A preparation method of a low potential near-infrared electrochemiluminescence immunosensor with tert-butylamine-borane as a co-reagent

Abstract

The application relates to a preparation method of a low-potential near-infrared electrochemiluminescence immunosensor taking t-butylamine-borane as a co-reagent. The application relates to a preparation of a low-potential near-infrared electrochemiluminescence (about 0.57 V vs Ag / AgCl) immunosensor, mainly including (1) a preparation method of water-soluble gold nanoclusters (Cit / MSA@Au NCs) capable of generating low-potential near-infrared electrochemiluminescence, taking double ligand citrate and thiomalic acid as stabilizers, (2) preparation of gold nanocluster-labeled secondary antibody (Cit / MSA@Au|Ab2), (3) preparation of a low-potential near-infrared electrochemiluminescence sensor based on a sandwich immunization principle and taking gold nanoclusters as a label, and (4) drawing of a working curve. The prepared low-potential near-infrared electrochemiluminescence immunosensor has high detection sensitivity, and other antigen proteins do not interfere with the sensing and detection of the target antigen of the application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical technology methods, and relates to a preparation method of a low-potential near-infrared electrochemiluminescence immunosensor taking tert-butylamine-borane as a co-reactant. BACKGROUND

[0002] At present, most of the electrochemiluminescence biochemical analysis and clinical diagnosis adopt ruthenium bipyridine as a marker, and the electrochemiluminescence radiation potential thereof is about 1.2 V. Traditional co-reactants mainly include tripropylamine, oxalate, and persulfate. Liu et al. studied the anodic electrochemiluminescence of Ru(bpy)3 2+ / 2-N-dibutylaminoethanol as a co-reactant. However, the oxidation potential of tripropylamine and 2-N-dibutylaminoethanol is relatively high, and there is strong electrochemical interference in a relatively wide potential window, which is not conducive to the stability of the electrode and the detection of biochemical reagents. Therefore, it is crucial to develop an electrochemiluminescence system with a lower radiation potential.

[0003] A broad-spectrum low-potential electrochemiluminescence system and a construction method thereof are disclosed in Chinese patent document CN111139066B. Fu et al. disclosed a low-potential electrochemiluminescence system taking hydrazine hydrate as a co-reactant and copper indium sulfide as a luminescent substance (Anal. Chem. 2019, 91, 10221). However, hydrazine hydrate has relatively high biological toxicity, and the electrochemiluminescence intensity of the carbonyl hydrazine system is much lower than that of the electrochemiluminescence radiation intensity of the hydrazine hydrate system.

[0004] At present, there are few low-potential electrochemiluminescence systems, and the electrochemiluminescence efficiency of the existing low-potential co-reactant system is low, and the co-reactant is toxic. Therefore, it is urgent to develop a low-potential electrochemiluminescence system that can match the hydrazine hydrate system, reduce electrochemical interference, improve the electrochemiluminescence intensity of the low-potential system, and avoid the influence of high potential on biochemical substances. SUMMARY

[0005] In view of the problems of the prior art, especially the few low-potential electrochemiluminescence systems, the low electrochemiluminescence efficiency, and the toxicity of the co-reactant, the present application provides a preparation method of a low-potential near-infrared electrochemiluminescence immunosensor taking tert-butylamine-borane as a co-reactant, which takes gold nanoclusters as a marker and tert-butylamine-borane as a co-reactant. The electrochemiluminescence potential of the immunosensor is located at 0.57 V, which can match the low-potential electrochemiluminescence system of the hydrazine hydrate system, and is safe and non-toxic.

[0006] Term explanation:

[0007] Antigens: The antigens (Ag) described in this invention refer to conventional antigens such as fetal protein antigen (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), rabbit treponemal antigen 15 (TP15), rabbit treponemal antigen 17 (TP17), or prostate-specific antigen.

[0008] Primary antibody: The primary antibody (Ab1) mentioned in this invention refers to the antibody produced in response to the antigen. The monoclonal antibody of this invention is more effective against the antigen.

[0009] Secondary antibody: The secondary antibody mentioned in this invention refers to the secondary antibody generated in response to the above-mentioned antigen and primary antibody.

[0010] U is defined as the amount of enzyme required to catalyze the reaction of 1 μmol of substrate within 1 minute under experimentally specified conditions, such as optimal temperature, optimal pH, and optimal substrate concentration.

