CN112526135A - Preparation method and application of photoelectrochemical biosensor for detecting prostate specific antigen - Google Patents
Preparation method and application of photoelectrochemical biosensor for detecting prostate specific antigen Download PDFInfo
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- 102000007066 Prostate-Specific Antigen Human genes 0.000 title claims abstract description 52
- 108010072866 Prostate-Specific Antigen Proteins 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- ADZWSOLPGZMUMY-UHFFFAOYSA-M silver bromide Chemical compound [Ag]Br ADZWSOLPGZMUMY-UHFFFAOYSA-M 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 210000002307 prostate Anatomy 0.000 claims abstract description 8
- 230000004044 response Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 54
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 40
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 36
- 239000012498 ultrapure water Substances 0.000 claims description 36
- 238000005406 washing Methods 0.000 claims description 32
- 238000001035 drying Methods 0.000 claims description 27
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 15
- 238000003760 magnetic stirring Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- PPNKDDZCLDMRHS-UHFFFAOYSA-N dinitrooxybismuthanyl nitrate Chemical compound [Bi+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O PPNKDDZCLDMRHS-UHFFFAOYSA-N 0.000 claims description 10
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 10
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 238000001354 calcination Methods 0.000 claims description 6
- 229910052979 sodium sulfide Inorganic materials 0.000 claims description 6
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000003599 detergent Substances 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 101710134784 Agnoprotein Proteins 0.000 claims description 2
- 239000002211 L-ascorbic acid Substances 0.000 claims description 2
- 235000000069 L-ascorbic acid Nutrition 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 2
- 230000005284 excitation Effects 0.000 claims description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 2
- -1 polytetrafluoroethylene Polymers 0.000 claims description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 239000000427 antigen Substances 0.000 abstract description 3
- 108091007433 antigens Proteins 0.000 abstract description 3
- 102000036639 antigens Human genes 0.000 abstract description 3
- 239000004005 microsphere Substances 0.000 abstract description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 abstract 4
- 230000003287 optical effect Effects 0.000 abstract 1
- 238000012113 quantitative test Methods 0.000 abstract 1
- 230000009870 specific binding Effects 0.000 abstract 1
- 238000011895 specific detection Methods 0.000 abstract 1
- 239000000758 substrate Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000004809 Teflon Substances 0.000 description 3
- 229920006362 Teflon® Polymers 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000011953 bioanalysis Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229910052946 acanthite Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001027 hydrothermal synthesis Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- FSJWWSXPIWGYKC-UHFFFAOYSA-M silver;silver;sulfanide Chemical compound [SH-].[Ag].[Ag+] FSJWWSXPIWGYKC-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
Abstract
The invention relates to a preparation method and application of a photoelectrochemical biosensor for detecting Prostate Specific Antigen (PSA), which is prepared by mixing Ag2The S-sensitized microspheric Ag/AgBr/BiOBr heterojunction is fixed on the surface of an ITO electrode and is used as an excellent optical activity substrate, so that the photocurrent response and the sensitivity thereof are effectively improved. Thioglycolic acid (TGA) is used as a linker to immobilize prostate specific antibodies, and the photocurrent signal of different concentrations of antigen is detected through specific binding between the antigen and the antibody, so that the quantitative test of PSA is completed. Under the best experimental conditions, the PEC immunosensor has good stability, wide linear range, high sensitivity and good reproducibility, can be used for detecting actual samples, realizes the specific detection of the prostate specific antigen, and provides a novel and feasible detection for the early detection of PSAThe detection method has potential application prospect in clinic.
Description
Technical Field
The invention relates to the technical field of bioanalysis chemistry, nanomaterials, immunoassay and photoelectrochemical biosensor, and provides a preparation method and application of a photoelectrochemical biosensor for detecting a prostate specific antigen. Synthesizing microspherical Ag/AgBr/BiOBr, Ag by hydrothermal method2S is taken as a sensitizer, and an antibody capture material is fixed on the surface of an electrode, thereby providing a preparation method and application of a non-standard photoelectrochemical biosensor for detecting prostate specific antigen.
