CN104931698B - The preparation method of a kind of stomach cancer marker gold nanoclusters Electrochemiluminescsensor sensor based on NP-NiGdAu and application - Google Patents

Abstract

<b />The invention relates to a preparation method and application of an NP-NiGdAu-based gastric cancer marker gold nanocluster electrochemiluminescence sensor, which belongs to the field of electrochemiluminescence sensors. Using gold nanoclusters as the electrochemiluminescent signal source, the nanoporous material NP-NiGdAu has excellent biocompatibility and large specific surface area to increase the immobilization capacity of antibodies. Graphene oxide was used to immobilize gold nanoclusters AuNCsBSA as a secondary antibody label. The detection of gastric cancer markers is realized according to the difference in the strength of the electrochemiluminescent signal of different concentrations of the analyte.

Description

Preparation method and application of a NP-NiGdAu-based gastric cancer marker gold nanocluster electrochemiluminescence sensor

technical field

The invention relates to a preparation method and application of an NP-NiGdAu-based gastric cancer marker gold nanocluster electrochemiluminescence sensor. It specifically relates to a kind of AuNCsBSA as a luminescent material, which uses the excellent biocompatibility and large specific surface area of nanoporous material NP-NiGdAu as a base material to increase the immobilization capacity of antibodies. Using GO/AuNCsBSA as a secondary antibody marker belongs to the technical field of electrochemiluminescence detection.

Background technique

During the occurrence and development of gastric cancer, gastric cancer cells usually produce substances such as enzymes, isozymes, hormones, and immunoglobulins. These substances related to gastric cancer are called gastric cancer markers. These markers not only exist in the cells or on the cell surface, but can also be secreted into the blood or body cavity. Therefore, gastric cancer markers can be detected in body fluids or mucosal tissues, and can be used for early warning or auxiliary diagnosis of gastric cancer, analysis of disease course, and guidance of treatment. , Monitoring recurrence or metastasis, judging prognosis, etc.

At present, the analytical methods for detecting gastric cancer markers mainly include radioimmunoassay, enzyme-linked immunoassay and kit method, but the reagents used have short validity periods, radioactive contamination, long detection cycle, low sensitivity, and cumbersome steps. In order to overcome the shortcomings of the above traditional analysis methods, the present invention designs an electrochemiluminescence immunoassay method with strong specificity, high sensitivity, no radioactive pollution, and quick and easy operation. Prior art literature: CN104391117A.

Contents of the invention

The purpose of the present invention is to solve the problems existing in the existing detection methods of gastric cancer markers, to provide a simple, fast and reliable preparation method and application of electrochemiluminescent sensors based on NP-NiGdAu and GO/AuNCsBSA, and to realize detection of gastric cancer markers. Rapid, sensitive, specific and efficient detection.

Technical scheme of the present invention is as follows:

1. A method for preparing a gastric cancer marker gold nanocluster electrochemiluminescent sensor based on NP-NiGdAu, comprising the following steps:

(1) Polish the glassy carbon electrode with a diameter of 4mm with 1.0μm, 0.3μm, and 0.05μm alumina polishing powder in sequence, and rinse it with ultrapure water;

(2) Drop-coat 6 μL, 0.5-2 mgmL ^-1 capture antibody substrate material NP-NiGdAu/Ab ₁ solution on the surface of the electrode, and store at 4°C until dry;

(3) Add 4 μL bovine serum albumin solution with a mass fraction of 1-3% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS solution with pH 6.4-8.4, and dry at 4°C;

(4) Add 6 μL of different concentrations of the antigen to be tested, incubate at 4°C for 30 minutes, rinse the surface of the electrode with PBS solution with pH 6.4~8.4, and dry at 4°C;

(5) Drop-coat 6 μL of the secondary antibody marker GO/AuNCsBSA/Ab ₂ solution, rinse the electrode surface with PBS solution of pH 6.4-8.4, and dry at 4°C.