[0011] This invention is achieved through the following technical solution:

[0012] A method for preparing a low-potential near-infrared electrochemiluminescence immunosensor includes the following steps:

[0013] (1) Using chloroauric acid as the gold source and citrate and thiomalic acid as reducing agents and stabilizers, Au is reduced by ligands. 3+ Water-soluble, dual-ligand-stabilized gold nanoclusters (Cit / MSA@Au NCs) were prepared.

[0014] (2) Preparation of dual-ligand stable gold nanocluster labeled secondary antibody Cit / MSA@Au|Ab2;

[0015] (3) A low-potential near-infrared electrochemiluminescence immunosensor was prepared by grafting and fixing Cit / MSA@Au|Ab2 onto the working electrode surface in the form of a sandwich immune complex formed on the working electrode surface.

[0016] According to a preferred embodiment of the present invention, the specific preparation method of the water-soluble dual-ligand-stabilized gold nanoclusters in step (1) is as follows:

[0017] a. Mix the chloroauric acid (H4AuCl4) solution and the thiomalic acid (MSA) ligand by ultrasonication until homogeneous;

[0018] b. The solution obtained in step a is heated and reacted to obtain gold nanoclusters coated with a single ligand thiomalic acid. The solution changes from colorless to brownish-yellow.

[0019] c. Add citrate (Cit) to the crude solution obtained in step (2), mix by sonication, and incubate at room temperature;

[0020] d, after the reaction is completed, centrifugal removal of the bottom of the large particles, the solution is purified with isopropanol, the resulting precipitate is a water-soluble double-ligand stabilized gold nanoclusters.

[0021] In the preparation of water-soluble double-ligand stabilized gold nanoclusters, the preferred conditions are as follows:

[0022] In step a, the molar ratio of gold to thiomalic acid is 1: (1-5), and further preferably, the molar ratio of gold to thiomalic acid is 1:3.

[0023] In step a, the concentration of H4AuCl4 solution is 80-100mM.

[0024] In step b, the heating temperature is 90-100℃, and the heating time is 130-150h.

[0025] In step c, the molar ratio of citrate to gold is 1:350-500.

[0026] In step c, the room temperature incubation time is 0.5-3h.

[0027] In step c, isopropanol purification is to add isopropanol to the solution, and centrifuge at 13300r / min.

[0028] In a preferred embodiment of the present application, the preparation method of water-soluble double-ligand stabilized gold nanoclusters comprises the following steps:

[0029] a, 610-650μL of 96mM H4AuCl4 solution is ultrasonically mixed with 10-30mL of 9.6mM thiomalic acid solution, and the mixture is heated to reflux at 90-100℃ for 130-150h to obtain thiomalic acid coated gold nanoclusters;

[0030] b, 0.1-0.3mM of citrate solution is mixed with the solution obtained in step (1), ultrasonically mixed, and incubated at room temperature for 1h to obtain citrate and thiomalic acid coated gold nanoclusters;

[0031] c, the solution obtained in step b is purified by centrifugation with isopropanol, and the resulting precipitate is a double-ligand coated gold nanocluster.

[0032] According to the present application, preferably, in step (2), the preparation of double-ligand stabilized gold nanocluster labeled secondary antibody Cit / MSA@Au|Ab2 is as follows:

[0033] The 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and hydroxyl succinimide are added to the water-soluble double-ligand stabilized gold nanoclusters (Cit / MSA@Au NCs), and the activation is performed for 20-30 min, and then the centrifugation is performed, and the obtained product is redissolved in a phosphate buffer solution, and then the corresponding secondary antibody is added, and the incubation is performed at 37 ℃, and the bovine serum albumin is used for blocking, and the obtained product is the Cit / MSA@Au|Ab2, and the Cit / MSA@Au|Ab2 is redissolved in the phosphate buffer solution.

[0034] According to the application, preferably, the concentration of the phosphate buffer solution is 0-0.02 mol / L, and the pH is 6.0-8.0, and the concentration of the redissolved product in the phosphate buffer solution is 1-10 μg / mL.

