Preparation and application of a dual signal amplification ECL sensor based on luminol luminophore and double metal oxide

Abstract

The application relates to preparation and application of a sandwich type ECL immunosensor of a H2O2 / O2 system for signal amplification by using luminol as a luminophore and a double metal oxide, and belongs to the field of biosensing and novelty combination of new functional materials. A nickel-manganese double metal oxide (NiMnO3@Au-luminol) loaded with luminol luminophore is combined with a gold-modified cobalt-cerium double metal oxide (CoCeOx@Au) to serve as an electrochemiluminescence sensing platform, a sandwich type electrochemiluminescence immunosensor is constructed, and the sensor is used for super-sensitive detection of a non-small cell lung cancer marker CYFRA21-1. The NiMnO3 and CoCeOx materials have great application potential due to simple preparation, suitable acid-base property and multiple valence states of metals. The NiMnO3 can catalyze O2 to generate O2 •− , the CoCeOx can promote H2O2 to decompose to generate •OH, both of the two free radicals can act on Luminol •− to generate a stronger luminescence signal; Au modification on the material surface can significantly improve the electron transfer rate, increase the number of antigen-antibody combination, enhance the ECL signal, and realize sensitive and accurate detection of biomolecules.

Description

Technical Field

[0001] This invention relates to the preparation and application of an electrochemiluminescence-based immune sensor, specifically a sandwich-type ECL immune sensor using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate in an H2O2 / O2 system. This invention belongs to the fields of novel functional materials and biosensing technology. Background Technology

[0002] Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lungs. It ranks among the top cancer deaths and seriously threatens human life, health and safety. The main reason for the high mortality rate of lung cancer is the lack of typical symptoms. Most cases are discovered at an advanced stage, missing the best treatment period. CYFRA21-1 is a soluble fragment of cytokeratin 19. As a typical tumor marker, it is of great significance for the early diagnosis and treatment of non-small cell lung cancer, especially squamous cell carcinoma.

[0003] Electrochemiluminescence (ECL) is a research method that combines electrochemical techniques with chemiluminescence technology. It is a relaxation phenomenon of energy and has attracted widespread attention in ion detection, immunoassay, and other fields in recent years. ECL sensors have advantages such as low background signal, high sensitivity, and good selectivity, and can quickly and accurately detect the content of analytes.

[0004] In recent years, bimetallic oxides have become a research hotspot due to their superior electrochemical performance compared to monometallic oxides. NiMnO3@Au-luminol has three main advantages as a labeling material: First, luminol, as a traditional luminescent material, exhibits a good ECL signal; second, NiMnO3 is a bimetallic composite material with multiple oxidation states, demonstrating excellent catalytic effects on the H2O2 / O2 system, and its preparation is simple and readily available; third, Au modification of NiMnO3 not only improves the material's conductivity and accelerates electron transfer but also increases the amount of antigen-antibody binding, thereby amplifying and stabilizing the luminescent signal and improving detection sensitivity and accuracy. CoCeOx@Au, as a substrate material, also plays an important role in catalyzing H2O2 decomposition and accelerating electron conduction.

[0005] Bimetallic oxide is a hot material in the field of electrocatalysis, which has very good electrochemical effect, and is expected to be widely used in the field of biosensing due to showing more valence state conversion and better catalytic performance than single metal oxide; at present, bimetallic oxides such as NiCo2O4, CoFe2O4 and CuFe2O4 are often used in electrochemiluminescence sensors; in the present application, NiMnO3 with multiple oxidation states and oxidase-like activity and bimetallic oxide CoCeOx are used to construct an immunosensor, hydrogen peroxide and dissolved oxygen (H2O2 / O2) are used as co-reactive active substances, NiMnO3 can stabilize and enhance the ECL emission of luminol in the solution; the mechanism is mainly that O2 •− is produced by electron transfer, and the produced O2 •− reacts with luminol •− to produce more excited 3-APA*, and the excited 3-APA* produces a luminescent signal in the process of transition back to the ground state, and CoCeOx catalyzes the decomposition of H2O2 to produce •OH, and •OH can also react with luminol •− to produce 3-APA* and further produce more light signals. SUMMARY

[0006] One of the purposes of the present application is to prepare a sandwich type ECL immunosensor of H2O2 / O2 system with NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate.

[0007] The second purpose of the present application is to use the sensor for high-sensitivity, specific and accurate detection of the typical marker CYFRA21-1 of non-small cell lung cancer.