2. Preparation of capture antibody substrate material NP-NiGdAu/Ab ₁ solution

(1) Preparation of NP-NiGdAu

Put the NiGdAl alloy into an excess of 1.5-2.5mol/L sodium hydroxide solution, chemically corrode it at room temperature for 32-74 hours, centrifugally wash it to neutrality, and dry it in a vacuum oven for 12-48 hours to obtain nanoporous NiGd ; Boil 80-120mL of chloroauric acid solution with a mass fraction of 0.005-0.015%, add 2-3mL of trisodium citrate aqueous solution with a mass fraction of 0.5-1.5%, heat and reflux for 10-20min, until the color of the solution turns to Wine red, the solution was cooled to room temperature, and the obtained gold nanoparticles were stored at 4°C in the dark.

Disperse 1-3 mg of NP-NiGd into 1 mL of ultrapure water, add 0.5-1.5 mL of gold nanoparticles, shake the resulting mixed solution at room temperature for 12-32 hours, and centrifuge to remove excess gold nanoparticles, and the product NP-NiGdAu was redispersed into 1mL ultrapure water to prepare the base material NP-NiGdAu solution;

(2) Preparation of capture antibody substrate material NP-NiGdAu/Ab1 solution

Add 10-100 μL, 1 mg/mL antibody Ab ₁ solution to the base material NP-NiGdAu solution, shake and incubate at 4°C for 24 hours, remove excess antibody Ab ₁ by centrifugation, and disperse the product into 1 mL , PBS solution with pH 7.4 to prepare the capture antibody substrate material NP-NiGdAu/Ab ₁ solution, and store it at 4°C for future use.

3. Preparation of secondary antibody marker GO/AuNCsBSA/Ab ₂ solution

(1) Preparation of graphene oxide GO

Add 0.2-0.4g of graphite and 1.5-2.1g of potassium permanganate into a 500mL three-neck flask with a magnet, add a mixture of concentrated sulfuric acid and concentrated phosphoric acid with a volume ratio of 9:1, 30-70 React at ℃ for 6-24 hours. After the reaction is over, pour the sample onto 30-50 mL of pre-frozen ice cubes, add 200-400 μL of hydrogen peroxide with a mass fraction of 30% under slow stirring, and react for 20-40 minutes. , centrifuged, and successively washed three times with 0.2mol/L hydrochloric acid solution, ethanol, and finally washed once with ether, and the obtained solid was vacuum-dried at 35°C to obtain a brown solid powder;

(2) Preparation of gold nanocluster AuNCsBSA

Mix 0.3~0.8mmol ethanol solution of chloroauric acid and 0.5mmol mercaptosuccinic acid MSA with 100mL methanol evenly by bubbling nitrogen, and add 20~30mL drop by drop under vigorous stirring, 0.1~0.3mol/L fresh configuration Sodium borohydride solution, the resulting dark brown precipitate was centrifuged and washed with a water/ethanol solution with a volume ratio of 1:4, and dried under vacuum to obtain MSAAuNCs solid; MSAAuNCs and BSA were mixed in a mass ratio of 1:10 Add 5mL of water, stir for 3-8min, adjust the pH of the solution to 12 with 1-3mol/L sodium hydroxide, stir at 37°C for 6-8h, dialyze the obtained product for 24h, freeze-dry to obtain AuNCsBSA;

(3) Preparation of secondary antibody marker GO/AuNCsBSA/Ab ₂ solution

Add 1 to 3 mg of graphene oxide GO and 2 mg of AuNCsBSA into 2 mL of ultrapure water, shake for 32 to 72 hours, centrifuge and redisperse into 1 mL of PBS buffer solution with pH 7.4, add 0.05 to 0.15 mg of the antibody to be tested Ab ₂ was incubated at 4°C for 12-32 hours, centrifuged and redispersed into 1 mL of PBS buffer solution with pH 7.4, and stored at 4°C for use.

