Construction method of self-energized photoelectrochemical immunosensor and application of self-energized photoelectrochemical immunosensor in genotoxic marker detection

Abstract

The invention provides a construction method of a self-energized photoelectrochemical immunosensor and application of the self-energized photoelectrochemical immunosensor in genotoxic marker detection, and relates to the technical field of chemical biosensors. Based on a specific immune recognition reaction of a genotoxic marker phosphorylated histone (gamma H2AX), CdIn2S4 nanospheres are used as a photo-anode, and a CdIn2S4 nanosphere is used as a photo-cathode; au is used as a photocathode substrate material for modifying a gamma H2AX primary antibody (Ab1), Bi-doped BiOI (Bi-BiOI) nanoflowers are used for marking a gamma H2AX secondary antibody (Ab2), and through layer-by-layer fixed modification and sandwich immunoreaction, the signal amplification type double-photoelectrode self-energized photoelectrochemical immunosensor is constructed. The sensor constructed by the invention has a wider linear range and an ultralow detection limit, and is of great significance to detection of genotoxic markers and screening of genotoxic substances.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical biosensors, and in particular to a method for constructing a self-powered photoelectrochemical immunosensor and its application in detecting genotoxicity markers. Background Art

[0002] Genotoxic substances are carcinogenic, teratogenic, and mutagenic, and pose a serious threat to the environment and human health. When DNA is damaged by genotoxic substances, especially when the most serious DNA damage, DNA double bond breaks (DSBs), occurs, the C-terminal S-139 of histone H2AX is phosphorylated to form phosphorylated histone H2AX, i.e., γH2AX. γH2AX rapidly aggregates at the DSBs site to form foci, and the number of γH2AX foci is proportional to the number of DSBs, making γH2AX a biomarker that specifically characterizes DNA damage and a specific effect indicator that indicates genotoxicity. Therefore, the genotoxicity of compounds can be screened and evaluated by detecting the γH2AX protein.

[0003] At present, a variety of technologies for detecting γH2AX have been developed both at home and abroad, including two-dimensional gel electrophoresis, protein blotting, immunofluorescence microscopy, enzyme-linked immunosorbent assay (ELISA), flow cytometry and high-performance liquid chromatography-mass spectrometry. Compared with traditional in vitro genotoxicity testing methods, the above technologies have better sensitivity, specificity and accuracy, but most of them rely on large-scale instruments and equipment or the experimental procedures are cumbersome and time-consuming, which can hardly meet the actual needs of rapid screening of large quantities of genotoxic pollutants.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for constructing a self-powered photoelectrochemical immunosensor to at least solve one of the technical problems existing in the prior art. The method for constructing a self-powered photoelectrochemical immunosensor provided by the present invention can detect γH2AX protein with high sensitivity and high specificity, and realize early rapid screening of genotoxic substances.

[0006] The second purpose of the present invention is to provide a method for constructing a self-powered photoelectrochemical immunosensor and a self-powered photoelectrochemical immunosensor constructed therefrom.

[0007] The third object of the present invention is to provide a method for constructing a self-powered photoelectrochemical immunosensor for use in the detection of genotoxic markers.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: In a first aspect, the present invention provides a method for constructing a self-powered photoelectrochemical immunosensor, comprising the following steps: Applying a γH2AX standard solution and a Bi-BiOI@APTES@Ab2 dispersion to an ITO / Au / 11-MUA / Ab1 / BSA electrode in sequence to form an ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode, using the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode as a working electrode, and the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode as a photocathode; Applying CdIn2S4 dispersion to the ITO electrode to form an ITO / CdIn2S4 electrode, using the ITO / CdIn2S4 electrode as a counter electrode and a reference electrode, and the ITO / CdIn2S4 as a photoanode; Among them, Ab1 is a γH2AX capture antibody, and Ab2 is a γH2AX recognition antibody.

[0009] Furthermore, the CdIn2S4 dispersion is prepared by the following steps: The mixed solution of Cd(NO3)2·4H2O, In(NO3)3·xH2O and TAA is heated, centrifuged, washed and dried in sequence to obtain CdIn2S4 nanosphere powder, and then the CdIn2S4 nanosphere powder is dispersed in a solvent to obtain the CdIn2S4 dispersion; Preferably, the amount of Cd(NO3)2·4H2O used is 1-3 mmol; Preferably, the amount of In(NO3)3·xH2O used is 2-6 mmol.

[0010] Furthermore, the preparation process of the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode includes: The γH2AX standard solution was applied to the ITO / Au / 11-MUA / Ab1 / BSA electrode, and after incubation, it was washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode; Afterwards, the Bi-BiOI@APTES@Ab2 dispersion was applied to the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode, and after incubation, it was washed with PBS to obtain an ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode; Preferably, the Bi-BiOI@APTES@Ab2 is incubated on the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode for 10-80 min.

[0011] Furthermore, the ITO / Au / 11-MUA / Abl / BSA electrode is prepared by the following steps: The ITO electrode is sequentially subjected to nano-gold coating deposition and carboxylation to obtain an ITO / Au / 11-MUA electrode, and then Ab1 is fixed to the ITO / Au / 11-MUA electrode to obtain the ITO / Au / 11-MUA / Ab1 / BSA electrode; Preferably, the nano-gold coating deposition comprises: using an ITO substrate as a working electrode, an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and HAuCl4 and H2SO4 as electrolytes, using a three-electrode system for electrodeposition, depositing a gold film on the ITO surface to obtain an ITO / Au electrode; Preferably, the carboxylation comprises: immersing the ITO / Au electrode in 11-mercaptoundecanoic acid solution and EDC / NHS solution in sequence to obtain an ITO / Au / 11-MUA electrode; Preferably, the immobilization process of Ab1 on the ITO / Au / 11-MUA electrode comprises: applying Ab1 and BSA solution to the ITO / Au / 11-MUA electrode in sequence to obtain an ITO / Au / 11-MUA / Ab1 / BSA electrode.

