CN113295867B - Preparation method and application of electrochemical immunosensor for amplifying palladium-silver copper mesoporous nanosphere signals - Google Patents
Preparation method and application of electrochemical immunosensor for amplifying palladium-silver copper mesoporous nanosphere signals Download PDFInfo
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Abstract
The invention belongs to the technical fields of novel nano composite materials, immunoassay and biosensing, and relates to a preparation method and application of an electrochemical immunosensor for amplifying palladium-silver-copper mesoporous nanospheres signals.
Description
Technical Field
The invention belongs to the technical fields of novel nanocomposite materials, immunoassay and biosensing, and provides a construction method of an electrochemical immunosensor for amplifying palladium-silver-copper mesoporous nanosphere signals, which is applied to detection of tumor marker antigens.
Background
Cancer has become one of the health problems of great concern, carcinoembryonic antigen is a broad-spectrum tumor marker, is a specific marker for early diagnosis of cancer, has important clinical value in the aspects of differential diagnosis, disease monitoring, curative effect evaluation and the like of malignant tumors, and therefore, requires a sensitive, rapid and accurate detection method. In recent years, the electrochemical instrument is convenient to use and rapid in detection, so that the research of the electrochemical immunosensor is focused on, and the electrochemical immunosensor is used for quantitatively detecting tumor marker antigens.
The electrochemical immunosensor integrates biological sensing and electrochemical analysis, is an analysis method based on antigen and antibody specific combination, has the advantages of high sensitivity, low detection limit and low preparation cost, and has important value for the actual detection of tumor markers.
The two-dimensional leaf-shaped metal-organic framework ZIF-L is an inorganic-organic hybrid crystalline material with two-dimensional regular morphology formed by self-assembly through coordination, has the characteristics of stable framework, easy regulation of composition and the like, and has the high chemical stability and the thermal stability of the traditional zeolite. After high-temperature roasting, the porosity of the carbonized ZIF-L material can be partially reserved, the carbonized ZIF-L material has higher specific surface area and rich pore structure, and meanwhile, the defect degree and the conductivity of the material are greatly improved. The ZIF-L has high nitrogen content and strong electronegativity after carbonization, is easy to bind alloy nano particles, can efficiently transfer charges, can provide a good microenvironment for incubation of capture antibodies, and can stably bind the capture antibodies, so that the construction stability of a working electrode sensing interface of an electrochemical immunosensor is improved; the palladium-silver-copper mesoporous nanospheres have rich mesoporous intercommunication pores and excellent hydrogen peroxide catalytic capability, can effectively amplify electric response signals, improve the sensitivity of an electrochemical immunosensor, and the synthesis process adopts a green, simple and efficient preparation method.
The electrochemical immunosensor for amplifying the palladium-silver-copper mesoporous nanosphere signal, which is constructed by using a layer-by-layer self-assembly technology and taking gold nanoparticles/carbonized ZIF-L as a substrate and palladium-silver-copper mesoporous nanospheres as detection antibody markers, has the advantages of wide detection range, low detection limit, simple operation, high detection speed and the like, has good reproducibility, stability and selectivity, and has important application value for early differential diagnosis of cancers.
Disclosure of Invention
The invention provides a construction method and application of an electrochemical immunosensor for amplifying palladium-silver-copper mesoporous nanosphere signals, and realizes sensitive detection of tumor marker antigens.
The invention aims to provide a construction method of an electrochemical immunosensor for amplifying palladium-silver copper mesoporous nanosphere signals.
The second purpose of the invention is to use the prepared electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification for detecting tumor marker antigens.
The technical scheme of the invention comprises the following steps:
1. the construction method of the electrochemical immunosensor for amplifying the palladium-silver copper mesoporous nanosphere signal comprises the following steps:
(1) Al for glassy carbon electrode with diameter of 3.0-5.0 mm 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Dripping gold nano particles/carbonized ZIF-L dispersion of 6.0 mu L and 1.0-2.0 mg/mL on the surface of the electrode, airing at room temperature, flushing the surface of the electrode with ultrapure water, and airing;
(3) Tumor marker capture antibody Ab with 6.0 [ mu ] L and 5.0-15.0 [ mu ] g/mL 1 Dripping the mixture on the surface of an electrode, flushing the mixture by ultrapure water, and airing the mixture in a refrigerator at a temperature of 4 ℃;
(4) Continuously dripping 4 mu L of 0.8-1.2 mg/mL of bovine serum albumin BSA solution on the surface of the electrode, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at a temperature of 4 ℃;
(5) Continuously dripping a series of tumor marker antigen solutions with different concentrations of 6.0 mu L, 10.0 fg/mL-100.0 ng/mL, flushing the electrode surface with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) Continuously dropwise adding 6.0 mu L and 1.0-3.0 mg/mL palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 And (3) flushing the surface of the electrode by using the dispersion liquid and ultrapure water, and airing in a refrigerator at a temperature of 4 ℃ to prepare the electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification.