[0035] According to the application, preferably, in the step (3), the preparation process of the low-potential near-infrared electrochemiluminescence immunosensor is as follows:

[0036] (i) : the polished glassy carbon electrode is placed in a 1.0 mM aqueous p-aminobenzoic acid solution, and the scanning is performed at a scanning speed of 10 mV / S in the range of 0.4 V-1.2 V for 2 cycles, so that the p-aminobenzoic acid is polymerized to the surface of the glassy carbon electrode, and the electrode is washed with 10 mM PBS of pH 7.4 to remove the unreacted p-aminobenzoic acid;

[0037] (ii) : the mixed solution of EDC and NHS is added to the surface of the glassy carbon electrode modified electrode obtained in (i), and the activation is performed for 20-30 min, and then the electrode is washed with 10 mM phosphate buffer solution of pH 7.4 to remove the unreacted activator;

[0038] (iii) : the aqueous solution of the primary antibody (Ab1) is added to the surface of the activated electrode obtained in (ii), and the incubation is performed for 3 h, the bovine serum albumin is used for blocking the unreacted active sites of the electrode, and the electrode is washed;

[0039] (iv) : the aqueous solution of the antigen (Ag) is added to the electrode surface treated in (iii), and the incubation is performed at room temperature for 90 min, the electrode is washed, and then the aqueous solution of the double-ligand stabilized gold nanocluster labeled secondary antibody Cit / MSA@Au|Ab2 is added to the electrode surface to perform the incubation for 1 h; based on the formation of the immunocomplex, the Cit / MSA@Au|Ab2 is grafted and fixed to the surface of the working electrode, and the preparation of the low-potential near-infrared electrochemiluminescence immunosensor is realized.

[0040] According to the application, preferably, the volume fraction of the bovine serum albumin in the step (iii) is 2%.

[0041] According to the application, preferably, the antigen (Ag) in step (iv) is alpha-fetoprotein antigen (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), rabbit treponema pallidum antigen 15 (TP15), rabbit treponema pallidum antigen 17 (TP17) or prostate specific antigen.

[0042] According to the application, preferably, the buffer solution in step (iii) is one of a phosphate buffer solution, a HEPES buffer solution, a tris-hydroxymethyl aminomethane hydrochloride-hydrochloric acid buffer solution or a borrate-robinson buffer solution.

[0043] In the preparation process of the low-potential near-infrared electrochemiluminescence immunosensor, the details are not described, and the prior art in the field is followed.

[0044] The application further provides a low-potential near-infrared electrochemiluminescence immunodetection method, comprising the following steps:

[0045] I: using the glassy carbon electrode modified with the immunocomplex and the gold nanocluster Cit / MSA@Au NCs prepared above as a working electrode, a platinum electrode as a counter electrode and an Ag / AgCl electrode as a reference electrode, driving the gold nanocluster Cit / MSA@Au NCs fixed on the surface of the glassy carbon electrode to generate electrochemiluminescence in a phosphate buffer solution containing 10 mM of tert-butylamine-borane;

[0046] II: performing electrochemiluminescence tests on each sample solution according to the method in steps I and II, and detecting the antigen concentration in the sample solution according to the light intensity signal at the maximum radiation wavelength on the obtained electrochemiluminescence curve and a working curve.

[0047] Principles of the application:

[0048] The application adopts the gold nanocluster coated with the double ligand thiomalic acid and citrate as a label; the carboxyl on the surface of the gold nanocluster can be activated by 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide to branch the amino on the surface of the second antibody, so that the second antibody is labeled.

[0049] The application adopts the method of forming a carbon-nitrogen bond under an electro-polymerization condition to branch p-aminobenzoic acid to the surface of the glassy carbon electrode, and further activates the carboxyl of the p-aminobenzoic acid on the surface of the glassy carbon electrode by 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide to complete the branching of the first antibody.

[0050] Technical features and advantages of the application:

[0051] 1. The method of this invention has high selectivity. The spectroscopic electrochemiluminescence immunosensor constructed in this invention is based on the specific recognition and binding between antigens and antibodies. Therefore, interfering proteins in the test solution cannot bind to the primary and secondary antibodies of the antigen, and do not interfere with the detection system of this invention.

[0052] 2. The method of the present invention is simple to operate and has good repeatability, which has important scientific significance and application value for the early clinical diagnosis of cancer.

[0053] 3. The electrochemiluminescence radiation potential of this invention is below 0.60V, and the radiation band is located at 815 nanometers. When combined with existing light intensity-based multi-component immunoassay technology, it can further enrich the content of multi-component research and detection index information. Attached Figure Description

[0054] Figure 1 The fluorescence spectrum of the gold nanoclusters prepared in Example 1 is shown.

[0055] Figure 2 The image shows the ultraviolet spectrum of the gold nanoclusters prepared in Example 2.

[0056] Figure 3 This is a high-magnification transmission electron microscope image of the gold nanoclusters prepared in Example 3.