[0012] Furthermore, before depositing the nano-gold coating, the ITO electrode is pretreated; Preferably, the pretreatment includes: cutting, first cleaning and second cleaning of the ITO electrode in sequence; Preferably, the first cleaning comprises: cleaning the ITO electrode with acetone, and then performing ultrasonic treatment on the ITO electrode; Preferably, the second cleaning comprises: using an ITO electrode as a working electrode, a platinum electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode, placing them in PBS, performing a potential cycle cleaning from 0 to +1 V, and then rinsing with deionized water.

[0013] Furthermore, the Bi-BiOI@APTES@Ab2 dispersion is prepared by the following steps: After the synthesis of the Bi-BiOI nanomaterial, it is aminated to obtain Bi-BiOI@APTES, and then Ab2 and Bi-BiOI@APTES are mixed and dispersed in PBS, the Bi-BiOI@APTES@Ab2 dispersion; Preferably, the synthesis of the Bi-BiOI nanomaterial comprises: using Bi(NO3)3·5H2O solution as solution A, using KI solution as solution B, using a mixed solution of solution A and solution B as solution C, and sequentially heating, centrifuging, washing and drying solution C to obtain the Bi-BiOI nanomaterial; Preferably, the amination comprises: dispersing the Bi-BiOI nanomaterial in anhydrous ethanol, and then adding (3-aminopropyl)triethoxysilane to obtain Bi-BiOI@APTES; Preferably, the (3-aminopropyl)triethoxysilane is used in an amount of 0.1-0.3 mL; Preferably, the mixing of Ab2 and Bi-BiOI@APTES comprises: adding BSA to a mixed solution of Bi-BiOI@APTES, Ab2 and EDC / NHS, and obtaining Bi-BiOI@APTES@Ab2 after centrifugation; Preferably, the concentration of Bi-BiOI@APTES is 0.5-10 mg / mL; Preferably, the volume ratio of Bi-BiOI@APTES, Ab2 and EDC / NHS is 1:1:(1-2).

[0014] Further, the amount of Bi(NO3)3·5H2O used was 1-5 mmol, and the amount of KI used was 1-5 mmol; The solvent of the solution A includes at least one of EG and H2O, wherein EG is 0-40 mL, H2O is 0-40 mL, and the total amount of EG and H2O in the solution A is 40 mL; The solvent of the solution B includes at least one of EG and H2O, wherein EG is 0-30 mL, H2O is 0-30 mL, and the total amount of EG and H2O in the solution B is 30 mL.

[0015] Furthermore, the concentration of the γH2AX standard solution is 0.0001 ng / mL to 1000 ng / mL.

[0016] In a second aspect, the present invention provides a self-powered photoelectrochemical immunosensor constructed by the construction method of the self-powered photoelectrochemical immunosensor, and the detection method of the self-powered photoelectrochemical immunosensor comprises: PEC detection: ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode was used as the working electrode, and ITO / CdIn2S4 electrode was used as the counter electrode and reference electrode. The ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode and the ITO / CdIn2S4 electrode were placed in a PBS electrolyte containing H2O2. A three-electrode system was used for photoelectric testing, PEC signals were measured, and working curves were drawn. Wherein, the pH of the PBS electrolyte containing H2O2 is 6-8.5.

[0017] In a third aspect, the present invention provides an application of the method for constructing a self-powered photoelectrochemical immunosensor in the detection of genotoxicity markers.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The method for constructing a self-powered photoelectrochemical immunosensor provided by the present invention is based on the specific immune recognition reaction of phosphorylated histone (γH2AX), a marker of genotoxicity, and uses CdIn2S4 nanospheres as photoanode, Au as photocathode substrate material to modify γH2AX primary antibody (Ab1), Bi-doped BiOI (Bi-BiOI) nanoflowers to label γH2AX secondary antibody (Ab2). Through layer-by-layer fixed modification and sandwich immune reaction, a signal amplification type dual-photoelectrode self-powered photoelectrochemical immunosensor is constructed. The sensor constructed by the present invention has a wide linear range and an ultra-low detection limit, which is of great significance for the detection of genotoxic markers and the screening of genotoxic substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The photocurrent detection signals of the self-powered photoelectrochemical immunosensor provided in Example 1 of the present invention for different concentrations of γH2AX: 0.0001 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 20 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL; Figure 2The linear relationship between the photocurrent change value ΔI and the Log value of the γH2AX concentration provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0021] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0022] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The first aspect of the present invention provides a method for constructing a self-powered photoelectrochemical immunosensor, comprising the following steps: Applying a γH2AX standard solution and a Bi-BiOI@APTES@Ab2 dispersion to an ITO / Au / 11-MUA / Ab1 / BSA electrode in sequence to form an ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode, using the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode as a working electrode, and the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode as a photocathode; Applying CdIn2S4 dispersion to the ITO electrode to form an ITO / CdIn2S4 electrode, using the ITO / CdIn2S4 electrode as a counter electrode and a reference electrode, and the ITO / CdIn2S4 as a photoanode; Among them, Ab1 is a γH2AX capture antibody, and Ab2 is a γH2AX recognition antibody.

[0025] The method for constructing a self-powered photoelectrochemical immunosensor provided by the present invention is a method for constructing a self-powered photoelectrochemical immunosensor based on CdIn2S4 and Au / Bi-BiOI. Based on the specific immune recognition reaction of the genotoxic marker phosphorylated histone (γH2AX), CdIn2S4 nanospheres are used as photoanodes, Au is used as photocathode substrate materials to modify γH2AX primary antibody (Ab1), Bi-doped BiOI (Bi-BiOI) nanoflowers are used to label γH2AX secondary antibody (Ab2), and a signal amplification type dual-photoelectrode self-powered photoelectrochemical immunosensor is constructed through layer-by-layer fixed modification and sandwich immune reaction.

