A highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers and a method for constructing the same

Abstract

The present invention relates to a highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers and a construction method thereof, belonging to the field of electrochemiluminescent analysis technology. The sensor uses D-penicillamine as a ligand and borane-tert-butylamine complex as a reducing agent to prepare low-luminescent gold nanoclusters. 2+ After aggregation induction, ultra-bright red fluorescent gold nanoclusters (Zn 2+ ‑AuNCs), the fluorescence intensity increased by about 20 times and the electrochemiluminescence intensity increased by nearly 2000 times, the fluorescence lifetime reached an ultra-long lifetime of 2000 ns and had certain phosphorescent properties. An ECL immunosensor was constructed using the gold cluster as a marker, which has excellent selectivity and ultra-high sensitivity to human carcinoembryonic antigen and has broad application prospects in the medical field.

Description

Technical Field

[0001] The invention relates to a highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers and a construction method thereof, and belongs to the field of electrochemiluminescent analysis technology methods. Background Art

[0002] Electrochemiluminescence (ECL) has attracted wide attention in the fields of clinical diagnosis, environmental monitoring and food safety due to its advantages of low background, high sensitivity and simple equipment. Among the various luminophores, noble metal nanoclusters (NCs) have been widely studied due to their low toxicity, good photostability, long life and good biocompatibility. However, compared with traditional fluorophores and colloidal quantum dots, the luminescence intensity of noble metal nanoclusters is still relatively weak, which seriously limits their development in the optical field. Synthetic strategies such as adjusting particle size, changing surface ligands, doping and aggregation induction are usually used to improve their luminescence intensity.

[0003] In recent years, aggregation-induced ionization (AIE) has attracted great attention because of its significant effect on improving the fluorescence intensity (PL), ECL intensity and quantum yield (QY) of gold nanoclusters. By solidifying the chemical environment on the surface of metal nanoclusters, non-radiative relaxation can be reduced, reducing its energy dissipation, and thus enhancing the luminescence intensity of metal nanoclusters. Tang first discovered that aggregates can significantly improve the luminescence intensity, increasing the emission efficiency by two orders of magnitude (Chem. Commun. 2001, 18, 1740-1741). Xie et al. introduced the idea of ​​aggregation induction into the synthesis of gold nanoclusters (AuNCs), inducing the controlled aggregation of Au(I)-sulfate complexes on the in-situ generated Au(0) core to form Au(0)@Au(I)-sulfate core-shell NCs, which produced strong luminescence (J.Am.Chem.Soc.2012, 134, 16662−16670). Hyeon et al. 4 (MHA) 4 Zn 2+ Successfully prepared ultra-bright green-blue fluorescence with a QY of up to 90% (J.Am.Chem.Soc. 2021, 143, 326−334). 2+ The surface environment of gold nanoclusters is solidified, and ligand-solvent interactions occur through an effective non-radiative process to increase PL and QY. This has broad application prospects for the development of water-soluble gold nanoclusters with good photostability and high fluorescence, and can be used for the preparation of biological markers and immunosensors.

[0004] The application of ECL in immunoassay began in 1991 with the development of the tris(bipyridine)ruthenium(II) complex (Ru(bpy) 3 2+) / tri-n-propylamine (TPrA) system. At the end of the 20th century, Roche Diagnostics Company introduced Ru(bpy) 3 2+ / TprA kit is commercialized and occupies 80% of the market, but its high potential (+1.4V) leads to more interference from electroactive substances in the reaction area. In recent years, the luminescent clusters used to construct ECL immunosensors are still limited. Wang et al. constructed an ECL immunosensor with l-cysteine ​​copper clusters (Cys-CuNCs) as luminescent bodies, but its monochromaticity is poor, the ECL signal response intensity is limited, and the sensitivity of the sensor is reduced (Anal.Chem. 2023, 95, 4155−4161). Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers and a construction method thereof.

[0006] The present invention firstly utilizes the AIE mechanism to prepare Zn 2+ Aggregation-induced gold nanoclusters (Zn 2+ -AuNCs), compared with the gold nanoclusters (AuNCs) before aggregation induction, the fluorescence increased by nearly 20 times, and the electrochemiluminescence intensity increased by nearly 2000 times.

[0007] Using it as a luminescent cluster to construct an ECL immunosensor, the ECL response intensity was greatly improved, making the ECL sensor highly sensitive and with a low potential (+1.0V), successfully reducing the degree of interference from electroactive substances and expanding its application in immunoassays.

[0008] The ECL immunosensor constructed by the present invention has high sensitivity and high selectivity, and the detection range of human carcinoembryonic antigen (CEA) is about 5 pg / mL-5 ng / mL, the detection range is wide, the detection limit (LOD) is 5 pg / mL, the detection limit is low, the cost is low, and the process is simple.

[0009] Terminology Note:

[0010] CEA primary antibody: a protein that can specifically bind to carcinoembryonic antigen (CEA), referred to as CEA-Ab 1 .

[0011] CEA secondary antibody: an antibody that can bind to CEA primary antibody, referred to as CEA-Ab 2 .

[0012] Grafting: After activation, the carboxyl group and the amino group are connected by chemical bonds.

[0013] DBAE: 2-(Dibutylamino)ethanol

[0014] TEOA: triethanolamine

[0015] H 2 O 2 : Hydrogen peroxide

[0016] N 2 H 4 :Hydrazine hydrate

[0017] TEA: triethylamine

[0018] TPRA: Tri-n-propylamine

[0019] Hepes: 4-Hydroxyethylpiperazineethanesulfonic acid

[0020] Tris-HCl: Tris-HCl

[0021] PB: Phosphate buffer

[0022] CB: Carbonate buffer

[0023] The present invention is achieved through the following technical solutions:

[0024] The method for constructing a highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers is as follows:

[0025] (1) After the working electrode is polished, washed, grafted with para-aminobenzoic acid ABA, and activated, CEA-Ab is added to the electrode surface. 1 Aqueous solution, incubated at constant temperature, formed GCE|ABA-Ab 1 After glycine blocking, CEA antigen aqueous solution was added dropwise and incubated at constant temperature to form GCE|ABA-Ab 1 <CEA;

[0026] (2) Zn 2+ -AuNCs-Ab 2 The aqueous solution was added dropwise to the electrode surface of step (1) and incubated at a constant temperature to form GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs, with Zn 2+ -AuNCs as markers to form highly sensitive electrochemiluminescent immunosensor GCE|ABA-Ab via sandwich immunoassay 1 <cea>Ab 2 |Zn 2+ -AuNC.

