Photoelectrochemical biosensor based on cascade amplification and application of photoelectrochemical biosensor in ultra-sensitive detection of cardiac troponin

Abstract

The invention provides a cascade amplification-based photoelectrochemical biosensor and application thereof in ultra-sensitive detection of cardiac troponin, and belongs to the technical field of biosensing. A nuclear track membrane containing a plurality of nano-pore channels is used as a base material, Z-type heterojunction g-C3N4 (at) CdS quantum dots are modified to the inner walls of the nano-pore channels of the nuclear track membrane, then an aptamer Apt1 and ssDNA1 double-chain heterocomplex is sequentially fixed to the inner walls of the nano-pore channels of the nuclear track membrane through an amidation reaction, and then ssDNA2-MB is modified to the inner walls of the pore channels to form a double-lock recognition interface. H1 and H2 hairpin DNA are respectively modified with Fe3O4 (at) Pt nano-enzyme and are pre-assembled in a nano-pore channel, and then non-specific sites are closed to obtain the cascade amplification-based photoelectrochemical biosensor, so that ultrasensitive (0.02 fmol / mL) and high-specificity detection of cTnI is realized, and the detection sensitivity of cTnI is greatly improved. Meanwhile, the operation process is simplified, and the anti-interference performance is improved. The invention further discloses the application of the photoelectrochemical biosensor in cardiac troponin ultra-sensitive detection.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and specifically relates to a high-sensitivity electrochemiluminescence biosensor that integrates CRISPR-rRNA specific recognition, hybridization chain reaction (HCR) signal amplification, methylene blue (MB) electron transfer enhancement, photoelectrochemical (PEC) signal regulation, and bimetallic nanozyme synergistic catalysis, to achieve ultra-sensitive and high-specificity detection of cTnI, while simplifying the operation process and improving the anti-interference performance of the photoelectrochemical biosensor, and its application in ultra-sensitive detection of cardiac troponin. Background Art

[0002] In recent years, acute myocardial infarction (AMI) has become the world's leading cause of death. It is caused by acute and persistent ischemia and hypoxia of the coronary arteries, resulting in myocardial damage and necrosis. At present, the biomarkers used in clinical diagnosis of AMI include myoglobin, creatine kinase isoenzymes, cardiac troponin, etc. Among them, cardiac troponin I (cTnI) is considered to be the "gold standard" for detecting AMI. Various sensing methods for detecting cTnI have been reported, such as surface plasmon resonance (SPR), optical sensing, enzyme-linked immunosorbent assay (ELISA) and electrochemical detection. However, the existing cTnI detection technology has the following bottlenecks: 1. Limited sensitivity; 2. Poor stability and insufficient anti-interference ability; 3. Slow detection time; 4. Cumbersome detection steps; 5. Dependence on load equipment. Therefore, a detection method with advantages such as high sensitivity, high stability, high specificity, convenience, rapidity, and easy design and construction is needed. In recent years, biosensors have attracted much attention due to their excellent detection characteristics. Compared with traditional immunosensors, biosensors have the advantages of high sensitivity, high specificity, convenience, rapidity, easy design and construction, etc., and are favored by scientific researchers and widely used in AMI diagnosis. Based on this, the present invention combines the advantages of small size, controllable structure, simple design, easy preparation and the excellent electrochemical properties of precious metal nanomaterials to construct an electrochemical biosensor and explore its application in the high-sensitivity and high-specificity detection of cTnI. Summary of the invention

[0003] The purpose of the present invention is to provide a photoelectrochemical biosensor based on CRISPR-HCR cascade amplification in nuclear pore membrane, methylene blue (MB) electron transfer enhancement and photoelectrochemical coordinated regulation to achieve ultra-sensitive and high-specificity detection of cTnI, while simplifying the operation process and improving the anti-interference performance, and its application in ultra-sensitive detection of cardiac troponin.