[0057] Figure 4 The image shows the electrochemiluminescence intensity of the gold nanoclusters prepared in Example 5.

[0058] Figure 5 The image shows the electrochemiluminescence spectrum of the gold nanoclusters prepared in Example 5.

[0059] Figure 6 The image shows the electrochemiluminescence intensity of the low-potential near-infrared immunosensors labeled with gold nanoclusters of different carcinoembryonic antigen concentrations prepared in Example 6.

[0060] Figure 7 The image shows the operating curve of the low-potential near-infrared immunosensor using gold nanoclusters of carcinoembryonic antigen as a label prepared in Example 7.

[0061] Figure 8 This is the selectivity spectrum of the sensor prepared in Example 8. Detailed Implementation

[0062] The present invention is further illustrated by the following examples, but is not limited thereto.

[0063] Example 1

[0064] (1) 625 microliters of 96mM H4AuCl4 solution was mixed with 20mL of 9.6mM thiomalic acid solution under ultrasonic, and then added into a three-necked flask, and then heated under reflux at 95 degrees Celsius for 144 hours to obtain unit thiomalic acid coated gold nanoclusters. The electrochemiluminescence radiation intensity of the unit thiomalic acid coated gold nanoclusters was detected by an electrochemiluminescence detection device. The solution changed from colorless to brown yellow; (2) 0.15mM citrate was weighed and mixed with the gold nanoclusters prepared in step (1) under ultrasonic, and incubated at room temperature for 1 hour to obtain gold nanoclusters coated with double ligands of citrate and thiomalic acid; (3) The solution obtained in step (2) was purified by centrifugation with isopropanol. The obtained precipitate was the double ligand coated gold nanoclusters. The gold nanocluster solution was stored in a refrigerator at 4 degrees Celsius. The fluorescence spectrum of the gold nanoclusters was tested at a fixed concentration of 1mg / mL, as shown in FIG. 3. Figure 1

[0065] Preparation of single double-ligand gold nanocluster labeled secondary antibody (Cit / MSA@Au|Ab2): 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide were added to the gold nanoclusters and activated for half an hour. After centrifugation, the solution was redissolved in a phosphate buffer solution. The corresponding secondary antibody was added and incubated at 37 degrees Celsius for 3 hours. Bovine serum albumin was used for blocking. The solution was redissolved in a phosphate buffer solution and stored in a refrigerator at 4 degrees Celsius. The obtained substance was Cit / MSA@Au|Ab2.

[0066] Preparation of a low-potential near-infrared electrochemiluminescence immunosensor using single double-ligand gold nanoclusters as labels:

[0067] (1): A glassy carbon electrode was treated and cleaned. The glassy carbon electrode was scanned at a scan rate of 10mV / S in the range of 0.4V-1.2V for 2 circles, so that p-aminobenzoic acid was polymerized to the surface of the glassy carbon electrode. The electrode was washed with 10mM pH 7.4 PBS to remove unreacted p-aminobenzoic acid;

[0068] (2): A mixed solution of EDC and NHS was added to the modified electrode surface of the glassy carbon electrode obtained in (1), and activated for half an hour. The electrode was washed with 10mM pH 7.4 phosphate buffer solution to remove unreacted activators;

[0069] (3): The primary antibody (Ab1) aqueous solution (1mL, 10μg / mL) was added to the activated electrode surface obtained in (2), and incubated for 3 hours. Bovine serum albumin was used to block the unreacted active sites of the electrode, and the electrode was washed;

[0070] ​(4): 20 μL of different concentrations of carcinoembryonic antigen (0.33, 2, 5, 10, 50, 200, 2000, 5000, 20000 fg / mL) were added to the electrode surface treated in (3), and incubated at room temperature for 90 min. The electrode was washed, and Cit / MSA@Au|Ab2 (1 mL, 10 μg / mL) was added to the electrode surface for incubation for 1 h, to obtain a low-potential near-infrared electrochemiluminescence immunosensor.

[0071] Example 2

[0072] The procedure was the same as in Example 1, except that the testing method for the bimetallic nanocluster solution was ultraviolet spectrogram, as shown in Figure 2 .

[0073] Example 3

[0074] The procedure was the same as in Example 1, except that the prepared bimetallic nanocluster solution was dropped on a copper mesh to observe the morphology. The high-magnification transmission electron micrograph of the prepared gold-silver bimetallic nanocluster is shown in Figure 3 .