[0008] The technical scheme of the present application is as follows:

[0009] 1. Preparation of a sandwich type ECL immunosensor of H2O2 / O2 system with NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate as follows:

[0010] (1) polish the glassy carbon electrode with polishing powder, then clean it with deionized water, and place the electrode in a 5 mmol / L potassium ferricyanide solution and scan at a potential of-0.2 ~ 0.6 V, so that the difference of peak potential is less than 100 mV;

[0011] (2) drop 6 μL of 1 ~ 4 mg / mL ultrasonically treated CoCeOx@Au solution on the electrode and dry it at 4 ℃;

[0012] (3) 6 μL, 0.5 ~ 2 μg / mL CYFRA21-1 antibody was dropped on the electrode, dried in the 4 ℃ refrigerator, washed with PBS to remove the excess antibody, and dried at 4 ℃;

[0013] (4) 6 μL, 1 ~ 4 % BSA solution was dropped on the electrode to block the non-specific binding sites, dried, and then washed with PBS to remove the excess BSA, and dried at 4 ℃;

[0014] (5) 6 μL, 0.001 ~ 150 ng / mL CYFRA21-1 antigen standard solution of different concentrations was dropped on the electrode, dried at 4 ℃, washed with PBS to remove the excess antigen, and dried at 4 ℃;

[0015] (6) 6 μL, 1 ~ 8 mg / mL NiMnO3@Au-luminol labeled secondary antibody was dropped on the electrode, dried at room temperature, washed with PBS to remove the excess secondary antibody, and dried at 4 ℃, to obtain a sandwich type ECL immunosensor of the H2O2 / O2 system with NiMnO3@Au-luminol as the label and CoCeOx@Au as the substrate.

[0016] 2. A sandwich type ECL immunosensor of the H2O2 / O2 system with NiMnO3@Au-luminol as the label and CoCeOx@Au as the substrate, wherein the preparation steps of the NiMnO3@Au-luminol material are as follows:

[0017] (1) Preparation of NiMnO3

[0018] First, 1-4 mM Ni(NO3)2•6H2O, 1-4 mM KMnO4 and 1-4 mM MnSO4•H2O were dispersed in 20-80 mL of deionized water and stirred for 15 min, then 5-20 mM urea was added, and the mixture was stirred for 30 min, then the mixture was transferred to a stainless steel autoclave and kept at 160 ℃ for 12 h, after the reactor was cooled to room temperature, the reacted solution was washed with deionized water and ethanol three times, the obtained precipitate was dried at 60 ℃ overnight to obtain the NiMnO3 precursor, finally, the obtained solid was calcined at 450 ℃ in air at a heating rate of 1-4 ℃ / min for 3 h to obtain the NiMnO3 composite material;

[0019] (2) Preparation of NiMnO3@Au-luminol

[0020] Mix 0.02-0.08 g CoCeOx powder and 100-400 μL 1 % of HAuCl4•3H2O in 1-4 mL deionized water, ultrasonic for 30 minutes, then dry in vacuum oven at room temperature for 12 h, add 1-4 mL freshly prepared NaBH4 aqueous solution (0.10 M) to the dried mixture, the solution is slightly shaken in a shaker for 20 minutes (250 rpm), the resulting precipitate is centrifuged at 8000 rpm, washed with water 3 times, 10-40 mg NiMnO3@Au is dispersed in 10-40 mL water, then 1-4 mL 0.2 M luminol solution is added, and after shaking in the dark for 12 h, centrifugation gives NiMnO3@Au-luminol;

[0021] (3) Preparation of NiMnO3@Au-luminol-Ab2

[0022] The prepared 2-8 mg NiMnO3@Au-lumino is dispersed in 1-4 mL Ab2 solution (2 mg / mL), shaken at 4°C for 24 h, and finally centrifuged to obtain NiMnO3@Au-luminol-Ab2, which is stored at 4°C for further use.

[0023] 3. A sandwich type ECL immunosensor of H2O2 / O2 system with NiMnO3@Au-luminol as label and CoCeOx@Au as substrate, the preparation steps of the CoCeOx@Au material are as follows:

[0024] (1) Preparation of CoCeOx

[0025] 1-4 mmol Ce(NO3)3•6H2O is dissolved in 10-40 mL of DMF, then 0.5-2 mmol of (CH3COO)2Co•4H2O is added to the above solution and stirred vigorously, and the solution is ultrasonicated for 10 minutes to make it uniform, in addition, 2-8 mmol of terephthalic acid is dissolved in 20-80 mL of DMF and stirred vigorously, then the two parts of the solution are mixed with 2-8 mL of water and ultrasonicated for 10 minutes to make it uniform, the obtained solution is transferred to a reactor and reacted at 80°C for 24 hours, after the reactor is cooled to room temperature, the product is washed with DMF and ethanol by centrifugation 3 times or more, then it is placed in a vacuum drying oven and dried overnight, finally it is placed in a muffle furnace and heated to 350°C at a rate of 3°C per minute, and calcined for 4 hours to obtain CoCeOx powder;

[0026] (2) Preparation of CoCeOx@Au

[0027] Generally, 20-80 mg of CoCeOx powder is suspended in 1-4 mL of deionized water and ultrasonicated for 5 minutes, 100-400 μL of 1% HAuCl4·3H2O is added dropwise to the mixture and ultrasonicated for 30 minutes, and then the mixture is dried in a vacuum oven at room temperature for 12 hours. 1-4 mL of freshly prepared NaBH4 aqueous solution (0.10 M) is added to the dried mixture, the solution is gently shaken in a shaker for 20 minutes (250 rpm), and the resulting precipitate is centrifuged at 8000 rpm and washed with water 3 times;

[0028] (3) Preparation of CoCeOx@Au-Ab1

[0029] 1-4 mg of CoCeOx@Au composite is weighed, 1-4 mL of CYFRA21-1 (10 μg / mL) and 1-4 mL of PBS (pH=6.5 ~ 9.1) are added, and after constant temperature oscillation at-4 ℃ for 12 hours, the resulting precipitate is centrifuged and 1-4 mL of PBS solution is added to obtain CoCeOx@Au-Ab1.