4. Detection method of the analyte

(1) The three-electrode system of the electrochemical workstation was used for testing, the Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the prepared electrochemical luminescence sensor was used as the working electrode. Connect together to set the high voltage of the photomultiplier tube to 800V, the scanning potential range of cyclic voltammetry is -1.8V～-0.6V, and the scanning rate is 0.1V/s;

(2) In 10mL PBS solution containing 26-46mmol/L potassium persulfate at pH 6.4-8.4, use the electrochemiluminescence system to detect the intensity of the electrochemiluminescence signal generated by different concentrations of the analyte antigen, and draw Working curve;

(3) The test sample solution is used instead of the test substance antigen for detection.

5. The above-mentioned test substance is selected from one of the following gastric cancer antigens: CEA, CA199, CA50, CA724, CA195, CA242, and CYFRA2H.

Beneficial results of the present invention

(1) Preparation method of electrochemiluminescence sensor, using AuNCsBSA as luminescent material, taking advantage of the good optical properties of AuNCsBSA, the sensor constructed has higher sensitivity;

(2) Using NP-NiGdAu as the capture antibody substrate and GO/AuNCsBSA as the secondary antibody label, NP-NiGdAu and GO/AuNCsBSA have good biocompatibility and large specific surface area effectively increase the immobilization capacity of antibodies.

(3) The electrochemiluminescence sensor prepared by the present invention is used for the detection of gastric cancer markers, and has simple operation, fast response, wide signal response range, and can realize simple, rapid, sensitive and specific detection.

Example 1 Preparation method of a NP-NiGdAu-based gastric cancer marker gold nanocluster electrochemiluminescence sensor

(1) Polish the glassy carbon electrode with a diameter of 4mm with 1.0μm, 0.3μm, and 0.05μm alumina polishing powder in sequence, and rinse it with ultrapure water;

(2) Drop-coat 6 μL, 0.5 mgmL ^-1 capture antibody substrate material NP-NiGdAu/Ab ₁ solution on the surface of the electrode, store at 4°C until dry;

(3) Add 4 μL bovine serum albumin solution with a mass fraction of 1% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS solution with pH 6.4, and dry at 4°C;

(4) Add 6 μL of different concentrations of the antigen to be tested, incubate at 4°C for 30 minutes, rinse the surface of the electrode with PBS solution of pH 6.4, and dry at 4°C;

(5) Drop-coat 6 μL of the secondary antibody marker GO/AuNCsBSA/Ab ₂ solution, rinse the electrode surface with PBS solution at pH 6.4, and dry at 4°C.

Example 2 A preparation method of an NP-NiGdAu-based gastric cancer marker gold nanocluster electrochemiluminescence sensor

(1) Polish the glassy carbon electrode with a diameter of 4mm with 1.0μm, 0.3μm, and 0.05μm alumina polishing powder in sequence, and rinse it with ultrapure water;

(2) Drop-coat 6 μL, 1 mgmL ^-1 capture antibody substrate material NP-NiGdAu/Ab ₁ solution on the surface of the electrode and store at 4°C until dry;

(3) Add 4 μL bovine serum albumin solution with a mass fraction of 2% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS solution with pH 7.4, and dry at 4°C;

(4) Add 6 μL of different concentrations of the antigen to be tested, incubate at 4°C for 30 minutes, rinse the surface of the electrode with PBS solution of pH 7.4, and dry at 4°C;

(5) Drop-coat 6 μL of the secondary antibody marker GO/AuNCsBSA/Ab ₂ solution, rinse the electrode surface with PBS solution with pH 7.4, and dry at 4°C.

Example 3 A preparation method of an NP-NiGdAu-based gastric cancer marker gold nanocluster electrochemiluminescence sensor

(1) Polish the glassy carbon electrode with a diameter of 4mm with 1.0μm, 0.3μm, and 0.05μm alumina polishing powder in sequence, and rinse it with ultrapure water;

(2) Drop-coat 6 μL, 2 mgmL ^-1 capture antibody substrate material NP-NiGdAu/Ab ₁ solution on the surface of the electrode, store at 4°C until dry;

(3) Add 4 μL bovine serum albumin solution with a mass fraction of 3% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS solution with pH 8.4, and dry at 4°C;

(4) Add 6 μL of different concentrations of the antigen to be tested, incubate at 4°C for 30 minutes, rinse the surface of the electrode with PBS solution with pH 8.4, and dry at 4°C;

(5) Drop-coat 6 μL of the secondary antibody marker GO/AuNCsBSA/Ab ₂ solution, rinse the electrode surface with PBS solution at pH 8.4, and dry at 4°C.