[0026] The present invention constructs a dual-photoelectrode self-powered photoelectrochemical sensor based on CdIn2S4 photoanode and Au / Bi-BiOI photocathode materials for the detection of γH2AX protein. Generally speaking, PEC sensors are divided into anode sensors and cathode sensors. The former usually has higher sensitivity but weaker anti-interference ability, while the latter has good anti-interference ability but lower sensitivity. In the dual-photoelectrode self-powered sensor of the present invention, the photoanode amplifies the signal and the photocathode senses the analyte, combining the strong anti-interference ability of the cathode and the high detection sensitivity of the anode, thereby overcoming their respective shortcomings. Test results show that the photoelectrochemical sensor has high sensitivity, low detection limit and good stability.

[0027] In some preferred embodiments, the CdIn2S4 dispersion is prepared by the following steps: The mixed solution of Cd(NO3)2·4H2O, In(NO3)3·xH2O and TAA is heated, centrifuged, washed and dried in sequence to obtain CdIn2S4 nanosphere powder, and then the CdIn2S4 nanosphere powder is dispersed in a solvent to obtain the CdIn2S4 dispersion; Preferably, the amount of Cd(NO3)2·4H2O used is 1-3 mmol, for example, 1 mmol, 2 mmol, 3 mmol, etc.; Preferably, the usage amount of the In(NO3)3·xH2O is 2-6 mmol, for example, 2 mmol, 4 mmol, 6 mmol, etc. The usage amount of the In(NO3)3·xH2O is preferably twice the usage amount of the Cd(NO3)2·4H2O.

[0028] Further preferably, the specific preparation process of the CdIn2S4 dispersion includes: Synthesis of CdIn2S4 nanospheres with narrow band gap width: 1-3 mmol Cd(NO3)2·4H2O, 2-6 mmol In(NO3)3·xH2O and 6 mmol TAA were dissolved in 80 mL deionized water. After continuous stirring for 30 min, the mixture was transferred to a 100 mL reactor, heated at 160°C for 12 h, and then naturally cooled to room temperature, collected by centrifugation, washed several times with ethanol and distilled water, and vacuum dried at 60°C for 12 h to obtain yellow CdIn2S4 nanosphere powder. The CdIn2S4 nanosphere powder was then dispersed in deionized water to obtain the CdIn2S4 dispersion.

[0029] In some preferred embodiments, the preparation process of the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode includes: The γH2AX standard solution was applied to the ITO / Au / 11-MUA / Ab1 / BSA electrode, and after incubation, it was washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode; Wherein, the concentration of the γH2AX standard solution is 0.0001 ng / mL to 1000 ng / mL, for example, it can be 0.0001 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 20 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, etc.; Afterwards, the Bi-BiOI@APTES@Ab2 dispersion was applied to the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode, and after incubation, it was washed with PBS to obtain an ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode; Preferably, the incubation time of the Bi-BiOI@APTES@Ab2 on the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode is 10-80 min, for example, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, etc.

[0030] In some preferred embodiments, the Bi-BiOI@APTES@Ab2 dispersion is prepared by the following steps: After the synthesis of the Bi-BiOI nanomaterial, it is aminated to obtain Bi-BiOI@APTES, and then Ab2 and Bi-BiOI@APTES are mixed and dispersed in PBS, the Bi-BiOI@APTES@Ab2 dispersion; Preferably, the synthesis of the Bi-BiOI nanomaterial comprises: using a mixed solution of Bi(NO3)3·5H2O, EG and H2O as solution A, using a mixed solution of KI, EG and H2O as solution B, using a mixed solution of solution A and solution B as solution C, and sequentially heating, centrifuging, washing and drying solution C to obtain the Bi-BiOI nanomaterial; Preferably, the amination comprises: dispersing the Bi-BiOI nanomaterial in anhydrous ethanol, and then adding (3-aminopropyl)triethoxysilane to obtain Bi-BiOI@APTES; Preferably, the (3-aminopropyl)triethoxysilane is used in an amount of 0.1-0.3 mL; Preferably, the mixing of Ab2 and Bi-BiOI@APTES comprises: adding BSA to a mixed solution of Bi-BiOI@APTES, Ab2 and EDC / NHS, and obtaining Bi-BiOI@APTES@Ab2 after centrifugation; Preferably, the concentration of Bi-BiOI@APTES is 0.5-10 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, etc.; Preferably, the volume ratio of Bi-BiOI@APTES, Ab2 and EDC / NHS is 1:1:(1-2), for example, it can be 1:1:1, 1:1:1.5, 1:1:2, etc.

[0031] Preferably, the solvent of the solution A comprises at least one of EG and H2O, wherein EG is 0-40 mL, H2O is 0-40 mL, and the total amount of EG and H2O in the solution A is 40 mL; Preferably, the solvent of the solution B comprises at least one of EG and H2O, wherein EG is 0-30 mL, H2O is 0-30 mL, and the total amount of EG and H2O in the solution B is 30 mL.

[0032] Further preferably, the present invention uses Bi-doped BiOI as the core to construct a signal-sensitized photoelectric immune signal probe (Bi-BiOI@APTES@Ab2), which specifically includes the following steps: Step 1: Synthesis of Bi-BiOI nanomaterials. 1-5 mmol Bi(NO3)3·5H2O was dissolved in a mixed solution of 0-40 mL EG and 0-40 mL H2O and magnetically stirred for 30 min (referred to as solution A, the total amount of EG and H2O in solution A was 40 mL), and 1-5 mmol KI was dissolved in a mixed solution of 0-30 mL EG and 0-30 mL H2O and magnetically stirred for 10 min (referred to as solution B, the total amount of EG and H2O in B was 30 mL). Solution B was added to solution A and magnetically stirred to form a yellow solution (solution C) immediately. Solution C was stirred for 60 min, transferred to a 100 mL Teflon liner, sealed, heated at 160°C for 12 h, cooled naturally to room temperature, collected by centrifugation, washed several times with ethanol and distilled water, and vacuum dried at 60°C for 12 h to obtain a brick red powder.

