A method for constructing a glucose-oxidation-induced pH-stimulus-responsive controlled-release electrochemiluminescence sensor

Abstract

This invention develops a pH-responsive controlled-release electrochemiluminescence sensor based on glucose oxidation and its application in the detection of CYFRA 21-1, belonging to the field of electrochemiluminescence sensor construction technology. The sensor constructed in this invention uses BSA / luminol-Ab2 / SiO2-PEI@Au NPs as the secondary antibody label and CeO2-Au as the substrate material. It utilizes the oxidation of glucose to gluconic acid under voltage, thereby lowering the pH. The sensor constructed in this invention has a wide detection range, high sensitivity, and low detection limit, providing a feasible solution for the detection of CYFRA 21-1.

Description

Technical Field

[0001] This invention relates to the field of electrochemiluminescence sensor construction technology, and specifically to a method for constructing a pH-stimulated controlled release electrochemiluminescence sensor based on glucose oxidation-induced release. Background Technology

[0002] Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lungs. It has the fastest-growing incidence and mortality rates, making it one of the cancers with the highest incidence and mortality rates worldwide. CYFRA21-1, a soluble fragment of cytokeratin 19, is a sensitive biomarker for lung cancer. Achieving high sensitivity and accurate detection of CYFRA21-1 is a key issue in the early diagnosis, monitoring, and prognosis of cancer. Many methods have been used for the quantitative analysis of cancer biomarkers, such as electrochemistry, photoelectrochemical methods, colorimetry, electrochemical chemiluminescence, and fluorescence. Among these, electrochemical chemiluminescence offers numerous advantages, including high sensitivity, simple optical equipment, wide detection range, low background light, low detection cost, good controllability, and simple operation. Therefore, electrochemical chemiluminescence sensors are promising for the sensitive detection of CYFRA21-1.

[0003] This invention utilizes the principle that glucose is oxidized to gluconic acid under voltage, thus lowering the pH, to construct a novel pH-responsive controlled-release electrochemiluminescence sensor, achieving sensitive detection of CYFRA21-1. BSA / luminol-Ab2 / SiO2-PEI@Au NPs are used as the secondary antibody label, and CeO2-Au is used as the substrate material. Test results show that this controlled-release electrochemiluminescence sensor has high sensitivity, low detection limit, and good stability. Based on these findings, the inventors completed this invention. Summary of the Invention

[0004] One of the technical objectives of this invention is to utilize the fact that glucose is oxidized to gluconic acid under voltage, thereby causing a decrease in pH, and to develop a method for constructing a pH-stimulated controlled release electrochemiluminescence sensor based on glucose oxidation-induced release.

[0005] The second technical objective of this invention is to use BSA / luminol-Ab2 / SiO2-PEI@Au NPs as secondary antibody markers and CeO2-Au as substrate material to construct a sensor. The constructed sensor has good stability and high sensitivity.

[0006] The third technical objective of this invention is to realize the construction of the controlled release electrochemiluminescence sensor and its effective detection in CYFRA21-1.

[0007] The technical solution of the present invention is as follows:

[0008] 1. A method for constructing a glucose oxidation-induced pH-responsive controlled release electrochemiluminescence sensor.

[0009] (1) First, the bare glassy carbon electrode was polished with aluminum powder and rinsed with ultrapure water. 10 μL of 6-10 mg / mL CeO2-Au solution was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature.

[0010] (2) Add 10 μL of CYFRA 21-1 antibody Ab1 (1 μg / mL) and 3 µL of BSA solution (1% by mass) to the electrode surface in sequence, rinse with ultrapure water, and air dry at room temperature;

[0011] (3) Add 10 μL of a series of CYFRA 21-1 antigen solutions of different concentrations (0.000001-100 ng / mL) to the electrode, incubate for 2 h, rinse with ultrapure water, and air dry at room temperature;

[0012] (4) Add 10 μL of secondary antibody labeling BSA / luminol-Ab2 / SiO2-PEI@Au NPs solution to the electrode, rinse with ultrapure water, and air dry at room temperature to obtain a controlled release electrochemiluminescence sensor.

[0013] 2. Preparation steps of CeO2-Au

[0014] (1) Dilute 0.5-1.5 mL of HAuCl4 solution with a mass fraction of 1% in 100 mL of ultrapure water. Then, add 2.0-3.0 mL of trisodium citrate dihydrate with a mass fraction of 1% to the above solution under vigorous stirring. Boil for 10 min and then stir for 10 min. After cooling to room temperature, obtain Au NPs solution and store at 4 °C.