[0004] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a polymer film bombarded by heavy ions is used as a substrate, and it is first sensitized by ultraviolet light, and then chemically etched to prepare nanopores of different shapes, and a thin film containing multiple nanopores is prepared; then the Z-type heterojunction g-C3N4@CdS quantum dots are modified to the inner wall of the nuclear pore membrane nanopore to form a g-C3N4@CdS heterojunction in the nanopore; then the nuclear pore membrane is activated by an activator, and the aptamer Apt1 and the ssDNA1 double-stranded hybrid are fixed to the inner wall of the nuclear pore membrane nanopore in sequence through an amidation reaction, and then ssDNA2-MB is modified on the inner wall of the pore to form a double-lock recognition interface, and then the Fe3O4@Pt nanozyme is modified with H1 and H2 hairpin DNA respectively, and pre-assembled in the nanopore; then polyethylene glycol and bovine serum albumin are used in sequence to block non-specific sites to obtain a photoelectrochemical biosensor based on cascade amplification.

[0005] The polymer film of the technical solution is a polyethylene terephthalate (PET) film, the shape of the nanopores is columnar, conical, or irregular, and the etching solution is a sodium hydroxide solution.

[0006] The Z-type heterojunction g-C3N4@CdS quantum dots of the technical solution significantly improve the visible light absorption efficiency due to the narrow bandgap characteristics, and the photocurrent baseline intensity reaches 745 μA. The modification of MB in the pores can accelerate the separation of photogenerated electron-hole pairs, increase the photocurrent response by 4 times, and inhibit hole recombination.

[0007] The etched nanopore surface of the technical solution contains carboxyl functional groups, which can be activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form amine-reactive ester intermediates, and then these active esters are further condensed with probes with amino groups by forming covalent bonds to obtain nanopores with modified probes, and then to obtain probe-functionalized nanopores.

[0008] In the technical solution, when cTnI is present, Apt1 specifically binds to cTnI, releases the ssDNA1 single chain, hybridizes with the ssDNA2-MB single chain, activates the trans-cutting function of Cas12a, cuts the ssDNA2-MB chain, and divides it into ssDNA2 short chain and ssDNA2 short chain-MB, ssDNA2 short chain-MB falls off naturally, and the photocurrent signal initially decreases rapidly; the ssDNA2 short chain and H1 and H2 are triggered to start the HCR reaction to generate a long-chain DNA structure, and the terminal-modified Fe3O4@Pt nanozyme catalyzes H2O2 to oxidize TMB to generate a precipitate, which covers the surface of the nuclear pore membrane and inhibits the photocurrent signal, and the photocurrent signal decreases rapidly again.

[0009] A preparation method of a photoelectrochemical biosensor based on cascade amplification, which uses an electrochemical method to detect a target object by using a probe-functionalized nanopore modified by a photosensitive material g-C3N4@CdS heterojunction as a detection material, and based on a probe hybridization reaction, the probe-functionalized nanopore modified by a photosensitive material g-C3N4@CdS heterojunction is prepared according to the following steps: (1) Preparation of a nuclear pore membrane: a polymer membrane bombarded by heavy ions is used as a substrate, and the film is first sensitized by irradiating the film with an ultraviolet lamp for 2 hours, and then the film is immersed in a sodium hydroxide solution for 5 minutes to chemically etch it to prepare nanopores of different shapes, thereby preparing a film containing multiple nanopores; (2) Coordinated amplification design of the nuclear pore membrane and the photoelectrochemical method: the nuclear pore membrane containing the nanopores is immersed in 3-5 ml of 5-10 The membrane was immersed in a solution containing Z-type heterojunction g-C3N4@CdS quantum dots at 400 mg / mL for 10 minutes, then rinsed and dried in air; (3) MB-HCR cascade signal amplification in the nanopore: the nuclear pore membrane was immersed in an activator for activation for 1 hour, and the nuclear pore membrane was immersed in a double-stranded hybrid solution of aptamer Apt1 and ssDNA1 for 1 hour through an amidation reaction, and then ssDNA2-MB was modified on the inner wall of the pore to form a dual specific recognition interface, and then H1 and H2 hairpin DNAs were respectively modified with Fe3O4@Pt nanozymes and preassembled in the nanopore; (4) Blocking the active site: the modified nanopore was washed with deionized water to remove the unreacted probe DNA, and then the active site was blocked with polyethylene glycol and bovine serum albumin solution in sequence, and then washed with deionized water to finally obtain a composite membrane containing a probe DNA nanopore with blocked active sites.

