Preparation method of electrochemical immunosensor for detecting tumor marker-loaded molecular sieve
A tumor marker and immunosensor technology is applied in the field of preparation of a molecular sieve-loaded electrochemical immunosensor for detecting tumor markers, and achieves the effects of good electrical conductivity, large surface area, and high catalytic performance.
Inactive Publication Date: 2018-03-27
NINGBO COLLEGE OF HEALTH SCI
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Abstract
The invention provides a preparation method of an electrochemical immunosensor for detecting a tumor marker-loaded molecular sieve. The preparation method comprises the following steps: polishing a glassy carbon electrode with Al2O3 polishing powder, and cleaning with ultra-pure water; dropwise adding a chitosan solution of piperazine-modified aminated graphene onto the surface of the electrode, rinsing and airing; dropwise adding 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solutions onto the surface of the electrode; coating the surface of a working electrode with a silver nanoparticle p-thiophenol functional sulfonated carbon nanotube in a dropping manner to detect an antibody Ab2 incubation solution; dropwise adding a SAPO-34-Pd/Co-Ab2 second antibody incubation solution to prepare the electrochemical immunosensor for detecting the tumor marker-loaded molecular sieve. By adopting the piperazine-modified aminated graphene, a relatively large surface area, higher conductivity, high biocompatibility and high stability of the graphene are kept.
Application Domain
Material analysis by electric/magnetic means
Technology Topic
Cvd grapheneMolecular sieve +17
Examples
- Experimental program(3)
Example Embodiment
[0049] Example 1:
[0050] A preparation method for detecting tumor marker-loaded molecular sieve electrochemical immunosensor, comprising the following steps:
[0051] Step (1), the glassy carbon electrode with a diameter of 4mm is polished with Al2O3 polishing powder, and cleaned with ultrapure water;
[0052] Take 8 μL, 1.5 mg/mL chitosan solution of piperazine-modified aminated graphene and add it dropwise to the electrode surface, dry it at room temperature, rinse the electrode surface with ultrapure water, and dry it; add 8 μL dropwise at a concentration of 0.8 mol/L 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide EDC and 0.3mol/L N-hydroxysuccinimide NHS solution to the electrode surface, incubate at 4°C for 40min , dry at room temperature;
[0053] Step (2), add 8 μL, 10 μg/mL primary tumor marker antibody dropwise on the surface of the working electrode modified in step (1), rinse the electrode surface with ultrapure water, and dry it in a refrigerator at 4°C;
[0054] Step (3), drop 8 μL of BSA solution with a mass fraction of 2% on the surface of the working electrode modified in step (2) to seal the non-specific active sites on the electrode surface, rinse the electrode surface with ultrapure water, and dry in a refrigerator at 4°C Dry;
[0055] Step (4), drop 8 μL of bovine serum albumin BSA solution with a mass fraction of 3% on the surface of the working electrode modified in step (3) to seal the non-specific active sites on the surface of the electrode, rinse with ultrapure water , dry in a refrigerator at 4°C; add 8 μL, 20ng/mL of a series of tumor marker antigen Ag solutions with different concentrations dropwise, rinse the electrode surface with ultrapure water, and dry in a refrigerator at 4°C;
[0056] Step (5), on the surface of the working electrode modified in step (4), first drop-coat 8 μ L, 2 mg/mL of silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotube detection antibody Ab2 incubation solution, drop-coated on the electrode surface placed in a 4°C refrigerator to dry; then 8 μL of the secondary antibody hatching solution of SAPO-34-Pd/Co-Ab2 was added dropwise, and placed in a 4°C refrigerator to dry to prepare a molecular sieve electrode for the detection of tumor markers. Chemoimmunosensors.