Embodiment 4 captures the preparation of antibody base material NP-NiGdAu/Ab ₁ solution

(1) Preparation of NP-NiGdAu

Put the NiGdAl alloy into an excess of 1.5mol/L sodium hydroxide solution, chemically corrode it at room temperature for 32h, centrifuge and wash to neutrality, and dry it in a vacuum oven for 12h to prepare nanoporous NiGd; mix 80mL, mass fraction Boil a 0.005% chloroauric acid solution, add 2 mL of trisodium citrate aqueous solution with a mass fraction of 0.5%, heat and reflux for 10 min, until the color of the solution turns wine red, and cool the solution to room temperature, the obtained gold nanoparticles, 4 Store in the dark at ℃.

Disperse 1 mg of NP-NiGd into 1 mL of ultrapure water, add 0.5 mL of gold nanoparticles, shake the resulting mixed solution at room temperature for 12 h, and centrifuge to remove excess gold nanoparticles, and redisperse the product NP-NiGdAu in 1mL ultrapure water, prepared base material NP-NiGdAu solution;

(2) Preparation of capture antibody substrate material NP-NiGdAu/Ab ₁ solution

In the base material NP-NiGdAu solution, add 10μL, 1mg/mL antibody Ab ₁ solution of the analyte, shake and incubate at 4°C for 24h, centrifuge to remove excess antibody Ab ₁ , and disperse the product to 1mL, pH7 .4 in the PBS solution, prepare the capture antibody substrate material NP-NiGdAu/Ab ₁ solution, and store it at 4°C for future use.

Embodiment 5 captures the preparation of antibody substrate material NP-NiGdAu/Ab ₁ solution

(1) Preparation of NP-NiGdAu

Put the NiGdAl alloy into an excess of 2mol/L sodium hydroxide solution, chemically corrode it at room temperature for 48 hours, centrifuge and wash it to neutrality, and dry it in a vacuum oven for 32 hours to obtain nanoporous NiGd; 0.01% chloroauric acid solution was boiled, added 2.5mL, the mass fraction was 1% trisodium citrate aqueous solution, heated and refluxed for 15min, until the color of the solution turned wine red, the solution was cooled to room temperature, and the obtained gold nanoparticles, 4 Store in the dark at ℃.

Disperse 2 mg of NP-NiGd into 1 mL of ultrapure water, add 1 mL of gold nanoparticles, shake the resulting mixed solution at room temperature for 24 h, and centrifuge to remove excess gold nanoparticles, and redisperse the product NP-NiGdAu into 1 mL In ultrapure water, the base material NP-NiGdAu solution is prepared;

(2) Preparation of capture antibody substrate material NP-NiGdAu/Ab ₁ solution

In the base material NP-NiGdAu solution, add 50μL, 1mg/mL antibody Ab ₁ solution of the analyte, shake and incubate at 4°C for 24h, centrifuge to remove excess antibody Ab ₁ , and disperse the product to 1mL, pH7 .4 in the PBS solution, prepare the capture antibody substrate material NP-NiGdAu/Ab ₁ solution, and store it at 4°C for future use.

Embodiment 6 captures the preparation of antibody substrate material NP-NiGdAu/Ab ₁ solution

(1) Preparation of NP-NiGdAu

Put the NiGdAl alloy into an excess of 2.5mol/L sodium hydroxide solution, chemically corrode it at room temperature for 74 hours, centrifuge and wash it to neutrality, and dry it in a vacuum oven for 48 hours to obtain nanoporous NiGd; Boil a 0.015% chloroauric acid solution, add 3 mL of trisodium citrate aqueous solution with a mass fraction of 1.5%, heat and reflux for 20 min, until the color of the solution turns wine red, and cool the solution to room temperature, the obtained gold nanoparticles, 4 Store in the dark at ℃.

