CN110907511B - Gold-curcumin nanoparticle quenched CdS hybrid TiO2Electrochemical luminescence sensor for detecting insulin by nanobelt - Google Patents

Gold-curcumin nanoparticle quenched CdS hybrid TiO2Electrochemical luminescence sensor for detecting insulin by nanobelt Download PDF

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CN110907511B
CN110907511B CN201911272160.0A CN201911272160A CN110907511B CN 110907511 B CN110907511 B CN 110907511B CN 201911272160 A CN201911272160 A CN 201911272160A CN 110907511 B CN110907511 B CN 110907511B
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杜宇
李小建
任祥
孙晓君
魏琴
杨兴龙
王雪莹
吴丹
马洪敏
王欢
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Abstract

The invention relates to gold-curcumin nanoparticle quenched CdS hybridized TiO2An electrochemical luminescence sensor for detecting insulin by a nanobelt. In the invention, potassium persulfate and hydrogen peroxide are used as a coreactant together to enhance CdS hybrid TiO2The electrochemical luminescence property of the nano-belt. In order to sensitively detect insulin, the invention designs a sandwich type quenching electrochemiluminescence immunosensor, which adopts gold-curcumin composite ZIF-8 as a quencher to reduce CdS hybridized TiO2The electrochemiluminescence intensity of the nanoribbons. The quenching mechanism is mainly that curcumin consumes hydroxyl free radicals generated in the electrochemical reaction process, and further reduces the luminous intensity of the luminescent material through energy transfer, so that double quenching of an electrochemical luminescence signal is realized. Different amounts of the second antibody marker gold-curcumin complex ZIF-8 can be combined according to different concentrations of insulin, so that the electrochemical luminescence intensity of the sensor is different. The linear range of the invention for detecting insulin is 0.3 pg/mL-20 ng/mL, and the detection limit is 0.09 pg/mL.

Description

Gold-curcumin nanoparticle quenched CdS hybrid TiO2Electrochemical luminescence sensor for detecting insulin by nanobelt
Technical Field
The invention relates to gold-curcumin nanoparticle quenched CdS hybridized TiO2An electrochemical luminescence sensor for detecting insulin by a nanobelt. In particular to CdS hybridized TiO2The nano-belt is taken as a luminescent material, the gold-curcumin nano-particles are taken as a quenching agent, and in order to enhance the quenching effect, the gold-curcumin nano-particles are compounded with ZIF-8 (gold-curcumin/ZIF-8), and belongs to the technical field of electrochemiluminescence detection.
Background
Insulin is a polypeptide hormone that can be used to regulate the metabolism of fats and carbohydrates. In clinic, the detection of insulin is very significant, because the content of insulin is an important index for evaluating endocrine function, and provides basis for detecting diabetes, insulinoma, insulin resistance syndrome and the like. Therefore, in the invention, a novel and sensitive electrochemical luminescence immunosensor is designed by taking insulin as a detection object.
The current main methods for detecting insulin include bioassay, immunoassay, instrumental assay (high performance liquid chromatography, capillary electrophoresis, nuclear magnetism, mass spectrometry, etc.), etc., wherein the immunoassay mainly includes radioimmunoassay, fluorescence immunoassay, enzyme linked immunosorbent assay, etc. The invention designs an Electrochemiluminescence (ECL) immunosensor which is used for sensitively detecting insulin and detecting the insulin in an actual sample. ECL analysis has high sensitivity and wide linear range; the reaction controllability and the space-time controllability are good; the instrument is simple and the analysis speed is high; the reagent is saved; the analysis has wide application range; can obtain various information simultaneously, is favorable for researching the advantages of rapid luminescence reaction, luminescence reaction mechanism and the like, and has been developed into a branch subject of analytical chemistry.
In the present invention, CdS is adopted to hybridize TiO2Nanobelt (CdS @ TiO)2) As a luminescent material, potassium persulfate and hydrogen peroxide together act as a co-reactant. Electroreduction of hydrogen peroxide to produce hydroxyl radicals (OH)•) Can promote S2O8 2-Generate more sulfate radical (SO)4 -•) Further enhancing CdS @ TiO2The electrochemiluminescence intensity. gold-curcumin/ZIF-8 as quencher to reduce CdS @ TiO for sensitive detection of insulin2ECL signal of (c). Therefore, the immunosensor designed by the invention not only can sensitively detect insulin, but also provides detection for other analytesA new method is provided. Quenching CdS @ TiO based on gold-curcumin nanoparticles at present2No methods for detecting insulin have been reported.