[0057] Wherein: the preparation steps of the silver nanoparticle p-thiophenol functionalized sulfonated carbon nanotube detection antibody Ab2 hatch solution are as follows:
[0058] ① Preparation of sulfonated carbon nanotubes
[0059] Weigh 2.0g of multi-walled carbon nanotubes and place them in a 500mL three-neck flask, add 88mL of concentrated sulfuric acid and 45mL of concentrated nitric acid in sequence, react in an oil bath at 180°C for 60min, cool to room temperature, transfer the resulting suspension into a 1000mL beaker, add Dilute with 400mL of ultrapure water, filter with suction, wash with ultrapure water until the filtrate becomes neutral, and dry the obtained solid in a vacuum oven at 90°C for 15 hours to obtain sulfonated carbon nanotubes;
[0060] ② Preparation of p-thiophenol functionalized sulfonated carbon nanotubes
[0061] Add 12 mg of sulfonated carbon nanotubes, 80 mL of 1.5 mol/L hydrochloric acid solution to a 250 mL round bottom flask, ultrasonically disperse for 2 hours, add 2.0 g of 4,4'-diaminodiphenyl disulfide, and cool to 0°C in an ice bath , slowly add 20mL sodium nitrite aqueous solution under stirring, react for 1.5h, heat up to 35°C, continue stirring for 2.5h, filter with 0.55μm polytetrafluoroethylene membrane, wash with ultrapure water 4 times, absolute ethanol Wash 4 times, ultrasonically disperse the obtained solid into 80mL ethanol, add 80mL acetonitrile and 620mg triphenylphosphine successively, stir for 2h, filter with 0.55μm polytetrafluoroethylene membrane, wash 4 times with absolute ethanol, the obtained solid Dry in a vacuum oven at 90°C for 15 hours to obtain p-thiophenol functionalized sulfonated carbon nanotubes;
[0062] Described sodium nitrite aqueous solution is that 300mg solid sodium nitrite is dissolved in 15mL ultrapure water and makes;
[0063] ③ Preparation of silver nanoparticles solution
[0064] Add 2mL, 50mmol/L silver nitrate and 2mL, 5% sodium citrate into 50mL ultrapure water under stirring, then add 50mg sodium borohydride, react under stirring for 20min, the solution turns brownish yellow, continue Stir until the color of the solution no longer changes, cool to room temperature, and centrifuge at a speed of 6000rpm for 10min, the resulting supernatant is a silver nanoparticle solution;
[0065] ④ Preparation of incubation solution of antibody Ab2 detected by thiophenol-functionalized sulfonated carbon nanotubes with silver nanoparticles
[0066] Take 10 mg of p-thiophenol functionalized sulfonated carbon nanotubes and add them to 35 mL of silver nanoparticle solution, shake at 130 rpm for 12 hours, centrifuge, and dry in a vacuum oven at 90°C to obtain silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotubes. carbon nanotubes;
[0067] Disperse 4 mg of silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotubes into 2 mL of ultrapure water, add 200 μL, 100 μg/mL of tumor marker detection antibody Ab2 solution and 1000 μL, 100 mmol/L of pH7.4 phosphoric acid Salt buffer solution, shaking and incubating in a constant temperature shaking incubator at 4°C for 15h; centrifuging at 8000rpm at 4°C for 20min to obtain the lower layer precipitate, adding 2mL, 100mmol/L phosphate buffer solution with pH=7.4 and washing once by centrifugation to obtain The lower layer was precipitated, and finally 2 mL of 100 mmol/L phosphate buffer solution at pH = 7.4 was added to prepare a silver nanoparticle-thiophenol-functionalized sulfonated carbon nanotube detection antibody Ab2 incubation solution, which was stored at 4°C for future use.
[0068] Wherein: the preparation process of the SAPO-34-Pd/Co-Ab2 secondary antibody hatch solution comprises the following steps:
[0069] (1) Preparation of chitosan solution of piperazine-modified aminated graphene
[0070] Add 1.5mg of piperazine-modified aminated graphene to 2mL, 0.3% chitosan solution, ultrasonically disperse for 20min, and store the solution at 4°C for later use;
[0071] Described 0.3% chitosan solution is that the chitosan of 2g is dissolved in the dilute hydrochloric acid solution of 15mL, 0.03mol/L;
[0072] (2) Preparation of SAPO-34-Pd/Co-Ab2 secondary antibody incubation
[0073] 1) Preparation of aminated molecular sieves
[0074] Weigh 1.0g SAPO-34 into a three-necked flask, add 1mL of 3-aminopropyltriethoxysilane and 20mL of absolute ethanol, heat the mixture to 80°C and keep it for 2.0h, then cool to room temperature, and the mixture is heated at 9000rpm Centrifuge for 10 minutes to prepare an aminated molecular sieve NH2-SAPO-34, wash with water and centrifuge three times, and finally dry the aminated molecular sieve in vacuum at 55°C;