2. The method for preparing the electrochemical immunosensor for amplifying palladium-silver-copper mesoporous nanosphere signals as claimed in claim 1, wherein the preparation of the gold nanoparticle/carbonized ZIF-L dispersion liquid comprises the following steps:
(1) Preparation of gold nanoparticle Dispersion
Adding 0.1-0.3 mL and 1.0wt% chloroauric acid tetrahydrate into a round bottom bottle of 50.0mL, adding 19.7-19.9 mL ultrapure water for dilution, and heating to micro-boiling under magnetic stirring; adding 0.2-0.4 mL and 10.0mg/mL sodium citrate into the solution, stirring and refluxing for 15min, and cooling to obtain gold nanoparticle dispersion;
wherein 10.0mg/mL sodium citrate is prepared by the following steps: weighing 10.0mg sodium citrate, adding into 1.0mL ultrapure water, and preparing 10.0mg/mL sodium citrate solution for later use;
(2) Preparation of carbonized ZIF-L
Weighing 200.0-400.0 mg zinc nitrate hexahydrate, adding into a small beaker, adding 20.0mL ultrapure water, stirring until the solution A is completely dissolved, adding 20.0mL ultrapure water and 600.0-700.0 mg methylimidazole into the other beaker, fully dissolving, adding into the solution A, magnetically stirring 4h, centrifugally washing the ultrapure water after the reaction is complete, vacuum freeze-drying the powder for 12h at-60 ℃ to obtain white powder, placing the white powder into a quartz boat, placing the quartz boat into a tube furnace, introducing nitrogen for 30min, heating to 900 ℃ at 5 ℃/min, keeping the temperature constant at 3h, and cooling to room temperature to obtain carbonized ZIF-L;
(3) Preparation of gold nanoparticle/carbonized ZIF-L dispersion
Weighing 1.0-3.0 mg, adding the prepared carbonized ZIF-L into 1.0mL ultrapure water, performing ultrasonic dispersion, dripping 1.0-3.0 mL gold nanoparticle dispersion into the dispersion medium, oscillating 12h, performing centrifugal washing on the ultrapure water for three times to obtain a lower-layer precipitate, adding 1.0mL ultrapure water, and uniformly dispersing to obtain gold nanoparticle/carbonized ZIF-L dispersion.
3. The method for preparing the electrochemical immunosensor for amplifying palladium-silver-copper mesoporous nanosphere signals as in claim 1, wherein the palladium-silver-copper mesoporous nanosphere/detection antibody Ab 2 The preparation of the dispersion liquid comprises the following steps:
(1) Preparation of palladium-silver-copper mesoporous nanospheres
Weighing 3.0-9.0 mg dioctadecyl dimethyl ammonium chloride, dissolving in an ethanol/water volume ratio of 2:8 cosolvent of 10.0mL, then adding 0.1-0.3 mL and 100.0mmol/L sodium hydroxide solution to adjust pH, then adding 0.48-mL and 10.0mmol/L sodium tetrachloropalladate solution into the solution, stirring for 10min, sequentially adding 0.24-mL, 10.0mmol/L silver nitrate solution and 0.08-mL and 10.0mmol/L copper nitrate solution, and keeping standing for 30min; adding 1.0mL and newly prepared 0.3mol/L ascorbic acid solution into the solution rapidly under stirring, reacting for 30min, and sequentially washing with acetic acid, ethanol and ultrapure water respectively to obtain palladium-silver-copper mesoporous nanospheres;
the preparation method of the used 0.3mol/L ascorbic acid solution comprises the following steps: weighing 52.8. 52.8mg ascorbic acid and dissolving in 1.0mL ultrapure water;
(2) Palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 Preparation of the Dispersion
2.0-6.0 mg of palladium-silver-copper mesoporous nanospheres are taken, and 1.0-2.0 mL and 20.0-30.0 mug/mL of tumor targets are addedAntibody Ab for detecting marker 2 Placing the dispersion liquid in a constant temperature oscillation incubator at 4 ℃ for oscillation incubation for 6.0-8.0 h, centrifuging for 5.0-10.0 min at 3000 rotating speed to obtain lower layer precipitate, adding phosphate buffer solution with pH of 1.0mL and 7.38 for centrifugal washing for three times, taking out the lower layer precipitate, adding phosphate buffer solution with pH of 1.0mL and 7.38, and dispersing uniformly to obtain palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 And (3) a dispersion.