[0030] 4. Test of CYFRA21-1, the steps are as follows:

[0031] (1) A three-electrode system of an electrochemical workstation is used for testing, an Ag / AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and the prepared electrochemiluminescence immunosensor is used as a working electrode. The electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of the photomultiplier tube is set to 550 V, the cyclic voltammetry scanning potential range is 0 ~ 0.6 V, and the scanning rate is 0.2 V / s;

[0032] (2) In 10 mL of phosphate buffer solution (PBS, 1 / 15 mol / L of KH2PO4 and 1 / 15 mol / L of Na2HPO4) containing 10-40 μL of H2O2, the working curve is drawn by detecting the electrochemiluminescence signal intensity generated by different concentrations of CYFRA21-1 standard solution through electrochemiluminescence method;

[0033] (3) The CYFRA21-1 sample solution to be tested is used instead of the CYFRA21-1 standard solution for determination.

[0034] Advantages of the present application

[0035] (1) Luminol with good luminescence effect is used as a luminophor, hydrogen peroxide / dissolved oxygen (H2O2 / O2) is used as a co-reactive active substance, strong and stable ECL emission can be generated in the solution, thereby improving the accuracy and specificity of CYFRA21-1 detection;

[0036] (2) The NiMnO3 material adopted by the application is simple to prepare, has excellent catalytic performance, has oxidase-like activity, and has great application potential in multiple fields; the valence state conversion of Ni 2+ / Ni 3+ and Mn 3+ / Mn 4+ makes the material have high catalytic activity and electrochemical activity, can promote the generation of O2 •− , the generated O2 •− reacts with luminol •− to generate a stronger luminescence signal, the Au-modified NiMnO3 not only improves the conductivity of the material and accelerates electron transfer, but also can increase the number of antigen-antibody combinations, enhance the ECL signal, and improve the sensitivity of CYFRA21-1 detection;

[0037] (3) The super-sensitive sandwich-type electrochemiluminescence immunosensor constructed by the CoCeOx@Au adopted by the application can be applied to the clinical detection of CYFRA21-1, has the advantages of simple operation, rapid detection, wide signal linear range (0.001 ~ 150 ng / mL) and low detection limit (0.3 pg / mL). DETAILED DESCRIPTION

[0038] Example 1 A sandwich-type ECL immunosensor of H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate, the preparation steps of the NiMnO3@Au-luminol material are as follows:

[0039] (1) Preparation of NiMnO3

[0040] First, 1 mM Ni(NO3)2•6H2O, 1 mM KMnO4 and 1 mM MnSO4•H2O were dispersed in 20 mL of deionized water and stirred for 15 min, then 5 mM urea was added, and the mixture was stirred for 30 min before being transferred to a stainless steel autoclave and kept at 160℃ for 12 hours. After the reactor was cooled to room temperature, the reacted solution was washed with deionized water and ethanol three times, and the obtained precipitate was dried at 60℃ overnight to obtain a NiMnO3 precursor. Finally, the obtained solid was calcined in air at 450℃ at a heating rate of 1℃ / min for 3 h to obtain a NiMnO3 composite material;

[0041] (2) Preparation of NiMnO3@Au-luminol

[0042] The prepared 0.02 g CoCeOx powder and 100 μL 1 % HAuCl4·3H2O were mixed in 1 mL deionized water, ultrasonic for 30 min, and then dried in a vacuum oven at room temperature for 12 h. 1 mL of freshly prepared NaBH4 aqueous solution (0.10 M) was added to the dried mixture, the solution was slightly shaken in a shaker for 20 min (250 rpm), the obtained precipitate was centrifuged at 8000 rpm, washed with water for 3 times, 10 mg of NiMnO3@Au was dispersed in 10 mL of water, 1 mL of 0.2 M luminol solution was added, and after shaking in the dark for 12 h, centrifugation was performed to obtain NiMnO3@Au-luminol;

[0043] (3) Preparation of NiMnO3@Au-luminol-Ab2

[0044] The prepared 2 mg of NiMnO3@Au-luminol was dispersed in 1 mL of Ab2 solution (2 mg / mL), shaken at 4 °C for 24 h, and finally centrifuged to obtain NiMnO3@Au-luminol-Ab2, which was stored at 4 °C for further use.