[0075] 2) Preparation of SAPO-34-Pd/Co
[0076] Under nitrogen protection and ice bath cooling, mix 5mL, 0.3mol/L cobalt nitrate, 4.5mL, 80mmol/L sodium citrate, 30mgNH2-SAPO-34 and 35mL ultrapure water for 30min;
[0077] 8mL, 2.5mol/L sodium borohydride solution was added to the above mixed solution at a rate of 25mL/h and vigorously stirred to obtain a Co nanoparticle solution;
[0078] Add 15mL, 50mmol/L sodium tetrachloropalladate Na2PdCl4 and 20mL, 0.25mol/L NaBH4 solution to the Co nanoparticle solution at the same time at a rate of 30mL/h. Dried under vacuum to obtain SAPO-34-Pd/Co;
[0079] 3) Preparation of SAPO-34-Pd/Co-Ab2 secondary antibody incubation solution
[0080] Disperse 2 mg of SAPO-34-Pd/Co into 1 mL of ultrapure water, add 200 μL of 100 μg/mL secondary antibody Ab2 and 1000 μL of 50 mmol/L pH7.4 phosphate buffer solution, and place in a constant temperature shaking incubator at 4 °C Shake and incubate for 12 hours, centrifuge at 4°C and 8000rpm for 25 minutes to obtain the lower precipitate; then add 2mL, 60mmol/L phosphate buffer solution with pH=7.4 and centrifuge once to obtain the lower precipitate; finally add 2mL, 60mmol/L pH = 7.4 phosphate buffer solution to prepare SAPO-34-Pd/Co-Ab2 secondary antibody incubation solution, which was stored at 4°C for future use.
[0081] The present invention also provides a molecular sieve electrochemical immunosensor for detecting tumor markers prepared according to the above preparation method, which is used for the detection of various tumor markers. The detection steps are as follows:
[0082] (1) The electrochemical workstation was used to test with a three-electrode system. The Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared immunosensor was used as the working electrode. 0 in phosphate buffered saline solution;
[0083] (2) Detect various tumor marker antigen standard solutions by time-current method, the input voltage is -0.6V, the sampling interval is 0.05s, and the running time is 500s;
[0084] (3) When the background current tends to be stable, inject 10 μL, 5 mol/L hydrogen peroxide solution into 20 mL of 60 mmol L-1, pH=7.0 phosphate buffer solution every 10 s, then record the current change and draw the working curve ;
[0085] (4) The test sample solution is replaced by the standard solution of various tumor marker antigens for detection.
Example Embodiment
[0086] Example 2:
[0087] A preparation method for detecting tumor marker-loaded molecular sieve electrochemical immunosensor, comprising the following steps:
[0088] Step (1), the glassy carbon electrode with a diameter of 4mm is polished with Al2O3 polishing powder, and cleaned with ultrapure water;
[0089] Take 8 μL, 2.5 mg/mL chitosan solution of piperazine-modified aminated graphene and add it dropwise to the electrode surface, dry it at room temperature, rinse the electrode surface with ultrapure water, and dry it; add 8 μL dropwise at a concentration of 1.0 mol/L 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide EDC and 0.4mol/L N-hydroxysuccinimide NHS solution to the electrode surface, incubate at 4°C for 40min , dry at room temperature;
[0090] Step (2), add 8 μL, 12 μg/mL primary tumor marker antibody dropwise on the surface of the working electrode modified in step (1), rinse the electrode surface with ultrapure water, and dry it in a refrigerator at 4°C;
[0091] Step (3), drop 8 μL of BSA solution with a mass fraction of 4% on the surface of the working electrode modified in step (2) to seal the non-specific active sites on the surface of the electrode, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4°C Dry;
[0092] Step (4), drop 8 μL of bovine serum albumin BSA solution with a mass fraction of 3.5% on the surface of the working electrode modified in step (3) to seal the non-specific active sites on the surface of the electrode, rinse with ultrapure water , dry in a refrigerator at 4°C; add 8 μL, 100 ng/mL of a series of tumor marker antigen Ag solutions with different concentrations dropwise, rinse the electrode surface with ultrapure water, and dry in a refrigerator at 4°C;
[0093] Step (5), on the surface of the working electrode modified in step (4), first drop-coat 8 μ L, 4 mg/mL of silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotube detection antibody Ab2 incubation solution, drop-coated on the electrode surface placed in a 4°C refrigerator to dry; then 8 μL of the secondary antibody hatching solution of SAPO-34-Pd/Co-Ab2 was added dropwise, and placed in a 4°C refrigerator to dry to prepare a molecular sieve electrode for the detection of tumor markers. Chemoimmunosensors.