[0010] Furthermore, in step 1, the thickness of the polymer membrane is 10-20 µm, the pore size of the surface pores is 0.1-5 µm, and the pore density is 1×10 3 ~1×10 15 / cm 2 , the wavelength of ultraviolet light is 300~400 nm, and the concentration of sodium hydroxide solution is 6mol / L~9 mol / L.

[0011] Furthermore, in step 2, a Z-type heterojunction g-C3N4@CdS quantum dots are prepared by a hydrothermal method. A mixture of Cd(CH3COO)2·2H2O and Na2S·9H2O is dissolved in deionized water, and then g-C3N4 powder is added to the solution, and then the solution is ultrasonically treated, and the mixture is transferred to a Teflon-lined autoclave for heating, washing, drying, and grinding.

[0012] Furthermore, in the step 3, the concentration of EDC is 10-20 mg / L, the concentration of NHS is 3-5 mg / L, and the concentration of all DNA aqueous solutions is (2-5 µM / L, pH 7.0-8.0); the synthesis method of ssDNA2-MB is to add 5-10 mM EDC and 5-10 mM NHS to 5-15 mg / mL MBs solution, react the solution at room temperature for 1 hour, rinse 3 times, then add 2-5 µM / L ssDNA2, incubate at room temperature for 2 hours, and then add bovine serum albumin aqueous solution with a concentration of (8 mg / mL-10 mg / mL, pH 7.0-8.0) to block non-specific binding sites for 1 hour to obtain ssDNA2-MB.

[0013] Furthermore, in step 4, the concentration of the polyethylene glycol aqueous solution is (8 mg / mL~10 mg / mL, pH 7.0~8.0), the concentration of the bovine serum albumin aqueous solution is (8 mg / mL~10 mg / mL, pH 7.0~8.0), the immersion time is 1 hour, and the process is carried out at room temperature.

[0014] In addition, the present invention also provides an application of a photoelectrochemical biosensor based on cascade amplification in ultrasensitive detection of cardiac troponin, and the main steps are as follows: placing a sealed composite film on ITO conductive glass as a working electrode, a silver / silver chloride electrode as a reference electrode, and a platinum wire as a counter electrode, based on a three-electrode system, in a PBS buffer solution with a concentration of 0.1~0.5 mol / L and a pH of 6~8, a xenon lamp as a light source, and a wavelength of 350~380 nm, adding a mixed solution of troponin and Cas12a-crRNA, and testing the electrochemical performance by chronoamperometry; a linear relationship curve is drawn according to the electrochemical test results, and the applicable detection range of the constructed biosensor is 0.02 fmol / mL~2 mmol / mL, and it has efficient analytical detection capabilities; according to the signal test results of multiple interfering substances, it is shown that the constructed biosensor has high specificity; according to the results of multiple cycle tests, the constructed biosensor has good stability; according to the signal responses of different batches of biosensors, the constructed biosensor has good reproducibility.

[0015] Among them, the concentration of the electrolyte solution is 0.1 mol / L~0.5 mol / L, and the concentration of the mixed solution of cardiac troponin and Cas12a-crRNA is 0.02 fmol / L~200 fmol / L.