Disclosure of Invention
The invention designs a quenching type electrochemiluminescence immunosensor for detecting insulin.
In the present invention, TiO2The nanobelt has larger specific surface area and can immobilize more CdS nano particles, so that the prepared CdS @ TiO2The nano material has stronger luminous performance. In order to further enhance the luminous performance, potassium persulfate and hydrogen peroxide are simultaneously used as a co-reactant, and the hydrogen peroxide is subjected to electroreduction to generate OH•Can promote S2O8 2-More SO is generated4 -•Further enhancing CdS @ TiO2The electrochemiluminescence intensity. However, when OH is present•In excess, OH•Will consume SO4 -•Hydrogen sulfate ions and oxygen are generated, so that the generated excited species are reduced, thereby reducing the ECL intensity of the light emitting material. For sensitive detection of insulin, gold-curcumin/ZIF-8 is used as a quencher to reduce CdS @ TiO2The electrochemiluminescence intensity. The antioxidant curcumin can consume OH through electron transfer•Reduction of SO in electrochemical reaction4 -•To thereby quench CdS @ TiO2The electrochemiluminescence intensity. And, the UV-visible absorption peak of gold-curcumin/ZIF-8 and CdS @ TiO2The fluorescence emission peak has certain spectral overlap, namely certain energy transfer also exists between the two, and the CdS @ TiO further is quenched2The electrochemical luminescence intensity of the CdS @ TiO is realized2Double quenching of electrochemiluminescence properties. When the surface of the electrode contains insulin with different concentrations, different amounts of the second antibody marker gold-curcumin/ZIF-8 can be combined on the surface of the electrode, so that the ECL strength is reduced to different degrees, and the purpose of detection is achieved.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
1. gold-curcumin nanoparticle quenched CdS hybrid TiO2Nano beltThe preparation method of the electrochemical luminescence sensor for detecting insulin comprises the following steps:
(1) pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder, and washing the glassy carbon electrode with ultrapure water;
(2) 6 mu L5-10 mg/mL CdS @ TiO2The chitosan solution is dripped on the surface of a bare glass carbon electrode and is stored at room temperature until being dried;
(3) 5 mu L of 500 mu g/mL primary antibody solution is dripped on the surface of the bare glassy carbon electrode, and the bare glassy carbon electrode is preserved in a refrigerator at 4 ℃ until being dried and washed by ultrapure water;
(4) dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(5) dripping 6 μ L of insulin with different concentrations on the surface of the electrode, storing in a refrigerator at 4 deg.C until dry, and cleaning with ultrapure water;
(6) dropwise coating 5 mu L of 1-5 mg/mL secondary antibody-gold-curcumin/ZIF-8 solution on the surface of an electrode, storing in a refrigerator at 4 ℃ until the solution is dried, and cleaning with ultrapure water to obtain the electrochemical luminescence biosensor for detecting insulin.
2. The gold-curcumin nano particle quenching CdS hybridized TiO2Preparation method of electrochemical luminescence sensor for detecting insulin by nanobelt, CdS @ TiO2The preparation method of the chitosan solution comprises the following steps:
0.1 g of cadmium acetate is dispersed in 30 mL of ultrapure water, 25-50 mg of titanium dioxide nanobelts are added, and ultrasonic treatment is carried out for 1 hour. Then, 5 mL of 5M Na was added2Stirring the S solution for 12 h, transferring the solution into a 100 mL reaction kettle, reacting for 16 h at 160 ℃, centrifuging, washing and drying to obtain CdS @ TiO2A nanoribbon.
The chitosan solution with the mass fraction of 0.5% is prepared by adding 0.5 g of chitosan into 100 mL of acetic acid with the volume fraction of 1% and stirring for 2 h.