4. The electrochemical immunosensor for amplifying palladium-silver-copper mesoporous nanosphere signals, prepared by the preparation method of claim 1, is used for detecting tumor markers, and comprises the following detection steps:
(1) Using an electrochemical workstation, using a saturated calomel electrode as a reference electrode, using a platinum wire electrode as a counter electrode, and using the prepared sensor as a working electrode to form a three-electrode system, and testing in a phosphate buffer solution with the pH value of 10.0mL and 7.38;
(2) The initial potential was-0.4. 0.4V with a sampling interval of 0.1s and the run time was 400.0s by time-current method;
(3) After the background current stabilized, 10.0. Mu.L, 5.0mol/L H was injected into 10.0mL, pH=7.38 phosphate buffer solution 2 O 2 Recording the change of the current after the current is stabilized again;
(4) Measuring tumor marker antigens with different concentrations, and recording current value changes corresponding to the tumor marker antigens with different concentrations;
(5) And obtaining the concentration of the tumor marker antigen in the sample to be detected by using a working curve method.
Advantageous results of the invention
(1) The electrochemical immunosensor for amplifying the palladium-silver-copper mesoporous nanosphere signal has the advantages of low detection limit, wide linear range, high sensitivity, simplicity in operation, low cost and the like, and has a good application prospect in quantitative detection of tumor markers with high sensitivity and high selectivity.
(2) According to the invention, the gold nano particles/carbonized ZIF-L are used as a base material, the porosity of the carbonized ZIF-L material can be partially reserved, the material has a higher specific surface area and a rich pore structure, meanwhile, the defect degree and conductivity of the material are greatly improved, after the gold nano particles are loaded, the loading quantity of detection antibodies can be improved, and meanwhile, the stability of the adhesion of the detection antibodies can be greatly improved, so that the sensitivity of an electrochemical immunosensor is improved.
(3) According to the invention, the palladium-silver-copper mesoporous nanospheres are used as detection antibody markers for constructing the immunosensor, and the palladium-silver-copper mesoporous nanospheres have catalytic performance due to rich mesoporous pores and excellent specific surface area, and the three metals have excellent catalytic performance, so that signals can be effectively amplified.
(4) The electrochemical immunosensor for amplifying the palladium-silver copper mesoporous nanosphere signals detects different tumor marker antigens, wherein the linear detection range of CEA is 10.0 fg/mL-100.0 ng/mL, and the detection limit is 3.4 fg/mL; the linear detection range of CA125 is 10.0 fg/mL-100.0 ng/mL, and the detection limit is 4.2 fg/mL; the linear detection range of SCCA is 10.0 fg/mL-100.0 ng/mL, and the detection limit is 5.8 fg/mL; the electrochemical immunosensor with palladium-silver copper mesoporous nanosphere signal amplification can achieve the purpose of accurately and quantitatively detecting tumor marker antigens.
Detailed Description
The invention will now be further illustrated by means of specific embodiments, without being limited thereto.
Embodiment 1 a method for preparing an electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification comprises the following steps:
(1) Al for glassy carbon electrode with diameter of 3.0. 3.0 mm 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Dripping gold nano particles/carbonized ZIF-L dispersion of 6.0 mu L and 1.0 mg/mL on the surface of the electrode, airing at room temperature, flushing the surface of the electrode with ultrapure water, and airing;
(3) Tumor marker capture antibody Ab with 6.0 mu L and 5.0 mu g/mL 1 Dripping the mixture on the surface of an electrode, flushing the mixture by ultrapure water, and airing the mixture in a refrigerator at a temperature of 4 ℃;
(4) Continuously dripping 4 mu L of 0.8 mg/mL of bovine serum albumin BSA solution on the surface of the electrode, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at a temperature of 4 ℃;
(5) Continuously dripping a series of tumor marker antigen solutions with different concentrations of 6.0 mu L, 10.0 fg/mL-100.0 ng/mL, flushing the electrode surface with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) Continuously dripping 6.0 mu L and 1.0 mg/mL palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 And (3) flushing the surface of the electrode by using the dispersion liquid and ultrapure water, and airing in a refrigerator at a temperature of 4 ℃ to prepare the electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification.