[0094] Wherein: the preparation steps of the silver nanoparticle p-thiophenol functionalized sulfonated carbon nanotube detection antibody Ab2 hatch solution are as follows:
[0095] ① Preparation of sulfonated carbon nanotubes
[0096] Weigh 2.5g of multi-walled carbon nanotubes and place them in a 500mL three-neck flask, add 88mL of concentrated sulfuric acid and 45mL of concentrated nitric acid in sequence, react in an oil bath at 180°C for 70min, cool to room temperature, transfer the resulting suspension into a 1000mL beaker, add Dilute with 400mL of ultrapure water, filter with suction, wash with ultrapure water until the filtrate becomes neutral, and dry the obtained solid in a vacuum oven at 100°C for 18 hours to obtain sulfonated carbon nanotubes;
[0097] ② Preparation of p-thiophenol functionalized sulfonated carbon nanotubes
[0098] Add 16 mg of sulfonated carbon nanotubes, 80 mL of 1.5 mol/L hydrochloric acid solution to a 250 mL round bottom flask, ultrasonically disperse for 2 hours, add 2.0 g of 4,4'-diaminodiphenyl disulfide, and cool to 0°C in an ice bath , slowly add 20mL sodium nitrite aqueous solution under stirring, react for 1.5h, heat up to 35°C, continue stirring for 3.5h, filter with 0.55μm polytetrafluoroethylene membrane, wash with ultrapure water 4 times, absolute ethanol Wash 4 times, ultrasonically disperse the obtained solid into 80mL ethanol, add 80mL acetonitrile and 620mg triphenylphosphine successively, stir for 2h, filter with 0.55μm polytetrafluoroethylene membrane, wash 4 times with absolute ethanol, the obtained solid Dry in a vacuum oven at 90°C for 15 hours to obtain p-thiophenol functionalized sulfonated carbon nanotubes;
[0099] Described sodium nitrite aqueous solution is that 300mg solid sodium nitrite is dissolved in 15mL ultrapure water and makes;
[0100] ③ Preparation of silver nanoparticles solution
[0101] Add 2mL, 50mmol/L silver nitrate and 2mL, 5% sodium citrate into 50mL ultrapure water under stirring, then add 55mg sodium borohydride, and react under stirring for 20min, the solution turns brownish yellow, and continues Stir until the color of the solution no longer changes, cool to room temperature, and centrifuge at a speed of 6000rpm for 10min, the resulting supernatant is a silver nanoparticle solution;
[0102] ④ Preparation of incubation solution of antibody Ab2 detected by thiophenol-functionalized sulfonated carbon nanotubes with silver nanoparticles
[0103] Take 15 mg of p-thiophenol functionalized sulfonated carbon nanotubes and add them to 45 mL of silver nanoparticle solution, shake at 130 rpm for 15 hours, centrifuge, and dry in a vacuum oven at 90 ° C to obtain silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotubes. carbon nanotubes;
[0104] Disperse 8 mg of silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotubes into 2 mL of ultrapure water, add 200 μL, 110 μg/mL of tumor marker detection antibody Ab2 solution and 1000 μL, 100 mmol/L of pH7.4 phosphoric acid Salt buffer solution, shaking and incubating in a constant temperature shaking incubator at 4°C for 15h; centrifuging at 8000rpm at 4°C for 20min to obtain the lower layer precipitate, adding 2mL, 100mmol/L phosphate buffer solution with pH=7.4 and washing once by centrifugation to obtain The lower layer was precipitated, and finally 2 mL of 100 mmol/L phosphate buffer solution at pH = 7.4 was added to prepare a silver nanoparticle-thiophenol-functionalized sulfonated carbon nanotube detection antibody Ab2 incubation solution, which was stored at 4°C for later use.