[0016] The detection principle of the present invention is as follows: a polymer film bombarded by heavy ions is used as a substrate, and it is first sensitized by ultraviolet light irradiation, and then chemically etched to prepare nanopores of different shapes, and a thin film containing multiple nanopores is prepared; Z-type heterojunction g-C3N4@CdS quantum dots are modified to the inner wall of the nanopore of the nuclear pore membrane as a photosensitive material to form a g-C3N4@CdS heterojunction in the nanopore, and then an activator is used to activate the nuclear pore membrane, and the aptamer Apt1 and the ssDNA1 double-stranded hybrid are fixed to the inner wall of the nanopore of the nuclear pore membrane in sequence through an amidation reaction, and then ssDNA2-MB is modified on the inner wall of the pore, and then Fe3O4@Pt nanozymes are modified with H1 and H2 hairpin DNAs respectively, which are preassembled in the nanopore, and then polyethylene glycol and bovine serum albumin solutions are used in sequence to seal the active sites for 1 hour, and finally a nuclear pore membrane containing probe DNA in the nanopore is obtained. When cTnI does not exist, the narrow bandgap characteristics of the Z-type heterojunction g-C3N4@CdS quantum dots significantly improve the visible light absorption efficiency, and the photocurrent intensity reaches 745 μA. At the same time, due to the modification of MB in the pores, the separation of photogenerated electron-hole pairs is accelerated, and the photocurrent response value reaches 1005 μA, while inhibiting hole recombination; when cTnI is present, Apt1 specifically binds to cTnI, releases the ssDNA1 single strand, hybridizes with the ssDNA2-MB single strand, activates the trans-cutting function of Cas12a, cuts the ssDNA2-MB chain, and divides it into ssDNA2 short chain and ssDNA2 short chain-MB, ssDNA2 short chain-MB falls off naturally, and the photocurrent signal initially drops rapidly; ssDNA2 short chain and H1, H2 initiate the HCR reaction to generate a long-chain DNA structure, and the end-modified Fe3O4@Pt nanozyme catalyzes H2O2 to oxidize TMB to generate a precipitate, which covers the surface of the nuclear pore membrane and inhibits the photocurrent signal. The photocurrent signal drops rapidly again, and drops to below 115 μA after target binding.

[0017] Innovations: 1. Synergistic enhancement of nuclear pore membrane confinement and MB-PEC: The nuclear pore membrane concentrates the target molecules through the confinement effect, combined with the enhanced electron transfer of MB and the efficient photoelectric conversion of the Z-type heterojunction, the photocurrent response intensity is increased to 5 times that of traditional PEC sensors, and the detection limit is as low as 0.02 fmol / mL; 2. CRISPR-HCR dual-stage amplification and target capture: CRISPR precise cutting and HCR chain reaction synergize, combined with nanozyme catalytic precipitation to inhibit photocurrent, the sensitivity is increased by 150 times compared with single CRISPR technology.

[0018] The present invention achieves ultra-sensitive (0.02 fmol / mL) and highly specific detection of cTnI through CRISPR-HCR cascade amplification within the nuclear pore membrane, methylene blue (MB) electron transfer enhancement and photoelectrochemical synergistic regulation, overcoming the limitations of traditional sensors in sensitivity, anti-interference and ease of operation. Its nuclear pore membrane design significantly improves the target capture efficiency, providing an efficient and reliable solution for the ultra-early diagnosis of myocardial infarction, and has significant clinical transformation potential and socio-economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a flow chart of a method for preparing a photoelectrochemical biosensor based on cascade amplification.

[0020] Figure 2 Schematic diagram of a photoelectrochemical biosensor based on cascade amplification for detecting targets.

[0021] Figure 3 (A) Scanning electron microscopy image and (B) nuclear pore membrane electron microscopy image of g-C3N4@CdS.

[0022] Figure 4 This is a photocurrent diagram of the preparation process of a photoelectrochemical biosensor based on cascade amplification.

[0023] Figure 5 The photocurrent diagram is the response to different concentrations of targets.