Background
Prostate cancer is the most common cancer in 105 countries. It is also the second most common cancer in men in the world and a common cause of male death in different countries, and therefore its early discovery is critical in saving the life of patients. Prostate Specific Antigen (PSA), a glycoprotein in semen that can be transferred into the blood, is considered as a major tumor marker for early diagnosis and prevention of prostate cancer. The PSA concentration in normal human serum should be less than 4 ng/mL, with higher PSA concentrations being at greater risk of cancer. Therefore, it is critical to establish an assay technology that can detect PSA rapidly and sensitively.
Up to now, a number of different detection methods have been available for PSA detection, such as fluorescence immunosensors, molecular imprinting techniques, Electrochemiluminescence (ECL) immunosensors, electrochemical immunosensors, Photoelectrochemical (PEC) sensors, etc. The PEC immunosensor is a novel detection method, and can be used for detecting tumor markers, microorganisms, toxins and the like. PEC immunosensors have also attracted considerable attention in the field of bioanalysis due to their high sensitivity, low cost, good selectivity, etc.
For the photoelectrochemical immunosensor, sensitivity is an important index for evaluating the performance thereof. Methods for improving sensitivity are various, and increasing the initial signal is one of the most effective methods. The single photosensitive material has low photocurrent conversion efficiency, and the semiconductor nano composite material with excellent photoelectric performance is utilized to improve photoelectric signals, so that the photoelectric signal conversion device has very important significance. BiOBr is widely used due to its stability and good light absorption. However, the photoelectric activity of the BiOBr is limited by poor separation capability and weak surface adsorption capability of electron-hole pairs. Therefore, the photoelectric activity of the BiOBr needs to be further optimized, and the coupling of the BiOBr with other materials to form a p-n heterojunction is an effective method for improving the photoelectric activity of the BiOBr. In the patent, Ag/AgBr/BiOBr is synthesized by an in-situ ion exchange method, has larger specific surface area and can be combined with more particles, thereby being beneficial to the separation and transfer of charges.
The invention uses photoelectrochemical analysis method to prepare Ag2The S quantum dots are used for sensitizing Ag/AgBr/BiOBr, the visible light absorption of the S quantum dots is enhanced, the photoelectric activity is obviously improved, and the prostate specific antibody and bovine serum are subjected to layer-by-layer self-assemblyAssemble of albumin and prostate specific antigen to Ag/AgBr/BiOBr/Ag2On the S composite material, the excellent photoelectric activity of Ag/AgBr/BiOBr and the specific combination between the prostate specific antigen antibodies construct a photoelectrochemical biosensor of the prostate specific antigen based on Ag/AgBr/BiOBr. The sensor has excellent photoelectrochemical activity, has the advantages of high sensitivity, wide linear range, low detection limit, rapid detection, relatively simple preparation process and the like, realizes the ultra-sensitive analysis of the prostate specific antigen, and provides a new method for effectively detecting the prostate specific antigen at present.
Disclosure of Invention
The invention provides a preparation method and application of a photoelectrochemical biosensor for detecting a prostate specific antigen, and realizes the ultra-sensitive detection of the prostate specific antigen. One of the purposes of the present invention is to provide a preparation method of a photoelectrochemical biosensor for detecting prostate specific antigen. The invention also aims to realize the ultra-sensitive detection of the prostate specific antigen by the prepared photoelectrochemistry immunosensor.
The technical scheme of the invention comprises the following steps:
(1) preparing hollow microspherical BiOBr;
(2) preparing microspherical Ag/AgBr/BiOBr;
(3) preparing a working curve of the photoelectrochemical biosensor for detecting the prostate specific antigen.
