Preparation method of low-potential near-infrared electrochemical luminescence immunosensor taking tert-butylamine-borane as co-reactant

Abstract

The invention relates to a preparation method of a low-potential near-infrared electrochemical luminescence immunosensor taking tert-butylamine-borane as a co-reactant. The invention relates to a preparation method of a low-potential near-infrared electrochemiluminescence (-0.57 V vs Ag / AgCl) immunosensor, which mainly comprises the following steps: (1) preparing a water-soluble gold nano-cluster (Cit / MSA at Au NCs) capable of generating low-potential near-infrared electrochemiluminescence by using double-ligand citrate and thiomalic acid as stabilizers, (2) preparing a gold nano-cluster labeled secondary antibody (Cit / MSA at AuAb2), and (3) preparing a gold nano-cluster labeled secondary antibody (Cit / MSA at AuAb2). (3) preparing a low-potential near-infrared electrochemical luminescence sensor by taking the gold nanocluster as a marker based on a sandwich immune principle; and (4) drawing a working curve. The prepared low-potential near-infrared electrochemical luminescence immunosensor has high detection sensitivity, and other antigen proteins do not interfere with the sensing detection of the target antigen.

Description

Technical Field

[0001] The invention belongs to the field of analytical technology methods and relates to a method for preparing a low-potential near-infrared electrochemical luminescence immunosensor using tert-butylamine-borane as a coreactant. Background Art

[0002] At present, most electrochemiluminescence biochemical analysis and clinical diagnosis use ruthenium bipyridine as a marker, and the potential of its electrochemiluminescence radiation is about 1.2 V. Traditional co-reactants are mainly tripropylamine, oxalate, persulfate, etc. Liu et al. used 2-N-dibutylaminoethanol as a co-reactant to study Ru(bpy)3 2+ / 2-N-dibutylaminoethanol's anodic electrochemiluminescence. However, the oxidation potentials of tripropylamine and 2-N-dibutylaminoethanol are both high, and there is strong electrochemical interference in the measurement within a wide potential window, which is not conducive to electrode stability and detection of biochemical reagents, because the development of an electrochemiluminescence system with a lower radiation potential is crucial.

[0003] Chinese patent document CN111139066B discloses a broad-spectrum low-potential electrochemiluminescence system and its construction method. Fu et al. disclosed a low-potential electrochemiluminescence system with hydrazine hydrate as a co-reactant and copper indium sulfur as a luminescent substance (Anal. Chem. 2019, 91, 10221). However, hydrazine hydrate has high biological toxicity, and the electrochemiluminescence intensity of the carbohydrazide system is much lower than the electrochemiluminescence radiation intensity of the hydrazine hydrate system.

[0004] Currently, there are few low-potential electrochemiluminescence systems, and the electrochemiluminescence efficiency of existing low-potential co-reactant systems is low, and the co-reactants are toxic. There is an urgent need to develop a low-potential electrochemiluminescence system that can be comparable to the hydrazine hydrate system, which can reduce electrochemical interference, increase the electrochemiluminescence intensity of the low-potential system, and avoid the influence of high potential on biochemical substances. Summary of the invention

[0005] In view of the shortcomings of the prior art, especially the problems of few low-potential electrochemiluminescence systems, low electrochemiluminescence efficiency and toxicity of co-reactants, the present invention uses gold nanoclusters as markers and tert-butylamine-borane as a co-reactant to provide a method for preparing a low-potential near-infrared electrochemiluminescence immunosensor using tert-butylamine-borane as a co-reactant. The electrochemiluminescence potential of the immunosensor is 0.57V, which is comparable to the low-potential electrochemiluminescence system of a hydrazine hydrate system and is safe and non-toxic.

[0006] Terminology Note:

[0007] Antigen: The antigen (Ag) described in the present invention refers to conventional antigens such as talofetoprotein 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.

[0008] Primary antibody: The primary antibody (Ab1) described in the present invention refers to an antibody produced in response to an antigen. The present invention has a better effect on the monoclonal antibody corresponding to the above antigen.