[0033] Step 2: Synthesis of aminated Bi-BiOI. 10 mg Bi-BiOI was dispersed in 10 mL of anhydrous ethanol and treated with 0.1-0.3 mL (3-aminopropyl)triethoxysilane (APTES, purity 99%) to aminate BiOI. After stirring at 350 rpm for 24 h at 30 °C, the aminated Bi-BiOI (i.e., Bi-BiOI@APTES) precipitate was collected by centrifugation and dispersed in PBS (pH 7.4).

[0034] Step 3: Synthesis of Bi-BiOI@APTES@Ab2 conjugates. The Bi-BiOI@APTES dispersion was first sonicated for 20 min, and then the mixed solution of Bi-BiOI@APTES (0.5-10 mg / mL), γH2AX recognition antibody (Ab2) dispersion (10 μg / mL) and EDC / NHS (10 mM / 2 mM) was stirred at 4 °C for 12 h. Then, 100 μL of BSA (0.1 wt %) solution was added and shaken at 250 rpm at 4 °C for 2 h to mask the nonspecific reaction sites of the electrode. After centrifugation, the obtained Bi-BiOI@APTES@Ab2 was redispersed in 2 mL PBS and stored at 4 °C.

[0035] In some preferred embodiments, the ITO / Au / 11-MUA / Abl / BSA electrode is prepared by the following steps: The ITO electrode is sequentially subjected to nano-gold coating deposition and carboxylation to obtain an ITO / Au / 11-MUA electrode, and then Ab1 is fixed to the ITO / Au / 11-MUA electrode to obtain the ITO / Au / 11-MUA / Ab1 / BSA electrode; Preferably, the nano-gold coating deposition comprises: using an ITO substrate as a working electrode, an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and HAuCl4 and H2SO4 as electrolytes, using a three-electrode system for electrodeposition, depositing a gold film on the ITO surface to obtain an ITO / Au electrode; Preferably, the carboxylation comprises: immersing the ITO / Au electrode in 11-mercaptoundecanoic acid solution and EDC / NHS solution in sequence to obtain an ITO / Au / 11-MUA electrode; Preferably, the immobilization process of Ab1 on the ITO / Au / 11-MUA electrode comprises: applying Ab1 and BSA solution to the ITO / Au / 11-MUA electrode in sequence to obtain an ITO / Au / 11-MUA / Ab1 / BSA electrode.

[0036] In some preferred embodiments, the ITO electrode is pretreated before the nano-gold coating is deposited; Preferably, the pretreatment includes: cutting, first cleaning and second cleaning of the ITO electrode in sequence; Preferably, the first cleaning comprises: cleaning the ITO electrode with acetone, and then performing ultrasonic treatment on the ITO electrode; Preferably, the second cleaning comprises: using an ITO electrode as a working electrode, a platinum electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode, placing them in PBS, performing a potential cycle cleaning from 0 to +1 V, and then rinsing with deionized water.

[0037] Further preferably, the preparation process of the ITO / Au / 11-MUA / Abl / BSA electrode specifically includes: Step 1: Pretreatment of ITO electrode: Cut the ITO glass into 1×2 cm 2 , and washed in acetone, then ultrasonically treated in acetone and water for 15 min each. The ITO electrode was used as the working electrode, the platinum electrode was used as the counter electrode, and the Ag / AgCl (containing 3 M KCl) electrode was used as the reference electrode. The electrode was placed in 50 mM PBS (pH 7.4, containing 0.1 M KCl) and washed in a potential cycle from 0 to +1 V, and then rinsed with deionized water.

[0038] Step 2: Electrodeposition of nanogold coating: The electrolyte consists of 3 mM HAuCl4 and 0.1 M H2SO4. In order to prevent oxygen from affecting the reduction reaction of gold during the electrodeposition process, the solution was bubbled with nitrogen for at least 15 min to remove dissolved oxygen. The experiment used a three-electrode system for electrodeposition. The ITO substrate was used as the working electrode, the reference electrode was an Ag / AgCl electrode, and the counter electrode was a platinum wire electrode. The pretreated ITO working electrode was placed in an electrochemical cell filled with chloroauric acid electrolyte together with the reference electrode and the counter electrode, and connected to the electrochemical workstation. The potential scan range of the electrochemical workstation was set to +0.2 V to -0.2 V ( vs . Ag / AgCl), and 10 cyclic voltammetry scans were performed at a scan rate of 10 mV / s. As the number of cycles increases, Au³⁺ in chloroauric acid is gradually reduced to gold under the action of potential and deposited on the ITO surface to form a uniform gold film.

[0039] Step 3: Carboxylation of ITO / Au electrode: 11-Mercaptoundecanoic acid (11-MUA) was modified as a self-assembled monolayer (SAM) on the Au / ITO electrode. 5 mM 11-MUA was prepared with anhydrous ethanol, during which N2 was bubbled to isolate oxygen and avoid oxidation; then, the ITO / Au electrode was immersed in the mixture for 20 h at 25 °C. Then, the electrode was thoroughly rinsed with ethanol and dried with N2. Then, the electrode was immersed in 50 mM EDC and NHS solution for 1 h to activate the carboxylic acid groups on the electrode surface to convert the terminal carboxyl groups of 11-MUA into NHS esters, and then the electrode was rinsed with deionized water and dried with N2 to obtain the ITO / Au / 11-MUA electrode.

