A method for constructing a self-powered photoelectrochemical immune sensing platform for detecting norovirus capsid protein and application thereof

Abstract

The application belongs to the field of analysis and detection, and discloses a construction method and application of a self-powered photoelectrochemical immune sensing platform for detecting norovirus capsid protein. The application aims to design a photo-catalytic fuel cell-based self-powered system different from a three-electrode system, construct a carbon-rich graphite phase carbon nitride photo-anode material BSA / Ab / GA / PEI / C-CN / ITO, and construct a label-free self-powered immune sensor with a strong reduction capacity cathode to realize high-sensitivity and high-selectivity detection of norovirus capsid protein, promote the practical application of graphite phase carbon nitride material in the field of photoelectric sensing detection, and propose a new idea of virus detection based on a label-free self-powered immune analysis method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of photoelectrochemistry and analytical detection technology, and relates to a construction method and use of a nanobody-based photoelectrochemical immunosensor for detecting norovirus capsid protein. BACKGROUND

[0002] Norovirus (NoV) is considered to be the main cause of acute viral gastroenteritis, which has seriously aggravated the disease burden and caused huge economic losses worldwide (Medicine 2017, 96, e8139). NoV is a highly infectious virus (<100 copies / mL) with strong thermal stability and resistance to common disinfectants, which can be transmitted through direct contact between people, food or water contaminated by feces, and aerosols (Trends Microbiol. 2004, 12, 279-287). Since its diagnosis in 2002, NoV has shown a trend of multiple occurrences and increasing incidence worldwide. Since the capsid protein (VP1) is the most abundant protein that constitutes the virus particle and is the source of infection of the virus, VP1 is a potential target for diagnosing NoV infection (Nature Commun. 2020, 11, 2759). There is an urgent need to develop an analytical technique to achieve rapid, high-sensitivity and high-specificity screening of norovirus VP1.

[0003] Self-powered sensing has become a research hotspot due to its miniaturization, convenience, low cost and ability to integrate with other technologies (Nano Energy 2019, 61, 173-193; Anal. Chem. 2021, 93, 8393-8398). Unlike the traditional three-electrode system, this self-powered sensor provides the energy for detection through the spontaneous electrochemical reaction of the anode and cathode, without the need for an external power source or bias voltage, and is expected to achieve power-free and on-site detection. Photocatalytic fuel cells (PFCs) as a self-powered energy conversion element can simultaneously convert light energy and chemical energy into electrical energy, thereby improving energy utilization (Anal. Chem. 2022, 94, 1654-1660). In the sensing and detection platform, light is used as the excitation source, which has the advantages of strong energy, good stability and environmental friendliness. The analyte has a direct or indirect influence on the conversion process and affects the output signal, such as open-circuit voltage, current or power, and is widely used in antibiotic, pollutant and biomarker detection.

[0004] So far, there have been few reports on the construction of self-powered photoelectrochemical immunosensors for detecting norovirus capsid protein VP1. The present application uses a self-powered PFCs platform constructed by selecting a light anode with strong oxidation and a cathode with strong reduction to achieve high specificity and high sensitivity detection of norovirus capsid protein VP1. SUMMARY

[0005] The application aims to design a self-powered PFCs immunosensor different from a three-electrode system, construct a series of carbon-rich graphite phase carbon nitride (C-CN) photoelectric active materials, and build a photoelectric sensing platform for analyzing norovirus capsid protein VP1. By self-doping carbon in carbon nitride, the absorption range of bulk CN is widened, the utilization rate of visible light is enhanced, the effective separation of photo-generated electron-hole pairs is promoted, and excellent photoelectrochemical performance is obtained. In addition, antibodies are introduced as specific recognition elements to realize high selectivity and high sensitivity detection of norovirus capsid protein VP1.

[0006] The application achieves the above technical purpose through the following technical means.

[0007] A construction method of a self-powered photoelectrochemical immunosensing platform for detecting norovirus capsid protein is carried out according to the following steps:

[0008] (1) Preparation of C-CN photoelectric active material:

[0009] The locust bean gum or glucose is weighed and dissolved in deionized water, and stirring is maintained at a certain temperature; melamine, urea or dicyandiamide is weighed into the above solution, and after continuous stirring, freeze-drying is performed to obtain a supramolecular precursor; finally, the precursor powder is placed in a crucible for one-step calcination to obtain the C-CN material.