[0024] Figure 6 It is a specific identification map.

[0025] Figure 7 This is a stability exploration diagram.

[0026] Example DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto, and all changes made to the technical solution of the present invention by professionals in this field should fall within the protection scope of the present invention.

[0028] In the following examples, the nucleic acid sequence table involved is shown in Table 1

[0029] Example 1. Preparation method of a photoelectrochemical biosensor based on cascade amplification (1) Preparation of nuclear pore membrane: A polymer membrane bombarded with heavy ions is used as a substrate. The film is first sensitized by ultraviolet light for 2 hours, and then the film is immersed in a sodium hydroxide solution for 5 minutes to chemically etch it to prepare nanopores of different shapes, and a film containing multiple nanopores is prepared; (2) Nuclear pore membrane and photoelectrochemical coordinated amplification design: The nuclear pore membrane containing nanopores is immersed in 5 ml of 10 The membrane was immersed in a solution containing Z-type heterojunction g-C3N4@CdS quantum dots at 400 mg / mL for 10 minutes, then rinsed and dried in air; (3) MB-HCR cascade signal amplification in the nanopore: The nuclear pore membrane was immersed in an activator for activation for 1 hour, and the nuclear pore membrane was immersed in a double-stranded hybrid solution of aptamer Apt1 and ssDNA1 in turn through an amidation reaction for 1 hour, and then ssDNA2-MB was modified on the inner wall of the pore to form a dual specific recognition interface, and then H1 and H2 hairpin DNAs were respectively modified with Fe3O4@Pt nanozymes and preassembled in the nanopore; (4) Blocking the active site: The modified nanopore was washed with deionized water to remove the unreacted probe DNA, and then the active site was blocked with polyethylene glycol and bovine serum albumin solution in turn, and then washed with deionized water to finally obtain a composite membrane containing a probe DNA nanopore with blocked active sites.

[0030] The polymer film of the technical solution is a polyethylene terephthalate (PET) film, the shape of the nanopores is columnar, conical, or irregular, and the etching solution is a sodium hydroxide solution.

[0031] The Z-type heterojunction g-C3N4@CdS quantum dots of the technical solution significantly improve the visible light absorption efficiency due to the narrow bandgap characteristics, and the photocurrent baseline intensity reaches 745 μA. Modified MB can accelerate the separation of photogenerated electron-hole pairs, increase the photocurrent response by 4 times, and inhibit hole recombination.

[0032] The etched nanopore surface of the technical solution contains carboxyl functional groups, which can be activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form amine-reactive ester intermediates. These active esters are then further condensed with probes with amino groups by forming covalent bonds to obtain nanopores with modified probes, and then nanopores with probe DNA functionalization are obtained.

[0033] In the technical solution, when cTnI is present, Apt1 specifically binds to cTnI, releases the ssDNA1 single strand, hybridizes with the ssDNA2 single strand, activates the trans-cutting function of Cas12a, and releases the triggering ssDNA2 strand; the triggering ssDNA2 strand and H1 and H2 start the HCR reaction to generate a long-chain DNA structure, and the terminal-modified Fe3O4@Ptt nanozyme catalyzes H2O2 to oxidize TMB to generate a precipitate, which covers the surface of the nuclear pore membrane and inhibits the photocurrent signal.

[0034] Furthermore, in step 1, the thickness of the polymer membrane is 15 µm, the pore size of the surface pores is 5 µm, and the pore density is 1×10 3 Pieces / cm 2 , the wavelength of ultraviolet light is 360 nm, and the concentration of sodium hydroxide solution is 6 mol / L.

[0035] Furthermore, in step 2, a Z-type heterojunction g-C3N4@CdS quantum dots are prepared by a hydrothermal method. A mixture of Cd(CH3COO)2·2H2O and Na2S·9H2O is dissolved in deionized water, and then g-C3N4 powder is added to the solution, and then the solution is ultrasonically treated, and the mixture is transferred to a Teflon-lined autoclave for heating, washing, drying, and grinding.