Disperse 3 mg of NP-NiGd into 1 mL of ultrapure water, add 1.5 mL of gold nanoparticles, shake the resulting mixed solution at room temperature for 32 h, and centrifuge to remove excess gold nanoparticles, and redisperse the product NP-NiGdAu in 1mL ultrapure water, prepared base material NP-NiGdAu solution;

(2) Preparation of capture antibody substrate material NP-NiGdAu/Ab ₁ solution

In the base material NP-NiGdAu solution, add 100μL, 1mg/mL antibody Ab ₁ solution of the analyte, shake and incubate at 4°C for 24h, centrifuge to remove excess antibody Ab ₁ , and disperse the product to 1mL, pH7 .4 in the PBS solution, prepare the capture antibody substrate material NP-NiGdAu/Ab ₁ solution, and store it at 4°C for future use.

Example 7 Preparation of Secondary Antibody Marker GO/AuNCsBSA/Ab ₂ Solution

(1) Preparation of graphene oxide GO

Add 0.2g of graphite and 1.5g of potassium permanganate into a 500mL three-neck flask with a magnet, add a mixture of concentrated sulfuric acid and concentrated phosphoric acid with a volume ratio of 9:1, and react at 30°C for 6 hours. After the end, pour the sample on the pre-frozen 30mL ice cube, add 200μL hydrogen peroxide with a mass fraction of 30% under slow stirring, react for 20min, centrifuge, and successively use 0.2mol/L hydrochloric acid solution , washed three times with ethanol, and finally washed once by centrifugation with ether, and the obtained solid was vacuum-dried at 35° C. to obtain a brown solid powder;

(2) Preparation of gold nanocluster AuNCsBSA

0.3mmol chloroauric acid ethanol solution and 0.5mmol mercaptosuccinic acid MSA were mixed uniformly with 100mL methanol by nitrogen bubbling, and 20mL, 0.1mol/L freshly configured sodium borohydride solution was added dropwise under vigorous stirring, The resulting dark brown precipitate was centrifuged and washed with a water/ethanol solution with a volume ratio of 1:4, and dried under vacuum to obtain a solid MSAAuNCs; MSAAuNCs and BSA were added to 5 mL of water at a mass ratio of 1:10, and stirred for 3 min , adjust the pH of the solution to 12 with 1mol/L sodium hydroxide, stir at 37°C for 6h, dialyze the obtained product for 24h, and freeze-dry to obtain AuNCsBSA;

(3) Preparation of secondary antibody marker GO/AuNCsBSA/Ab ₂ solution

Add 1 mg of graphene oxide GO and 2 mg of AuNCsBSA into 2 mL of ultrapure water, shake for 32 hours, centrifuge and redisperse into 1 mL of PBS buffer solution with pH 7.4, add 0.05 mg of the antibody Ab ₂ to be tested, and incubate at 4 °C Incubate for 12 hours, centrifuge and redisperse into 1 mL of PBS buffer solution, pH 7.4, and store at 4°C for future use.

Example 8 Preparation of Secondary Antibody Marker GO/AuNCsBSA/Ab ₂ Solution

(1) Preparation of graphene oxide GO

Add 0.3g of graphite and 1.7g of potassium permanganate into a 500mL three-necked flask with a magnet, add a mixture of concentrated sulfuric acid and concentrated phosphoric acid with a volume ratio of 9:1, and react at 50°C for 18 hours. After the end, pour the sample on the pre-frozen 40mL ice cube, add 300μL hydrogen peroxide with a mass fraction of 30% under slow stirring, react for 30min, centrifuge, and successively use 0.2mol/L hydrochloric acid solution , washed three times with ethanol, and finally washed once by centrifugation with ether, and the obtained solid was vacuum-dried at 35° C. to obtain a brown solid powder;