[0105] Wherein: the preparation process of the SAPO-34-Pd/Co-Ab2 secondary antibody hatch solution comprises the following steps:
[0106] (1) Preparation of chitosan solution of piperazine-modified aminated graphene
[0107] Add 2.5mg of piperazine-modified aminated graphene to 2mL, 0.3% chitosan solution, ultrasonically disperse for 30min, and store the solution at 4°C for later use;
[0108] Described 0.3% chitosan solution is that the chitosan of 2g is dissolved in the dilute hydrochloric acid solution of 15mL, 0.03mol/L;
[0109] (2) Preparation of SAPO-34-Pd/Co-Ab2 secondary antibody incubation
[0110] 1) Preparation of aminated molecular sieves
[0111] Weigh 2.5g SAPO-34 into a three-necked flask, add 4mL of 3-aminopropyltriethoxysilane and 20mL of absolute ethanol, heat the mixture to 80°C and keep it for 3.0h, then cool to room temperature, and the mixture is heated at 9000rpm Centrifuge for 10 minutes to prepare an aminated molecular sieve NH2-SAPO-34, wash with water and centrifuge three times, and finally vacuum dry the aminated molecular sieve at 65°C;
[0112] 2) Preparation of SAPO-34-Pd/Co
[0113] Under nitrogen protection and ice bath cooling, mix 8mL, 0.3mol/L cobalt nitrate, 4.5mL, 80mmol/L sodium citrate, 50mgNH2-SAPO-34 and 35mL ultrapure water for 30min;
[0114] 8mL, 2.5mol/L sodium borohydride solution was added to the above mixed solution at a rate of 25mL/h and vigorously stirred to obtain a Co nanoparticle solution;
[0115] Add 15mL, 80mmol/L sodium tetrachloropalladate Na2PdCl4 and 20mL, 0.25mol/L NaBH4 solution to the Co nanoparticle solution at the same time at a rate of 30mL/h, the mixture is stirred for another 2h, then separated and washed, and heated at 45°C Dried under vacuum to obtain SAPO-34-Pd/Co;
[0116] 3) Preparation of SAPO-34-Pd/Co-Ab2 secondary antibody incubation solution
[0117]Disperse 4 mg of SAPO-34-Pd/Co into 1 mL of ultrapure water, add 200 μL of 140 μg/mL secondary antibody Ab2 and 1000 μL of 70 mmol/L pH7.4 phosphate buffer solution, and place in a constant temperature shaking incubator at 4 °C Shake and incubate for 12 hours, centrifuge at 4°C and 8000rpm for 25 minutes to obtain the lower precipitate; then add 2mL, 60mmol/L phosphate buffer solution with pH=7.4 and centrifuge once to obtain the lower precipitate; finally add 2mL, 60mmol/L pH = 7.4 phosphate buffer solution to prepare SAPO-34-Pd/Co-Ab2 secondary antibody incubation solution, which was stored at 4°C for future use.
[0118] The present invention also provides a molecular sieve electrochemical immunosensor for detecting tumor markers prepared according to the above preparation method, which is used for the detection of various tumor markers. The detection steps are as follows:
[0119] (1) The electrochemical workstation was used to test with a three-electrode system. The Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared immunosensor was used as the working electrode. 90 phosphate buffer solution for testing;
[0120] (2) Detect various tumor marker antigen standard solutions by time-current method, the input voltage is -0.6V, the sampling interval is 0.05s, and the running time is 500s;
[0121] (3) When the background current tends to be stable, inject 10 μL, 5 mol/L hydrogen peroxide solution into 20 mL of 60 mmol L-1, pH=7.0 phosphate buffer solution every 10 s, then record the current change and draw the working curve ;
[0122] (4) The test sample solution is replaced by the standard solution of various tumor marker antigens for detection.
Example Embodiment
[0123] Example 3:
[0124] A preparation method for detecting tumor marker-loaded molecular sieve electrochemical immunosensor, comprising the following steps:
[0125] Step (1), the glassy carbon electrode with a diameter of 4mm is polished with Al2O3 polishing powder, and cleaned with ultrapure water;
[0126] Take 8 μL, 2.0 mg/mL chitosan solution of piperazine-modified aminated graphene and add it dropwise to the electrode surface, dry it at room temperature, rinse the electrode surface with ultrapure water, and dry it; add 8 μL dropwise at a concentration of 0.9 mol/L 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide EDC and 0.35mol/L N-hydroxysuccinimide NHS solution to the electrode surface, incubate at 4°C for 40min , dry at room temperature;
[0127] Step (2), add 8 μL, 11 μg/mL primary tumor marker antibody dropwise on the surface of the working electrode modified in step (1), rinse the electrode surface with ultrapure water, and dry it in a refrigerator at 4°C;
[0128] Step (3), drop 8 μL of BSA solution with a mass fraction of 3% on the surface of the working electrode modified in step (2) to seal the non-specific active sites on the electrode surface, rinse the electrode surface with ultrapure water, and dry in a refrigerator at 4°C Dry;
[0129] Step (4), drop 8 μL of bovine serum albumin BSA solution with a mass fraction of 3.2% on the surface of the working electrode modified in step (3) to seal the non-specific active sites on the surface of the electrode, rinse with ultrapure water , dry in a refrigerator at 4°C; add 8 μL, 60 ng/mL of a series of tumor marker antigen Ag solutions with different concentrations dropwise, rinse the electrode surface with ultrapure water, and dry in a refrigerator at 4°C;
[0130] Step (5), on the surface of the working electrode modified in step (4), first drop-coat 8 μL, 3 mg/mL of silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotube detection antibody Ab2 incubation solution, drop-coated on the electrode surface placed in a 4°C refrigerator to dry; then 8 μL of the secondary antibody hatching solution of SAPO-34-Pd/Co-Ab2 was added dropwise, and placed in a 4°C refrigerator to dry to prepare a molecular sieve electrode for the detection of tumor markers. Chemoimmunosensors.