[0045] Example 2 A sandwich type ECL immunosensor of H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate, the preparation steps of the NiMnO3@Au-luminol material are as follows:

[0046] (1) Preparation of NiMnO3

[0047] First, 2 mM Ni(NO3)2·6H2O, 2 mM KMnO4 and 2 mM MnSO4·H2O were dispersed in 40 mL of deionized water, stirred for 15 min, and then 10 mM urea was added. After stirring for 30 min, the mixture was transferred to a stainless steel autoclave, kept at 160 °C for 12 h, and the reactor was cooled to room temperature. The prepared solution was washed with deionized water and ethanol three times, and the obtained precipitate was dried at 60 °C overnight to obtain the NiMnO3 precursor. Finally, the obtained solid was calcined at 450 °C in air at a heating rate of 2 °C / min for 3 h to obtain the NiMnO3 composite material;

[0048] (2) Preparation of NiMnO3@Au-luminol

[0049] The prepared 0.04 g CoCeOx powder and 200 μL 1 % HAuCl4·3H2O were mixed in 2 mL deionized water, ultrasonic for 30 min, and then dried in a vacuum oven at room temperature for 12 h. 2 mL of freshly prepared NaBH4 aqueous solution (0.10 M) was added to the dried mixture, and the solution was gently shaken in a shaker for 20 min (250 rpm). The obtained precipitate was centrifuged at 8000 rpm, washed with water for 3 times. 20 mg of NiMnO3@Au was dispersed in 20 mL of water, and then 2 mL of 0.2 M luminol solution was added. After shaking in the dark for 12 h, centrifugation was performed to obtain NiMnO3@Au-luminol;

[0050] (3) Preparation of NiMnO3@Au-luminol-Ab2

[0051] The prepared 4 mg of NiMnO3@Au-luminol was dispersed in 2 mL of Ab2 solution (2 mg / mL) and shaken at 4°C for 24 h. Finally, centrifugation was performed to obtain NiMnO3@Au-luminol-Ab2, which was stored at 4°C for further use.

[0052] Example 3 A sandwich-type ECL immunosensor of H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate, wherein the preparation steps of the NiMnO3@Au-luminol material are as follows:

[0053] (1) Preparation of NiMnO3

[0054] First, 4 mM Ni(NO3)2·6H2O, 4 mM KMnO4 and 4 mM MnSO4·H2O were dispersed in 80 mL of deionized water and stirred for 15 min. Then, 20 mM urea was added, and the mixture was stirred for 30 min. The mixture was then transferred to a stainless steel autoclave and kept at 160°C for 12 h. After the reactor was cooled to room temperature, the reacted solution was washed with deionized water and ethanol three times. The obtained precipitate was dried at 60°C overnight to obtain a NiMnO3 precursor. Finally, the obtained solid was calcined in air at 450°C at a heating rate of 4°C / min for 3 h to obtain a NiMnO3 composite material.

[0055] (2) Preparation of NiMnO3@Au-luminol

[0056] CoCeOx powder 0.08 g and 400 μL of 1 % HAuCl4.3H2O were mixed in 4 mL of deionized water, ultrasonicated for 30 minutes and then dried in a vacuum oven at room temperature for 12 h. To the dried mixture, 4 mL of freshly prepared aqueous NaBH4 solution (0.10 M) was added. The solution was gently shaken in a shaker for 20 minutes (250 rpm). The resulting precipitate was centrifuged at 8000 rpm and washed with water three times. 40 mg of NiMnO3@Au was dispersed in 40 mL of water and 4 mL of 0.2 M luminol solution was added. After shaking in the dark for 12 h, NiMnO3@Au-luminol was obtained by centrifugation.

[0057] (3) Preparation of NiMnO3@Au-luminol-Ab2

[0058] The prepared 8 mg of NiMnO3@Au-luminol was dispersed in 4 mL of Ab2 solution (2 mg / mL) and shaken at 4 °C for 24 h. Finally, NiMnO3@Au-luminol-Ab2 was obtained by centrifugation and stored at 4 °C for further use.

[0059] Example 4 A sandwich-type ECL immunosensor of H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate, wherein the preparation steps of the CoCeOx@Au material are as follows:

[0060] (1) Preparation of CoCeOx

[0061] 1 mmol of Ce(NO3)3.6H2O was dissolved in 10 mL of DMF. Then, 0.5 mmol of (CH3COO)2Co.4H2O was added to the above solution and stirred vigorously. The solution was ultrasonicated for 10 minutes to make it uniform. In addition, 2 mmol of terephthalic acid was dissolved in 20 mL of DMF and stirred vigorously. Then, the two parts of the solution were mixed with 2 mL of water and ultrasonicated for 10 minutes to make it uniform. The resulting solution was transferred to a reactor and reacted at 80 °C for 24 hours. After the reactor was cooled to room temperature, the product was washed with DMF and ethanol by centrifugation three or more times. Then, it was placed in a vacuum drying oven and dried overnight. Finally, it was placed in a muffle furnace and heated to 350 °C at a rate of 3 °C per minute. After 4 hours of calcination, CoCeOx powder was obtained.