[0015] (2) Dissolve 6.6-6.8 mmol L-asparagine in 40 mL of ultrapure water, then add 6.6-6.8 mmol CeCl3∙7H2O to the above solution and stir for 15 min before transferring to a reaction vessel. React at 160 °C for 24 h. Then wash with ultrapure water and ethanol. The white solid obtained after vacuum drying is calcined at 360 °C for 1 h to finally obtain bright yellow CeO2. Mix 0.006-0.010 g CeO2 with 1 mL Au NPs solution, place at 4 °C and shake for 4 h, then centrifuge and wash with ultrapure water to obtain CeO2-Au.

[0016] 3. Preparation of secondary antibody markers BSA / luminol-Ab2 / SiO2-PEI@Au NPs

[0017] (1) Dissolve 0.4-0.6 g of hexadecyltrimethylammonium bromide in 200 mL of ultrapure water, then add 1.7 mL of 2.0 M NaOH solution and stir at 80 °C for 20 min. Then add 2.0-3.0 mL of tetraethyl orthosilicate to the solution and stir for 2 h until a white precipitate is obtained. Filter the prepared product and wash it with ultrapure water and methanol. After vacuum drying, a white solid is obtained. Dissolve the obtained product in 1.0-2.0 mL of 37% hydrochloric acid, then add 75 mL of methanol and place it in a flask. Reflux in an oil bath for 10 h. Then wash it 6 times alternately with ultrapure water and methanol. After drying at 60 °C for 4 h, SiO2 is obtained. Put 0.1-0.3 g of the prepared SiO2 and 0.2-0.4 g of polyethyleneimine into a beaker, then add 40 mL of ultrapure water and stir for 6 h. After centrifugation, wash with ultrapure water and finally vacuum dry at 60 °C for 6 h. h yields SiO2-PEI;

[0018] (2) Mix 0.52-0.54 mg luminol with 10 mL of PBS solution at pH 7.4. Then, add 100-300 µL of glutaraldehyde solution, 300 µL of CYFRA 21-1 antibody Ab2 solution at 1 µg / mL, 200 µL of BSA solution, 0.02 g of SiO2-PEI, and 0.5-1.5 mL of Au NPs solution to the mixture in sequence. After each addition of a solution, place the mixture at 4°C and shake for 6 h. After centrifugation, wash with ultrapure water to obtain the secondary antibody label BSA / luminol-Ab2 / SiO2-PEI@Au NPs.

[0019] 4. Detection of CYFRA 21-1

[0020] (1) The test was conducted using an electrochemical workstation with a three-electrode system. The silver / silver chloride electrode was used as the reference electrode, the platinum wire electrode as the counter electrode, and the constructed sensor as the working electrode. The test was conducted in 10 mL of PBS buffer solution with pH 7.4 containing 0.50-0.60 mol / L glucose and 0.018 mmol / L hydrogen peroxide.

[0021] (2) The intensity of electrochemiluminescence signals generated by different concentrations of CYFRA 21-1 was detected, and the scanning voltage was set to 0-0.6 V;

[0022] (3) Based on the linear relationship between the obtained electrochemiluminescence intensity and the concentration of CYFRA 21-1, plot the working curve.

[0023] The beneficial effects of this invention are:

[0024] (1) The inventors of this invention utilize the oxidation of glucose to gluconic acid under voltage to lower the pH and apply it to the construction of a pH-stimulated controlled release electrochemiluminescence sensor. This method has no toxic byproducts and has clinical application prospects.

[0025] (2) In this invention, BSA / luminol-Ab2 / SiO2-PEI@Au NPs are used as secondary antibody markers and CeO2-Au is used as substrate material in the construction of the sensor, which improves the sensitivity and stability of the sensor.

[0026] (3) The controlled release electrochemiluminescence sensor constructed in this invention is used for the detection of CYFRA21-1. This sensor can achieve high sensitivity and specificity detection. Implementation

[0027] Example 1: A method for constructing a glucose oxidation-induced pH-stimulated controlled release electrochemiluminescence sensor.

[0028] (1) First, the bare glassy carbon electrode was polished with aluminum powder and rinsed with ultrapure water. 10 μL of 6 mg / mL CeO2-Au solution was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature.