[0075] Example 4

[0076] The procedure was the same as in Example 1, except that the prepared gold nanocluster solution was fixed at a concentration of 2 mg / mL, 10 μL of which was added to the electrode surface, and the electrode surface-fixed gold nanocluster (Cit / MSA@Au|GCE) was prepared by air-drying at room temperature. The electrochemiluminescence light intensity graph was measured, as shown in Figure 4 .

[0077] Example 5

[0078] The procedure was the same as in Example 4, except that the electrochemical method was a cyclic voltammetry-driven electrochemiluminescence spectroscopy method, as shown in Figure 5 .

[0079] Example 6

[0080] The procedure was the same as in Example 1, except that the above-prepared glassy carbon electrode with surface-modified immunocomplex and gold nanocluster Cit / MSA@Au NCs was used as a working electrode, a platinum electrode was used as a counter electrode, and an Ag / AgCl electrode was used as a reference electrode. In a phosphate buffer solution containing 10 mM t-butylamine-borane, the gold nanocluster Cit / MSA@Au NCs fixed on the glassy carbon electrode surface were driven to produce electrochemiluminescence by cyclic voltammetry;

[0081] Example 7

[0082] The electrochemiluminescence light intensity of each sample solution was tested according to the above-mentioned method. The antigen concentration in the sample solution was determined according to the light intensity signal at the maximum radiation wavelength on the obtained electrochemiluminescence light intensity curve and the working curve.

[0083] Example 8

[0084] The selectivity of the sensor was determined according to the above-mentioned method. The difference was that the antigen in step (4) was blank, bovine serum albumin, alpha-fetal protein antigen, carcinoembryonic antigen, prostate specific antigen, carbohydrate antigen 125, rabbit treponema pallidum antigen 15, rabbit treponema pallidum antigen 17 or prostate specific antigen, and a mixture of the seven detection substances. The selectivity spectrum of the sensor in this example is shown in Fig. 8. According to Fig. 8, the sensor prepared in this example had good selectivity for carcinoembryonic antigen, and other antigen proteins did not interfere with the detection of the target antigen of the sensor. Figure 8 Figure 8 The selectivity of the sensor was determined according to the above-mentioned method. The difference was that the antigen in step (4) was blank, bovine serum albumin, alpha-fetal protein antigen, carcinoembryonic antigen, prostate specific antigen, carbohydrate antigen 125, rabbit treponema pallidum antigen 15, rabbit treponema pallidum antigen 17 or prostate specific antigen, and a mixture of the seven detection substances. The selectivity spectrum of the sensor in this example is shown in Fig. 8. According to Fig. 8, the sensor prepared in this example had good selectivity for carcinoembryonic antigen, and other antigen proteins did not interfere with the detection of the target antigen of the sensor.​

Claims

1. A detection method for non-disease diagnostic and therapeutic purposes using low-potential near-infrared electrochemiluminescence immunoassay, employing a low-potential near-infrared electrochemiluminescence immunoassay sensor. The low-potential near-infrared electrochemiluminescence immunosensor was prepared according to the following method: (1) Using chloroauric acid as the gold source and citrate and thiomalic acid as reducing agents and stabilizers, Au is reduced by ligands. 3 + Water-soluble, dual-ligand-stabilized gold nanoclusters Cit / MSA@Au NCs were prepared. (2) Preparation of dual-ligand-stabilized gold nanocluster-labeled secondary antibody Cit / MSA@Au|Ab2; (3) A low-potential near-infrared electrochemiluminescence immunosensor was prepared by grafting and fixing Cit / MSA@Au|Ab2 onto the working electrode surface in the form of a sandwich immune complex formed on the working electrode surface. The detection method includes the following steps: I: Using a glassy carbon electrode with surface-modified immune complexes and dual-ligand-stabilized gold nanoclusters Cit / MSA@Au NCs as the working electrode, a platinum electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode, electrochemiluminescence was generated by cyclic voltammetry in PBS containing 10 mM tert-butylamine-borane on the surface of the glassy carbon electrode. II: Perform electrochemiluminescence intensity testing on each sample solution according to the method in step I. Detect the antigen concentration in the sample solution based on the light intensity signal at the maximum radiation point on the obtained electrochemiluminescence curve and the working curve.