Wherein the step (1) of preparing the hollow microspherical BiOBr comprises the following steps:
0.5 ~ 1.5 mmol Bi(NO3)3·5H2dispersing O in 20 mL of glycol, and slowly adding 0.05-0.15 g of polyvinylpyrrolidone (PVP; MW-40K) into the solution under magnetic stirring, wherein the solution is marked as solution A; 0.05-0.15 mmol of KBr is dispersed in 20 mL of glycol and marked as solution B; slowly dripping the solution B into the solution A under magnetic stirring, and changing the color from transparent to white; transferring the final mixture into 50 ml polytetrafluoroethylene reaction kettle, reacting at 120 deg.C for 12 h, centrifuging the obtained mixture, washing with deionized water and anhydrous ethanol for 3 times, and drying at 60 deg.C；
Wherein the preparation of the microspherical Ag/AgBr/BiOBr in the step (2) comprises the following steps:
dispersing the prepared 0.5-1.5 mmol BiOBr in AgNO solution containing 0.05-0.15 mmol3In 80 mL of ethylene glycol, the mixture is stirred vigorously and reacts for 12 hours under the magnetic stirring, the obtained mixture is centrifugally washed with deionized water and absolute ethyl alcohol for 3 times respectively, and the mixture is dried at 60 ℃;
wherein the working curve of the photoelectrochemical biosensor for detecting the prostate specific antigen without the standard type prepared in the step (3) comprises the following steps:
firstly, ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
dripping 8-12 mu L of Ag/AgBr/BiOBr suspension of 4-8 mg/mL on an ITO electrode, naturally drying at room temperature, and calcining at 400 ℃ for 60 min;
thirdly, continuously dropwise adding 3-5 mu L of 0.03-0.09 mol/L sodium sulfide solution on the surface of the electrode, reacting for 20-40 minutes, washing with ultrapure water, and naturally drying;
continuing to dropwise add 3-5 mu L of 0.1 mol/L TGA solution, reacting for 20-40 min, and washing the surface of the electrode with ultrapure water;
continuously dropwise adding 3-5 mu L of L-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide on the surface of the modified electrode, reacting for 20-40 min, washing with ultrapure water, and naturally drying;
sixthly, dripping 3-5 mu L of prostate specific antibody solution with the concentration of 10 mu g/mL, reacting for 20-40 min, washing the surface of the electrode by using ultrapure water, and drying in a refrigerator at the temperature of 4 ℃;
seventhly, continuously dropwise adding 3-5 mu L of BSA solution with the mass fraction of 1% to seal non-specific active sites on the surface of the electrode, reacting for 20-40 min, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
continue to drop 3-5 mu L of PSA (pressure swing adsorption) with the concentration of 0.001-50 ng/mL, wash the surface of the electrode with ultrapure water, and dry the electrode in a refrigerator at 4 ℃ to obtain the Ag/AgBr/BiOBr/Ag-based electrode2S, storing the non-standard photoelectrochemical biosensor of the prostate specific antigen in a refrigerator at 4 ℃ for later use;
the 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3 mol/L of N-hydroxysuccinimide;
the raw materials used in the present invention are all available from chemical or biopharmaceutical companies.
Advantageous results of the invention
(1) The invention is based on Ag/AgBr/BiOBr/Ag2S constructs a novel photoelectrochemical immunosensor for the detection of prostate specific antigens. Using Ag2A method for S-sensitizing microspheric Ag/AgBr/BiOBr in-situ growth heterojunction;
(2) the immunosensor has high sensitivity for PSA detection, the linear range is from 0.001 ng/mL to 50 ng/mL, and the detection limit is low and is 0.25 pg/mL-1. The prepared immunosensor has high stability and good reproducibility;
(3) the invention provides a novel and feasible detection method for the early detection of PSA, has simple operation and rapid detection, and can be used for the detection of actual samples.
Detailed Description
The invention will now be further illustrated by, but not limited to, the following specific embodiments
Example 1 a hollow microspheroidal bibir is prepared by the following steps:
0.5 mmol Bi(NO3)3·5H2dispersing O in 20 mL of glycol, and slowly adding 0.05g of polyvinylpyrrolidone (PVP; MW-40K) into the solution under magnetic stirring, wherein the label is solution A; 0.05 mmol KBr was dispersed in 20 mL ethylene glycol and labeled as solution B; slowly dripping the solution B into the solution A under magnetic stirring, and changing the color from transparent to white; the final mixture was transferred to a 50 ml teflon reaction kettle and reacted at 120 ℃ for 12 hours, and the resulting mixture was centrifugally washed with deionized water, absolute ethanol 3 times each, and then dried at 60 ℃.