Embodiment 2 a method for preparing an electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification comprises the following steps:
(1) Al for glassy carbon electrode with diameter of 4.0. 4.0 mm 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Dripping gold nano particles/carbonized ZIF-L dispersion of 6.0 mu L and 1.5 mg/mL on the surface of the electrode, airing at room temperature, flushing the surface of the electrode with ultrapure water, and airing;
(3) Tumor marker capture antibody Ab with 6.0 mu L and 10.0 mu g/mL 1 Dripping the mixture on the surface of an electrode, flushing the mixture by ultrapure water, and airing the mixture in a refrigerator at a temperature of 4 ℃;
(4) Continuously dripping 4 mu L of 1.0 mg/mL of bovine serum albumin BSA solution on the surface of the electrode, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at a temperature of 4 ℃;
(5) Continuously dripping a series of tumor marker antigen solutions with different concentrations of 6.0 mu L, 10.0 fg/mL-100.0 ng/mL, flushing the electrode surface with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) Continuously dripping 6.0 mu L and 2.0mg/mL palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 And (3) flushing the surface of the electrode by using the dispersion liquid and ultrapure water, and airing in a refrigerator at a temperature of 4 ℃ to prepare the electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification.
Embodiment 3 a method for preparing an electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification comprises the following steps:
(1) Al for glassy carbon electrode with diameter of 5.0. 5.0mm 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Dripping gold nano particles/carbonized ZIF-L dispersion of 6.0 mu L and 2.0mg/mL on the surface of the electrode, airing at room temperature, flushing the surface of the electrode with ultrapure water, and airing;
(3) Tumor marker capture antibody Ab with 6.0 mu L and 15.0 mu g/mL 1 Dripping the mixture on the surface of an electrode, flushing the mixture by ultrapure water, and airing the mixture in a refrigerator at a temperature of 4 ℃;
(4) Continuously dripping 4 mu L of 1.2mg/mL of bovine serum albumin BSA solution on the surface of the electrode, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at a temperature of 4 ℃;
(5) Continuously dripping a series of tumor marker antigen solutions with different concentrations of 6.0 mu L, 10.0 fg/mL-100.0 ng/mL, flushing the electrode surface with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) Continuously dripping 6.0 mu L and 3.0mg/mL palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 And (3) flushing the surface of the electrode by using the dispersion liquid and ultrapure water, and airing in a refrigerator at a temperature of 4 ℃ to prepare the electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification.
The preparation of the gold nanoparticle/carbonized ZIF-L dispersion described in example 4 includes the following steps:
(1) Preparation of gold nanoparticle Dispersion
Adding 0.1. 0.1 mL and 1.0wt% chloroauric acid tetrahydrate into a round bottom bottle of 50.0mL, adding 19.9mL ultrapure water for dilution, and heating to micro-boiling under magnetic stirring; adding 0.2 mL and 10.0mg/mL sodium citrate into the solution, stirring and refluxing for 15min, and cooling to obtain gold nanoparticle dispersion;
wherein 10.0mg/mL sodium citrate is prepared by the following steps: weighing 10.0mg sodium citrate, adding into 1.0mL ultrapure water, and preparing 10.0mg/mL sodium citrate solution for later use;
(2) Preparation of carbonized ZIF-L
Weighing 200.0 mg zinc nitrate hexahydrate, adding 20.0mL ultrapure water into a small beaker, stirring until the zinc nitrate hexahydrate is completely dissolved to obtain solution A, adding 20.0mL ultrapure water and 600.0 mg methylimidazole into the other beaker, fully dissolving, adding the solution A, magnetically stirring 4h, centrifugally washing the solution with the ultrapure water for three times after the reaction is complete, vacuum freeze-drying the solution at-60 ℃ for 12h to obtain white powder, placing the white powder into a quartz boat, placing the quartz boat into a tube furnace, introducing nitrogen for 30min, heating the solution to 900 ℃ at 5 ℃/min, keeping the temperature constant for 3h, and cooling the solution to room temperature to obtain carbonized ZIF-L;
(3) Preparation of gold nanoparticle/carbonized ZIF-L dispersion
Weighing 1.0 mg, adding the prepared carbonized ZIF-L into 1.0mL ultrapure water, performing ultrasonic dispersion, dripping 1.0mL gold nanoparticle dispersion into the dispersion medium, oscillating 12h, performing centrifugal washing on the ultrapure water for three times to obtain a lower-layer precipitate, adding 1.0mL ultrapure water, and uniformly dispersing to obtain gold nanoparticle/carbonized ZIF-L dispersion.