[0040] Step 4: Immobilization of γH2AX capture antibody (Ab1) on ITO / Au / 11-MUA electrode: γH2AX capture antibody (Ab1) solution (20 µg / mL) was freshly prepared in PBS (pH 7.4). Then 10 µL of this solution was drop-casted on the ITO / Au / 11-MUA electrode. After the electrode was incubated at 37°C for 1 h, it was rinsed with PBS (pH 7.4) to remove any free antibody molecules. Ab1 was covalently bound to the -COOH end of ITO / Au / 11-MUA through an amide bond (CO-NH) using its own amino group, thereby being immobilized on the electrode surface. Then, 5 µL of BSA (0.5 wt%) solution was drop-coated on the electrode surface to mask nonspecific reaction sites. After incubation at 4°C for 1 h, the electrode surface was washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA immunoelectrode.

[0041] Step 5: Preparation of self-powered sensor: 10 μL of γH2AX standard solution with different concentrations (0.0001 ng / mL to 1000 ng / mL) was drop-coated on the surface of the ITO / Au / 11-MUA / Ab1 / BSA electrode, incubated at 37°C for 1 h, and washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode. After that, 10 μL of the prepared Bi-BiOI@APTES@Ab2 dispersion was drop-coated on the surface of the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode, incubated at 37°C for 10-80 min, and then washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode. In addition, 10 μL of 10 mg / mL CdIn2S4 was dropped on the surface of another ITO electrode and dried at 60 °C to obtain an ITO / CdIn2S4 electrode.

[0042] The second aspect of the present invention provides a self-powered photoelectrochemical immunosensor constructed by a method for constructing a self-powered photoelectrochemical immunosensor. The detection method of the self-powered photoelectrochemical immunosensor comprises: PEC detection: ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode was used as the working electrode, and ITO / CdIn2S4 electrode was used as the counter electrode and reference electrode. The ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode and the ITO / CdIn2S4 electrode were placed in a PBS electrolyte containing H2O2. A three-electrode system was used for photoelectric testing, PEC signals were measured, and working curves were drawn. In the present invention, it is preferred that: The ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode was used as the working electrode, and the ITO / CdIn2S4 electrode was used as the counter electrode and reference electrode in a 30 mL PBS (pH 6-8.5, 0.1 M) electrolyte containing 5 mM H2O2. The light source (450 nm) illuminated the two electrode surfaces simultaneously and switched at a frequency of 10 seconds. The PEC signal was measured using the current-time mode at 0 V potential using a CHI660E electrochemical workstation.

[0043] Wherein, the pH value of the PBS electrolyte containing H2O2 is 6-8.5, for example, it can be 6, 6.5, 7, 7.5, 8, 8.5, etc.

[0044] The third aspect of the present invention provides a method for constructing a self-powered photoelectrochemical immunosensor and its application in the detection of genotoxicity markers.

[0045] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0046] Example 1 This embodiment provides a method for constructing a self-powered photoelectrochemical immunosensor, comprising the following steps: (I) Synthesis of CdIn2S4 nanospheres: 1 mmol Cd(NO3)2·4H2O, 2 mmol In(NO3)3·xH2O and 6 mmol TAA were dissolved in 80 mL deionized water. After continuous stirring for 30 min, the mixture was transferred to a 100 mL reactor, heated at 160°C for 12 h, then naturally cooled to room temperature, collected by centrifugation, washed several times with ethanol and distilled water, and vacuum dried at 60°C for 12 h to obtain yellow CdIn2S4 nanosphere powder. The CdIn2S4 nanosphere powder was then dispersed in deionized water to obtain the CdIn2S4 dispersion.

[0047] (II) Construction of photoelectric immune signal probe with signal sensitization based on Bi-doped BiOI: Step 1: Synthesis of Bi-BiOI nanomaterials. 2 mmol Bi(NO3)3·5H2O was dissolved in 40 mL EG and magnetically stirred for 30 min (referred to as solution A), and 2 mmol KI was dissolved in 30 mL EG and magnetically stirred for 10 min (referred to as solution B). Solution B was added to solution A and magnetically stirred to immediately form a yellow solution (solution C). Solution C was stirred for 60 min, transferred to a 100 mL Teflon liner, sealed, heated at 160°C for 12 h, cooled naturally to room temperature, collected by centrifugation, washed several times with ethanol and distilled water, and vacuum dried at 60°C for 12 h to obtain a brick-red powder.

[0048] Step 2: Synthesis of aminated Bi-BiOI. 10 mg Bi-BiOI was dispersed in 10 mL of anhydrous ethanol and treated with 0.1 mL (3-aminopropyl)triethoxysilane (APTES, purity 99%) to aminate BiOI. After stirring at 350 rpm for 24 h at 30 °C, the aminated Bi-BiOI (i.e., Bi-BiOI@APTES) precipitate was collected by centrifugation and dispersed in PBS (pH 7.4).

[0049] Step 3: Synthesis of Bi-BiOI@APTES@Ab2 conjugates. The Bi-BiOI@APTES dispersion was first sonicated for 20 min, and then the mixed solution of Bi-BiOI@APTES (2 mg / mL, 2 mL), γH2AX recognition antibody (Ab2) dispersion (10 μg / mL, 2 mL) and EDC / NHS (10 mM / 2 mM, 2 mL) was stirred at 4 °C for 12 h. Then, 100 μL of BSA (0.1 wt %) solution was added and shaken at 250 rpm at 4 °C for 2 h to mask the nonspecific reaction sites of the electrode. After centrifugation, the obtained Bi-BiOI@APTES@Ab2 was redispersed in 2 mL PBS and stored at 4 °C.