Example 2 hollow microspherical BiOBr was prepared as follows:
1.0 mmol Bi(NO3)3·5H2dispersing O in 20 mL of ethylene glycol, and slowly adding 0.10 g of polyvinylpyrrolidone (PVP; MW-40K) into the solution under magnetic stirring, wherein the label is solution A; 0.10 mmol KBr was dispersed in 20 mL ethylene glycol and labeled as solution B; slowly dripping the solution B into the solution A under magnetic stirring, and changing the color from transparent to white; the final mixture was transferred to a 50 ml teflon reaction kettle and reacted at 120 ℃ for 12 hours, and the resulting mixture was centrifugally washed with deionized water, absolute ethanol 3 times each, and then dried at 60 ℃.
Example 3 hollow microspherical BiOBr was prepared as follows:
1.5 mmol Bi(NO3)3·5H2dispersing O in 20 mL of ethylene glycol, and slowly adding 0.15 g of polyvinylpyrrolidone (PVP; MW-40K) into the solution under magnetic stirring, wherein the label is solution A; 0.15mmol KBr was dispersed in 20 mL ethylene glycol and labeled as solution B; slowly dripping the solution B into the solution A under magnetic stirring, and changing the color from transparent to white; the final mixture was transferred to a 50 ml teflon reaction kettle and reacted at 120 ℃ for 12 hours, and the resulting mixture was centrifugally washed with deionized water, absolute ethanol 3 times each, and then dried at 60 ℃.
Example 4 microspherical Ag/AgBr/BiOBr was prepared as follows:
dispersing the prepared 0.5 mmol BiOBr in a solution containing 0.05 mmol AgNO3In 80 mL of ethylene glycol, the reaction was vigorously stirred for 12 hours under magnetic stirring, and the resulting mixture was centrifugally washed with deionized water and absolute ethanol 3 times each, and dried at 60 ℃.
Example 5 microspherical Ag/AgBr/BiOBr was prepared as follows:
dispersing the prepared 1.0 mmol BiOBr in a solution containing 0.10 mmol AgNO3In 80 mL of ethylene glycol, the reaction was vigorously stirred for 12 hours under magnetic stirring, and the resulting mixture was centrifugally washed with deionized water and absolute ethanol 3 times each, and dried at 60 ℃.
Example 6 microspherical Ag/AgBr/BiOBr was prepared as follows:
dispersing the prepared 1.5 mmol BiOBr in a solution containing 0.15mmol AgNO3In 80 mL of ethylene glycol (E) of the solution,the reaction was carried out under vigorous stirring for 12 hours under magnetic stirring, and the resulting mixture was washed by centrifugation with deionized water and absolute ethanol 3 times each, and dried at 60 ℃.
Example 7 working curve of a photoelectrochemical biosensor for the detection of prostate specific antigen without a standard was prepared by the following steps:
firstly, ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
dropping 8 mu L of 4 mg/mL Ag/AgBr/BiOBr suspension on an ITO electrode, naturally airing at room temperature, and calcining at 400 ℃ for 60 min;
thirdly, continuously dripping 3 mu L and 0.03 mol/L sodium sulfide solution on the surface of the electrode, reacting for 20-40 minutes, washing with ultrapure water, and naturally drying;
continuing to dropwise add 3 mu L of 0.1 mol/L TGA solution, reacting for 20-40 min, and washing the surface of the electrode by using ultrapure water;
continuously dropwise adding 3 mu L of L-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide on the surface of the modified electrode, reacting for 20-40 min, washing with ultrapure water, and naturally drying;
sixthly, dripping 3 mu L of prostate specific antibody solution with the concentration of 10 mu g/mL, reacting for 20-40 min, washing the surface of the electrode by ultrapure water, and drying in a refrigerator at the temperature of 4 ℃;
seventhly, continuously dropwise adding 3 mu L of BSA solution with the mass fraction of 1% to seal non-specific active sites on the surface of the electrode, reacting for 20-40 min, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
eighthly, continuously dropwise adding 3 mu L and 0.001-50 ng/mL of PSA, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the Ag/AgBr/BiOBr/Ag-based electrode2S, storing the non-standard photoelectrochemical biosensor of the prostate specific antigen in a refrigerator at 4 ℃ for later use;
the 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3 mol/L of N-hydroxysuccinimide.