[0062] (2) Preparation of CoCeOx@Au

[0063] Generally, 20 mg of CoCeOx powder was suspended in 1 mL of deionized water for 5 minutes, 100 μL of 1 % HAuCl4•3H2O was added dropwise to the mixture and ultrasonicated for 30 min, then the mixture was dried in a vacuum oven at room temperature for 12 h, 1 mL of freshly prepared NaBH4 aqueous solution (0.10 M) was added to the dried mixture, the solution was gently shaken in a shaker for 20 min (250 rpm), and the resulting precipitate was centrifuged at 8000 rpm and washed with water 3 times;

[0064] (3) Preparation of CoCeOx@Au-Ab1

[0065] 1 mg of CoCeOx@Au composite was weighed, 1 mL of CYFRA21-1 (10 μg / mL) and 1 mL of PBS (pH=6.5~9.1) were added, and after constant temperature oscillation at -4 ℃ for 12 h, centrifugal treatment was performed, and 1 mL of PBS solution was added to the obtained precipitate to obtain CoCeOx@Au-Ab1.

[0066] Example 5 A sandwich type ECL immunosensor of H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate, and the preparation steps of the CoCeOx@Au material are as follows:

[0067] (1) Preparation of CoCeOx

[0068] 2 mmol of Ce(NO3)3•6H2O was dissolved in 20 mL of DMF, then 1 mmol of (CH3COO)2Co•4H2O was added to the solution and stirred vigorously, and the solution was ultrasonicated for 10 min to make it uniform, in addition, 4 mmol of terephthalic acid was dissolved in 40 mL of DMF and stirred vigorously, then the two parts of the solution were mixed with 4 mL of water and ultrasonicated for 10 min to make them uniform, the obtained solution was transferred to a reactor and reacted at 80 ℃ for 24 h, after the reactor was cooled to room temperature, the product was washed with DMF and ethanol by centrifugation 3 times or more, then it was placed in a vacuum drying oven and dried overnight, finally it was placed in a muffle furnace and heated to 350 ℃ at a rate of 3 ℃ per min, and calcined for 4 h to obtain CoCeOx powder;

[0069] (2) Preparation of CoCeOx@Au

[0070] Generally, 40 mg of CoCeOx powder was suspended in 2 mL of deionized water for 5 minutes, 200 μL of 1 % HAuCl4•3H2O was added dropwise to the mixture and ultrasonicated for 30 min, then the mixture was dried in a vacuum oven at room temperature for 12 h, 2 mL of freshly prepared NaBH4 aqueous solution (0.10 M) was added to the dried mixture, the solution was gently shaken in a shaker for 20 min (250 rpm), and the resulting precipitate was centrifuged at 8000 rpm and washed with water 3 times;

[0071] (3) Preparation of CoCeOx@Au-Ab1

[0072] 2 mg of CoCeOx@Au composite was weighed, 2 mL of CYFRA21-1 (10 μg / mL) and 2 mL of PBS (pH = 6.5~ 9.1) were added, and after constant temperature oscillation at -4 ℃ for 12 h, centrifugal treatment was performed, and 2 mL of PBS solution was added to the obtained precipitate to obtain CoCeOx@Au-Ab1.

[0073] Example 6 A sandwich type ECL immunosensor of H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate, and the preparation steps of the CoCeOx@Au material are as follows:

[0074] (1) Preparation of CoCeOx

[0075] 4 mmol of Ce(NO3)3•6H2O was dissolved in 40 mL of DMF, then 2 mmol of (CH3COO)2Co•4H2O was added to the solution and stirred vigorously, and the solution was ultrasonicated for 10 min to make it uniform, in addition, 8 mmol of terephthalic acid was dissolved in 80 mL of DMF and stirred vigorously, then the two parts of the solution were mixed with 8 mL of water and ultrasonicated for 10 min to make them uniform, the obtained solution was transferred to a reactor and reacted at 80 ℃ for 24 h, after the reactor was cooled to room temperature, the product was washed with DMF and ethanol by centrifugation 3 times or more, then it was placed in a vacuum drying oven and dried overnight, finally it was placed in a muffle furnace and heated to 350 ℃ at a rate of 3 ℃ per min, and after 4 h of calcination, CoCeOx powder was obtained;

[0076] (2) Preparation of CoCeOx@Au

[0077] Under normal circumstances, 80 mg of CoCeOx powder is suspended in 4 mL of deionized water and sonicated for 5 minutes. 400 μL of 1% HAuCl4•3H2O is added dropwise to the above mixture and sonicated for 30 minutes. Then, the mixture is dried at room temperature in a vacuum oven for 12 h. 4 mL of freshly prepared NaBH4 aqueous solution (0.10 M) is added to the dried mixture, and the solution is gently shaken in a shaker for 20 minutes (250 rpm). The resulting precipitate is centrifuged at 8000 rpm and washed three times with water.