2. The detection method according to claim 1, characterized in that, The preparation process of the dual-ligand stabilized gold nanoclusters Cit / MSA@Au NCs in step (1) is as follows: (a) The chloroauric acid H4AuCl4 solution and the thiomalic acid MSA ligand were ultrasonically mixed until homogeneous; (b) The solution prepared in step (a) was placed in a three-necked flask and heated at 95 degrees Celsius for 10-200 h to obtain gold nanoclusters coated with single ligand thiomalic acid; the solution changed from colorless to brownish-yellow. (c) Add citrate Cit to the crude solution prepared in step (b) and continue stirring for 10-120 min; (d) The resulting solution is then centrifuged to remove large particles at the bottom; the solution is further purified by centrifugation with isopropanol, and the resulting precipitate is the mono-dual-ligand-stabilized gold nanoclusters obtained by this method.

3. The detection method according to claim 1, characterized in that, In step (1), during the preparation of the unit dual-ligand metal nanoclusters, the conditions are as follows: In step (a), the molar ratio of gold to thiomalic acid is 1:(1-5); In step (a), the concentration of the chloroauric acid solution is 80-100 mM; The heating time in step (b) is 10-200 hours; In step (c), the molar ratio of sodium citrate to H4AuCl4 in step (1) is 1:200-1:

400.

4. The detection method according to claim 2, characterized in that, The preparation process of the dual-ligand-stabilized gold nanoclusters in step (1) is as follows: (a) 625 μL of 96 mM H4AuCl4 solution and 20 mL of 9.6 mM thiomalic acid solution were ultrasonically mixed and added to a three-necked flask. The mixture was then heated under reflux at 95 degrees Celsius for 144 h to obtain gold nanoclusters coated with single thiomalic acid. The electrochemiluminescence radiation intensity of the gold nanoclusters coated with single thiomalic acid was detected by an electrochemiluminescence detection device. The solution changed from colorless to brownish-yellow. (b) Weigh 0.15 mM citrate and mix it with the gold nanoclusters prepared in step (a), sonicate to mix, and incubate at room temperature for 1 h to obtain gold nanoclusters co-coated with dual-ligand citrate and thiomalic acid. (c) Finally, the gold nanoclusters were centrifuged at 13,300 rpm with isopropanol to collect the precipitate and stored in a refrigerator at 4 degrees Celsius.

5. The detection method according to claim 1, characterized in that, The process of preparing the gold nanocluster-labeled secondary antibody Cit / MSA@Au|Ab2 in step (2) is as follows: 1-Ethyl-(3-dimethylaminopropionic acid)carbodiimide hydrochloride and hydroxysuccinimide were added to gold nanoclusters Cit / MSA@Au NCs for half an hour of activation. After centrifugation, the resulting precipitate was reconstituted and incubated with the corresponding secondary antibody at 37°C for 3 hours. Then, it was blocked with bovine serum albumin. The resulting substance is Cit / MSA@Au|Ab2, which is soluble in PBS and can be stored at 4°C.

6. The detection method according to claim 1, characterized in that, The process of preparing the low-potential near-infrared electrochemiluminescence immunosensor in step (3) is as follows: (i): The polished glassy carbon electrode was placed in a 1.0 mM aqueous solution of p-aminobenzoic acid and scanned for 2 cycles in the range of 0.4 V-1.2 V at a scanning speed of 10 mV / S to polymerize p-aminobenzoic acid onto the surface of the glassy carbon electrode. The electrode was then cleaned with 10 mM pH 7.4 PBS to remove unreacted p-aminobenzoic acid. (ii): Add ethyl-(3-dimethylaminopropionic acid)carbodiimide hydrochloride and hydroxysuccinimide solution to the glassy carbon electrode modified electrode surface obtained in (i), activate for half an hour, and wash the electrode with 10mM pH 7.4 PBS to remove unreacted activators. (iii): Add the primary antibody Ab1 solution to the surface of the activated electrode obtained in ii, incubate for 3 h, then block the unreacted active sites of the electrode with bovine serum albumin, and clean the electrode. (iv): Antigen Ag was dropped onto the electrode surface after treatment iii and incubated at room temperature for 90 min; the electrode was cleaned, and Cit / MSA@Au|Ab2 was dropped onto the electrode surface and incubated for 1 h; Cit / MSA@Au|Ab2 was grafted and fixed onto the working electrode surface based on the formation of immune complexes, thus realizing the preparation of a low-potential near-infrared electrochemiluminescence immunosensor.

7. The detection method according to claim 6, characterized in that, The volume fraction of bovine serum albumin mentioned in step (3) is 2%; the antigen Ag is alpha-fetoprotein antigen (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), rabbit treponemal antigen 15, rabbit treponemal antigen 17, or prostate-specific antigen.

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