Example 8 a working curve of a photoelectrochemical biosensor for the detection of prostate specific antigen without a standard was prepared by the following steps:
firstly, ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
dropping 10 mu L of Ag/AgBr/BiOBr suspension liquid with the concentration of 6 mg/mL on an ITO electrode, naturally airing at room temperature, and calcining at 400 ℃ for 60 min;
thirdly, continuously dripping 4 mu L and 0.06 mol/L sodium sulfide solution on the surface of the electrode, reacting for 20-40 minutes, washing with ultrapure water, and naturally drying;
continuing to dropwise add 4 mu L of 0.1 mol/L TGA solution, reacting for 20-40 min, and washing the surface of the electrode by using ultrapure water;
continuously dropwise adding 4 mu L of L-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide on the surface of the modified electrode, reacting for 20-40 min, washing with ultrapure water, and naturally drying;
sixthly, dripping 4 mu L of prostate specific antibody solution with the concentration of 10 mu g/mL, reacting for 20-40 min, washing the surface of the electrode by ultrapure water, and drying in a refrigerator at the temperature of 4 ℃;
seventhly, continuously dropwise adding 4 mu L of BSA solution with the mass fraction of 1% to seal non-specific active sites on the surface of the electrode, reacting for 20-40 min, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
eighthly, continuously dropwise adding 4 mu L and 0.001-50 ng/mL of PSA, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the Ag/AgBr/BiOBr/Ag-based electrode2S, storing the non-standard photoelectrochemical biosensor of the prostate specific antigen in a refrigerator at 4 ℃ for later use;
the 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3 mol/L of N-hydroxysuccinimide.
Example 9 a working curve of a photoelectrochemical biosensor for the detection of prostate specific antigen without a standard was prepared by the following steps:
firstly, ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
dripping 12 mu L of Ag/AgBr/BiOBr suspension liquid with the concentration of 8mg/mL on an ITO electrode, naturally airing at room temperature, and calcining for 60 min at 400 ℃;
thirdly, continuously dripping 5 mu L of 0.09 mol/L sodium sulfide solution on the surface of the electrode, reacting for 20-40 minutes, washing with ultrapure water, and naturally drying;
continuously dropwise adding 5 mu L of 0.1 mol/L TGA solution, reacting for 20-40 min, and washing the surface of the electrode by using ultrapure water;
continuously dropwise adding 3 mu L of L-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide on the surface of the modified electrode, reacting for 20-40 min, washing with ultrapure water, and naturally drying;
sixthly, dripping 5 mu L of prostate specific antibody solution with the concentration of 10 mu g/mL, reacting for 20-40 min, washing the surface of the electrode by ultrapure water, and drying in a refrigerator at the temperature of 4 ℃;
seventhly, continuously dropwise adding 5 mu L of BSA solution with the mass fraction of 1% to seal non-specific active sites on the surface of the electrode, reacting for 20-40 min, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
eighthly, continuously dropwise adding 5 mu L of PSA (pressure swing adsorption) with the concentration of 0.001-50 ng/mL, washing the surface of an electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the Ag/AgBr/BiOBr/Ag-based electrode2S prostate specific antigen, stored in a refrigerator at 4 ℃ for future use.
The 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3 mol/L of N-hydroxysuccinimide;
example 10 the method of preparing a non-standard photoelectrochemical biosensor for detecting prostate specific antigen and the use of the same according to claim l, wherein the method is used for detecting prostate specific antigen by the following steps:
firstly, a three-electrode system of an electrochemical workstation is used for testing, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared prostate specific antigen photoelectrochemical biosensor is used as a working electrode, and the test is carried out in a PBS buffer solution;
secondly, detecting the prostate specific antigen of the standard product with different concentrations by adopting a time-current method, setting the voltage to be 0V, the running time to be 50 s, using an LED as an excitation light source, recording the change of current, and drawing a working curve;
diluting the sample to be detected, and then replacing the standard product to detect, and obtaining the content of the prostate specific antigen in the sample to be detected according to the photocurrent response intensity and the working curve;
the PBS buffer solution is 10-15 mL of phosphate buffer solution containing 0.1 mol/L ascorbic acid and the pH value of the phosphate buffer solution is 5.0-8.5.