[0009] Secondary antibody: The secondary antibody described in the present invention refers to a secondary antibody produced corresponding to the above-mentioned antigen and primary antibody.

[0010] Definition of U: Under the conditions specified in the experiment, such as optimal temperature, optimal pH, and optimal substrate concentration, the amount of enzyme required to catalyze the reaction of 1 μmol of substrate within 1 minute is regarded as an international unit of enzyme activity U.

[0011] The present invention is achieved through the following technical solutions:

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

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

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

[0015] (3) Cit / MSA@Au|Ab2 was grafted and fixed to the surface of the working electrode in the form of a sandwich immune complex formed on the surface of the working electrode to prepare a low-potential near-infrared electrochemiluminescence immunosensor.

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

[0017] a. Ultrasonic mixing of chloroauric acid (H4AuCl4) solution and thiomalic acid (MSA) ligand;

[0018] b. heating the solution obtained in step a to react, thereby obtaining gold nanoclusters coated with single ligand thiomalic acid, and the solution changes from colorless to brownish yellow;

[0019] c. adding citrate (Cit) to the crude solution obtained in step (2), mixing by ultrasonication, and incubating at room temperature;

[0020] d. After the reaction is completed, the large particles at the bottom are removed by centrifugation, and the solution is purified with isopropanol. The resulting precipitate is the water-soluble dual-ligand stabilized gold nanoclusters.

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

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

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

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

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

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

[0027] In step c, isopropanol is purified by adding isopropanol to the solution and centrifuging at 13,300 rpm.

[0028] In a preferred embodiment of the present invention, a method for preparing water-soluble dual-ligand stabilized gold nanoclusters comprises the following steps:

[0029] a. Ultrasonic mixing of 610-650 μL of 96 mM H4AuCl4 solution and 10-30 mL of 9.6 mM thiomalic acid solution was performed, and the mixture was heated under reflux at 90-100°C for 130-150 h to obtain thiomalic acid-coated gold nanoclusters;

[0030] b. Mixing a 0.1-0.3 mM citrate solution with the solution obtained in step (1), mixing by ultrasonic mixing, and incubating at room temperature for 1 hour to obtain gold nanoclusters coated with citrate and thiomalic acid as dual ligands;

[0031] c. Purify the solution obtained in step b by centrifugation with isopropanol, and the resulting precipitate is the dual-ligand coated gold nanoclusters.

[0032] According to the present invention, preferably, in step (2), a secondary antibody Cit / MSA@Au|Ab2 labeled with dual-ligand stabilized gold nanoclusters is prepared by the following process:

[0033] 1-Ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide were added to water-soluble dual-ligand stabilized gold nanoclusters (Cit / MSA@Au NCs), activated for 20-30 minutes, centrifuged and redissolved in phosphate buffer solution, added with corresponding secondary antibody, incubated at 37°C, blocked with bovine serum albumin, and the obtained substance was Cit / MSA@Au|Ab2; Cit / MSA@Au|Ab2 was redissolved in phosphate buffer solution.

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

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

[0036] (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 to 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 washed with 10 mM PBS (pH 7.4) to remove unreacted p-aminobenzoic acid.

[0037] (ii): Add a mixed solution of EDC and NHS to the surface of the glassy carbon electrode modified electrode obtained in (i) and activate it for 20-30 minutes. Wash the electrode with a 10mM pH 7.4 phosphate buffer solution to remove the unreacted activator.

[0038] (iii): Add the primary antibody (Ab1) aqueous solution dropwise to the activated electrode surface obtained in (ii), incubate for 3 h, block the unreacted active sites of the electrode with bovine serum albumin, and then wash the electrode;

[0039] (iv): Add the antigen (Ag) aqueous solution to the electrode surface treated in (iii), incubate at room temperature for 90 minutes, wash the electrode, and then add the secondary antibody Cit / MSA@Au|Ab2 aqueous solution labeled with dual ligand-stabilized gold nanoclusters to the electrode surface for incubation for 1 hour; based on the formation of immune complexes, Cit / MSA@Au|Ab2 is grafted and fixed to the working electrode surface to realize the preparation of low-potential near-infrared electrochemiluminescence immunosensor.