3. The gold-curcumin nano particle quenching CdS hybridized TiO2The preparation method of the electrochemical luminescence sensor for detecting insulin by using the nanobelt comprises the steps of preparing the second antibody-gold-curcumin/ZIF-8 solutionThe method comprises the following steps:
(1) preparation of gold-curcumin nanoparticles
8 mg of curcumin was first dispersed in 5 mL of dimethyl sulfoxide to form a clear and homogeneous solution. Then, 2 mL of curcumin-dimethylsulfoxide solution was added to 45 mL of ultrapure water, and stirred under reflux at 100 ℃. Add 100. mu.L of 0.1M NaOH, heat the solution to 100 ℃ and add 1 mL of 2% HAuCl4And stirring the solution for 1 h, cooling to room temperature, dialyzing by using a 12 kDa dialysis bag, and removing unreacted impurities and small particles to obtain the gold-curcumin solution.
(2) Preparation of gold-curcumin/ZIF-8 nanoflower
Mixing 1 mL of 2-methylimidazole with 2-5 mL of gold-curcumin solution, and stirring for 10 min. Then, 1 mL of 20 mM zinc acetate solution and 0.05 g of bovine serum albumin were added thereto, and the mixture was stirred for 4 hours. And finally, centrifuging, washing and vacuum drying the obtained gold-curcumin/ZIF-8 nanoflower.
(3) Preparation of secondary antibody-gold-curcumin/ZIF-8 solution
The carboxyl on the surface of ZIF-8 is activated by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to react with the amino on the surface of a secondary antibody, so that the antibody is fixed on the surface of gold-curcumin/ZIF-8. 2 mg of gold-curcumin/ZIF-8 was dispersed in 1 mL of pH 7.4 phosphate buffer and sonicated for 30 min. Then, 500. mu.L of 100 mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 500. mu.L of 400 mM N-hydroxysuccinimide were added and stirred for 1 h. Then, 500. mu.L of a 500. mu.g/mL secondary antibody solution was added to the above solution, and stirred at 4 ℃ for 6 hours. Finally, 100 μ L of 1% BSA was added to block non-specific sites, centrifuged to give the second antibody-gold-curcumin/ZIF-8, which was dispersed in 1 mL phosphate buffer (pH = 7.4) and stored in a refrigerator at 4 ℃ until use.
4. The gold-curcumin nano particle quenching CdS hybrid TiO prepared by the preparation method2The electrochemical luminescence sensor for detecting insulin by a nanobelt is used for detecting insulin and comprises the following steps:
(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum electrode and the prepared electrochemical luminescence sensor as working electrodes in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 800V, the scanning voltage to be 0-1.6V and the scanning speed to be 0.15V/s;
(2) detecting the intensity of an electrochemiluminescence signal generated by insulin with different concentrations by an electrochemiluminescence method by using a phosphate buffer solution containing 1-8 mmol/L hydrogen peroxide and 20-120 mM potassium persulfate;
the phosphate buffer solution has pH = 5.0-8.5 and is prepared from 1/15 mol/L Na2HPO4And 1/15 mol/L KH2PO4Preparing;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the logarithm of the insulin concentration.
Advantageous results of the invention
(1) Hydrogen peroxide and potassium persulfate are simultaneously used as CdS @ TiO2The co-reactant greatly enhances the electrochemical luminescence property of the luminescent material, and the enhancing mechanism is mainly through OH•Can promote S2O8 2-Produce more sulfate radical SO4 -•；
(2) The antioxidant curcumin is chelated with the gold nanoparticles, so that the water solubility of the curcumin is improved, and the application range of the curcumin in the field of bioanalysis is widened;
(3) the gold-curcumin is compounded with the ZIF-8, so that the composite material contains more gold-curcumin, and the quenching efficiency of the composite material is improved. In the present invention, the quenching mechanism is mainly due to curcumin consuming OH generated from hydrogen peroxide during the electrochemical reaction•And the ultraviolet absorption peak of gold-curcumin/ZIF-8 and CdS @ TiO2The emission peak of (a) has good spectral overlap, which indicates that a certain ECL resonance energy transfer exists between the luminescent material and the quencher, so that the intensity of the luminescent material is further reduced;
(4) the quenching type electrochemical luminescence sensor prepared by the invention is used for detecting insulin, has the advantages of small using amount of luminescent substances and short response time, and can realize simple, quick and high-sensitivity detection. The linear range of the invention for detecting insulin is 0.3 pg/mL-20 ng/mL, and the detection limit is 0.09 pg/mL.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of CdS @ TiO2The chitosan solution of (2)
0.1 g of cadmium acetate is dispersed in 30 mL of ultrapure water, 25 mg of titanium dioxide nanobelt is added, and ultrasonic treatment is carried out for 1 h. Then, 5 mL of 5M Na was added2Stirring the S solution for 12 h, transferring the solution into a 100 mL reaction kettle, reacting for 16 h at 160 ℃, centrifuging, washing and drying to obtain CdS @ TiO2A nanoribbon.