[0050] (III) Preparation of self-powered photoelectrochemical immunosensor based on CdIn2S4 nanospheres and Bi-BiOI nanomaterials: Step 1: Pretreatment of ITO electrode: Cut the ITO glass into 1×2 cm 2 , and washed in acetone, then ultrasonically treated in acetone and water for 15 min each. The ITO electrode was used as the working electrode, the platinum electrode was used as the counter electrode, and the Ag / AgCl (3 M KCl) electrode was used as the reference electrode. The electrode was placed in 50 mM PBS (pH 7.4, containing 0.1 M KCl) and washed in a potential cycle from 0 to +1 V, and then rinsed with deionized water.

[0051] Step 2: Electrodeposition of nanogold coating: The electrolyte consists of 3 mM HAuCl4 and 0.1 M H2SO4. In order to prevent oxygen from affecting the reduction reaction of gold during the electrodeposition process, the solution was bubbled with nitrogen for at least 15 min to remove dissolved oxygen. The experiment used a three-electrode system for electrodeposition. The ITO substrate was used as the working electrode, the reference electrode was an Ag / AgCl electrode, and the counter electrode was a platinum wire electrode. The pretreated ITO working electrode was placed in an electrochemical cell filled with chloroauric acid electrolyte together with the reference electrode and the counter electrode, and connected to the electrochemical workstation. The potential scan range of the electrochemical workstation was set to +0.2 V to -0.2 V ( vs. Ag / AgCl) and performed 10 cyclic voltammetry scans at a scan rate of 10 mV / s. As the number of cycles increases, Au³⁺ in chloroauric acid is gradually reduced to gold under the action of potential and deposited on the ITO surface to form a uniform gold film.

[0052] Step 3: Carboxylation of ITO / Au electrode: 11-Mercaptoundecanoic acid (11-MUA) was modified as a self-assembled monolayer (SAM) on the Au / ITO electrode. 5 mM 11-MUA was prepared with anhydrous ethanol, during which N2 was bubbled to isolate oxygen and avoid oxidation; then, the Au / ITO electrode was immersed in the mixture for 20 h at 25 °C. Then, the electrode was thoroughly rinsed with ethanol and dried with N2. Then, the electrode was immersed in 50 mM EDC and NHS solution for 1 h to activate the carboxylic acid groups on the electrode surface to convert the terminal carboxyl groups of 11-MUA into NHS esters, and then the electrode was rinsed with deionized water and dried with N2 to obtain the ITO / Au / 11-MUA electrode.

[0053] Step 4: Immobilization of γH2AX capture antibody (Ab1) on ITO / Au / 11-MUA electrode: γH2AX capture antibody (Ab1) solution (20 µg / mL) was freshly prepared in PBS (pH 7.4). Then 10 µL of this solution was drop-casted on the ITO / Au / 11-MUA electrode. After the electrode was incubated at 37°C for 1 h, it was rinsed with PBS (pH 7.4) to remove any free antibody molecules. Ab1 was covalently bound to the -COOH end of ITO / Au / 11-MUA through an amide bond (CO-NH) using its own amino group, thereby being immobilized on the electrode surface. Then, 5 µL of BSA (0.5 wt%) solution was drop-coated on the electrode surface to mask nonspecific reaction sites. After incubation at 4°C for 1 h, the electrode surface was washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA immunoelectrode.

[0054] Step 5: Preparation of self-powered sensor: 10 μL of γH2AX standard solution with different concentrations (0.0001 ng / mL to 1000 ng / mL) was drop-coated on the surface of the ITO / Au / 11-MUA / Ab1 / BSA electrode, incubated at 37°C for 1 h, and washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode. After that, 10 μL of the prepared Bi-BiOI@APTES@Ab2 dispersion was drop-coated on the surface of the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode, incubated at 37°C for 10-80 min, and then washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode. In addition, 10 μL of 10 mg / mL CdIn2S4 was dropped on the surface of another ITO electrode and dried at 60 °C to obtain an ITO / CdIn2S4 electrode.

[0055] Among them, the concentrations of γH2AX standard solutions are: 0.0001 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 20 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL.

[0056] Example 2 This embodiment provides a method for constructing a self-powered photoelectrochemical immunosensor, which differs from Embodiment 1 in that: 2 mmol Bi(NO3)3·5H2O was dissolved in 20 mL EG and 20 mL H2O and magnetically stirred for 30 min (referred to as solution A), and 2 mmol KI was dissolved in 15 mL EG and 15 mL H2O and magnetically stirred for 10 min (referred to as solution B); The other preparation conditions are the same as in Example 1.

[0057] Example 3 This embodiment provides a method for constructing a self-powered photoelectrochemical immunosensor, which differs from Embodiment 1 in that: 2 mmol Bi(NO3)3·5H2O was dissolved in 10 mL EG and 30 mL H2O and magnetically stirred for 30 min (referred to as solution A), and 2 mmol KI was dissolved in 10 mL EG and 20 mL H2O and magnetically stirred for 10 min (referred to as solution B); The other preparation conditions are the same as in Example 1.

[0058] Example 4 This embodiment provides a method for constructing a self-powered photoelectrochemical immunosensor, which differs from Embodiment 1 in that: 2 mmol Bi(NO3)3·5H2O was dissolved in 30 mL EG and 10 mL H2O and magnetically stirred for 30 min (referred to as solution A), and 2 mmol KI was dissolved in 20 mL EG and 10 mL H2O and magnetically stirred for 10 min (referred to as solution B); The other preparation conditions are the same as in Example 1.

[0059] Example 5 This embodiment provides a method for constructing a self-powered photoelectrochemical immunosensor, which differs from Embodiment 1 in that: 2 mmol Bi(NO3)3·5H2O was dissolved in 40 mL H2O and magnetically stirred for 30 min (referred to as solution A), and 2 mmol KI was dissolved in 30 mL H2O and magnetically stirred for 10 min (referred to as solution B); The other preparation conditions are the same as in Example 1.