[0010] The amount ratio of the locust bean gum or glucose to melamine, urea or dicyandiamide is 2-100 mg: 1.0-4.0 g;

[0011] The stirring temperature is 70-90℃;

[0012] The calcination temperature is 400-700℃, the heating rate is 2-5℃ / min, and the calcination time is 2-6h.

[0013] (2) Preparation of C-CN / ITO photoanode:

[0014] The C-CN material prepared in step (1) is dispersed in deionized water, and a naphthol solution is added dropwise, and ultrasonic dispersion is performed to obtain a stable and uniform suspension; then the obtained suspension is drop-coated on the surface of indium tin oxide (ITO) conductive glass, and dried under an infrared lamp to obtain a C-CN / ITO electrode.

[0015] The concentration of the naphthol solution is 0.5wt%.

[0016] In the suspension, the concentration of the C-CN material is 1.0-5.0 mg / mL; the volume ratio of deionized water to naphthol solution is 25:1;

[0017] During drop-coating, the amount of the suspension used is 50 μL;

[0018] (3) Preparation of PEI / C-CN / ITO photoanode:

[0019] A certain amount of polyethyleneimine (PEI) solution was taken and modified on the surface of the C-CN / ITO electrode prepared in step (2), and a PEI / C-CN / ITO electrode was obtained after drying at room temperature.

[0020] The concentration of the polyethyleneimine (PEI) solution was 0.1 wt%, and the amount used was 10-30 μL.

[0021] (4) Preparation of GA / PEI / C-CN / ITO photoanode:

[0022] A certain amount of glutaraldehyde (GA) solution was dropped and coated on the surface of the PEI / C-CN / ITO electrode prepared in step (3) as a crosslinking agent, and after drying at room temperature, the electrode was washed with a phosphate buffer solution (PBS) and air-dried at room temperature, thereby obtaining a GA / PEI / C-CN / ITO photoanode.

[0023] The mass fraction of the GA solution was 2%-5%, and the amount used was 10-30 μL; the concentration of PBS was 6.7 mM.

[0024] (5) Preparation of Ab / GA / PEI / C-CN / ITO electrode:

[0025] A certain amount of antibody (Ab) of norovirus capsid protein VP1 was dropped and coated on the surface of the GA / PEI / C-CN / ITO electrode prepared in step (4), and after incubation at a certain temperature, the electrode was washed with PBS and air-dried at room temperature, thereby obtaining an Ab / GA / PEI / C-CN / ITO photoanode.

[0026] The titer of the VP1 antibody Ab was 1:5000-1:10000, the amount used was 10 μL, the incubation time was 12-24 h, and the incubation temperature was 4°C.

[0027] (6) Preparation of BSA / Ab / GA / PEI / C-CN / ITO photoanode:

[0028] The Ab / GA / PEI / C-CN / ITO electrode prepared in step (5) was immersed in a bovine serum albumin (BSA) solution for a period of time, washed with PBS and air-dried at room temperature, thereby obtaining a photoelectrochemical immunosensor, i.e., a BSA / Ab / GA / PEI / C-CN / ITO electrode.

[0029] The mass fraction and amount of the BSA solution were 2-6 wt% and 10 μL, respectively, and the reaction time was 10-50 min.

[0030] The application of the self-powered PFCs immunosensor prepared from the photoanode of the application in detecting norovirus capsid protein VP1 is as follows:

[0031] Step 1: determination of a standard curve:

[0032] A series of norovirus capsid protein VP1 with different concentrations were dropped on the surface of a BSA / Ab / GA / PEI / C-CN / ITO electrode, and after incubation for a certain time, natural drying, PBS flushing and air drying at room temperature, the obtained electrode was VP1 / BSA / Ab / GA / PEI / C-CN / ITO.

[0033] The prepared VP1 / BSA / Ab / GA / PEI / C-CN / ITO photoanode was used as a working electrode, Pt@C dispersion (5 mg Pt@C dispersed in 500 μL water, 500 μL anhydrous ethanol and 40 μL 5wt% naphthol) was modified to a glassy carbon electrode (5 μL, Pt@C / GCE) or Pt wire as a reference or counter electrode, and PBS was used as an electrolyte, and through testing the open circuit voltage (E OCP ) - time curve of the self-powered PFCs immunosensor platform, a series of concentration-E OCP correspondence was obtained, and then a standard curve of VP1 was obtained.