[0078] (3) Preparation of CoCeOx@Au-Ab1

[0079] Weigh 4 mg of CoCeOx@Au complex, add 4 mL of CYFRA21-1 (10 μg / mL) and 4 mL of PBS (pH=6.5~9.1), incubate at -4 ℃ with shaking for 12 h, then centrifuge. Add 4 mL of PBS solution to the precipitate to obtain CoCeOx@Au-Ab1.

[0080] Example 7: The fabrication of a sandwich-type ECL immunosensor using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate in an H2O2 / O2 system is as follows:

[0081] (1) Polish the glassy carbon electrode with polishing powder, then clean it with deionized water, place the electrode in a 5 mmol / L potassium ferricyanide solution, and scan it at a potential of -0.2 ~ 0.6 V so that the difference in peak potential is less than 100 mV;

[0082] (2) Add 6 μL of 1 mg / mL of sonicated CoCeOx@Au solution to the electrode and dry it at 4 °C;

[0083] (3) Add 6 μL of 0.5 μg / mL CYFRA21-1 antibody to the electrode, dry it at 4 °C, wash it with PBS to remove excess antibody, and dry it at 4 °C.

[0084] (4) Add 6 μL of 1% BSA solution to the electrode to block non-specific binding sites. After drying, wash away excess BSA with PBS and dry at 4 °C.

[0085] (5) Add 6 μL of a series of CYFRA21-1 antigen standard solutions of different concentrations (0.001 ~ 150 ng / mL) to the electrode, dry at 4 °C, wash with PBS to remove excess antigen, and dry at 4 °C.

[0086] (6) 6 μL, 1 mg / mL NiMnO3@Au-luminol labeled secondary antibody was added on the electrode, dried at room temperature, then washed with PBS to remove the excess secondary antibody, dried at 4 ℃, to prepare a kind of NiMnO3@Au-luminol labeled sandwich-type ECL immunosensor of H2O2 / O2 system with CoCeOx@Au as substrate.

[0087] Example 8: A kind of NiMnO3@Au-luminol labeled sandwich-type ECL immunosensor of H2O2 / O2 system with CoCeOx@Au as substrate was prepared as follows:

[0088] (1) The glassy carbon electrode was polished with polishing powder, then cleaned with deionized water, and then placed in a 5 mmol / L potassium ferricyanide solution and scanned at a potential of-0.2 ~ 0.6 V, so that the difference of peak potential was less than 100 mV;

[0089] (2) 6 μL, 2 mg / mL CoCeOx@Au solution after ultrasonic was added on the electrode, dried at 4 ℃;

[0090] (3) 6 μL, 1 μg / mL CYFRA21-1 antibody was added on the electrode, dried in a 4 ℃ refrigerator, then washed with PBS to remove the excess antibody, and dried at 4 ℃;

[0091] (4) 6 μL, 2% BSA solution was added on the electrode to block the non-specific binding sites, then washed with PBS to remove the excess BSA, and dried at 4 ℃;

[0092] (5) 6 μL, 0.001 ~ 150 ng / mL CYFRA21-1 antigen standard solution with different concentrations was added on the electrode, dried at 4 ℃, then washed with PBS to remove the excess antigen, and dried at 4 ℃;

[0093] (6) 6 μL, 4 mg / mL NiMnO3@Au-luminol labeled secondary antibody was added on the electrode, dried at room temperature, then washed with PBS to remove the excess secondary antibody, dried at 4 ℃, to prepare a kind of NiMnO3@Au-luminol labeled sandwich-type ECL immunosensor of H2O2 / O2 system with CoCeOx@Au as substrate.

[0094] Example 9: A kind of NiMnO3@Au-luminol labeled sandwich-type ECL immunosensor of H2O2 / O2 system with CoCeOx@Au as substrate was prepared as follows:

[0095] (1) The glassy carbon electrode was polished with polishing powder, then cleaned with deionized water, and placed in a 5 mmol / L potassium ferricyanide solution and scanned at a potential of -0.2 ~ 0.6 V, so that the difference between the peak potentials was less than 100 mV;

[0096] (2) 6 μL of 4 mg / mL ultrasonic CoCeOx@Au solution was dropped on the electrode and dried at 4°C;

[0097] (3) 6 μL of 2 μg / mL CYFRA21-1 antibody was dropped on the electrode, dried in a 4°C refrigerator, and then cleaned with PBS to remove excess antibody, and dried at 4°C;

[0098] (4) 6 μL of 4% BSA solution was dropped on the electrode to block non-specific binding sites, and then cleaned with PBS to remove excess BSA, and dried at 4°C;

[0099] (5) 6 μL of a series of CYFRA21-1 antigen standard solutions with different concentrations of 0.001 ~ 150 ng / mL was dropped on the electrode, dried at 4°C, and then cleaned with PBS to remove excess antigen, and dried at 4°C;

[0100] (6) 6 μL of 8 mg / mL NiMnO3@Au-luminol labeled secondary antibody was dropped on the electrode, dried at room temperature, and then cleaned with PBS to remove excess secondary antibody, and dried at 4°C, to prepare a sandwich type ECL immunosensor of the H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate.