[0060] Example 6 This embodiment provides a method for constructing a self-powered photoelectrochemical immunosensor, which differs from Embodiment 1 in that: Step (i) In the synthesis of CdIn2S4 nanospheres, the amount of Cd(NO3)2·4H2O used is 2 mmol and the amount of In(NO3)3·xH2O used is 4 mmol; In step 1 of step (ii), the amount of Bi(NO3)3·5H2O used is 1 mmol, and the amount of KI used is 1 mmol; In step 2 of step (ii), the amount of (3-aminopropyl)triethoxysilane used was 0.3 mL; In step 3 of step (ii), Bi-BiOI@APTES was 0.5 mg / mL, and the amount of EDC / NHS mixed solution used was 4 mL; The other preparation conditions are the same as in Example 1.

[0061] Example 7 This embodiment provides a method for constructing a self-powered photoelectrochemical immunosensor, which differs from Embodiment 1 in that: In step (i) of the synthesis of CdIn2S4 nanospheres, the amount of Cd(NO3)2·4H2O used was 3 mmol and the amount of In(NO3)3·xH2O used was 6 mmol; In step 1 of step (ii), the amount of Bi(NO3)3·5H2O used is 5 mmol, and the amount of KI used is 5 mmol; In step 2 of step (ii), the amount of (3-aminopropyl)triethoxysilane used was 0.2 mL; In step 3 of step (ii), Bi-BiOI@APTES was 10 mg / mL, and the amount of EDC / NHS mixed solution used was 3 mL; The other preparation conditions are the same as in Example 1.

[0062] Application Examples 1-7 Application Examples 1-7 respectively use the self-powered photoelectrochemical immunosensors prepared in Examples 1-7 to perform sample detection. The detection method includes the following steps: The ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode was used as the working electrode, and the ITO / CdIn2S4 electrode was used as the counter electrode and reference electrode in 30 mL PBS (pH 7.4, 0.1 M) electrolyte containing 5 mM H2O2. The photoelectric test was carried out using a three-electrode system, and the light source (450 nm) irradiated the two electrode surfaces simultaneously, switching at a frequency of 10 seconds. Using a CHI660E electrochemical workstation, the PEC signal was measured using the current-time mode at 0 V potential, and the working curve was drawn. The sample solution to be tested was used instead of the γH2AX standard solution for detection, and the results can be obtained through the working curve.

[0063] Comparative Example 1 This comparative example provides a method for constructing a self-powered photoelectrochemical immunosensor, which is different from Example 1 in that: Step (ii) is not performed, that is, the photoelectric immune signal probe with signal sensitization constructed with Bi-doped BiOI as the core is not prepared.

[0064] Comparative application example 1 Comparative Application Example 1 The self-powered photoelectrochemical immunosensor prepared in Comparative Example 1 was used to perform sample detection, and the detection method was the same as that in Application Example 1.

[0065] Test Case Test method: PEC test was performed on 0.0001 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 20 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL γH2AX standard solutions, and the current difference ΔI (ΔI = I i -I blank , i = 1, 2, ... 9) were linearly fitted with the Log values ​​of the corresponding concentrations to obtain the working curve and determine the linear range of detection; according to S / N=3, the detection limit was calculated; in order to verify the accuracy, reliability and practical application potential of the sensor, the sensor of Application Example 1 was used to detect γH2AX in actual samples of cell lysate, and the spike recovery method was used to calculate the spike recovery rate and relative standard deviation.

[0066] The test results are shown in Table 1 and Table 2.

[0067] Table 1

[0068] Table 2

[0069] It can be seen from the data in Table 1 that, compared with the comparative application examples, the self-powered photoelectrochemical immunosensor obtained in the application examples of the present invention has a wider detection linear range, a lower detection limit, and better detection performance. Among them, the self-powered photoelectrochemical immunosensor of Application Example 1 has a wider detection linear range, a lower detection limit, and better detection performance, which proves that the self-powered photoelectrochemical immunosensor constructed by the present invention has ultra-high sensitivity and better detection performance, and can be used for the accurate detection of the low-abundance genotoxicity marker γH2AX and the reliable screening of genotoxic compounds.

[0070] From the data in Table 2, it can be seen that γH2AX was not detected in the cell lysate. When 80 pg / mL, 120 pg / mL, and 240 pg / mL γH2AX standards were added, the spike recovery rate of the sensor in Application Example 1 was 98.4%-103.2%, and the RSD was less than 3.3%, indicating that the immunosensor has good accuracy, reliability, and practicality.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a self-powered photoelectrochemical immunosensor, characterized in that: The following steps are involved: Applying a γH2AX standard solution and a Bi-BiOI@APTES@Ab2 dispersion to an ITO / Au / 11-MUA / Ab1 / BSA electrode in sequence to form an ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode, using the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode as a working electrode, and the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode as a photocathode; Applying CdIn2S4 dispersion to the ITO electrode to form an ITO / CdIn2S4 electrode, using the ITO / CdIn2S4 electrode as a counter electrode and a reference electrode, and the ITO / CdIn2S4 as a photoanode; Among them, Ab1 is a γH2AX capture antibody, and Ab2 is a γH2AX recognition antibody.

2. The method for constructing a self-powered photoelectrochemical immunosensor according to claim 1, characterized in that: The CdIn2S4 dispersion is prepared by the following steps: The mixed solution of Cd(NO3)2·4H2O, In(NO3)3·xH2O and TAA is heated, centrifuged, washed and dried in sequence to obtain CdIn2S4 nanosphere powder, and then the CdIn2S4 nanosphere powder is dispersed in a solvent to obtain the CdIn2S4 dispersion; Preferably, the amount of Cd(NO3)2·4H2O used is 1-3 mmol; Preferably, the amount of In(NO3)3·xH2O used is 2-6 mmol.