[0027] Preferably, in step (1), the working electrode is a glassy carbon electrode, polishing is performed by polishing with alumina powders having diameters of 0.3 μm and 0.05 μm in sequence, and grafting of p-aminobenzoic acid (ABA) is performed by immersing the treated glassy carbon electrode in a 0.1 M pH 7.4 PB buffer solution containing 1 mM ABA to form GCE|ABA by electrodeposition; the electrodeposition conditions are as follows: a scanning speed of 0.05 mV / s; a scanning potential interval of +0.40 V to +1.20 V; a number of cycles of 2; and activation is performed by dripping 10 μL of a mixed solution of EDC and NHS onto the electrode surface, wherein the EDC concentration in the mixed solution is 1 mg / mL and the NHS concentration is 1 mg / mL, activation is performed at 37°C for 15 min, and the electrode is rinsed with deionized water.

[0028] Further preferably, the solvent of the mixed solution of EDC and NHS is 10 mM pH 6 PB buffer solution.

[0029] According to the preferred embodiment of the present invention, in step (1), CEA-Ab 1 The concentration of the aqueous solution is 5-15 μg / mL, and the amount added is 5-15 μL.

[0030] Preferably, according to the present invention, in step (1), the amount of the CEA antigen aqueous solution added is 5-15 μL, and the CEA concentration in the CEA antigen aqueous solution is 0.005 ng / mL to 10 ng / mL.

[0031] Preferably, according to the present invention, in step (1), the concentration of the glycine solution used for glycine blocking is 0.01 g / mL, and the solvent is 0.1 M pH 7.4 PB buffer solution.

[0032] According to the preferred embodiment of the present invention, in step (1), the constant temperature incubation is 37°C for 2-3 h to form GCE|ABA-Ab 1 , rinse the electrode with deionized water.

[0033] According to the preferred embodiment of the present invention, in step (2), Zn 2+ -AuNCs-Ab 2 The concentration of the aqueous solution is 5-15 μg / mL, the amount of the solution added is 5-15 μL, and the constant temperature incubation is 37°C for 2-3 h.

[0034] According to the preferred embodiment of the present invention, in step (2), Zn 2+ -AuNCs-Ab 2 The synthesis steps of the aqueous solution are as follows:

[0035] 1) To the purified Zn 2+ 6 μL of phosphate buffered saline (PBS) containing 1 mg / mL 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and 1 mg / mL hydroxysuccinimide (NHS) was added to the aggregation-induced gold nanoclusters and activated in an incubator at 37 °C for 15 min.

[0036] 2) Add 80 μL of 10 μg / mL CEA-Ab 2 The solution was activated in a 37 °C incubator for 2-3 h, and finally 10 μL of 0.01 g / mL glycine solution was added to block nonspecific binding sites. The mixture was incubated at 37 °C for 1-2 h, and the precipitate was collected by centrifugation at 3000 rpm and redissolved in an equal volume of water to obtain Zn 2+ -AuNCs-Ab 2 Aqueous solution.

[0037] Furthermore, Zn 2+ Aggregation-induced gold nanoclusters are based on HAuCl 4 ∙3H 2 The method is prepared by using O as a gold source, D-penicillamine as a ligand, borane-tert-butylamine complex as a reducing agent and zinc nitrate hexahydrate as an aggregation inducer.

[0038] The above Zn 2+ The preparation method of aggregation-induced gold nanoclusters comprises the following steps:

[0039] S1: adding chloroauric acid solution, D-penicillamine solution, and reducing agent borane-tert-butylamine complex solution to a container filled with ultrapure water in sequence and mixing them evenly to obtain a mixture;

[0040] S2 Add sodium hydroxide solution to the mixture, adjust the pH, continue stirring, add zinc nitrate hexahydrate solid until it is completely dissolved, and the reaction is complete. 2+ -AuNCs were purified by centrifugation at 12000 rpm three times with isopropanol, and the obtained solid was redissolved in an equal volume of water to obtain ultra-bright red fluorescent water-soluble gold nanoclusters Zn 2+ -AuNCs.

[0041] Preferably according to the present invention, in step S1, the molar concentration of the chloroauric acid solution is 92-100 mM.

[0042] Preferably according to the present invention, in step S1, the molar concentration of the D-penicillamine solution is 95-105 mM.

[0043] Preferably according to the present invention, in step S1, the molar concentration of the reducing agent borane-tert-butylamine complex solution is 95-105 mM.

[0044] Preferably, in step S1, the volume ratio of the chloroauric acid solution to ultrapure water is (0.1-0.3):(1-10), and the volume ratio of the chloroauric acid solution to the D-penicillamine solution and the borane-tert-butylamine complex solution is (150-450):(350-450):(200-400).

[0045] Preferably, according to the present invention, in step S2, the pH is adjusted by adding 1 M sodium hydroxide solution to adjust the solution pH to 8.0.

[0046] Preferably according to the present invention, in step S2, the amount of zinc nitrate hexahydrate added is such that the concentration of zinc nitrate hexahydrate in the system is 20 mM-140 mM, and the most preferred concentration is 100 mM.

[0047] Preferably according to the present invention, in step S2, the reaction temperature is 25°C.

[0048] Most preferably, Zn 2+ The optimized synthesis scheme of the preparation method of aggregation-induced gold nanoclusters is as follows:

[0049] (1) Add 200 μL of 96 mM HAuCl to 5 mL of ultrapure water. 4 ∙3H 2 O solution, stirred at room temperature for 5 min;

[0050] (2) Add 400 μL of 100 mM D-penicillamine solution to step (1) and stir at room temperature for 5 min;

[0051] (3) Add 300 μL of 100 mM mild reducing agent borane-tert-butylamine complex solution to step (2) and stir at room temperature for 30 min;

[0052] (4) Add 1 M sodium hydroxide solution to step (3), adjust the pH of the solution to 8.0, and continue stirring;

[0053] (5) Add 0.1700 g of zinc nitrate hexahydrate solid to step (4) until it is completely dissolved. After the reaction is complete, the obtained Zn 2+ -AuNCs were purified by centrifugation at 12000 rpm three times with isopropanol, and the obtained solid was redissolved in an equal volume of water to obtain ultra-bright red fluorescent water-soluble Zn 2+ Aggregation-induced gold nanoclusters Zn 2+ -AuNCs are stably stored at 4 °C.