(2) Preparation of gold nanocluster AuNCsBSA

0.5mmol chloroauric acid ethanol solution and 0.5mmol mercaptosuccinic acid MSA were mixed uniformly with 100mL methanol by nitrogen bubbling, and 25mL, 0.2mol/L freshly configured sodium borohydride solution was added dropwise under vigorous stirring, The resulting dark brown precipitate was centrifuged and washed with a water/ethanol solution with a volume ratio of 1:4, and dried under vacuum to obtain a solid MSAAuNCs; MSAAuNCs and BSA were added to 5 mL of water at a mass ratio of 1:10, and stirred for 5 min , adjust the pH of the solution to 12 with 2mol/L sodium hydroxide, stir at 37°C for 7h, dialyze the obtained product for 24h, and freeze-dry to obtain AuNCsBSA;

(3) Preparation of secondary antibody marker GO/AuNCsBSA/Ab ₂ solution

Add 2 mg of graphene oxide GO and 2 mg of AuNCsBSA into 2 mL of ultrapure water, shake for 48 hours, centrifuge and redisperse into 1 mL of PBS buffer solution with pH 7.4, add 0.1 mg of the antibody Ab ₂ to be tested, and incubate at 4 °C Incubate for 24 hours, centrifuge and redisperse into 1 mL of PBS buffer solution, pH 7.4, and store at 4°C for later use.

Example 9 Preparation of Secondary Antibody Marker GO/AuNCsBSA/Ab ₂ Solution

(1) Preparation of graphene oxide GO

Add 0.4g of graphite and 2.1g of potassium permanganate into a 500mL three-necked flask with a magnet, add a mixture of concentrated sulfuric acid and concentrated phosphoric acid with a volume ratio of 9:1, and react at 70°C for 24 hours. After the end, pour the sample on the pre-frozen 50mL ice cube, add 400μL hydrogen peroxide with a mass fraction of 30% under slow stirring, react for 40min, centrifuge, and successively use 0.2mol/L hydrochloric acid solution , washed three times with ethanol, and finally washed once by centrifugation with ether, and the obtained solid was vacuum-dried at 35° C. to obtain a brown solid powder;

(2) Preparation of gold nanocluster AuNCsBSA

0.8mmol chloroauric acid ethanol solution and 0.5mmol mercaptosuccinic acid MSA were mixed uniformly with 100mL methanol by nitrogen bubbling, and 30mL, 0.3mol/L freshly configured sodium borohydride solution was added dropwise under vigorous stirring, The resulting dark brown precipitate was centrifuged and washed with a water/ethanol solution with a volume ratio of 1:4, and dried under vacuum to obtain a solid MSAAuNCs; MSAAuNCs and BSA were added to 5 mL of water at a mass ratio of 1:10, and stirred for 8 min , adjust the pH of the solution to 12 with 3mol/L sodium hydroxide, stir at 37°C for 8h, dialyze the obtained product for 24h, and freeze-dry to obtain AuNCsBSA;

(3) Preparation of secondary antibody marker GO/AuNCsBSA/Ab ₂ solution

Add 3 mg of graphene oxide GO and 2 mg of AuNCsBSA into 2 mL of ultrapure water, shake for 72 hours, centrifuge and redisperse into 1 mL of PBS buffer solution with pH 7.4, add 0.15 mg of the antibody Ab ₂ to be tested, and incubate at 4°C Incubate for 32 hours, centrifuge and redisperse into 1 mL of PBS buffer solution with pH 7.4, and store at 4°C for future use.

The detection of embodiment 10 CEA

(1) The three-electrode system of the electrochemical workstation was used for testing, the Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the prepared electrochemical luminescence sensor was used as the working electrode. Connect together to set the high voltage of the photomultiplier tube to 800V, the scanning potential range of cyclic voltammetry is -1.8V～-0.6V, and the scanning rate is 0.1V/s;

(2) In 10 mL of PBS solution containing 26-46 mmol/L potassium persulfate at pH 6.4-8.4, use an electrochemiluminescence system to detect the intensity of electrochemiluminescence signals generated by different concentrations of CEA antigens, and draw a working curve ;

(3) The sample solution to be tested was replaced by CEA antigen for detection, and the measured linear range was 0.1pg/mL~5ng/mL, and the detection limit was 0.03pg/mL.