[0034] Step 2: a certain amount of a to-be-tested liquid was dropped on the surface of a BSA / Ab / GA / PEI / C-CN / ITO photoanode, and after incubation for a certain time, natural drying at room temperature, PBS flushing and air drying at room temperature, the prepared electrode was recorded as VP1 / BSA / Ab / GA / PEI / C-CN / ITO.

[0035] The prepared VP1 / BSA / Ab / GA / PEI / C-CN / ITO electrode was used as a working electrode, Pt@C / GCE cathode was used as a reference or counter electrode, and PBS was used as an electrolyte, and through testing the E OCP -time curve of the self-powered PFCs immunosensor platform, the E OCP value was brought into the standard curve in step 1 to obtain the concentration of VP1 in PBS.

[0036] In step 1, the amount and concentration of VP1 were 10-20 μL and 15 fg / mL-75 pg / mL, respectively.

[0037] In step 2, the amount of the to-be-tested liquid was 10-20 μL.

[0038] In steps 1 and 2, the incubation time was 10-50 min.

[0039] The linear range of the self-powered PFCs immunosensor constructed by the application is wide to 15fg / mL-15ng / mL, and the detection limit is low to 5fg / mL.

[0040] The application has the following advantages:

[0041] (1) The application designs a portable, low-cost, miniaturized self-powered PFCs immunosensor system with only anode and cathode and without external power supply, which meets the energy-saving, environment-friendly and ecological concept.

[0042] (2) The application replaces or substitutes nitrogen atoms in the carbon nitride skeleton to realize the regulation of the electronic structure of carbon nitride, exhibits significantly enhanced visible light utilization rate and rapid charge transport / separation capacity, explores the superior photoelectric performance, evaluates the sensitivity of the sensing system to norovirus protein detection, and promotes the application of graphite phase carbon nitride materials in the field of photoelectric sensing.

[0043] (3) Compared with the traditional detection method, the photoelectrochemical immunodetection method for norovirus capsid protein VP1 in the application has high detection sensitivity and selectivity, simpler instrument equipment, less reagent consumption, and low detection cost. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 X-ray diffraction (XRD) pattern of C-CN material, wherein a is CN and b is C-CN.

[0045] Figure 2 Solid state UV diffuse reflectance (DRS) pattern of C-CN material, wherein a is CN and b is C-CN. 13 C nuclear magnetic resonance (NMR) spectrum, wherein a is CN and b is C-CN.

[0046] Figure 3 Electron paramagnetic resonance (EPR) pattern of C-CN material, wherein a is CN and b is C-CN.

[0047] Figure 4 Solid state UV diffuse reflectance (DRS) pattern of C-CN material, wherein a is CN and b is C-CN.

[0048] Figure 5 Transient photocurrent response pattern of C-CN photoanode, wherein a is CN and b is C-CN.

[0049] Figure 6 Polarization curve pattern of C-CN photoanode, wherein a is without light and b is with light.

[0050] Figure 7 Open-circuit voltage pattern of PFCs composed of Pt@C cathode and C-CN photoanode, wherein a is without light and b is with light.

[0051] Figure 8 Open-circuit voltage versus time plots of PFCs composed of Pt@C cathode and different photoanode materials, where a is C-CN / ITO, b is PEI / C-CN / ITO, c is Ab / GA / PEI / C-CN / ITO, d is BSA / Ab / GA / PEI / C-CN / ITO, e is VP1 / BSA / Ab / GA / PEI / C-CN / ITO.

[0052] Figure 9 Open-circuit voltage plots of PFCs composed of Pt@C cathode and C-CN materials for detecting Norovirus capsid protein VP1, where a is open-circuit voltage versus time plots for detecting different concentrations of VP1, b is the linear relationship plot of VP1 concentration versus open-circuit voltage. DETAILED DESCRIPTION

[0053] The present application is further described in conjunction with the following examples to make those skilled in the art better understand the present application, but the scope of protection of the present application is not limited to the following examples.

[0054] Example 1:

[0055] (1) Preparation of CN powder by one-step calcination method:

[0056] 3 g of melamine was dissolved in 15 mL of deionized water, stirred for 1 h until completely dissolved, and freeze-dried to obtain a precursor. Finally, the precursor powder was placed in a crucible and calcined at 550℃ for 2 h under an argon atmosphere, with a heating rate of 5℃ / min. After cooling, CN material was obtained.