The preparation of the gold nanoparticle/carbonized ZIF-L dispersion of example 5 includes the following steps:
(1) Preparation of gold nanoparticle Dispersion
Adding 0.2. 0.2 mL and 1.0. 1.0wt% chloroauric acid tetrahydrate into a round bottom bottle of 50.0. 50.0mL, adding 19.8. 19.8 mL ultrapure water for dilution, and heating to micro-boiling under magnetic stirring; adding 0.3mL and 10.0mg/mL sodium citrate into the solution, stirring and refluxing for 15min, and cooling to obtain gold nanoparticle dispersion;
wherein 10.0mg/mL sodium citrate is prepared by the following steps: weighing 10.0mg sodium citrate, adding into 1.0mL ultrapure water, and preparing 10.0mg/mL sodium citrate solution for later use;
(2) Preparation of carbonized ZIF-L
Weighing 300.0 mg zinc nitrate hexahydrate, adding 20.0mL ultrapure water into a small beaker, stirring until the zinc nitrate hexahydrate is completely dissolved to obtain solution A, adding 20.0mL ultrapure water and 656.0 mg methylimidazole into another beaker, fully dissolving, adding the solution A into the beaker, magnetically stirring 4h, centrifugally washing the solution after the reaction is completed, and then performing vacuum freeze-drying at-60 ℃ for 12h to obtain white powder, placing the white powder into a quartz boat, placing the quartz boat into a tube furnace, introducing nitrogen for 30min, heating to 900 ℃ at 5 ℃/min, keeping the temperature constant at 3h, and cooling to room temperature to obtain carbonized ZIF-L;
(3) Preparation of gold nanoparticle/carbonized ZIF-L dispersion
Weighing 2.0mg of the prepared carbonized ZIF-L, adding into 1.0mL of ultrapure water, performing ultrasonic dispersion, dripping 2.0mL of gold nanoparticle dispersion into the dispersion medium, oscillating 12h, performing centrifugal washing on the ultrapure water for three times to obtain a lower-layer precipitate, adding 1.0mL of ultrapure water, and uniformly dispersing to obtain gold nanoparticle/carbonized ZIF-L dispersion liquid.
The preparation of the gold nanoparticle/carbonized ZIF-L dispersion of example 6 includes the following steps:
(1) Preparation of gold nanoparticle Dispersion
Adding 0.3. 0.3mL and 1.0. 1.0wt% chloroauric acid tetrahydrate into a round bottom bottle of 50.0. 50.0mL, adding 19.7. 19.7 mL ultrapure water for dilution, and heating to micro-boiling under magnetic stirring; adding 0.4mL and 10.0mg/mL sodium citrate into the solution, stirring and refluxing for 15min, and cooling to obtain gold nanoparticle dispersion;
wherein 10.0mg/mL sodium citrate is prepared by the following steps: weighing 10.0mg sodium citrate, adding into 1.0mL ultrapure water, and preparing 10.0mg/mL sodium citrate solution for later use;
(2) Preparation of carbonized ZIF-L
Weighing 400.0mg zinc nitrate hexahydrate, adding 20.0mL ultrapure water into a small beaker, stirring until the zinc nitrate hexahydrate is completely dissolved to obtain solution A, adding 20.0mL ultrapure water and 700.0mg methylimidazole into another beaker, fully dissolving, adding the solution A, magnetically stirring 4h, centrifugally washing the solution with the ultrapure water for three times after the reaction is complete, vacuum freeze-drying the solution at-60 ℃ for 12h to obtain white powder, placing the white powder into a quartz boat, placing the quartz boat into a tube furnace, introducing nitrogen for 30min, heating the quartz boat to 900 ℃ at 5 ℃/min, keeping the temperature constant for 3h, and cooling the quartz boat to room temperature to obtain carbonized ZIF-L;
(3) Preparation of gold nanoparticle/carbonized ZIF-L dispersion
Weighing 3.0mg, adding the prepared carbonized ZIF-L into 1.0mL ultrapure water, performing ultrasonic dispersion, dripping 3.0mL gold nanoparticle dispersion into the dispersion medium, oscillating 12h, performing centrifugal washing on the ultrapure water for three times to obtain a lower-layer precipitate, adding 1.0mL ultrapure water, and uniformly dispersing to obtain gold nanoparticle/carbonized ZIF-L dispersion.