[0054] The Zn prepared by the present invention 2+ aggregation-induced gold nanoclusters are placed in a three-electrode system. Using 0.1 M Hepes with a pH of 7.4 as the buffer solution and 10 mM 2-(dibutylamino)ethanol (DBAE) as the co-reactant, the Zn 2+ -AuNCs-based luminescence system generates a strong electrochemiluminescence signal by scanning and exciting the three-electrode system using cyclic voltammetry.

[0055] The preferred technical solution of the present invention:

[0056] A method for constructing a highly sensitive electrochemiluminescence immunosensor based on aggregation-induced gold clusters as markers is as follows:

[0057] a. The glassy carbon electrode (GCE) is successively polished on suede with alumina powders with diameters of 0.3 μm and 0.05 μm, and the electrode surface is washed and dried for later use;

[0058] b. The treated glassy carbon electrode is immersed in a 0.1 M PB buffer solution containing 1 mM ABA, and GCE|ABA is formed by electrodeposition; Electrodeposition conditions: The scanning rate is 0.05 mV / s; The scanning potential range is +0.40 V to +1.20 V; The number of cycles is 2;

[0059] c. Drop 10 μL of a mixed solution of EDC and NHS on the surface of the modified electrode obtained in step b. The concentration of EDC in the mixed solution is 1 mg / mL, and the concentration of NHS is 1 mg / mL. Activate at 37 °C for 15 min, and rinse the electrode with deionized water;

[0060] d. Drop 10 μL of a 10 μg / mL CEA primary antibody aqueous solution (CEA-Ab 1 ) on the surface of the electrode obtained in step c, incubate at 37 °C for 2 - 3 h to form GCE|ABA-Ab 1 , and rinse the electrode with deionized water;

[0061] e. Drop 10 μL of Gly on the surface of the electrode obtained in step d to block the unreacted active sites on the electrode, incubate at 37 °C for 1 - 2 h, and rinse the electrode with deionized water;

[0062] f. Drop a CEA antigen aqueous solution (CEA) on the surface of the electrode obtained in step e, incubate at 37 °C for 1 - 2 h to form GCE|ABA-Ab 1 <CEA, rinse the electrode with deionized water, and then add 10 μL of 10 μg / mL Zn 2+ -AuNCs-Ab 2 The aqueous solution was added dropwise to the electrode surface and incubated at 37 °C for 2-3 h to form GCE|ABA-Ab. 1 <cea>Ab 2 |Zn 2+ -AuNCs, rinse the electrode with deionized water and blow dry to obtain the electrochemiluminescent immunosensor GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNC.

[0063] A highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers is constructed using the above method.

[0064] A highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers, including Zn 2+ -AuNCs-Ab 2 , GCE|ABA-Ab after activation 1 And through the interaction between antigen and antibody, GCE|ABA-Ab 1 and Zn 2+ -AuNCs-Ab 2 Linked human carcinoembryonic antigen (CEA).

[0065] Application of highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers for antigen detection.

[0066] The method for antigen detection based on a high-sensitivity electrochemiluminescent immunosensor with aggregation-induced gold clusters as markers comprises the following steps:

[0067] A three-electrode system was adopted, with the constructed highly sensitive ECL immunosensor as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode, and cyclic voltammetry scans were carried out in a Hepes buffer solution containing a coreactant 2-(dibutylamino)ethanol DBAE.

[0068] Specific

[0069] The method for antigen detection based on a high-sensitivity electrochemiluminescence immunosensor with aggregation-induced gold clusters as markers, the specific steps are as follows:

[0070] I: A highly sensitive electrochemiluminescent immunosensor containing a known concentration of CEA antigen was constructed according to the above construction method, using Hepes as a buffer solution and 2-(dibutylamino)ethanol DBAE as a co-reactant. In a three-electrode system, the electrochemical reaction was triggered by cyclic voltammetry, the electrical triggering and light collection were synchronized, and the CCD was used to capture the ECL whole process image in real time to obtain the total spectrum. The ECL signal was collected using an ECL signal detection device, and a linear relationship curve between the maximum emission intensity of ECL and the concentration of CEA antigen was established.

[0071] II: constructing an ECL immunosensor of unknown CEA antigen concentration according to the above construction method, collecting ECL signals using the above method, and obtaining the antigen concentration in the sample solution to be tested according to the linear relationship curve;

[0072] Preferably, according to the present invention, in step I, in the above-mentioned three-electrode system, the constructed high-sensitivity electrochemiluminescence immunosensor is used as the working electrode, the platinum wire electrode is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode; the scanning voltage range is 0 ~+1.6V, the scanning number of circles is 1 circle, and the scanning speed is 0.05 V / s.

[0073] Preferably according to the present invention, in step I, the concentration of the Hepes buffer solution is 0.1 M, pH=7.4, and the concentration of 2-(dibutylamino)ethanol is 8-12 mM.

[0074] The present invention uses EDC and NHS to activate the carboxyl group in the gold cluster surface ligand DPA, grafts the amino group in the CEA secondary antibody, and successfully forms Zn 2+ -AuNCs-Ab 2 .

[0075] The present invention uses an electrodeposition method to deposit p-aminobenzoic acid on the surface of the working electrode glassy carbon electrode, activates the surface carboxyl groups of the p-aminobenzoic acid with EDC and NHS, and then grafts the amino groups on the surface of the CEA primary antibody to form GCE|ABA-Ab. 1 .