3. The method for constructing a self-powered photoelectrochemical immunosensor according to claim 1, characterized in that: The preparation process of the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode includes: The γH2AX standard solution was applied to the ITO / Au / 11-MUA / Ab1 / BSA electrode, and after incubation, it was washed with PBS to obtain the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode; Afterwards, the Bi-BiOI@APTES@Ab2 dispersion was applied to the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode, and after incubation, it was washed with PBS to obtain an ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode; Preferably, the Bi-BiOI@APTES@Ab2 is incubated on the ITO / Au / 11-MUA / Ab1 / BSA / γH2AX electrode for 10-80 min.

4. The method for constructing a self-powered photoelectrochemical immunosensor according to claim 1, characterized in that: The ITO / Au / 11-MUA / Abl / BSA electrode is prepared by the following steps: The ITO electrode is sequentially subjected to nano-gold coating deposition and carboxylation to obtain an ITO / Au / 11-MUA electrode, and then Ab1 is fixed to the ITO / Au / 11-MUA electrode to obtain the ITO / Au / 11-MUA / Ab1 / BSA electrode; Preferably, the nano-gold coating deposition comprises: using an ITO substrate as a working electrode, an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and HAuCl4 and H2SO4 as electrolytes, using a three-electrode system for electrodeposition, depositing a gold film on the ITO surface to obtain an ITO / Au electrode; Preferably, the carboxylation comprises: immersing the ITO / Au electrode in 11-mercaptoundecanoic acid solution and EDC / NHS solution in sequence to obtain an ITO / Au / 11-MUA electrode; Preferably, the immobilization process of Ab1 on the ITO / Au / 11-MUA electrode comprises: applying Ab1 and BSA solution to the ITO / Au / 11-MUA electrode in sequence to obtain an ITO / Au / 11-MUA / Ab1 / BSA electrode.

5. The method for constructing a self-powered photoelectrochemical immunosensor according to claim 4, characterized in that: Before depositing the nano-gold coating, pre-treating the ITO electrode; Preferably, the pretreatment includes: cutting, first cleaning and second cleaning of the ITO electrode in sequence; Preferably, the first cleaning comprises: cleaning the ITO electrode with acetone, and then performing ultrasonic treatment on the ITO electrode; Preferably, the second cleaning comprises: using an ITO electrode as a working electrode, a platinum electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode, placing them in PBS, performing a potential cycle cleaning from 0 to +1 V, and then rinsing with deionized water.

6. The method for constructing a self-powered photoelectrochemical immunosensor according to claim 1, characterized in that: The Bi-BiOI@APTES@Ab2 dispersion is prepared by the following steps: After the synthesis of the Bi-BiOI nanomaterial, it is aminated to obtain Bi-BiOI@APTES, and then Ab2 and Bi-BiOI@APTES are mixed and dispersed in PBS, the Bi-BiOI@APTES@Ab2 dispersion; Preferably, the synthesis of the Bi-BiOI nanomaterial comprises: using Bi(NO3)3·5H2O solution as solution A, using KI solution as solution B, using a mixed solution of solution A and solution B as solution C, and sequentially heating, centrifuging, washing and drying solution C to obtain the Bi-BiOI nanomaterial; Preferably, the amination comprises: dispersing the Bi-BiOI nanomaterial in anhydrous ethanol, and then adding (3-aminopropyl)triethoxysilane to obtain Bi-BiOI@APTES; Preferably, the (3-aminopropyl)triethoxysilane is used in an amount of 0.1-0.3 mL; Preferably, the mixing of Ab2 and Bi-BiOI@APTES comprises: adding BSA to a mixed solution of Bi-BiOI@APTES, Ab2 and EDC / NHS, and obtaining Bi-BiOI@APTES@Ab2 after centrifugation; Preferably, the concentration of Bi-BiOI@APTES is 0.5-10 mg / mL; Preferably, the volume ratio of Bi-BiOI@APTES, Ab2 and EDC / NHS is 1:1:(1-2).

7. The method for constructing a self-powered photoelectrochemical immunosensor according to claim 6, characterized in that: The amount of Bi(NO3)3·5H2O used is 1-5 mmol, and the amount of KI used is 1-5 mmol; The solvent of the solution A includes at least one of EG and H2O, wherein EG is 0-40 mL, H2O is 0-40 mL, and the total amount of EG and H2O in the solution A is 40 mL; The solvent of the solution B includes at least one of EG and H2O, wherein EG is 0-30 mL, H2O is 0-30 mL, and the total amount of EG and H2O in the solution B is 30 mL.

8. The method for constructing a self-powered photoelectrochemical immunosensor according to claim 1, characterized in that: The concentration of the γH2AX standard solution is 0.0001 ng / mL to 1000 ng / mL.

9. The self-powered photoelectrochemical immunosensor constructed by the method for constructing a self-powered photoelectrochemical immunosensor according to any one of claims 1 to 8, characterized in that: The detection method of the self-powered photoelectrochemical immunosensor comprises: PEC detection: ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode was used as the working electrode, and ITO / CdIn2S4 electrode was used as the counter electrode and reference electrode. The ITO / Au / 11-MUA / Ab1 / BSA / γH2AX / Ab2@APTES@Bi-BiOI electrode and the ITO / CdIn2S4 electrode were placed in a PBS electrolyte containing H2O2. A three-electrode system was used for photoelectric testing, PEC signals were measured, and working curves were drawn. Wherein, the pH of the PBS electrolyte containing H2O2 is 6-8.

5.

10. Application of the method for constructing the self-powered photoelectrochemical immunosensor according to any one of claims 1 to 8 in the detection of genotoxicity markers.

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