Example 7 Palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 The preparation of the dispersion liquid comprises the following steps:
(1) Preparation of palladium-silver-copper mesoporous nanospheres
Weighing 3.0. 3.0mg dioctadecyl dimethyl ammonium chloride, dissolving in an ethanol/water volume ratio of 10.0mL is 2:8 cosolvent, then adding 0.1 mL and 100.0mmol/L sodium hydroxide solution to adjust the pH to 7.4, then adding 0.48mL and 10.0mmol/L sodium tetrachloropalladate solution into the solution, stirring for 10min, sequentially adding 0.24mL, 10.0mmol/L silver nitrate solution and 0.08mL and 10.0mmol/L copper nitrate solution, and keeping standing for 30min; adding 1.0mL and newly prepared 0.3mol/L ascorbic acid solution into the solution rapidly under stirring, reacting for 30min, and sequentially washing with acetic acid, ethanol and ultrapure water respectively to obtain palladium-silver-copper mesoporous nanospheres;
the preparation method of the used 0.3mol/L ascorbic acid solution comprises the following steps: weighing 52.8. 52.8mg ascorbic acid and dissolving in 1.0mL ultrapure water;
(2) Palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 Preparation of the Dispersion
2.0mg palladium-silver-copper mesoporous nanospheres are taken, and 1.0mL and 20.0 mug/mL tumor marker detection antibody Ab is added 2 Placing the dispersion liquid in a constant temperature oscillation incubator at 4 ℃ for oscillation incubation for 6.0 h, centrifuging for 5.0 min at 3000 rotating speed to obtain lower layer precipitate, adding phosphate buffer solution with pH of 1.0mL and 7.38, centrifuging and washing for three times, taking out the lower layer precipitate, adding phosphate buffer solution with pH of 1.0mL and 7.38, and dispersing uniformly to obtain palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 And (3) a dispersion.
Example 8 Palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 The preparation of the dispersion liquid comprises the following steps:
(1) Preparation of palladium-silver-copper mesoporous nanospheres
Weighing 6.0. 6.0mg dioctadecyl dimethyl ammonium chloride, dissolving in a cosolvent with an ethanol/water volume ratio of 2:8 of 10.0mL, adding a sodium hydroxide solution with a concentration of 0.2: 0.2 mL and a concentration of 100.0mmol/L to adjust the pH to 7.9, adding a sodium tetrachloropalladate solution with a concentration of 0.48: 0.48mL and a concentration of 10.0mmol/L to the solution, stirring for 10min, sequentially adding a silver nitrate solution with a concentration of 0.24: 0.24mL and a concentration of 10.0mmol/L and a copper nitrate solution with a concentration of 0.08: 0.08mL and a concentration of 10.0mmol/L, and keeping standing for 30min; adding 1.0mL and newly prepared 0.3mol/L ascorbic acid solution into the solution rapidly under stirring, reacting for 30min, and sequentially washing with acetic acid, ethanol and ultrapure water respectively to obtain palladium-silver-copper mesoporous nanospheres;
the preparation method of the used 0.3mol/L ascorbic acid solution comprises the following steps: weighing 52.8. 52.8mg ascorbic acid and dissolving in 1.0mL ultrapure water;
(2) Palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 Preparation of the Dispersion
Taking 4.0 mg palladium-silver-copper mesoporous nanospheres, adding 1.5 mL and 25.0 mug/mL tumor marker detection antibody Ab 2 Placing the dispersion liquid in a constant temperature oscillation incubator at 4 ℃ for oscillation incubation for 7.0 h, centrifuging at 3000 rotating speed for 8.0 min to obtain lower layer precipitate, adding 1.0mL and pH=7.38 phosphate buffer solution, centrifuging and washing for three times, taking out the lower layer precipitate, adding 1.0mL and pH=7.38 phosphate buffer solution, and dispersing uniformly to obtain palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 And (3) a dispersion.
Example 9 Palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 The preparation of the dispersion liquid comprises the following steps:
(1) Preparation of palladium-silver-copper mesoporous nanospheres
9.0mg dioctadecyl dimethyl ammonium chloride is weighed and dissolved in a cosolvent with the ethanol/water volume ratio of 2:8 of 10.0mL, then 0.3mL and 100.0mmol/L sodium hydroxide solution is added to adjust the pH value to 8.2, 0.48mL and 10.0mmol/L sodium tetrachloropalladate solution is added to the solution, after stirring for 10min, 0.24mL and 10.0mmol/L silver nitrate solution and 0.08mL and 10.0mmol/L copper nitrate solution are sequentially added, and the mixture is kept stand for 30min; adding 1.0mL and newly prepared 0.3mol/L ascorbic acid solution into the solution rapidly under stirring, reacting for 30min, and sequentially washing with acetic acid, ethanol and ultrapure water respectively to obtain palladium-silver-copper mesoporous nanospheres;
the preparation method of the used 0.3mol/L ascorbic acid solution comprises the following steps: weighing 52.8. 52.8mg ascorbic acid and dissolving in 1.0mL ultrapure water;
(2) Palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 Preparation of the Dispersion
Taking 6.0mg palladium-silver-copper mesoporous nanospheres, adding 2.0mL and 30.0 mug/mL tumor marker detection antibody Ab 2 Placing the dispersion liquid in a constant temperature oscillation incubator at 4 ℃ for oscillation incubation for 8.0h, centrifuging for 10.0min at 3000 rotating speed to obtain lower layer precipitate, adding 1.0mL and pH=7.38 phosphate buffer solution, centrifuging and washing for three times, taking out the lower layer precipitate, adding 1.0mL and pH=7.38 phosphate buffer solution, and dispersing uniformly to obtain palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 And (3) a dispersion.