Example 11 Detection of CA199

(1) The three-electrode system of the electrochemical workstation was used for testing, the Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the prepared electrochemical luminescence sensor was used as the working electrode. Connect together to set the high voltage of the photomultiplier tube to 800V, the scanning potential range of cyclic voltammetry is -1.8V～-0.6V, and the scanning rate is 0.1V/s;

(2) In 10 mL of PBS solution containing 26-46 mmol/L potassium persulfate at pH 6.4-8.4, use an electrochemiluminescence system to detect the intensity of electrochemiluminescence signals generated by different concentrations of CA199 antigen, and draw a working curve ;

(3) The sample solution to be tested was replaced by the CA199 antigen for detection. The measured linear range was 0.1pg/mL~5ng/mL, and the detection limit was 0.03pg/mL.

Example 12 Detection of CA50

(1) The three-electrode system of the electrochemical workstation was used for testing, the Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the prepared electrochemical luminescence sensor was used as the working electrode. Connect together to set the high voltage of the photomultiplier tube to 800V, the scanning potential range of cyclic voltammetry is -1.8V～-0.6V, and the scanning rate is 0.1V/s;

(2) In 10 mL of PBS solution containing 26-46 mmol/L potassium persulfate at pH 6.4-8.4, use an electrochemiluminescence system to detect the intensity of electrochemiluminescence signals generated by different concentrations of CA50 antigens, and draw a working curve ;

(3) The sample solution to be tested was replaced by the CA50 antigen for detection, and the measured linear range was 0.1pg/mL~5ng/mL, and the detection limit was 0.03pg/mL.

Claims (3)