[0101] Example 10: CYFRA21-1 detection, the steps are as follows:

[0102] (1) The three-electrode system of the electrochemical workstation was used for testing, an Ag / AgCl electrode was used as a reference electrode, a platinum wire electrode was used as a counter electrode, and the prepared electrochemiluminescence immunosensor was used as a working electrode. The electrochemical workstation and the chemiluminescence detector were connected together, the high voltage of the photomultiplier tube was set to 550 V, the cyclic voltammetry scanning potential range was 0 ~ 0.6 V, and the scanning rate was 0.2 V / s;

[0103] (2) In 10 mL of phosphate buffer solution (PBS, 1 / 15 mol / L of KH2PO4 and 1 / 15 mol / L of Na2HPO4) containing 10 μL of H2O2, the working curve was drawn by detecting the electrochemiluminescence signal intensity generated by different concentrations of CYFRA21-1 standard solution through electrochemiluminescence method.

[0104] (3) The to-be-tested CYFRA21-1 sample solution is used instead of the CYFRA21-1 standard solution for determination.

[0105] Example 11: Test of CYFRA21-1, the steps are as follows:

[0106] (1) The test is performed using a three-electrode system of an electrochemical workstation, an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and the prepared electrochemiluminescence immunosensor as a working electrode. The electrochemical workstation and a chemiluminescence detector are connected together, the high voltage of a photomultiplier tube is set to 550 V, the cyclic voltammetry scanning potential range is 0 ~ 0.6 V, and the scanning rate is 0.2 V / s;

[0107] (2) In 10 mL of a phosphate buffer solution (PBS, 1 / 15 mol / L of KH2PO4 and 1 / 15 mol / L of Na2HPO4) containing 20 μL of H2O2 and having a pH of 8.0, the working curve is drawn by detecting the electrochemiluminescence signal intensity generated by different concentrations of CYFRA21-1 standard solution through an electrochemiluminescence method;

[0108] (3) The to-be-tested CYFRA21-1 sample solution is used instead of the CYFRA21-1 standard solution for determination.

[0109] Example 12: Test of CYFRA21-1, the steps are as follows:

[0110] (1) The test is performed using a three-electrode system of an electrochemical workstation, an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and the prepared electrochemiluminescence immunosensor as a working electrode. The electrochemical workstation and a chemiluminescence detector are connected together, the high voltage of a photomultiplier tube is set to 550 V, the cyclic voltammetry scanning potential range is 0 ~ 0.6 V, and the scanning rate is 0.2 V / s;

[0111] (2) In 10 mL of a phosphate buffer solution (PBS, 1 / 15 mol / L of KH2PO4 and 1 / 15 mol / L of Na2HPO4) containing 40 μL of H2O2 and having a pH of 9.1, the working curve is drawn by detecting the electrochemiluminescence signal intensity generated by different concentrations of CYFRA21-1 standard solution through an electrochemiluminescence method;

[0112] (3) The to-be-tested CYFRA21-1 sample solution is used instead of the CYFRA21-1 standard solution for determination.

Claims

1. A sandwich-type ECL immunosensor of H2O2 / O2 system with NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate, characterized in that The construction comprises the following steps: (1) polishing the glassy carbon electrode with polishing powder, then cleaning it with deionized water, placing the electrode in a 5 mmol / L potassium ferricyanide solution, and scanning at a potential of -0.2 ~ 0.6 V to make the difference between peak potentials less than 100 mV; (2) adding 6 μL of 1 ~ 4 mg / mL ultrasonically treated CoCeOx@Au solution to the electrode and drying at 4 ℃; (3) adding 6 μL of 0.5 ~ 2 μg / mL CYFRA21-1 antibody to the electrode, drying in a 4 ℃ refrigerator, then cleaning with PBS to remove excess antibody, and drying at 4 ℃; (4) adding 6 μL of 1 ~ 4 % BSA solution to the electrode to block non-specific binding sites, drying, then cleaning with PBS to remove excess BSA, and drying at 4 ℃; (5) adding 6 μL of 0.001 ~ 150 ng / mL CYFRA21-1 antigen standard solution of different concentrations to the electrode, drying at 4 ℃, then cleaning with PBS to remove excess antigen, and drying at 4 ℃; (6) adding 6 μL of 1 ~ 8 mg / mL NiMnO3@Au-luminol labeled secondary antibody to the electrode, drying at room temperature, then cleaning with PBS to remove excess secondary antibody, and drying at 4 ℃, to obtain a sandwich type ECL immunosensor of the H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate.