The chitosan solution with the mass fraction of 0.5% is prepared by adding 0.5 g of chitosan into 100 mL of acetic acid with the volume fraction of 1% and stirring for 2 h.
EXAMPLE 2 preparation of CdS @ TiO2The chitosan solution of (2)
0.1 g of cadmium acetate is dispersed in 30 mL of ultrapure water, 50 mg of titanium dioxide nanobelt is added, and ultrasonic treatment is carried out for 1 h. Then, 5 mL of 5M Na was added2Stirring the S solution for 12 h, transferring the solution into a 100 mL reaction kettle, reacting for 16 h at 160 ℃, centrifuging, washing and drying to obtain CdS @ TiO2A nanoribbon.
The chitosan solution with the mass fraction of 0.5% is prepared by adding 0.5 g of chitosan into 100 mL of acetic acid with the volume fraction of 1% and stirring for 2 h.
EXAMPLE 3 preparation of anti-gold-curcumin/ZIF-8 solution
(1) Preparation of gold-curcumin nanoparticles
8 mg of curcumin was first dispersed in 5 mL of dimethyl sulfoxide to form a clear and homogeneous solution. Then, 2 mL of curcumin-dimethylsulfoxide solution was added to 45 mL of ultrapure water, and stirred under reflux at 100 ℃. Add 100. mu.L of 0.1M NaOH, heat the solution to 100 ℃ and add 1 mL of 2% HAuCl4Stirring the solution for 1 h, cooling to room temperature, and dialyzing with 12 kDa dialysis bagAnd (4) separating and removing unreacted impurities and small particles to obtain the gold-curcumin solution.
(2) Preparation of gold-curcumin/ZIF-8 nanoflower
1 mL of 2-methylimidazole was mixed with 2 mL of gold-curcumin solution and stirred for 10 min. Then, 1 mL of 20 mM zinc acetate solution and 0.05 g of bovine serum albumin were added thereto, and the mixture was stirred for 4 hours. And finally, centrifuging, washing and vacuum drying the obtained gold-curcumin/ZIF-8 nanoflower.
(3) Preparation of secondary antibody-gold-curcumin/ZIF-8 solution
The carboxyl on the surface of ZIF-8 is activated by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to react with the amino on the surface of a secondary antibody, so that the antibody is fixed on the surface of gold-curcumin/ZIF-8. 2 mg of gold-curcumin/ZIF-8 was dispersed in 1 mL of pH 7.4 phosphate buffer and sonicated for 30 min. Then, 500. mu.L of 100 mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 500. mu.L of 400 mM N-hydroxysuccinimide were added and stirred for 1 h. Then, 500. mu.L of a 500. mu.g/mL secondary antibody solution was added to the above solution, and stirred at 4 ℃ for 6 hours. Finally, 100 μ L of 1% BSA was added to block non-specific sites, centrifuged to give the second antibody-gold-curcumin/ZIF-8, which was dispersed in 1 mL phosphate buffer (pH = 7.4) and stored in a refrigerator at 4 ℃ until use.