[0076] The present invention uses Zn 2+ The highly sensitive ECL immunosensor with aggregation-induced gold clusters as markers has the best specificity for human carcinoembryonic antigen (CEA), while the specificity for alpha-fetoprotein antigen (AFP), carbohydrate antigen (CA125) and prostate-specific antigen (PSA) is poor using the same immunoassay method.

[0077] The technical features and advantages of the present invention are as follows:

[0078] 1. The present invention first successfully prepared Zn 2+ Aggregation-induced generation of highly fluorescent water-soluble gold nanoclusters (Zn 2+ -AuNCs), whose electrochemiluminescence intensity is higher than that of Zn 2+ The aggregation-induced gold nanoclusters (AuNCs) increased by 2000 times, the fluorescence intensity increased by 20 times, and the fluorescence lifetime and quantum yield were also greatly improved.

[0079] 2. Zn prepared by the present invention 2+ -AuNCs has good photostability and can be stably stored at 4°C for nearly half a year; it has high sensitivity and uses Zn 2+ It induces aggregation of low-luminescent AuNCs, solidifies the chemical environment on the surface of the luminescent body, reduces non-radiative vibrational relaxation, greatly improves the ECL efficiency, and makes up for the defects of low-luminescent metal clusters.

[0080] 3. Zn prepared by the present invention 2+ -AuNCs can be used as high-quality luminophores to prepare ECL immunosensors. The sensors have high stability and high sensitivity. The ECL response signal corresponding to the luminescence wavelength of 767nm spans more than 4 orders of magnitude. It has high sensitivity for human carcinoembryonic antigen (CEA) detection, and the linear range is 5pg / mL-5ng / mL. It makes up for the limitations of low sensitivity and poor stability of the reported ECL immunosensors marked with metal nanoclusters, and optimizes the application of ECL immunosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The gold nanoclusters AuNCs and Zn prepared in Example 1 2+ -Fluorescence spectrum and UV-visible absorption spectrum of AuNCs.

[0082] Figure 2 The gold nanoclusters AuNCs and Zn prepared in Example 1 2+ -Fluorescence lifetime diagram of AuNCs.

[0083] Figure 3 The gold nanoclusters Zn prepared in Example 1 2+ -X-ray diffraction pattern of AuNCs.

[0084] Figure 4 The ligands D-penicillamine DPA and Zn in Example 1 2+ -Infrared spectrum of AuNCs.

[0085] Figure 5 The gold nanoclusters AuNCs and Zn prepared in Example 1 2+ -ECL-potential curve characterization of AuNCs; electrolyte: 0.1M pH 7.4 Hepes and 10 mM DBAE; potential scanning range: 0-1.6V; scanning rate: 50 mV / s.

[0086] Figure 6 The gold nanoclusters AuNCs and Zn prepared in Example 1 2+ -ECL total spectrum of AuNCs; electrolyte: 0.1MpH 7.4 Hepes and 10 mM DBAE; potential scanning interval: 0-1.6V; scanning rate: 50 mV / s.

[0087] Figure 7 The gold nanocluster Zn prepared in Example 1 2+ -AuNCs and Zn prepared in Example 2 2+ -AuNCs-Ab 2 ECL-potential curve characterization; electrolyte: 0.1 M pH 7.4 Hepes and 10 mM DBAE; potential scanning range: 0 - 1.6 V; scanning rate: 50 mV / s.

[0088] Figure 8 For Example 3, with gold nanocluster Zn 2+ -AuNCs as the marker, the ECL-potential curve characterizations of the electrochemiluminescence immunosensors prepared when the concentrations of human carcinoembryonic antigen (CEA) were 5 pg / mL, 10 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, and 10 ng / mL respectively; electrolyte: 0.1 M pH 7.4 Hepes and 10 mM DBAE; potential scanning range: 0 - 1.6 V; scanning rate: 50 mV / s.

[0089] Fig. 9 For Example 3, with gold nanocluster Zn 2+ -AuNCs as the marker, the working curves of the electrochemiluminescence immunosensors prepared when the concentrations of human carcinoembryonic antigen (CEA) were 5 pg / mL, 10 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, and 10 ng / mL respectively.

[0090] Fig.10 Comparison chart of the maximum ECL signal values corresponding to different antigens with a concentration of 100 pg / mL in Experimental Example 1;

[0091] Fig.11 ECL-potential curve characterization of the construction process of the electrochemiluminescence sensor in Comparative Example 1. Curve a is the bare glassy carbon electrode, curve b is GCE|ABA, curve c is GCE|ABA-Ab 1 and curve d is GCE|ABA-Ab 1 <CEA, and curve e is GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs;

[0092] Fig.12 For the electrochemical impedance spectroscopy (EIS) in the construction process of the electrochemiluminescence sensor in Comparative Example 2; Curve a is the bare glassy carbon electrode, Curve b is GCE|ABA, Curve c is GCE|ABA-Ab 1 , Curve d is GCE|ABA-Ab 1 <CEA, Curve e is GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs;

[0093] Fig.13 Zn prepared in Comparative Example 3 2+ -ECL potential curve characterization of AuNCs in DBAE as co-reactant and different buffer solution electrolytes; potential scanning range: 0-1.6V; scanning rate: 50 mV / s; the inset is an enlarged view of curve c because the intensity is too weak to be covered.

[0094] Fig.14 The Zn prepared in Comparative Example 4 2+ -ECL potential curves of AuNCs in an electrolyte with 0.1 M pH 7.4 Hepes as buffer and different co-reactants; potential scanning range: 0-1.6 V; scanning rate: 50 mV / s; the inset is an enlarged view of curves c, f, d, and e because the intensity is too weak to be covered. DETAILED DESCRIPTION

[0095] The present invention is further illustrated by the following examples, but is not limited thereto.

[0096] Example 1

[0097] Zn 2+ Aggregation-induced gold nanoclusters Zn 2+ -Preparation method of AuNC:

[0098] 1) First, 0.0150 g of ligand D-penicillamine and 0.0089 g of reducing agent borane-tert-butylamine complex were dissolved in 1 mL of water to prepare D-penicillamine solution and borane-tert-butylamine complex solution respectively;

[0099] 2) Add 200 μL of 96 mM HAuCl to 5 mL of deionized water. 4 ·3H 2 O, stirred at room temperature for 5 min; 400 μL D-penicillamine solution was added and stirred for 5 min, the solution quickly changed from light yellow to colorless; finally, 300 μL borane-tert-butylamine complex was added and stirred for 30 min, the solution slowly changed from colorless to brown yellow, and AuNCs solution was obtained;

[0100] 3) Adjust the pH of AuNCs solution to 8.0 with 1 M NaOH, add 0.1700 g zinc nitrate hexahydrate solid and stir until it is completely dissolved, and continue stirring at room temperature for 30 min to obtain Zn 2+ -AuNC solution.