[0029] (2) Add 10 μL of CYFRA 21-1 antibody Ab1 (1 μg / mL) and 3 µL of BSA solution (1% by mass) to the electrode surface in sequence, rinse with ultrapure water, and air dry at room temperature;

[0030] (3) Add 10 μL of a series of CYFRA 21-1 antigen solutions of different concentrations (0.000001-100 ng / mL) to the electrode, incubate for 2 h, rinse with ultrapure water, and air dry at room temperature;

[0031] (4) Add 10 μL of secondary antibody labeling BSA / luminol-Ab2 / SiO2-PEI@Au NPs solution to the electrode, rinse with ultrapure water, and air dry at room temperature to obtain a controlled release electrochemiluminescence sensor.

[0032] Example 2: A method for constructing a glucose oxidation-induced pH-stimulated controlled release electrochemiluminescence sensor.

[0033] (1) First, the bare glassy carbon electrode was polished with aluminum powder and rinsed with ultrapure water. 10 μL of 8 mg / mL CeO2-Au solution was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature.

[0034] (2) Add 10 μL of CYFRA 21-1 antibody Ab1 (1 μg / mL) and 3 µL of BSA solution (1% by mass) to the electrode surface in sequence, rinse with ultrapure water, and air dry at room temperature;

[0035] (3) Add 10 μL of a series of CYFRA 21-1 antigen solutions of different concentrations (0.000001-100 ng / mL) to the electrode, incubate for 2 h, rinse with ultrapure water, and air dry at room temperature;

[0036] (4) Add 10 μL of secondary antibody labeling BSA / luminol-Ab2 / SiO2-PEI@Au NPs solution to the electrode, rinse with ultrapure water, and air dry at room temperature to obtain a controlled release electrochemiluminescence sensor.

[0037] Example 3: A method for constructing a glucose oxidation-induced pH-stimulated controlled release electrochemiluminescence sensor.

[0038] (1) First, the bare glassy carbon electrode was polished with aluminum powder and rinsed with ultrapure water. 10 μL of 10 mg / mL CeO2-Au solution was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature.

[0039] (2) Add 10 μL of CYFRA 21-1 antibody Ab1 (1 μg / mL) and 3 µL of BSA solution (1% by mass) to the electrode surface in sequence, rinse with ultrapure water, and air dry at room temperature;

[0040] (3) Add 10 μL of a series of CYFRA 21-1 antigen solutions of different concentrations (0.000001-100 ng / mL) to the electrode, incubate for 2 h, rinse with ultrapure water, and air dry at room temperature;

[0041] (4) Add 10 μL of secondary antibody labeling BSA / luminol-Ab2 / SiO2-PEI@Au NPs solution to the electrode, rinse with ultrapure water, and air dry at room temperature to obtain a controlled release electrochemiluminescence sensor.

[0042] Example 4 Preparation of CeO2-Au

[0043] (1) Dilute 0.5 mL of HAuCl4 solution with a mass fraction of 1% in 100 mL of ultrapure water. Then, add 2.0 mL of trisodium citrate dihydrate with a mass fraction of 1% to the above solution under vigorous stirring. Boil for 10 min and then stir for 10 min. After cooling to room temperature, Au NPs solution is obtained and stored at 4 °C.

[0044] (2) Dissolve 6.6 mmol L-asparagine in 40 mL of ultrapure water, then add 6.6 mmol CeCl3∙7H2O to the above solution and stir for 15 min before transferring to a reaction vessel. React at 160 °C for 24 h. Then wash with ultrapure water and ethanol. The white solid obtained after vacuum drying is calcined at 360 °C for 1 h to obtain bright yellow CeO2. Mix 0.006 g CeO2 with 1 mL Au NPs solution, place at 4 °C and shake for 4 h, then centrifuge and wash with ultrapure water to obtain CeO2-Au.

[0045] Example 5 Preparation of CeO2-Au

[0046] (1) Dilute 1.0 mL of HAuCl4 solution with a mass fraction of 1% in 100 mL of ultrapure water. Then, add 2.5 mL of trisodium citrate dihydrate with a mass fraction of 1% to the above solution under vigorous stirring. Boil for 10 min and then stir for 10 min. After cooling to room temperature, Au NPs solution is obtained and stored at 4 °C.

[0047] (2) Dissolve 6.7 mmol L-asparagine in 40 mL of ultrapure water, then add 6.7 mmol CeCl3∙7H2O to the above solution and stir for 15 min before transferring to a reaction vessel. React at 160 °C for 24 h. Then wash with ultrapure water and ethanol. The white solid obtained after vacuum drying is calcined at 360 °C for 1 h to obtain bright yellow CeO2. Mix 0.008 g CeO2 with 1 mL Au NPs solution, place at 4 °C and shake for 4 h, then centrifuge and wash with ultrapure water to obtain CeO2-Au.