EXAMPLE 4 preparation of an electrochemiluminescence sensor for detecting insulin
(1) Pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder, and washing the glassy carbon electrode with ultrapure water;
(2) 6 mu L5 mg/mL CdS @ TiO2The chitosan solution is dripped on the surface of a bare glass carbon electrode and is stored at room temperature until being dried;
(3) 5 mu L of 500 mu g/mL primary antibody solution is dripped on the surface of the bare glassy carbon electrode, and the bare glassy carbon electrode is preserved in a refrigerator at 4 ℃ until being dried and washed by ultrapure water;
(4) dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(5) dripping 6 μ L of insulin with different concentrations on the surface of the electrode, storing in a refrigerator at 4 deg.C until dry, and cleaning with ultrapure water;
(6) and dripping 5 mu L of 1 mg/mL secondary antibody-gold-curcumin/ZIF-8 solution on the surface of the electrode, storing in a refrigerator at 4 ℃ until the solution is dried, and cleaning with ultrapure water to obtain the electrochemical luminescence biosensor for detecting insulin.
EXAMPLE 5 preparation of an electrochemiluminescence sensor for detecting insulin
(1) Pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder, and washing the glassy carbon electrode with ultrapure water;
(2) 6 mu L10 mg/mL CdS @ TiO2The chitosan solution is dripped on the surface of a bare glass carbon electrode and is stored at room temperature until being dried;
(3) 5 mu L of 500 mu g/mL primary antibody solution is dripped on the surface of the bare glassy carbon electrode, and the bare glassy carbon electrode is preserved in a refrigerator at 4 ℃ until being dried and washed by ultrapure water;
(4) dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(5) dripping 6 μ L of insulin with different concentrations on the surface of the electrode, storing in a refrigerator at 4 deg.C until dry, and cleaning with ultrapure water;
(6) and dripping 5 mu L of 5 mg/mL secondary antibody-gold-curcumin/ZIF-8 solution on the surface of the electrode, storing in a refrigerator at 4 ℃ until the solution is dried, and cleaning with ultrapure water to obtain the electrochemical luminescence biosensor for detecting insulin.
EXAMPLE 6 detection of insulin
(1) Connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum electrode and the prepared electrochemical luminescence sensor as working electrodes in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 800V, the scanning voltage to be 0-1.6V and the scanning speed to be 0.15V/s;
(2) detecting the intensity of an electrochemiluminescence signal generated by insulin with different concentrations by an electrochemiluminescence method by using a phosphate buffer solution containing 1 mmol/L hydrogen peroxide and 20 mM potassium persulfate;
the phosphate buffer solution, whichpH =7.4, using 1/15 mol/L Na2HPO4And 1/15 mol/L KH2PO4Preparing;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the logarithm of the insulin concentration.
EXAMPLE 7 detection method of insulin
(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum electrode and the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation with the chemiluminescence detector, setting the high voltage of a photomultiplier to be 800V, the scanning voltage to be 0-1.6V and the scanning speed to be 0.15V/s;
(2) detecting the intensity of an electrochemiluminescence signal generated by insulin with different concentrations by an electrochemiluminescence method by using a phosphate buffer solution containing 8 mmol/L hydrogen peroxide and 120 mM potassium persulfate;
the phosphate buffer solution, pH =8.5, was treated with 1/15 mol/L Na2HPO4And 1/15 mol/L KH2PO4Preparing;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the logarithm of the insulin concentration.
Example 9 detection of insulin in serum
(1) The mean recovery of insulin from the samples was determined by standard addition methods by adding insulin at different concentrations to the diluted serum and the results are shown in table 1.
TABLE 1 insulin in standard hair test samples
Figure DEST_PATH_IMAGE002
The detection results in table 1 show that the recovery rate of the detection result of insulin in the sample is 99.6-120%, which indicates that the method can be applied to the detection of practical biological samples and the result is accurate and reliable.