2. The sandwich type ECL immunosensor of the H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate according to claim 1, wherein the preparation steps of the NiMnO3@Au-luminol material are as follows: (1) Preparation of NiMnO3 First, 1-4 mM Ni(NO3)2•6H2O, 1-4 mM KMnO4 and 1-4 mM MnSO4•H2O were dispersed in 20-80 mL deionized water and stirred for 15 min, then 5-20 mM urea was added, and the mixture was stirred for 30 min before being transferred to a stainless steel autoclave and kept at 160 ℃ for 12 h. After the reactor cooled to room temperature, the reacted solution was washed with deionized water and ethanol three times, and the obtained precipitate was dried at 60 ℃ overnight to obtain the NiMnO3 precursor. Finally, the obtained solid was calcined at 450 ℃ in air at a heating rate of 1-4 ℃ / min for 3 h to obtain the NiMnO3 composite material; (2) Preparation of NiMnO3@Au-luminol ​ ​ Mix 0.02-0.08 g CoCeOx powder and 100-400 μL 1 % of HAuCl4•3H2O in 1-4 mL deionized water, ultrasonic for 30 minutes, then dry in a vacuum oven at room temperature for 12 h, add 1-4 mL of freshly prepared 0.10 M NaBH4 aqueous solution to the dried mixture, gently shake the solution in a shaker at 250 rpm for 20 minutes, centrifuge the obtained precipitate at 8000 rpm, wash with water 3 times, disperse 10-40 mg NiMnO3@Au in 10-40 mL water, then add 1-4 mL of 0.2 M luminol solution, shake in the dark for 12 h, then centrifuge to obtain NiMnO3@Au-luminol; (3) Preparation of NiMnO3@Au-luminol-Ab2 Disperse the prepared 2-8 mg NiMnO3@Au-luminol in 2 mg / mL 1-4 mL Ab2 solution, shake at 4 ℃ for 24 h, and finally centrifuge to obtain NiMnO3@Au-luminol-Ab2, store at 4 °C for further use.

3. A sandwich type ECL immunosensor of H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate according to claim 1, wherein the CoCeOx@Au material is prepared by the following steps: (1) Preparation of CoCeOx Dissolve 1-4 mmol Ce(NO3)3•6H2O in 10-40 mL DMF, then add 0.5-2 mmol (CH3COO)2Co•4H2O to the above solution and stir vigorously, ultrasonic for 10 minutes to make the solution uniform, in addition, dissolve 2-8 mmol of terephthalic acid in 20-80 mL of DMF, stir vigorously, then mix the two solutions with 2-8 mL of water, ultrasonic for 10 minutes to uniform, transfer the obtained solution to a reactor, react at 80 ℃ for 24 hours, after the reactor is cooled to room temperature, the product is washed with DMF and ethanol by centrifugation 3 times or more, then put it into a vacuum drying oven, dry overnight, finally put it into a muffle furnace, heat to 350 ℃ at a rate of 3 ℃ per minute, calcine for 4 hours to obtain CoCeOx powder; (2) Preparation of CoCeOx@Au CoCeOx@Au-Ab1 was prepared by the following steps: 20-80 mg CoCeOx powder was suspended in 1-4 mL deionized water for 5 min, 100-400 μL 1% HAuCl4·3H2O was added dropwise, and the mixture was ultrasonicated for 30 min, and then dried in a vacuum oven at room temperature for 12 h. To the dried mixture, 1-4 mL of freshly prepared 0.10 M NaBH4 aqueous solution was added, and the solution was gently shaken at 250 rpm in a shaker for 20 min. The obtained precipitate was centrifuged at 8000 rpm, and washed with water for 3 times; (3) Preparation of CoCeOx@Au-Ab1 1-4 mg CoCeOx@Au composite was weighed, 1-4 mL CYFRA21-1 with a concentration of 10 μg / mL and 1-4 mL PBS with a pH of 6.5-9.1 were added, and the mixture was incubated at -4 ℃ for 12 h. After centrifugal treatment, 1-4 mL PBS solution was added to the obtained precipitate to obtain CoCeOx@Au-Ab1.

4. The sandwich-type ECL immunosensor of H2O2 / O2 system using NiMnO3@Au-luminol as a label and CoCeOx@Au as a substrate according to claim 1, wherein the detection steps are as follows: (1) The three-electrode system of the electrochemical workstation was used for testing, Ag / AgCl electrode was used as the reference electrode, platinum wire electrode was used as the counter electrode, and the prepared electrochemiluminescence immunosensor was used as the working electrode. The electrochemical workstation and the chemiluminescence detector were connected together, the high voltage of the photomultiplier tube was set to 550 V, the cyclic voltammetry scanning potential range was 0-0.6 V, and the scanning rate was 0.2 V / s; (2) In 10 mL phosphate buffer solution containing 10-40 μL H2O2 and having a pH of 6.5-9.1, the working curve was drawn by detecting the electrochemiluminescence signal intensity generated by different concentrations of CYFRA21-1 standard solution by electrochemiluminescence method, and the concentrations of KH2PO4 and Na2HPO4 in the phosphate buffer solution were both 1 / 15 mol / L; (3) The CYFRA21-1 sample solution to be tested was used instead of the CYFRA21-1 standard solution for determination.