[0048] Example 6 Preparation of CeO2-Au

[0049] (1) Dilute 1.5 mL of HAuCl4 solution with a mass fraction of 1% in 100 mL of ultrapure water, and then add 3.0 mL of trisodium citrate dihydrate with a mass fraction of 1% under vigorous stirring. Boil for 10 min and then stir for 10 min. After cooling to room temperature, Au NPs solution is obtained and stored at 4 °C.

[0050] (2) Dissolve 6.8 mmol L-asparagine in 40 mL of ultrapure water, then add 6.8 mmol CeCl3∙7H2O to the above solution and stir for 15 min. Transfer to a reaction vessel and react at 160 °C for 24 h. Then wash with ultrapure water and ethanol. The white solid obtained after vacuum drying is calcined at 360 °C for 1 h to obtain bright yellow CeO2. Mix 0.010 g CeO2 with 1 mL Au NPs solution, place at 4 °C and shake for 4 h. Then centrifuge and wash with ultrapure water to obtain CeO2-Au.

[0051] Example 7 Preparation of secondary antibody markers BSA / luminol-Ab2 / SiO2-PEI@Au NPs

[0052] (1) Dissolve 0.4 g of hexadecyltrimethylammonium bromide in 200 mL of ultrapure water, then add 1.7 mL of 2.0 M NaOH solution and stir at 80 °C for 20 min. Then add 2.0 mL of tetraethyl orthosilicate to the solution and stir for 2 h until a white precipitate is obtained. Then filter the prepared product and wash it with ultrapure water and methanol. After vacuum drying, a white solid is obtained. Dissolve the obtained product in 1.0 mL of 37% hydrochloric acid, then add 75 mL of methanol and place it in a flask and reflux in an oil bath for 10 h. Then wash it with ultrapure water and methanol alternately 6 times. After drying at 60 °C for 4 h, SiO2 is obtained. Put 0.1 g of the prepared SiO2 and 0.2 g of polyethyleneimine into a beaker, then add 40 mL of ultrapure water and stir for 6 h. After centrifugation, wash with ultrapure water and finally vacuum dry at 60 °C for 6 h to obtain SiO2-PEI.

[0053] (2) Mix 0.52 mg luminol with 10 mL of PBS solution with pH=7.4. Then, add 100 µL of glutaraldehyde solution, 300 µL of CYFRA 21-1 antibody Ab2 solution with 1 µg / mL, 200 µL of BSA solution, 0.02 g of SiO2-PEI, and 0.5 mL of Au NPs solution to the mixture in sequence. After each addition of a solution, place the mixture at 4 °C and shake for 6 h. After centrifugation, wash with ultrapure water to obtain the secondary antibody label BSA / luminol-Ab2 / SiO2-PEI@Au NPs.

[0054] Example 8 Preparation of secondary antibody markers BSA / luminol-Ab2 / SiO2-PEI@Au NPs

[0055] (1) Dissolve 0.5 g of hexadecyltrimethylammonium bromide in 200 mL of ultrapure water, then add 1.7 mL of 2.0 M NaOH solution and stir at 80 °C for 20 min. Then add 2.5 mL of tetraethyl orthosilicate to the solution and stir for 2 h until a white precipitate is obtained. Then filter the prepared product and wash it with ultrapure water and methanol. After vacuum drying, a white solid is obtained. Dissolve the obtained product in 1.5 mL of 37% hydrochloric acid, then add 75 mL of methanol and place it in a flask and reflux in an oil bath for 10 h. Then wash it with ultrapure water and methanol alternately 6 times. After drying at 60 °C for 4 h, SiO2 is obtained. Put 0.2 g of the prepared SiO2 and 0.3 g of polyethyleneimine into a beaker, then add 40 mL of ultrapure water and stir for 6 h. After centrifugation, wash with ultrapure water and finally vacuum dry at 60 °C for 6 h to obtain SiO2-PEI.

[0056] (2) Mix 0.53 mg luminol with 10 mL of PBS solution with pH=7.4. Then, add 200 µL of glutaraldehyde solution, 300 µL of CYFRA 21-1 antibody Ab2 solution with 1 µg / mL, 200 µL of BSA solution, 0.02 g of SiO2-PEI, and 1.0 mL of Au NPs solution to the mixture in sequence. After each addition of a solution, place the mixture at 4 °C and shake for 6 h. After centrifugation, wash with ultrapure water to obtain the secondary antibody label BSA / luminol-Ab2 / SiO2-PEI@Au NPs.