[0101] The AuNCs obtained in step 2) and the Zn 2+ -AuNC fluorescence spectrum and UV-visible absorption spectrum are shown in Figure 1 , fluorescence lifetime see Figure 2 , XPS spectrum Figure 3 .

[0102] Figure 1 Display, Zn 2+ The fluorescence intensity of -AuNCs is about 20 times that of AuNCs, and Zn 2+ -AuNCs showed ultrabright red fluorescence.

[0103] Figure 2 Display, Zn 2+ -AuNCs fluorescence lifetime shows a significant advantage, about 18 times that of AuNCs.

[0104] Figure 3 Zn 2+ -AuNCs XPS spectrum, the two peaks at 1021 eV and 1044 eV are Zn(COOH) 2 The peak of Zn 2p in Zn 2+ -Zn in AuNCs 2+ Playing a bridging role, AuNCs form an aggregate, Zn 2+ With the -COO in the ligand DPA - Coordination solidification of gold cluster surface environment.

[0105] In Example 1, the ligand D-penicillamine DPA and Zn 2+ -Infrared spectra of AuNCs are shown in Figure 4 It can be seen that the present invention successfully synthesized Zn with D-penicillamine as a ligand. 2+ -AuNCs.

[0106] A three-electrode system was used, with a glassy carbon electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, a platinum wire as the counter electrode, 0.1 M pH 7.4 Hepes as the buffer solution, and 10 mM DBAE as the co-reactant. The AuNCs obtained in step 2) and the Zn obtained in step 3) were 2+ -AuNC are luminescent materials, and the luminescent material Zn 2+ -AuNCs were monodispersed in the above solution, and the luminophore concentration was 1.6 mg / mL. Cyclic voltammetry was used to scan and excite the three-electrode system to generate electrochemical luminescence signals. The generated signals were recorded at 800 V using an MPI-EIIECL analyzer. The obtained ECL spectrum is shown in the figure below: Figure 5 As shown, Zn 2+ -AuNCs ECL spectrum intensity is about 2000 times that of AuNCs, indicating that the Zn 2+ -AuNCs significantly increased the ECL signal intensity, and the total ECL spectrum was shown in Figure 6 .

[0107] Example 2

[0108] Zn 2+ -AuNCs-Ab 2 Preparation of aqueous solution:

[0109] 1) Add 350 μL of purified Zn prepared in Example 1 2+ Aggregation-induced gold nanoclusters Zn 2+ 6 μL of phosphate buffered saline (PBS) containing 1 mg / mL 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and 1 mg / mL hydroxysuccinimide (NHS) was added to the AuNCs and activated in an incubator at 37 °C for 15 min.

[0110] 2) Then add 80 μL of 10 μg / mL CEA-Ab 2 The solution was activated in a 37 °C incubator for 2 h, and finally 10 μL of 0.01 g / mL glycine solution was added to block nonspecific binding sites. The mixture was incubated at 37 °C for 2 h, and the precipitate was collected by centrifugation at 3000 rpm and redissolved in an equal volume of water to obtain Zn 2+ -AuNCs-Ab 2 Aqueous solution.

[0111] Zn 2+ -AuNC-Ab 2 The steps of electrochemiluminescence testing are as follows:

[0112] A three-electrode system was used, with a glassy carbon electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, a platinum wire as the counter electrode, 0.1 M pH 7.4 Hepes as the buffer solution, 10 mM DBAE as the co-reactant, and Zn 2+ -AuNC-Ab 2 It is a luminophore, and the concentration of the luminophore is 1.6 mg / mL;

[0113] The three-electrode system was scanned and excited using cyclic voltammetry to generate electrochemical luminescence signals, and the generated signals were recorded using an MPI-EII ECL analyzer at a voltage of 800 V. The obtained ECL spectrum is shown in the figure below: Figure 7 As shown, it can be seen that Zn 2+ -AuNCs-Ab 2 The luminescence potential is +0.9V, which is low potential luminescence. 2+ -AuNCs-Ab 2 ECL intensity is lower than Zn 2+ -AuNCs, indicating Ab 2 Binding with metal clusters hinders charge transfer, indicating that the antibody for CEA labeling is successfully grafted onto Zn 2+ Aggregation-induced gold nanoclusters on the surface.

[0114] Example 3

[0115] A method for constructing a highly sensitive electrochemiluminescence immunosensor based on aggregation-induced gold clusters as labels is as follows:

[0116] a. Polish the glassy carbon electrode (GCE) successively with alumina powders with diameters of 0.3 μm and 0.05 μm on suede, wash and dry the electrode surface for standby;

[0117] b. Immerse the treated glassy carbon electrode in 0.1 M PB buffer solution containing 1 mM ABA, and form GCE|ABA by electrodeposition; Electrodeposition conditions: scanning rate is 0.05 mV / s; scanning potential range is +0.40V - +1.20V; number of cycles is 2;

[0118] c. Drop 10 μL of the mixed solution of EDC and NHS on the surface of the modified electrode obtained in step b. The concentration of EDC in the mixed solution is 1mg / mL, and the concentration of NHS is 1mg / mL. Activate at 37 °C for 15 min, and rinse the electrode with deionized water;

[0119] d. Drop 10 μL of 10 μg / mL CEA primary antibody aqueous solution on the surface of the electrode obtained in step c, incubate at 37 °C for 2 - 3 h to form GCE|ABA-Ab 1 , rinse the electrode with deionized water;

[0120] e. Drop 10 μL of 0.01g / mL glycine solution on the surface of the electrode obtained in step d to block the unreacted active sites on the electrode, incubate at 37 °C for 1 h, and rinse the electrode with deionized water;

[0121] f. Drop CEA antigen aqueous solution on the surface of the electrode obtained in step e, incubate at 37 °C for 2 h to form GCE|ABA-Ab 1 <CEA, rinse the electrode with deionized water, and then add 10 μL of 10 μg / mL Zn prepared in Example 2 2+ -AuNCs-Ab 2 aqueous solution dropwise onto the electrode surface, incubate at 37 °C for 2h to form GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs, rinse the electrode with deionized water and blow dry to obtain a highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers, denoted as: GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNC.