[0057] (2) Preparation of C-CN powder by supramolecular self-assembly and high-temperature calcination method:

[0058] 25 mg of locust bean gum was dissolved in 15 mL of deionized water, and after stirring at 80℃ for 30 min, a viscous gel was formed. 3 g of melamine was added to the viscous gel and stirred for 1 h. After freeze-drying, a supramolecular precursor was obtained. Finally, the precursor powder was placed in a crucible and calcined at 550℃ for 2 h under an argon atmosphere, with a heating rate of 5℃ / min. After cooling, C-CN material was obtained.

[0059] (3) Preparation of anode photoelectrochemical immunosensor:

[0060] First, the ITO conductive glass was pre-processed by ultrasonic cleaning in anhydrous ethanol for half an hour, and then dried under an infrared lamp for use.

[0061] A dispersion of 1.0 mg / mL C-CN was prepared with deionized water as solvent, and a 0.5wt% naphthol solution (40 μL) was added dropwise into the dispersion, which was then placed in an ultrasonic machine for dispersion to obtain a stable and uniform suspension. Then, 50 μL of the suspension was drop-coated on a pretreated ITO (fixed area of 1 x 0.5 cm 2 ) conductive glass surface, and the electrode prepared was marked as C-CN / ITO.

[0062] Next, a polyethyleneimine solution (0.1wt%, 10 μL) was modified on the surface of the C-CN / ITO electrode, and a PEI / C-CN / ITO electrode was obtained after drying.

[0063] A glutaraldehyde solution (2.5wt%, 10 μL) was modified as a crosslinking agent on the surface of the PEI / C-CN / ITO electrode, and a GA / PEI / C-CN / ITO electrode was obtained after drying and PBS washing.

[0064] An antibody of norovirus capsid protein VP1 (1:8000, 10 μL) was drop-coated on the surface of the electrode, and the electrode obtained after 4°C incubation for 12 h and PBS washing was marked as Ab / GA / PEI / C-CN / ITO.

[0065] Subsequently, the surface of the electrode was coated with 5wt% bovine serum albumin (10 μL) to block non-specific binding sites, and a photoelectrochemical immunosensor, i.e., a BSA / Ab / GA / PEI / C-CN / ITO electrode, was obtained after PBS washing.

[0066] (5) Preparation of target analyte VP1:

[0067] Concentrations of 15 fg / mL, 75 fg / mL, 0.15 pg / mL, 0.75 pg / mL, 1.5 pg / mL, 4.5 pg / mL, 7.5 pg / mL, 15 pg / mL, 30 pg / mL, 45 pg / mL, 75 pg / mL, etc. were prepared, respectively, for detection.

[0068] (6) Photoelectrochemical detection method and conditions:

[0069] Electrochemical experiments were performed on a CHI660E electrochemical workstation (Shanghai Chenhua Instrument Co. Ltd.) using modified ITO electrode as working electrode, Pt@C dispersion (5 mg Pt@C dispersed in 500 μL water, 500 μL anhydrous ethanol and 40 μL 5 wt% naphthol) modified to glassy carbon electrode (5 μL, Pt@C / GCE) as reference or counter electrode to form a two-electrode system in the same quartz electrolytic cell. A three-electrode system was used to verify the thermodynamic feasibility of the system, with modified ITO electrode as working electrode, Pt wire electrode as counter electrode, and saturated Ag / AgCl electrode as reference electrode. The excitation light source was a 300 W xenon lamp (PLS-SXE300, Beijing Po Fei Technology Co. Ltd., wavelength range 320-780 nm). Electrochemical experiments were all carried out at room temperature in PBS electrolyte (6.7 mM) without applying any bias.

[0070] The product obtained in Example 1 was characterized as follows:

[0071] Figure 1 X-ray diffraction (XRD) patterns of C-CN materials, where a is CN and b is C-CN. From a, diffraction peaks of (100) and (002) crystal planes of CN were observed; in b, the diffraction peak of (002) crystal plane of C-CN was found to be weak, and the (100) crystal plane almost disappeared, which might be because the modified CN was thinner than the bulk CN.