[0057] Example 9 Preparation of secondary antibody markers BSA / luminol-Ab2 / SiO2-PEI@Au NPs

[0058] (1) Dissolve 0.6 g of hexadecyltrimethylammonium bromide in 200 mL of ultrapure water, then add 1.7 mL of 2.0 M NaOH solution and stir at 80 °C for 20 min. Then add 3.0 mL of tetraethyl orthosilicate to the solution and stir for 2 h until a white precipitate is obtained. Then filter the prepared product and wash it with ultrapure water and methanol. After vacuum drying, a white solid is obtained. Dissolve the obtained product in 2.0 mL of 37% hydrochloric acid, then add 75 mL of methanol and place it in a flask and reflux in an oil bath for 10 h. Then wash it with ultrapure water and methanol alternately 6 times. After drying at 60 °C for 4 h, SiO2 is obtained. Put 0.3 g of the prepared SiO2 and 0.4 g of polyethyleneimine into a beaker, then add 40 mL of ultrapure water and stir for 6 h. After centrifugation, wash with ultrapure water and finally vacuum dry at 60 °C for 6 h to obtain SiO2-PEI.

[0059] (2) Mix 0.54 mg luminol with 10 mL of PBS solution with pH=7.4. Then add 300 µL of glutaraldehyde solution, 300 µL of CYFRA 21-1 antibody Ab2 solution with 1 µg / mL, 200 µL of BSA solution, 0.02 g of SiO2-PEI, and 1.5 mL of Au NPs solution to the mixture in sequence. After each addition of a solution, place the mixture at 4 °C and shake for 6 h. After centrifugation, wash with ultrapure water to obtain the secondary antibody label BSA / luminol-Ab2 / SiO2-PEI@Au NPs.

[0060] Example 10 Detection of CYFRA 21-1

[0061] (1) The test was conducted using an electrochemical workstation with a three-electrode system. The silver / silver chloride electrode was used as the reference electrode, the platinum wire electrode as the counter electrode, and the constructed sensor as the working electrode. The test was conducted in 10 mL of PBS buffer solution with pH 7.4 containing 0.50 mol / L glucose and 0.018 mmol / L hydrogen peroxide.

[0062] (2) The intensity of electrochemiluminescence signals generated by different concentrations of CYFRA 21-1 was detected, and the scanning voltage was set to 0-0.6 V;

[0063] (3) Based on the linear relationship between the obtained electrochemiluminescence intensity and the concentration of CYFRA 21-1, plot the working curve.

[0064] Example 11 Detection of CYFRA 21-1

[0065] (1) The test was conducted using an electrochemical workstation with a three-electrode system. The silver / silver chloride electrode was used as the reference electrode, the platinum wire electrode as the counter electrode, and the constructed sensor as the working electrode. The test was conducted in 10 mL of PBS buffer solution with pH 7.4 containing 0.55 mol / L glucose and 0.018 mmol / L hydrogen peroxide.

[0066] (2) The intensity of electrochemiluminescence signals generated by different concentrations of CYFRA 21-1 was detected, and the scanning voltage was set to 0-0.6 V;

[0067] (3) Based on the linear relationship between the obtained electrochemiluminescence intensity and the concentration of CYFRA 21-1, plot the working curve.

[0068] Example 12 Detection of CYFRA 21-1

[0069] (1) The test was conducted using an electrochemical workstation with a three-electrode system. The silver / silver chloride electrode was used as the reference electrode, the platinum wire electrode as the counter electrode, and the constructed sensor as the working electrode. The test was conducted in 10 mL of PBS buffer solution with pH 7.4 containing 0.60 mol / L glucose and 0.018 mmol / L hydrogen peroxide.

[0070] (2) The intensity of electrochemiluminescence signals generated by different concentrations of CYFRA 21-1 was detected, and the scanning voltage was set to 0-0.6 V;

[0071] (3) Based on the linear relationship between the obtained electrochemiluminescence intensity and the concentration of CYFRA 21-1, plot the working curve.