[0131] Wherein: the preparation steps of the silver nanoparticle p-thiophenol functionalized sulfonated carbon nanotube detection antibody Ab2 hatch solution are as follows:
[0132] ① Preparation of sulfonated carbon nanotubes
[0133] Weigh 2.2g of multi-walled carbon nanotubes and place them in a 500mL three-necked flask, add 88mL of concentrated sulfuric acid and 45mL of concentrated nitric acid in sequence, react in an oil bath at 180°C for 65min, cool to room temperature, transfer the resulting suspension into a 1000mL beaker, add Dilute with 400mL of ultrapure water, filter with suction, wash with ultrapure water until the filtrate becomes neutral, and dry the obtained solid in a vacuum oven at 95°C for 17 hours to obtain sulfonated carbon nanotubes;
[0134] ② Preparation of p-thiophenol functionalized sulfonated carbon nanotubes
[0135] Add 14 mg of sulfonated carbon nanotubes, 80 mL of 1.5 mol/L hydrochloric acid solution to a 250 mL round-bottom flask, ultrasonically disperse for 2 hours, add 2.0 g of 4,4'-diaminodiphenyl disulfide, and cool to 0°C in an ice bath , slowly add 20mL sodium nitrite aqueous solution under stirring, react for 1.5h, heat up to 35°C, continue stirring for 3.0h, filter with 0.55μm polytetrafluoroethylene membrane, wash with ultrapure water 4 times, absolute ethanol Wash 4 times, ultrasonically disperse the obtained solid into 80mL ethanol, add 80mL acetonitrile and 620mg triphenylphosphine successively, stir for 2h, filter with 0.55μm polytetrafluoroethylene membrane, wash 4 times with absolute ethanol, the obtained solid Dry in a vacuum oven at 90°C for 15 hours to obtain p-thiophenol functionalized sulfonated carbon nanotubes;
[0136] Described sodium nitrite aqueous solution is that 300mg solid sodium nitrite is dissolved in 15mL ultrapure water and makes;
[0137] ③ Preparation of silver nanoparticles solution
[0138] Add 2mL, 50mmol/L silver nitrate and 2mL, 5% sodium citrate into 50mL ultrapure water under stirring, then add 52mg sodium borohydride, and react under stirring for 20min, the solution turns brownish yellow, and continues Stir until the color of the solution no longer changes, cool to room temperature, and centrifuge at a speed of 6000rpm for 10min, the resulting supernatant is a silver nanoparticle solution;
[0139] ④ Preparation of incubation solution of antibody Ab2 detected by thiophenol-functionalized sulfonated carbon nanotubes with silver nanoparticles
[0140] Take 13 mg of p-thiophenol functionalized sulfonated carbon nanotubes and add them to 40 mL of silver nanoparticle solution, shake at 130 rpm for 14 hours, centrifuge, and dry in a vacuum oven at 90 ° C to obtain silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotubes. carbon nanotubes;
[0141] Disperse 6 mg of silver nanoparticles p-thiophenol functionalized sulfonated carbon nanotubes into 2 mL of ultrapure water, add 200 μL, 105 μg/mL of tumor marker detection antibody Ab2 solution and 1000 μL, 100 mmol/L of pH7.4 phosphoric acid Salt buffer solution, shaking and incubating in a constant temperature shaking incubator at 4°C for 15h; centrifuging at 8000rpm at 4°C for 20min to obtain the lower layer precipitate, adding 2mL, 100mmol/L phosphate buffer solution with pH=7.4 and washing once by centrifugation to obtain The lower layer was precipitated, and finally 2 mL of 100 mmol/L phosphate buffer solution at pH = 7.4 was added to prepare a silver nanoparticle-thiophenol-functionalized sulfonated carbon nanotube detection antibody Ab2 incubation solution, which was stored at 4°C for later use.