Example 10 detection procedure for tumour marker CEA is as follows:
(1) Using an electrochemical workstation, using a saturated calomel electrode as a reference electrode, using a platinum wire electrode as a counter electrode, and using the prepared sensor as a working electrode to form a three-electrode system, and testing in a phosphate buffer solution with the pH value of 10.0mL and 7.38;
(2) The initial potential was-0.4. 0.4V with a sampling interval of 0.1s and the run time was 400.0s by time-current method;
(3) After the background current stabilized, 10.0. Mu.L, 5.0mol/L H was injected into 10.0mL, pH=7.38 phosphate buffer solution 2 O 2 Recording the change of the current after the current is stabilized again;
(4) Measuring tumor marker antigens with different concentrations, and recording current value changes corresponding to the tumor marker antigens with different concentrations;
(5) By using a working curve method, the linear detection range of the immunosensor for carcinoembryonic antigen is 10.0 fg/mL-100.0 ng/mL, and the detection limit is 3.4 fg/mL.
Example 11 detection of tumor marker CA125
CA125 in the sample was detected as in example 10, with a linear range of 10. 10 fg/mL-100 ng/mL and a detection limit of 4.2 fg/mL.
Example 12 detection of tumor marker SCCA
SCCA in a sample was tested according to the method of example 10, with a linear range of 10. 10 fg/mL-100 ng/mL and a limit of 5.8 fg/mL.
Claims (4)
1. The preparation method of the electrochemical immunosensor for amplifying the palladium-silver copper mesoporous nanosphere signal is characterized by comprising the following steps of:
(1) Al for glassy carbon electrode with diameter of 3.0-5.0 mm 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Dripping 6.0 mu L of gold nano particles/carbonized ZIF-L dispersion with the concentration of 1.0-2.0 mg/mL on the surface of an electrode, airing at room temperature, flushing the surface of the electrode with ultrapure water, and airing;
(3) Tumor marker capture antibody Ab with concentration of 6.0 mu L and 5.0-15.0 mu g/mL 1 Dripping the solution onto the surface of the electrode, flushing with ultrapure water, and airing in a refrigerator at 4 ℃;
(4) Continuously dripping 4 mu L of 0.8-1.2 mg/mL bovine serum albumin BSA solution on the surface of the electrode, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(5) Continuously dripping a series of tumor marker antigen solutions with different concentrations of 6.0 mu L and 10.0 fg/mL-100.0 ng/mL, flushing the electrode surface with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) Continuously dripping 6.0 mu L and 1.0-3.0 mg/mL palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 Washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to prepare the electrochemical immunosensor with palladium-silver-copper mesoporous nanosphere signal amplification;
the preparation of the gold nanoparticle/carbonized ZIF-L dispersion liquid comprises the following steps:
(1) Preparation of gold nanoparticle Dispersion
Adding 0.1-0.3 mL of 1.0wt% chloroauric acid tetrahydrate into a 50.0mL round bottom bottle, adding 19.7-19.9 mL of ultrapure water for dilution, and heating to micro boiling under magnetic stirring; adding 0.2-0.4 mL and 10.0mg/mL sodium citrate into the solution, stirring and refluxing for 15min, and cooling to obtain gold nanoparticle dispersion liquid;
wherein 10.0mg/mL sodium citrate is formulated as follows: weighing 10.0mg of sodium citrate, and adding the sodium citrate into 1.0mL of ultrapure water to prepare 10.0mg/mL of sodium citrate solution for later use;
(2) Preparation of carbonized ZIF-L
Weighing 200.0-400.0 mg of zinc nitrate hexahydrate, adding 20.0mL of ultrapure water into a small beaker, stirring until the zinc nitrate hexahydrate is completely dissolved to obtain solution A, adding 20.0mL of ultrapure water and 600.0-700.0 mg of methylimidazole into the other beaker, fully dissolving, adding the solution A into the solution A, magnetically stirring the solution A for 4 hours, centrifugally washing the solution after the reaction is completed, then performing vacuum freeze-drying at-60 ℃ for 12 hours to obtain white powder, placing the white powder into a quartz boat, placing the quartz boat into a tube furnace, introducing nitrogen for 30 minutes, heating the quartz boat to 900 ℃ at 5 ℃/min, keeping the temperature constant for 3 hours, and cooling the quartz boat to room temperature to obtain carbonized ZIF-L;