[0072] Figure 2 Solid state 13 Nuclear magnetic resonance (NMR) spectra of C, where a is CN and b is C-CN. From a and b, two strong peaks corresponding to the chemical shifts of C1 atoms in the tri-s-triazine ring [CN2-(NH X )] and C2 atoms (CN3) were observed, indicating that the basic chemical structures of CN and C-CN were similar, and the heptazine ring structure still existed in C-CN. Compared with a, another weak peak in C-CN was attributed to the sp 2 C=C electron-withdrawing group C3 atom, indicating that the N atom in the C-N=C bond was replaced by a C atom to form a C-C=C bond.

[0073] Figure 3 Electron paramagnetic resonance (EPR) spectra of C-CN materials, where a is CN and b is C-CN. From b, it can be seen that the C-CN material has more isolated electron-hole pairs than a, proving that the modified material has defects.

[0074] Figure 4The solid-state UV diffuse reflectance (DRS) spectra of C-CN materials, where a is CN and b is C-CN. The introduction of locust bean gum broadens the absorption range of CN materials and enhances the absorption intensity in the visible and ultraviolet regions, indicating that C-CN materials can generate more photoelectrons and holes under light excitation, which is conducive to promoting the improvement of photoelectrochemical performance.

[0075] Figure 5 The transient photocurrent response diagram of C-CN photoanode, where a is CN and b is C-CN. Compared with a, C-CN materials exhibit higher photocurrent values, indicating that the formation of carbon-rich structures inhibits the recombination of CN bulk phase and surface photo-generated carriers, promotes the separation and transfer of photo-generated electrons and holes, and makes C-CN have better photoelectrochemical performance, which lays a foundation for constructing high-sensitivity self-powered PFCs immunosensor.

[0076] Figure 6 The polarization curve diagram of C-CN material, where a is without light and b is with light. Under light, the photocurrent response of C-CN anode material is significantly enhanced, which is consistent with the transient photocurrent results, and the enhanced photocurrent is conducive to the oxidation of water.

[0077] Figure 7 The open-circuit voltage diagram of PFCs composed of Pt@C cathode and C-CN photoanode, where a is without light and b is with light. By comparing the changes of open-circuit voltage (E OCP ) values before and after light, it is found that the E OCP of the self-powered PFCs system reaches 377 mV under light, which is significantly higher than the E OCP without light. This indicates that in the self-powered system, the irradiation of sunlight promotes the transfer of photo-generated electrons from the photoanode to the Pt@C cathode, promotes the cathode oxygen reduction reaction, and produces an enhanced output signal.

[0078] Figure 8 The open-circuit voltage and time diagram of PFCs composed of Pt@C cathode and different photoanode materials, where a is C-CN / ITO, b is PEI / C-CN / ITO, c is Ab / GA / PEI / C-CN / ITO, d is BSA / Ab / GA / PEI / C-CN / ITO, and e is VP1 / BSA / Ab / GA / PEI / C-CN / ITO. From Figure 8 it can be seen that after modifying PEI on the surface of the anode, the E OCP value increases, indicating that the transmission ability of electrons on the surface of the photoanode is enhanced. After modifying GA, Ab, and BSA respectively, the E OCP gradually decreases, indicating that there is steric hindrance on the surface of the electrode, which accelerates the recombination of carriers. When the target detection material VP1 is introduced, the E OCPContinued to reduce, VP1 was specific recognition and formation of macromolecular protein Ab caused steric hindrance, weaken electron transfer. These results show that the successful construction of self-powered PFCs immunosensor, and is expected to achieve efficient norovirus VP1 detection.

[0079] Figure 9 Pt@C cathode and C-CN anode constitute PFCs detection of norovirus VP1 open-circuit voltage diagram, wherein a is the detection of different concentrations of VP1 open-circuit voltage and time diagram, wherein the VP1 concentration was 15fg / mL, 75fg / mL, 0.15pg / mL, 0.75pg / mL, 1.5pg / mL, 4.5pg / mL, 7.5pg / mL, 15pg / mL, 30pg / mL, 45pg / mL, 75pg / mL, b is the VP1 concentration logarithm-open-circuit voltage linear relationship diagram. From Figure 9 a can be seen, with the increase of VP1 concentration, the E OCP of the immunosensor gradually decreased, and the logarithm of VP1 concentration and E OCP showed a certain linear relationship, the linear range was 15fg / mL-75pg / mL, the linear equation was I = -46.86lgC VP1 + 378.70 (R 2 = 0.9969), the detection limit was as low as 5fg / mL.