1. A method for preparing a gastric cancer marker gold nanocluster electrochemiluminescent sensor based on NP-NiGdAu, comprising the following steps: (1) Preparation of NP-NiGdAu Put the NiGdAl alloy into an excess of 1.5-2.5mol/L sodium hydroxide solution, chemically corrode it at room temperature for 32-74 hours, centrifugally wash it to neutrality, and dry it in a vacuum oven for 12-48 hours to obtain nanoporous NiGd ; Boil 80-120mL of chloroauric acid solution with a mass fraction of 0.005-0.015%, add 2-3mL of trisodium citrate aqueous solution with a mass fraction of 0.5-1.5%, heat and reflux for 10-20min, until the color of the solution turns to wine red, cool the solution to room temperature, and store the obtained gold nanoparticles in the dark at 4°C; disperse 1 to 3 mg of NP-NiGd into 1 mL of ultrapure water, add 0.5 to 1.5 mL of gold nanoparticles, and obtain the mixed The solution was shaken at room temperature for 12 to 32 hours, centrifuged to remove excess gold nanoparticles, and the product NP-NiGdAu was redispersed into 1 mL of ultrapure water to prepare the base material NP-NiGdAu solution; (2) Preparation of capture antibody substrate material NP-NiGdAu/Ab ₁ solution Add 10-100 μL, 1 mg/mL antibody Ab ₁ solution to the base material NP-NiGdAu solution, shake and incubate at 4°C for 24 hours, remove excess antibody Ab ₁ by centrifugation, and disperse the product into 1 mL , PBS solution with pH 7.4 to prepare the capture antibody substrate material NP-NiGdAu/Ab ₁ solution, and store it at 4°C for later use; (3) Preparation of graphene oxide GO Add 0.2-0.4g of graphite and 1.5-2.1g of potassium permanganate into a 500mL three-neck flask with a magnet, add a mixture of concentrated sulfuric acid and concentrated phosphoric acid with a volume ratio of 9:1, 30-70 React at ℃ for 6-24 hours. After the reaction is over, pour the sample onto 30-50 mL of pre-frozen ice cubes, add 200-400 μL of hydrogen peroxide with a mass fraction of 30% under slow stirring, and react for 20-40 minutes. , centrifuged, and successively washed three times with 0.2mol/L hydrochloric acid solution, ethanol, and finally washed once with ether, and the obtained solid was vacuum-dried at 35°C to obtain a brown solid powder; (4) Preparation of gold nanocluster AuNCsBSA Mix 0.3~0.8mmol ethanol solution of chloroauric acid and 0.5mmol mercaptosuccinic acid MSA with 100mL methanol evenly by bubbling nitrogen, and add 20~30mL drop by drop under vigorous stirring, 0.1~0.3mol/L fresh configuration Sodium borohydride solution, the resulting dark brown precipitate was centrifuged and washed with a water/ethanol solution with a volume ratio of 1:4, and dried under vacuum to obtain MSAAuNCs solid; MSAAuNCs and BSA were mixed in a mass ratio of 1:10 Add 5mL of water, stir for 3-8min, adjust the pH of the solution to 12 with 1-3mol/L sodium hydroxide, stir at 37°C for 6-8h, dialyze the obtained product for 24h, freeze-dry to obtain AuNCsBSA; (5) Preparation of secondary antibody marker GO/AuNCsBSA/Ab ₂ solution Add 1 to 3 mg of graphene oxide GO and 2 mg of AuNCsBSA into 2 mL of ultrapure water, shake for 32 to 72 hours, centrifuge and redisperse into 1 mL of PBS buffer solution with pH 7.4, add 0.05 to 0.15 mg of the antibody to be tested Ab ₂ , incubate at 4°C for 12-32h, centrifuge and redisperse into 1mL PBS buffer solution with pH 7.4, store at 4°C for use; (6) Preparation of gastric cancer marker gold nanocluster electrochemiluminescence sensor a. Polish the glassy carbon electrode with a diameter of 4mm with 1.0μm, 0.3μm, and 0.05μm alumina polishing powder in sequence, and rinse it with ultrapure water; b. Drop-spray 6μL, 0.5~2mgmL ^-1 capture antibody substrate material NP-NiGdAu/Ab ₁ solution on the surface of the electrode, and store at 4°C until dry; c. Add 4 μL bovine serum albumin solution with a mass fraction of 1-3% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS solution with pH 6.4-8.4, and dry at 4°C; d. Add 6 μL of different concentrations of the test substance antigen dropwise, incubate at 4°C for 30 minutes, rinse the electrode surface with PBS solution with pH 6.4~8.4, and dry at 4°C; e. Drop-coat 6 μL of secondary antibody marker GO/AuNCsBSA/Ab ₂ solution, rinse the electrode surface with PBS solution with pH 6.4~8.4, and dry at 4°C. 2. The purposes of a kind of gastric cancer marker gold nanocluster electrochemiluminescence sensor based on NP-NiGdAu prepared by the preparation method as claimed in claim 1, it is characterized in that, the gastric cancer marker gold nanometer based on NP-NiGdAu The cluster electrochemiluminescence sensor is used for the detection of the analyte, and the detection steps are as follows: (1) The three-electrode system of the electrochemical workstation was used for testing, the Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the prepared electrochemical luminescence sensor was used as the working electrode. Connect together to set the high voltage of the photomultiplier tube to 800V, the scanning potential range of cyclic voltammetry is -1.8V～-0.6V, and the scanning rate is 0.1V/s; (2) In 10mL PBS solution containing 26-46mmol/L potassium persulfate at pH 6.4-8.4, use the electrochemiluminescence system to detect the intensity of the electrochemiluminescence signal generated by different concentrations of the analyte antigen, and draw Working curve; (3) The test sample solution is used instead of the test substance antigen for detection. 3. a kind of preparation method of the gastric cancer marker gold nanocluster electrochemiluminescent sensor based on NP-NiGdAu as claimed in claim 1, described analyte, is selected from one of following gastric cancer antigen: CEA, CA199, CA50 , CA724, CA195, CA242, CYFRA2H.