[0142] Wherein: the preparation process of the SAPO-34-Pd/Co-Ab2 secondary antibody hatch solution comprises the following steps:
[0143] (1) Preparation of chitosan solution of piperazine-modified aminated graphene
[0144] Add 2.0mg of piperazine-modified aminated graphene to 2mL, 0.3% chitosan solution, ultrasonically disperse for 25min, and store the solution at 4°C for later use;
[0145] Described 0.3% chitosan solution is that the chitosan of 2g is dissolved in the dilute hydrochloric acid solution of 15mL, 0.03mol/L;
[0146] (2) Preparation of SAPO-34-Pd/Co-Ab2 secondary antibody incubation
[0147] 1) Preparation of aminated molecular sieves
[0148] Weigh 1.5g SAPO-34 into a three-necked flask, add 3mL of 3-aminopropyltriethoxysilane and 20mL of absolute ethanol, heat the mixture to 80°C and keep it for 2.5h, then cool to room temperature, and the mixture is heated at 9000rpm Centrifuge for 10 min to prepare an aminated molecular sieve NH2-SAPO-34, wash with water and centrifuge three times, and finally vacuum dry the aminated molecular sieve at 60°C;
[0149] 2) Preparation of SAPO-34-Pd/Co
[0150] Under nitrogen protection and ice bath cooling, mix 6mL, 0.3mol/L cobalt nitrate, 4.5mL, 80mmol/L sodium citrate, 40mgNH2-SAPO-34 and 35mL ultrapure water for 30min;
[0151] 8mL, 2.5mol/L sodium borohydride solution was added to the above mixed solution at a rate of 25mL/h and stirred vigorously to obtain a Co nanoparticle solution;
[0152] Add 15mL, 65mmol/L sodium tetrachloropalladate Na2PdCl4 and 20mL, 0.25mol/L NaBH4 solution to the Co nanoparticle solution at a rate of 30mL/h at the same time, the mixture is stirred for another 2h, then separated and washed, and heated at 45°C Dried under vacuum to obtain SAPO-34-Pd/Co;
[0153] 3) Preparation of SAPO-34-Pd/Co-Ab2 secondary antibody incubation solution
[0154] Disperse 3 mg of SAPO-34-Pd/Co into 1 mL of ultrapure water, add 200 μL of 120 μg/mL secondary antibody Ab2 and 1000 μL of 60 mmol/L pH7.4 phosphate buffer solution, and place in a constant temperature shaking incubator at 4 °C Shake and incubate for 12 hours, centrifuge at 4°C and 8000rpm for 25 minutes to obtain the lower precipitate; then add 2mL, 60mmol/L phosphate buffer solution with pH=7.4 and centrifuge once to obtain the lower precipitate; finally add 2mL, 60mmol/L pH = 7.4 phosphate buffer solution to prepare SAPO-34-Pd/Co-Ab2 secondary antibody incubation solution, which was stored at 4°C for future use.
[0155] The present invention also provides a molecular sieve electrochemical immunosensor for detecting tumor markers prepared according to the above preparation method, which is used for the detection of various tumor markers. The detection steps are as follows:
[0156] (1) The electrochemical workstation was used to test with a three-electrode system. The Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared immunosensor was used as the working electrode. 80 phosphate buffer solution for testing;
[0157] (2) Detect various tumor marker antigen standard solutions by time-current method, the input voltage is -0.6V, the sampling interval is 0.05s, and the running time is 500s;
[0158](3) When the background current tends to be stable, inject 10 μL, 5 mol/L hydrogen peroxide solution into 20 mL of 60 mmol L-1, pH=7.0 phosphate buffer solution every 10 s, then record the current change and draw the working curve ;
[0159] (4) The test sample solution is replaced by the standard solution of various tumor marker antigens for detection.
PUM
| Property | Measurement | Unit |
| Diameter | 4.0 | mm |
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Owner:WARSAW ORTHOPEDIC INC
Degradable cigarette filters
Owner:PHILIP MORRIS USA INC
Metal microgrid transparent electrode and method for producing the same
Owner:BEIHANG UNIV