Claims

1. A method for constructing a glucose oxidation-induced pH stimuli-responsive controlled release electrochemiluminescence sensor, characterized in that, The steps are as follows: (1) Polish the bare glassy carbon electrode with aluminum powder and rinse it with ultrapure water. Add 10 μL of CeO2-Au solution with a concentration of 6-10 mg / mL to the electrode surface, rinse with ultrapure water, and air dry at room temperature. (2) Add 10 μL of CYFRA 21-1 antibody Ab1 (1 μg / mL) and 3 μL of BSA solution (1% by mass) to the electrode surface in sequence, rinse with ultrapure water, and air dry at room temperature. (3) Add 10 μL of a series of CYFRA 21-1 antigen solutions of different concentrations (0.000001-100 ng / mL) to the electrode, incubate for 2 h, rinse with ultrapure water, and air dry at room temperature; (4) 10 μL of secondary antibody labeling BSA / luminol-Ab2 / SiO2-PEI@Au NPs solution was added to the electrode, rinsed with ultrapure water, and dried at room temperature to obtain a controlled release electrochemiluminescence sensor; The secondary antibody marker BSA / luminol-Ab2 / SiO2-PEI@Au NPs was prepared by the following method: (a) Dissolve 0.4-0.6 g of hexadecyltrimethylammonium bromide in 200 mL of ultrapure water, then add 17 mL of 2.0 M NaOH solution and stir at 80°C for 20 min. Add 2.0-3.0 mL of tetraethyl orthosilicate to the solution and stir for 2 h until a white precipitate is obtained. Filter the prepared product and wash with ultrapure water and methanol. After vacuum drying, a white solid is obtained. Dissolve the obtained product in 1.0-2.0 mL of 37% hydrochloric acid, then add 75 mL of methanol and place in a flask. Reflux in an oil bath for 10 h. Wash with ultrapure water and methanol alternately 6 times. Dry at 60°C for 4 h to obtain SiO2. Place 0.1-0.3 g of the prepared SiO2 and 0.2-0.4 g of polyethyleneimine in a beaker, then add 40 mL of ultrapure water and stir for 6 h. Centrifuge, wash with ultrapure water, and finally vacuum dry at 60°C for 6 h. h yields SiO2-PEI; (b) Mix 0.52-0.54 mg luminol with 10 mL of PBS solution at pH 7.4 to obtain a mixed solution; add 100-300 μL of glutaraldehyde solution, 300 μL of CYFRA 21-1 antibody Ab2 solution at 1 μg / mL, 200 μL of BSA solution, 0.02 g of SiO2-PEI, and 0.5-15 mL of Au NPs solution to the above mixed solution in sequence. After each addition of a solution, place the solution at 4℃ and shake for 6 h. After centrifugation, wash with ultrapure water to obtain the secondary antibody label BSA / luminol-Ab2 / SiO2-PEI@AuNPs; The method for constructing a glucose oxidation-induced pH-stimulated controlled-release electrochemiluminescence sensor can be used for the detection of CYFRA 21-1 for non-disease diagnostic and / or therapeutic purposes. The specific steps are as follows: (i) The test was conducted using an electrochemical workstation with a three-electrode system, with a silver / silver chloride electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the constructed sensor as the working electrode. The test was conducted in 10 mL of PBS buffer solution at pH 7.4 containing 0.50-0.60 mol / L glucose and 0.018 mmol / L hydrogen peroxide. (ii) Detect the intensity of electrochemiluminescence signals generated by different concentrations of CYFRA 21-1, with the scanning voltage set to 0-0.6V; (iii) Plot the working curve based on the linear relationship between the obtained electrochemiluminescence intensity and the concentration of CYFRA 21-1.

2. The method for constructing a glucose oxidation-induced pH stimuli-responsive controlled release electrochemiluminescence sensor according to claim 1, wherein, In step (1), the CeO2-Au is prepared by the following method: (1) Dilute 0.5-1.5 mL of HAuCl4 solution with a mass fraction of 1% in 100 mL of ultrapure water. Then, add 2.0-3.0 mL of trisodium citrate dihydrate with a mass fraction of 1% to the above solution under vigorous stirring. Boil for 10 min and then stir for 10 min. After cooling to room temperature, obtain Au NPs solution and store at 4℃. (2) Dissolve 6.6-6.8 mmol L-asparagine in 40 mL of ultrapure water to obtain an L-asparagine solution; then add 6.6-6.8 mmol CeCl3·7H2O to the above L-asparagine solution and stir for 15 min before transferring to a reaction vessel. React at 160 °C for 24 h. Then wash with ultrapure water and ethanol. The white solid obtained after vacuum drying is calcined at 360 °C for 1 h to obtain bright yellow CeO2. Mix 0.006-0.010 g CeO2 with 1 mL Au NPs solution and place at 4 °C with shaking for 4 h. Then centrifuge and wash with ultrapure water to obtain CeO2-Au.