[0122] The concentrations of the aqueous CEA antigen solution in step f were 5 pg / mL, 10 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, and 10 ng / mL, respectively, and the ECL immunosensor (GCE|ABA-Ab) at the corresponding CEA antigen concentration was obtained. 1 <cea>Ab 2 -Zn 2+ -AuNCs).

[0123] The method for antigen detection using a highly sensitive electrochemiluminescent immunosensor with a known concentration of CEA antigen is as follows:

[0124] A three-electrode system was used, with a high-sensitivity electrochemiluminescence immunosensor with a known concentration of CEA antigen as the working electrode, a platinum wire electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, 0.1 M pH 7.4 Hepes as the buffer solution, and 10 mM DBAE as the co-reactant. Cyclic voltammetry was used to scan and excite the three-electrode system to generate electrochemiluminescence signals, and the generated signals were recorded at 800 V using an MPI-EIIECL analyzer. The obtained ECL spectrum is shown in the figure below: Figure 8 As shown in the figure, the maximum emission potential of the ECL sensor is +0.9V-1.2V. As the concentration of CEA antigen increases, the corresponding ECL intensity gradually increases, and the two are proportional, indicating that the Zn-based 2+ -AuNCs as markers for highly sensitive ECL immunosensors with good application prospects.

[0125] The maximum ECL intensity and the corresponding CEA antigen concentration were plotted into a working curve, and it was found that the maximum ECL intensity signal value was linearly related to the logarithm of the CEA antigen concentration, such as Fig. 9 As shown, the linear range is 10 pg / mL-5 ng / mL, and the correlation coefficient R 2 The detection limit (LOD) was 0.9934 and 5 pg / mL, showing excellent reproducibility and high sensitivity. 2+ -The ECL response performance of the ECL immunosensor with AuNCs as the marker is higher than that of the currently reported sensors (such as the sensor reported in Anal. Chem. 2024, 96, 17, 6652–6658).

[0126] Experimental Example 1

[0127] The same construction method as described in Example 3, except that:

[0128] Different ECL immunosensors were prepared by replacing 100 pg / mL human carcinoembryonic antigen (CEA) with blank, 100 pg / mL alpha-fetoprotein antigen (AFP), 100 pg / mL prostate-specific antigen (PSA) and 100 pg / mL carbohydrate antigen 125 (CA125), respectively.

[0129] The test steps of different ECL immunosensors were the same as those in Example 3, and the concentration of the above antigens was 100 pg / mL, corresponding to the maximum ECL signal value, such as Fig.10 As shown in the figure, it can be seen that the ECL immunosensor exhibits a strong ECL signal response to human carcinoembryonic antigen (CEA), but has almost no response to AFP, PSA and CA125, indicating that the prepared Zn 2+ -AuNCs-labeled ECL immunosensor has excellent selectivity and ultra-high sensitivity.

[0130] Experimental Example 2

[0131] (1) A clean glassy carbon electrode (GCE) was polished on suede with alumina powder with a diameter of 0.3 μm and 0.05 μm, washed with distilled water, and dried with nitrogen gas for later use;

[0132] (2) Using a bare glassy carbon electrode as the working electrode, a three-electrode system was excited by cyclic voltammetry. The working electrode was a glassy carbon electrode, the reference electrode was an Ag / AgCl electrode, the counter electrode was a platinum wire, 0.1 M pH 7.4 Hepes was used as the buffer solution, 10 mM DBAE was used as the co-reactant, the potential window was 0 - +1.6 V, and the scanning speed was 50 mV / s. The ECL intensity-potential curve of the bare glassy carbon electrode was obtained as shown in the figure. Fig.11 Curve a shows that the bare glassy carbon electrode has no electrochemiluminescence in Hepes buffer containing 10 mM DBAE.

[0133] (3) The glassy carbon electrode treated in step (1) was immersed in 0.1 M pH 7.4 Hepes buffer containing 1 mM ABA, and ABA was modified onto the surface of GCE by electrodeposition to form GCE|ABA; the electrodeposition conditions were: scanning speed of 0.05 mV / s; scanning potential range of +0.40 V-+1.20 V; and the number of cycles was 2;

[0134] Using GCE|ABA as the working electrode, the test was performed according to step (2) to obtain the GCE|ABA ECL intensity-potential curve as shown in the figure: Fig.11 As shown in curve b, the GCE|ABA electrode also has no electrochemiluminescence in Hepes buffer containing 10 mM DBAE.

[0135] (4) GCE|ABA was obtained according to step (3). 10 μL of a mixed solution of 1 mg / mL EDC and 1 mg / mL NHS was added to the electrode surface to activate the surface carboxyl groups of ABA. The mixture was activated at 37 °C for 15 min. The remaining EDC and NHS were rinsed with deionized water. 10 μL of a 10 μg / mL CEA primary antibody aqueous solution was added. The mixture was incubated at 37 °C for 2-3 h. The remaining CEA primary antibody was rinsed with deionized water to form GCE|ABA-Ab. 1 .

[0136] Using GCE|ABA-Ab 1 as the working electrode, perform the test according to the method in step (2) to obtain the GCE|ABA-Ab 1 ECL intensity-potential curve as Fig.11 shown in curve c, indicating that GCE|ABA-Ab 1 electrode also has no electrochemiluminescence in Hepes buffer containing 10 mM DBAE.