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

[0041] According to the present invention, preferably, the antigen (Ag) described 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 present invention, preferably, the buffer solution in step (iii) is one of phosphate buffer solution, HEPES buffer solution, Tris-HCl-HCl buffer solution or Berritan-Robinson buffer solution.

[0043] The preparation process of the low potential near-infrared electrochemiluminescence immunosensor is not described in detail and is carried out according to the existing technology in the field.

[0044] The present invention also provides a low-potential near-infrared electrochemical luminescence immunoassay method, comprising the following steps:

[0045] I: Using the glassy carbon electrode prepared above with the immune complex and gold nanoclusters Cit / MSA@Au NCs as the working electrode, the platinum electrode as the counter electrode, and the Ag / AgCl electrode as the reference electrode, cyclic voltammetry was used to drive the gold nanoclusters Cit / MSA@Au NCs fixed on the surface of the glassy carbon electrode to produce electrochemiluminescence in a phosphate buffer solution containing 10 mM tert-butylamine-borane;

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

[0047] Principle of the present invention:

[0048] The present invention uses gold nanoclusters coated with dual ligand thiomalic acid and citrate as markers; the carboxyl groups on the surface of the gold nanoclusters can be grafted onto the amino groups on the surface of the second antibody after being activated by 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide, thereby achieving labeling of the second antibody.

[0049] The present invention adopts a method of forming a carbon-nitrogen bond under electropolymerization conditions to graft p-aminobenzoic acid onto the surface of a glassy carbon electrode, and further activates the carboxyl group of p-aminobenzoic acid on the surface of the glassy carbon electrode by using 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide to complete the grafting of the first antibody.

[0050] The technical features and advantages of the present invention are as follows:

[0051] 1. The method of the present invention has high selectivity. The spectral electrochemiluminescence immunosensor constructed by the present invention is based on the specific recognition and binding between antigen and antibody, so the interfering protein in the test solution cannot bind to the antigen first antibody and the second antibody, and has no interference with the detection system of the present invention.

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

[0053] 3. The electrochemiluminescence radiation potential of the present invention is lower than 0.60V, and the radiation band is located at 815 nanometers. When combined with the existing light intensity-based multi-component immunoassay technology, it can further enrich the content of multi-component research and the detection index information. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0055] Figure 2 This is the ultraviolet spectrum of the gold nanoclusters prepared in Example 2.

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

[0057] Figure 4 This is the electrochemiluminescence intensity diagram of the gold nanoclusters prepared in Example 5.

[0058] Figure 5 This is the electrochemiluminescence spectrum of the gold nanoclusters prepared in Example 5.

[0059] Figure 6 This is an electrochemiluminescence intensity diagram of the low-potential near-infrared immunosensor labeled with gold nanoclusters at different carcinoembryonic antigen concentrations prepared in Example 6.

[0060] Figure 7 This is the working curve of the low potential near-infrared immunosensor using the gold nanoclusters of carcinoembryonic antigen prepared in Example 7 as markers.

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

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

[0063] Example 1

[0064] (1) Ultrasonically mix 625 microliters of 96mM H4AuCl4 solution and 20mL of 9.6mM thiomalic acid solution, add them to a three-necked flask, and then heat and 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 is detected by electrochemiluminescence detection equipment, and the solution changes from colorless to brown-yellow; (2) Weigh 0.15mM citrate and mix it with the gold nanoclusters prepared in step (1), ultrasonically mix it, and incubate it at room temperature for 1 hour to obtain gold nanoclusters co-coated with dual ligand citrate and thiomalic acid; (3) The solution obtained in step (2) is centrifuged and purified with isopropanol, and the resulting precipitate is the dual ligand-coated gold nanoclusters. Stored in a refrigerator at 4°C, the fluorescence spectrum of the gold nanocluster solution is tested at a fixed concentration of 1mg / mL, as shown Figure 1 shown.