(3) Preparation of gold nanoparticle/carbonized ZIF-L dispersion
Weighing 1.0-3.0 mg of the prepared carbonized ZIF-L, adding into 1.0mL of ultrapure water, performing ultrasonic dispersion, dripping 1.0-3.0 mL of gold nanoparticle dispersion into the dispersion medium, oscillating for 12h, centrifugally washing with ultrapure water for three times to obtain a lower-layer precipitate, adding 1.0mL of ultrapure water, and uniformly dispersing to obtain gold nanoparticle/carbonized ZIF-L dispersion;
palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 The preparation of the dispersion liquid comprises the following steps:
(1) Preparation of palladium-silver-copper mesoporous nanospheres
Weighing 3.0-9.0 mg of dioctadecyl dimethyl ammonium chloride, dissolving in 10.0mL of ethanol with a water volume ratio of 2:8 cosolvent, adding 0.1-0.3 mL of 100.0mmol/L sodium hydroxide solution to adjust pH, adding 0.48mL of 10.0mmol/L sodium tetrachloropalladate solution into the solution, stirring for 10min, sequentially adding 0.24mL of 10.0mmol/L silver nitrate solution and 0.08mL of 10.0mmol/L copper nitrate solution, and keeping standing for 30min; adding 1.0mL of newly prepared 0.3mol/L ascorbic acid solution into the solution rapidly under stirring, reacting for 30min, and sequentially washing with acetic acid, ethanol and ultrapure water respectively to obtain palladium-silver-copper mesoporous nanospheres;
the preparation method of the used 0.3mol/L ascorbic acid solution comprises the following steps: 52.8mg of ascorbic acid was weighed and dissolved in 1.0mL of ultrapure water;
(2) Palladium-silver-copper mesoporous nanospheres/detection antibodies Ab 2 Preparation of the Dispersion
2.0-6.0 mg of palladium-silver-copper mesoporous nanospheres are taken, 1.0-2.0 mL and 20.0-30.0 mug/mL of tumor marker detection antibody Ab are added 2 After the dispersion liquid is placed in a constant temperature oscillation incubator at 4 ℃ for oscillation incubation for 6.0-8.0 h, centrifuging for 5.0-10.0 min at 3000 rotating speed to obtain lower layer sediment, adding 1.0mL of phosphate buffer solution with pH=7.38, centrifuging and washing for three times, taking the lower layer sediment, adding 1.0mL of phosphate buffer solution with pH=7.38, and uniformly dispersing to obtain palladium-silver-copper mesoporous nanospheres/detection antibody Ab 2 And (3) a dispersion.
2. The method for preparing the electrochemical immunosensor for amplifying palladium-silver-copper mesoporous nanosphere signals according to claim 1, wherein the tumor marker is selected from one of the following: CEA, CA125, SCCA.
3. The electrochemical immunosensor for amplifying palladium-silver-copper mesoporous nanosphere signals, prepared by the preparation method of claim 1, is used for detecting tumor markers, and comprises the following detection steps:
(1) Using an electrochemical workstation, using a saturated calomel electrode as a reference electrode, using a platinum wire electrode as a counter electrode, and using the prepared sensor as a working electrode to form a three-electrode system, and testing in 10.0mL of phosphate buffer solution with pH=7.38;
(2) Using a time-current method, wherein the initial potential is-0.4V, the sampling interval is 0.1s, and the running time is 400.0s;
(3) After the background current stabilized, 10.0. Mu.L, 5.0mol/L H was injected into 10.0mL of phosphate buffer solution at pH=7.38 2 O 2 Recording the change of the current after the current is stabilized again;
(4) Measuring tumor marker antigens with different concentrations, and recording current value changes corresponding to the tumor marker antigens with different concentrations;
(5) And obtaining the concentration of the tumor marker antigen in the sample to be detected by using a working curve method.
4. An electrochemical immunosensor for amplifying a palladium-silver-copper mesoporous nanosphere signal prepared by the preparation method as claimed in claim 3, for detecting a tumor marker, wherein the tumor marker is selected from one of the following: CEA, CA125, SCCA.
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| CN108786880A (en) * | 2018-05-28 | 2018-11-13 | 安徽工大化工科技有限公司 | A kind of method of PdAgCu/N-C catalysts 1,3-butadiene selective hydrogenation |
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| CN108918853A (en) * | 2018-04-08 | 2018-11-30 | 山东理工大学 | A kind of Pd@Ag@CeO2The preparation method and application of the immunosensor of label |
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