[0137] (5) The GCE|ABA-Ab obtained in step (4) 1 , add 10 μL of 0.01 g / mL glycine solution to the electrode surface to block the remaining active sites, block at 37 °C for 2 h, rinse the remaining glycine with deionized water, add 10 μL of 1 ng / mL CEA antigen aqueous solution, incubate at 37 °C for 2 h, and rinse the remaining CEA antigen with deionized water to form GCE|ABA-Ab 1 <CEA,

[0138] Using GCE|ABA-Ab 1 <CEA as the working electrode, perform the test according to the method in step (2) to obtain the GCE|ABA-Ab 1 <CEA ECL intensity-potential curve as Fig.11 shown in curve d, indicating that GCE|ABA-Ab 1 <CEA electrode also has no electrochemiluminescence in Hepes buffer containing 10 mM DBAE.

[0139] (6) The GCE|ABA-Ab obtained in step (5) 1 <CEA, add 10 μL of Zn 2+ -AuNCs-Ab 2 (prepared by the method in Example 2), incubate at 37 °C for 1 h, and rinse the remaining Zn 2+ -AuNCs-Ab 2 , to form GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs.

[0140] Take GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs modified glassy carbon electrode was used as the working electrode to obtain GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs ECL intensity-potential curves Fig.11 As shown in curve e, the ECL intensity is significantly improved, eliminating the influence of other steps on the signal value of the ECL immunosensor. The signal generated by the MPI-EII ECL analyzer is about 16K when the photomultiplier tube voltage is 800V, indicating that the Zn 2+ -AuNCs-labeled ECL immunosensor has ultra-high sensitivity to CEA, indicating that the ECL immunosensor prepared by the present invention has broad application prospects in CEA.

[0141] Experimental Example 3

[0142] The bare glassy carbon electrode, GCE|ABA, and GCE|ABA-Ab formed in the synthesis step of Comparative Example 1 1 、GCE|ABA-Ab 1 <CEA、GCE|ABA-Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs, a three - electrode system was used respectively (the above - modified glassy carbon electrode as the working electrode, the reference electrode as the Ag / AgCl electrode, and the counter electrode as the platinum wire);

[0143] With 0.1 M pH 7.4 PB buffer solution containing 5 mM K 3 Fe(CN) 6 / K 4 Fe(CN) 6 as the electrolyte, the obtained electrochemical impedance spectra are as Fig.12 shown.

[0144] Fig.12 It shows that the resistance of the bare glassy carbon electrode is almost 0 (curve a); compared with the bare glassy carbon electrode, after modifying the glassy carbon electrode with ABA, a large amount of negative charges exist on the GCE surface, and there is a repulsive interaction with the negative charges carried by Fe(CN) 6 3- / Fe(CN) 6 4- which enhances the surface resistance of the electrode (curve b); grafting Ab 1 on the GCE|ABA surface reduces the amount of negative charges and thus the resistance decreases (curve c); grafting non - conductive CEA antigen and Ab 1 on the GCE|ABA - Ab 2 |Zn 2+ -AuNCs hinders the reaction and charge transport of Fe(CN) 6 3- / Fe(CN) 6 4- on the electrode surface, making the resistance of GCE|ABA - Ab 1 <CEA (curve d) and GCE|ABA - Ab 1 <cea>Ab 2 |Zn 2+ -AuNCs (curve e) resistance values ​​increase successively.

[0145] Experimental Example 4

[0146] The method described in Example 1 was used to conduct a comparative experiment by changing the buffer solution:

[0147] A three-electrode system was used, with a glassy carbon electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, a platinum wire as the counter electrode, 0.1 M pH 7.4 Hepes, 0.1 M pH 8.0 Tris-HCl, 0.1 M pH 7.4 PB, and 0.1 M pH 9.0 CB as the buffer solutions, 10 mM DBAE as the co-reactant, and the Zn obtained in step 3 of Example 1 2+ -AuNCs were used as the luminescent material, and 1.6 mg / mL Zn 2+ -AuNCs are monodispersed in the above solution.

[0148] Cyclic voltammetry was used with a potential window of 0 - +1.6 V and a scan rate of 50 mV / s to obtain the ECL intensity-potential curve of the glassy carbon electrode. Fig.13 The curve shows that the type of buffer solution has a great influence on the ECL signal response of the material. When the co-reactant is DBAE, Zn 2+ -AuNCs has the strongest ECL intensity in 0.1 M pH 7.4 Hepes, which is about 220,000 (the photomultiplier tube is set to 800 V and the amplification level is 3)

[0149] Experimental Example 5

[0150] The method described in Example 1 was used to conduct a comparative experiment by changing the co-reactant:

[0151] A three-electrode system was used, with a glassy carbon electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire as the counter electrode. 0.1 M pH 7.4 Hepes was used as the buffer solution, and 10 mM DBAE, TEOA, TPRA, and H 2 O 2 、TEA、N 2 H 4 As a co-reactant, Zn obtained in step 3 of Example 1 2+ -AuNCs were used as the luminescent material, and 1.6 mg / mL Zn 2+ -AuNCs are monodispersed in the above solution.

[0152] Cyclic voltammetry was used with a potential window of 0 - +1.6 V and a scan rate of 50 mV / s to obtain the ECL intensity-potential curve of the glassy carbon electrode. Fig.14 The curve shows that the type of co-reactant also has a great influence on the ECL signal response of the material. When the buffer solution is 0.1 M pH 7.4 Hepes, Zn 2+ -AuNCs had the strongest ECL intensity in 10 mM DBAE and the weakest ECL intensity in 10 mM TEA.< / cea> < / cea> < / cea> < / cea> < / cea> < / cea> < / cea> < / cea> < / cea> < / cea> < / cea> < / cea> < / cea> < / cea>