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

[0066] Preparation of low potential near-infrared electrochemiluminescent immunosensor using unit biligand gold nanoclusters as markers:

[0067] (1): Treat the glassy carbon electrode and clean it. Scan 2 circles in the range of 0.4V-1.2V at a scanning speed of 10mV / S to polymerize the p-aminobenzoic acid on the surface of the glassy carbon electrode. Wash the electrode with 10mM pH 7.4 PBS to remove the unreacted p-aminobenzoic acid;

[0068] (2): Add a mixed solution of EDC and NHS to the surface of the glassy carbon electrode modified electrode obtained in (1) and activate it for half an hour. Wash the electrode with a 10 mM pH 7.4 phosphate buffer solution to remove the unreacted activator;

[0069] (3): A primary antibody (Ab1) aqueous solution (1 mL, 10 μg / mL) was added dropwise to the surface of the activated electrode obtained in (2), incubated for 3 h, and then the unreacted active sites of the electrode were blocked with bovine serum albumin, and the electrode was washed;

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

[0071] Example 2

[0072] The steps are the same as those in Example 1, except that the test method for the bimetallic nanocluster solution is the ultraviolet spectrum. Figure 2 shown.

[0073] Example 3

[0074] The steps are the same as those in Example 1, except that the prepared bimetallic nanocluster solution is dropped onto a copper mesh to observe the morphology. The high-magnification transmission electron microscope photograph of the prepared gold-silver bimetallic nanocluster is shown in FIG. Figure 3 shown.

[0075] Example 4

[0076] The steps are the same as those in Example 1, except that the prepared gold nanocluster solution is fixed at a concentration of 2 mg / mL, 10 μL is added dropwise to the electrode surface, and dried at room temperature to obtain gold nanoclusters fixed on the electrode surface (Cit / MSA@Au|GCE), and its electrochemical luminescence intensity diagram is measured, as shown in FIG. Figure 4 shown.

[0077] Example 5

[0078] The steps are the same as those in Example 4, except that the electrochemical method is an electrochemical luminescence spectroscopy method driven by cyclic voltammetry, such as Figure 5 shown.

[0079] Example 6

[0080] The steps are the same as those in Example 1, except that: the glassy carbon electrode prepared above with the surface modified with immune complexes and gold nanoclusters Cit / MSA@Au NCs is used as the working electrode, the platinum electrode is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode. In a phosphate buffer solution containing 10 mM tert-butylamine-borane, cyclic voltammetry is used to drive the gold nanoclusters Cit / MSA@Au NCs fixed on the surface of the glassy carbon electrode to produce electrochemical luminescence;

[0081] Example 7

[0082] The steps are the same as those in Example 6. Each sample solution to be tested is subjected to an electrochemiluminescence intensity test. The antigen concentration in the sample solution to be tested is detected based on the light intensity signal at the maximum radiation wavelength on the obtained electrochemiluminescence intensity curve and the working curve.

[0083] Example 8

[0084] The steps are the same as those in Example 1 to determine the selectivity of the sensor. The difference is that in step (4), the antigens are blank, bovine serum albumin, alpha-fetoprotein antigen, carcinoembryonic antigen, prostate-specific antigen, carbohydrate antigen 125, rabbit syphilis treponema antigen 15, rabbit syphilis treponema antigen 17 or prostate-specific antigen. And a mixture of seven test substances. The selectivity spectrum of the sensor in this example is shown in Figure 8 As shown. Figure 8 As shown, the sensor prepared in this embodiment has good selectivity for carcinoembryonic antigen, and other antigen proteins do not interfere with the target antigen sensing detection of the invention.

Claims

1. A method for preparing a low potential near-infrared electrochemiluminescence immunosensor, comprising the following steps: (1) Using chloroauric acid as the gold source, citrate and thiomalic acid as the reducing agent and stabilizer, Au is reduced by ligand 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) Cit / MSA@Au|Ab2 was grafted and fixed to the surface of the working electrode in the form of a sandwich immune complex formed on the surface of the working electrode to prepare a low-potential near-infrared electrochemiluminescence immunosensor.