Claims

1. A method for constructing a self-powered photoelectrochemical immunosensing platform for detecting norovirus capsid proteins, characterized in that, Includes the following steps: (1) Preparation of C-CN photoelectric active materials: Locust bean gum or glucose was weighed and dissolved in deionized water, and stirred at a certain temperature. Melamine, urea or dicyandiamide was weighed into the above solution, and after stirring, it was freeze-dried to obtain a supramolecular precursor. Finally, the precursor powder was placed in a crucible and calcined in one step to obtain C-CN material. (2) Preparation of C-CN / ITO photoanode: The C-CN material prepared in step (1) was dispersed in deionized water and naphthol solution was added dropwise. The mixture was ultrasonically dispersed to obtain a stable and uniform suspension. The resulting suspension was then dropwise coated onto the surface of indium tin oxide (ITO) conductive glass and dried under an infrared lamp to obtain the C-CN / ITO electrode. (3) Preparation of PEI / C-CN / ITO photoanode: A polyethyleneimine (PEI) solution was transferred and modified onto the surface of the C-CN / ITO electrode prepared in step (2). After drying at room temperature, a PEI / C-CN / ITO electrode was obtained. (4) Preparation of GA / PEI / C-CN / ITO photoanode: Glutaraldehyde (GA) solution was drop-coated onto the surface of the PEI / C-CN / ITO electrode prepared in step (3) as a crosslinking agent. After drying at room temperature, it was rinsed with phosphate buffer solution (PBS) and air-dried at room temperature to obtain the GA / PEI / C-CN / ITO photoanode. (5) Preparation of Ab / GA / PEI / C-CN / ITO electrodes: The antibody Ab of norovirus capsid protein VP1 was drop-coated onto the surface of the GA / PEI / C-CN / ITO electrode prepared in step (4), incubated at a certain temperature, rinsed with PBS and air-dried at room temperature to obtain Ab / GA / PEI / C-CN / ITO photoanode. (6) Preparation of BSA / Ab / GA / PEI / C-CN / ITO photoanodes: The Ab / GA / PEI / C-CN / ITO electrode prepared in step (5) was immersed in bovine serum albumin (BSA) solution, kept for a reaction time, rinsed with PBS and air-dried at room temperature to obtain the photoelectrochemical immunosensor, namely the BSA / Ab / GA / PEI / C-CN / ITO electrode. (7) A self-powered photoelectrochemical immunosensing platform was constructed by using BSA / Ab / GA / PEI / C-CN / ITO electrode as working electrode, Pt@C / GCE cathode as reference or counter electrode, and PBS as electrolyte.

2. The construction method as described in claim 1, characterized in that, In step (1), The ratio of locust bean gum or glucose to melamine, urea, or dicyandiamide is 2-100 mg: 1.0-4.0 g; the stirring temperature is 70-90 ℃. The calcination temperature is 400~700℃, the heating rate is 2~5 ºC / min, and the calcination time is 2~6 h.

3. The construction method as described in claim 1, characterized in that, In step (2), The concentration of the naphthol solution is 0.5 wt%. The concentration of C-CN material in the suspension is 1.0~5.0 mg / mL; the volume ratio of deionized water to naphthol solution is 25:1; and the volume of the suspension used during drop casting is 50 μL.

4. The construction method as described in claim 1, characterized in that, In step (3), the concentration of the polyethyleneimine (PEI) solution is 0.1 wt%, and the amount used is 10~30 μL.

5. The construction method as described in claim 1, characterized in that, In step (4), the GA solution has a mass fraction of 2% to 5% and a volume of 10 to 30 μL; the PBS concentration is 6.7 mM.

6. The construction method as described in claim 1, characterized in that, In step (5), the antibody Ab titer of the norovirus capsid protein VP1 is 1:5000~1:10000, and the amount used is 10 µL.

7. The construction method as described in claim 1, characterized in that, In step (5), the incubation time is 12~24 h and the incubation temperature is 4 °C.

8. The construction method as described in claim 1, characterized in that, In step (6), the mass fraction of the BSA solution is 2-6 wt%, the amount used is 10 µL, and the reaction time is 10-50 min.

9. The use of the self-powered photoelectrochemical immunosensing platform constructed by the construction method according to any one of claims 1 to 8 for the detection of norovirus capsid protein VP1.

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