Claims

1. A method for constructing a highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers, the steps are as follows: (1) After the working electrode is polished, washed, grafted with para-aminobenzoic acid ABA, and activated for pretreatment, a CEA-Ab1 aqueous solution is added to the electrode surface and incubated at a constant temperature to form GCE|ABA-Ab1. After glycine blocking, a CEA antigen aqueous solution is added and incubated at a constant temperature to form GCE|ABA-Ab1. <CEA; (2) Zn 2+ -AuNCs-Ab2 aqueous solution was added dropwise to the electrode surface in step (1) and incubated at a constant temperature to form GCE|ABA-Ab1 <cea>Ab2|Zn 2+ -AuNCs, with Zn 2+ -AuNCs as markers to form highly sensitive electrochemiluminescent immunosensor GCE|ABA-Ab1 through sandwich immunoassay <cea>Ab2|Zn 2+ -AuNC;< / cea> < / cea> Zn 2+ -The concentration of AuNCs-Ab2 aqueous solution is 5-15 μg / mL, the amount of addition is 5-15 μL, and the constant temperature incubation is 37 ℃ for 2-3 h; Zn 2+ The synthesis steps of the AuNCs-Ab2 aqueous solution are as follows: 1) To the purified Zn 2+ 6 μL of phosphate buffered saline (PBS) containing 1 mg / mL 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and 1 mg / mL hydroxysuccinimide (NHS) was added to the aggregation-induced gold nanoclusters and activated in an incubator at 37 °C for 15 min. 2) Add 80 μL of 10 μg / mL CEA-Ab2 aqueous solution, activate in a 37 ℃ incubator for 2-3 h, and finally add 10 μL of 0.01g / mL glycine solution to block nonspecific binding sites, incubate at 37 ℃ for 1-2 h, collect the precipitate by centrifugation at 3000 rpm and redissolve in an equal volume of water to obtain Zn 2+ -AuNCs-Ab2 aqueous solution; Zn 2+ Aggregation-induced gold nanoclusters were prepared using HAuCl4∙3H2O as gold source, D-penicillamine as ligand, borane-tert-butylamine complex as reducing agent, and zinc nitrate hexahydrate as aggregation inducer. Zn 2+ The preparation method of aggregation-induced gold nanoclusters is as follows: a. Add 200 μL of 96 mM HAuCl4∙3H2O solution to 5 mL of ultrapure water and stir at room temperature for 5 min. b. Add 400 μL of 100 mM D-penicillamine solution to step a and stir at room temperature for 5 min. c. Add 300 μL of 100 mM mild reducing agent borane-tert-butylamine complex solution to step b and stir at room temperature for 30 min; d. Add 1 M sodium hydroxide solution to step c, adjust the pH of the solution to 8.0, and continue stirring; e. Add 0.1700 g of zinc nitrate hexahydrate solid to step d until it is completely dissolved. After the reaction is complete, the obtained Zn 2+ -AuNCs were purified by centrifugation at 12000 rpm three times with isopropanol, and the obtained solid was redissolved in an equal volume of water to obtain ultra-bright red fluorescent water-soluble Zn 2+ Aggregation-induced gold nanoclusters Zn 2+ -AuNCs are stably stored at 4 °C.

2. The construction method according to claim 1, characterized in that: In step (1), the working electrode is a glassy carbon electrode, polishing is performed by polishing with alumina powder with a diameter of 0.3 μm and 0.05 μm in sequence, and grafting para-aminobenzoic acid ABA is performed by immersing the treated glassy carbon electrode in a 0.1 M pH 7.4 PB buffer solution containing 1 mM ABA, and forming GCE|ABA by electrodeposition; the electrodeposition conditions are as follows: the scanning speed is 0.05 mV / s; the scanning potential range is +0.40 V to +1.20 V; the number of cycles is 2; activation is performed by dropping 10 μL of a mixed solution of EDC and NHS onto the electrode surface, the EDC concentration in the mixed solution is 1 mg / mL, and the NHS concentration is 1 mg / mL, the activation is performed at 37°C for 15 min, and the electrode is rinsed with deionized water; The concentration of CEA-Ab1 aqueous solution is 5-15 μg / mL, the drop volume is 5-15 μL, the drop volume of CEA antigen aqueous solution is 5-15 μL, the CEA concentration in the CEA antigen aqueous solution is 0.005 ng / mL ~ 10 ng / mL, the concentration of glycine solution used for glycine blocking is 0.01g / mL, the solvent is 0.1 M Ph 7.4 PB buffer solution, the constant temperature incubation is 37 ℃ for 2-3 h to form GCE|ABA-Ab1, and the electrode is rinsed with deionized water.

3. The construction method according to claim 1, characterized in that: In step S2, the pH is adjusted by adding 1 M sodium hydroxide solution to adjust the solution pH to 8.0, the amount of zinc nitrate hexahydrate added is such that the concentration of zinc nitrate hexahydrate in the system is 20 mM-140 mM, and the reaction temperature is 25°C.

4. A highly sensitive electrochemiluminescent immunosensor based on aggregation-induced gold clusters as markers, constructed using the method described in any one of claims 1 to 3.

5. Application of the high-sensitive electrochemiluminescence immunosensor with the aggregation-induced gold cluster as a marker according to claim 4 for antigen detection; a three-electrode system is used, with the constructed high-sensitive ECL immunosensor as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode, and cyclic voltammetry scanning is performed in a Hepes buffer solution containing a coreactant 2-(dibutylamino)ethanol DBAE.

6. A method for antigen detection based on the high-sensitivity electrochemiluminescence immunosensor using the aggregation-induced gold cluster as a marker according to claim 5, the specific steps being as follows: I: A highly sensitive electrochemiluminescent immunosensor containing a known concentration of CEA antigen was constructed according to the above construction method, using Hepes as a buffer solution and 2-(dibutylamino)ethanol DBAE as a co-reactant. In a three-electrode system, the electrochemical reaction was triggered by cyclic voltammetry, the electrical triggering and light collection were synchronized, and the CCD was used to capture the ECL whole process image in real time to obtain the total spectrum. The ECL signal was collected using an ECL signal detection device, and a linear relationship curve between the maximum emission intensity of the ECL and the concentration of the CEA antigen was established. II: constructing an ECL immunosensor of unknown CEA antigen concentration according to the above construction method, collecting ECL signals using the above method, and obtaining the antigen concentration in the sample solution to be tested according to the linear relationship curve; In step I, in the three-electrode system, the constructed highly sensitive electrochemiluminescent immunosensor is used as the working electrode, the platinum wire electrode is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode; the scanning voltage range is 0 ~ +1.6V, the scanning circle number is 1 circle, and the scanning speed is 0.05 V / s; the concentration of the Hepes buffer solution is 0.1M, pH=7.4, and the concentration of 2-(dibutylamino)ethanol is 8-12mM.

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