2. The method for preparing the immunosensor according to claim 1, characterized in that: The preparation process of dual-ligand stabilized gold nanoclusters (Cit / MSA@Au NCs) in step (1) is as follows: (a) mixing chloroauric acid (H4AuCl4) solution and thiomalic acid (MSA) ligand uniformly by ultrasonication; (b) placing the solution prepared in step (a) in a three-necked flask and heating at 95 degrees Celsius for 10-200 hours to obtain gold nanoclusters coated with a single ligand thiomalic acid; the solution changes from colorless to brownish yellow; (c) adding citrate (Cit) to the crude solution prepared in step (b) and continuing 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 using isopropanol, and the resulting precipitate is the unit biligand-stabilized gold nanoclusters obtained by this method.

3. The method for preparing the immunosensor according to claim 2, characterized in that: In the preparation process of the unit biligand metal nanoclusters in step (1), the conditions are as follows: In step (a), the molar ratio of gold to thiomalic acid is 1:(1-5); The concentration of the chloroauric acid solution in step (a) is 80-100 mM; The heating time in step (b) is 10-200h; The molar ratio of sodium citrate in step (c) to H4AuCl4 in step (1) is 1:200-1:

400.

4. The method for preparing the immunosensor 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 microliters of 96 mM H4AuCl4 solution and 20 mL of 9.6 mM thiomalic acid solution were ultrasonically mixed, added to a three-necked flask, and then heated to 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, and the solution changed from colorless to brown-yellow; (b) 0.15 mM citrate was weighed and mixed with the gold nanoclusters prepared in step (a), ultrasonically mixed, and incubated at room temperature for 1 hour to obtain gold nanoclusters co-coated with dual ligand citrate and thiomalic acid; (c) Finally, the prepared quantum dots were centrifuged at 13,300 rpm with isopropanol to collect the precipitate and store it in a refrigerator at 4 degrees Celsius.

5. The method for preparing the immunosensor 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 the gold nanoclusters Cit / MSA@Au NCs for activation for half an hour. The precipitate obtained after centrifugation was redissolved and the corresponding secondary antibody was added for incubation at 37°C for 3 hours, and then blocked with bovine serum albumin. The obtained substance is Cit / MSA@Au|Ab2, which is soluble in PBS and stored in a refrigerator at 4 degrees.

6. The method for preparing the immunosensor according to claim 1, characterized in that: The process of preparing the low potential near-infrared electrochemiluminescence immunosensor in step (4) 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 to 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 washed with 10 mM PBS (pH 7.4) to remove unreacted p-aminobenzoic acid. (ii): Add ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride and hydroxysuccinimide solution to the surface of the glassy carbon electrode modified electrode obtained in (i) and activate for half an hour. Wash the electrode with 10 mM pH 7.4 PBS to remove the unreacted activator; (iii): Add the primary antibody (Ab1) solution dropwise to the activated electrode surface obtained in (ii), incubate for 3 h, then block the unreacted active sites of the electrode with bovine serum albumin, and wash the electrode; (iv): Add antigen (Ag) to the electrode surface treated in (iii) and incubate at room temperature for 90 minutes; wash the electrode, and then add Cit / MSA@Au|Ab2 to the electrode surface and incubate for 1 hour; graft and fix Cit / MSA@Au|Ab2 to the working electrode surface based on the formation of immune complexes to achieve the preparation of low-potential near-infrared electrochemiluminescence immunosensor.

7. The method for preparing the immunosensor according to claim 6, characterized in that: The volume fraction of bovine serum albumin in step (3) is 2%; The antigen (Ag) 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.

8. A method for detecting low potential near-infrared electrochemiluminescence, comprising using the sandwich immunosensor according to any one of claims 1 to 9, comprising the following steps: I: Using a glassy carbon electrode modified with 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, cyclic voltammetry was used to drive the gold nanoclusters (Cit / MSA@Au NCs) immobilized on the surface of the glassy carbon electrode to produce electrochemiluminescence in PBS containing 10 mM tert-butylamine-borane; II: Perform an electrochemiluminescence intensity test on each sample solution according to the method in step I, and detect the antigen concentration in the sample solution according to the light intensity signal at the maximum radiation point on the obtained electrochemiluminescence curve and the working curve.

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