Claims (3)

1. AGold-curcumin nanoparticle quenched CdS hybrid TiO2The preparation method of the electrochemical luminescence sensor for detecting insulin by the nanobelt is characterized by comprising the following preparation steps:
(1) pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder, and washing the glassy carbon electrode with ultrapure water;
(2) 6 mu L5-10 mg/mL CdS @ TiO2The chitosan solution is dripped on the surface of a bare glass carbon electrode and is stored at room temperature until being dried;
(3) 5 mu L of 500 mu g/mL primary antibody solution is dripped on the surface of the glassy carbon electrode prepared in the step (2), and the glassy carbon electrode is preserved in a refrigerator at 4 ℃ until being dried and washed by ultrapure water;
(4) dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(5) dripping 6 μ L of insulin with different concentrations on the surface of the electrode, storing in a refrigerator at 4 deg.C until dry, and cleaning with ultrapure water;
(6) dropwise coating 5 mu L of 1-5 mg/mL secondary antibody-gold-curcumin/ZIF-8 solution on the surface of an electrode, storing in a refrigerator at 4 ℃ until the solution is dried, and cleaning with ultrapure water to obtain the electrochemical luminescence biosensor for detecting insulin;
the preparation method of the second antibody-gold-curcumin/ZIF-8 solution comprises the following steps:
(a) preparation of gold-curcumin nanoparticles
Firstly, 8 mg of curcumin is dispersed into 5 mL of dimethyl sulfoxide to form a transparent and uniform solution; then, 2 mL of curcumin-dimethyl sulfoxide solution is added into 45 mL of ultrapure water, and reflux stirring is carried out at 100 ℃; add 100. mu.L of 0.1M NaOH, heat the solution to 100 ℃ and add 1 mL of 2% HAuCl4Stirring the solution for 1 h, cooling to room temperature, dialyzing by using a 12 kDa dialysis bag, and removing unreacted impurities and small particles to obtain a gold-curcumin solution;
(b) preparation of gold-curcumin/ZIF-8 nanoflower
Mixing 1 mL of 2-methylimidazole with 2-5 mL of gold-curcumin solution, and stirring for 10 min; then respectively adding 1 mL of 20 mM zinc acetate solution and 0.05 g of bovine serum albumin, and stirring for 4 h; finally, centrifuging, washing and vacuum drying the obtained gold-curcumin/ZIF-8 nanoflower;
(c) preparation of secondary antibody-gold-curcumin/ZIF-8 solution
Activating carboxyl on the surface of ZIF-8 and reacting with amino on the surface of a secondary antibody by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to fix the antibody on the surface of gold-curcumin/ZIF-8; dispersing 2 mg of gold-curcumin/ZIF-8 in 1 mL of phosphate buffer solution with pH value of 7.4, and carrying out ultrasonic treatment for 30 min; then, 500. mu.L of 100 mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 500. mu.L of 400 mM N-hydroxysuccinimide were added and stirred for 1 h; then adding 500 mu L of 500 mu g/mL secondary antibody solution into the solution, and stirring for 6 h at 4 ℃; finally, 100 μ L of 1% BSA was added to block non-specific sites, centrifuged to give the second antibody-gold-curcumin/ZIF-8, which was dispersed in 1 mL of phosphate buffer solution pH =7.4 and stored in a refrigerator at 4 ℃ until use.
2. The gold-curcumin nanoparticle quenched CdS hybrid TiO of claim 12The preparation method of the electrochemical luminescence sensor for detecting insulin by the nanobelt is characterized in that CdS @ TiO2The preparation steps of the nanobelt are as follows:
dispersing 0.1 g of cadmium acetate in 30 mL of ultrapure water, adding 25-50 mg of titanium dioxide nanobelts, and carrying out ultrasonic treatment for 1 h; then, 5 mL of 5M Na was added2Stirring the S solution for 12 h, transferring the solution into a 100 mL reaction kettle, reacting for 16 h at 160 ℃, centrifuging, washing and drying to obtain CdS @ TiO2A nanoribbon.
3. A gold-curcumin nanoparticle quenched CdS hybrid TiO prepared according to the preparation method of claim 12The application of the electrochemical luminescence sensor for detecting insulin by a nanobelt is characterized in that the electrochemical luminescence sensor is applied to the detection of insulin, and the detection steps are as follows:
(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum electrode and the prepared electrochemical luminescence sensor as working electrodes in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 800V, the scanning voltage to be 0-1.6V and the scanning speed to be 0.15V/s;
(2) detecting the intensity of an electrochemiluminescence signal generated by insulin with different concentrations by an electrochemiluminescence method by using a phosphate buffer solution containing 1-8 mmol/L hydrogen peroxide and 20-120 mM potassium persulfate;
the phosphate buffer solution has pH = 5.0-8.5 and is prepared from 1/15 mol/L Na2HPO4And 1/15 mol/L KH2PO4Preparing;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the logarithm of the insulin concentration.
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