Claims (3)
1. A preparation method and application of a photoelectrochemical biosensor for detecting prostate specific antigen are characterized by comprising the following steps:
(1) ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
(2) dripping 8-12 mu L of Ag/AgBr/BiOBr suspension of 4-8 mg/mL on an ITO electrode, naturally airing at room temperature, and calcining for 60 minutes at 400 ℃;
(3) continuously dropwise adding 3-5 mu L of 0.03-0.09 mol/L sodium sulfide solution on the surface of the electrode, reacting for 20-40 minutes, washing with ultrapure water, and naturally drying;
(4) continuously dropwise adding 3-5 mu L of 0.1 mol/L TGA solution, reacting for 20-40 minutes, and then washing the surface of the electrode with ultrapure water;
(5) continuously dropwise adding 3-5 mu L of L-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide on the surface of the modified electrode, reacting for 20-40 min, washing with ultrapure water, and naturally drying;
(6) dripping 3-5 mu L of prostate specific antibody solution of 10 mu g/mL, reacting for 20-40 minutes, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(7) continuously dropwise adding 3-5 mu L of BSA solution with the mass fraction of 1% to seal the non-specific active sites on the surface of the electrode, reacting for 20-40 minutes, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(8) continuously dropwise adding 3-5 mu L of prostate specific antigen 0.001-50 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the Ag/AgBr/BiOBr/Ag-based electrode2S, storing the non-standard photoelectrochemical biosensor of the prostate specific antigen in a refrigerator at 4 ℃ for later use;
the 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3 mol/L of N-hydroxysuccinimide.
2. The method for preparing the photoelectrochemical biosensor for detecting the prostate specific antigen according to claim 1, wherein the Ag/AgBr/BiOBr/Ag is2The preparation of the S electrode is characterized by comprising the following steps:
（1）0.5 ~ 1.5 mmol Bi(NO3)3·5H2dispersing O in 20 mL of glycol, and slowly adding 0.05-0.15 g of polyvinylpyrrolidone (PVP; MW-40K) into the solution under magnetic stirring, wherein the solution is marked as solution A; 0.05-0.15 mmol of KBr is dispersed in 20 mL of glycol and marked as solution B; slowly dripping the solution B into the solution A under magnetic stirring, and changing the color from transparent to white; transferring the final mixture to a 50 ml polytetrafluoroethylene reaction kettle, reacting for 12 h at 120 ℃, centrifugally washing the obtained mixture with deionized water and absolute ethyl alcohol for 3 times respectively, and then drying at 60 ℃;
(2) dispersing the prepared 0.5-1.5 mmol BiOBr in AgNO solution containing 0.05-0.15 mmol3In 80 mL of ethylene glycol, the mixture is stirred vigorously and reacts for 12 hours under the magnetic stirring, the obtained mixture is centrifugally washed with deionized water and absolute ethyl alcohol for 3 times respectively, and the mixture is dried at 60 ℃;
(3) dripping 8-12 mu L of 4-8 mg/mL Ag/AgBr/BiOBr suspension into the solutionNaturally airing the ITO electrode at room temperature, calcining the ITO electrode for 60 minutes at 400 ℃, naturally cooling the ITO electrode to the room temperature, continuously dropwise adding 3-5 mu L and 0.03-0.09 mol/L sodium sulfide solution on the surface of the ITO electrode, reacting for 20-40 minutes, washing the ITO electrode with ultrapure water, and naturally airing to obtain Ag/AgBr/BiOBr/Ag2And an S electrode.
3. The method for preparing the photoelectrochemical biosensor for detecting the prostate specific antigen according to claim i and the application thereof are used for detecting the prostate specific antigen, and the detection steps are as follows:
(1) testing by using a three-electrode system of an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared prostate specific antigen photoelectrochemical biosensor as a working electrode, and testing in a PBS (phosphate buffer solution);
(2) detecting the prostate specific antigen of the standard product with different concentrations by adopting a time-current method, setting the voltage to be 0V, the running time to be 50 s, recording the change of current by using an LED as an excitation light source, and drawing a working curve;
(3) diluting a sample to be detected, and then replacing the standard substance in the step (2) for detection, and obtaining the content of the prostate specific antigen in the sample to be detected according to the photocurrent response intensity and the working curve;
the PBS buffer solution is 10-15 mL of phosphate buffer solution containing 0.1 mol/L ascorbic acid and the pH value of the phosphate buffer solution is 5.0-8.5.
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