kind of preparation method and application of the Amperometric Immunosensor based on polypyrrole nanosheet composite material |
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