[0036] Furthermore, in step 3, the concentration of EDC is 10 mg / L, the concentration of NHS is 3 mg / L, and the concentration of all DNA aqueous solutions is (2 µM / L, pH 7.0).

[0037] Furthermore, in step 4, the concentration of the polyethylene glycol aqueous solution is (8 mg / mL, pH 7.0), the concentration of the bovine serum albumin aqueous solution is (8 mg / mL, pH 7.0), the immersion time is 1 hour, and the process is carried out at room temperature.

[0038] Example 2. In addition, the present invention also provides an application of a photoelectrochemical biosensor based on cascade amplification in ultrasensitive detection of cardiac troponin, and the main steps are as follows: placing the sealed composite film on ITO conductive glass as a working electrode, a silver / silver chloride electrode as a reference electrode, and a platinum wire as a counter electrode, based on a three-electrode system, in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, a xenon lamp as a light source, and a wavelength of 360 nm. Add a mixed solution of troponin and Cas12a-crRNA, and use a chronoamperometry method to test the electrochemical performance; a linear relationship curve is drawn according to the electrochemical test results, and the applicable detection limit of the constructed biosensor is 0.02 fmol / mL, which has efficient analytical detection capabilities; according to the signal test results of a variety of interfering substances, it is shown that the constructed biosensor has high specificity; according to the results of multiple cycle tests, the constructed biosensor has good stability; according to the signal response of different batches of biosensors, the constructed biosensor has good reproducibility.

[0039] Among them, the concentration of the electrolyte solution is 0.1 mol / L, and the concentration of the mixed solution of cardiac troponin and Cas12a-crRNA is 0.02 fmol / L.

[0040] Example 3 This example uses scanning electron microscopy (SEM) as a powerful technique to help observe the morphology and structure of the prepared materials. Figure 3 As shown in Figure a, the structure of the g-C3N4@CdS composite material clearly shows that g-C3N4 is successfully coated on the surface of spherical CdS. Although the composite of the two monomers makes the CdS surface rough, the spherical structure of the single CdS remains almost unchanged and establishes good contact at the interface. This unique hierarchical structure gives g-C3N4@CdS a large specific surface area and provides more active sites for the reaction. Figure 3 As shown in Figure b, uniform pores are distributed on the nuclear pore membrane.

[0041] Example 4, experimental verification of the principle: In order to prove the feasibility of this technical solution, this implementation case used a method of preparing a photoelectrochemical biosensor using different modified electrodes. Figure 4As shown, after coating the ITO conductive glass with a core pore membrane with a Z-type heterojunction g-C3N4@CdS, a photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and a photocurrent value of 745 μA was observed, indicating that the core pore membrane with a Z-type heterojunction g-C3N4@CdS has good photocurrent performance; after activation with EDC and NHS, a photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and a photocurrent value of 730 μA was observed; after modification of apt1-ssDNA1, a photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and a photocurrent value of 715 μA was observed; after modification of ssDNA2-MB, a photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and a photocurrent value of 1005 μA was observed. µA photocurrent value; when modified with H1-H2-Fe3O4@Pt, the photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and a photocurrent value of 985 µA was observed; when blocked, the photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and a photocurrent value of 965 µA was observed; when incubated with the target, the photocurrent response was carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, and a photocurrent value of 115 µA was observed, which was much smaller than the photocurrent value before incubation, indicating that when the target is present, it specifically recognizes the probe, thereby changing the transfer state of the photogenerated electron-hole pairs on the electrode surface, causing a change in the photocurrent.

[0042] Example 5, Sensitivity Experiment: In order to evaluate the sensitivity of the present technical solution in detecting the target, the photocurrent of the target with different concentrations was measured under the optimal experimental conditions. Figure 5 As shown, the photocurrent decreases as the concentration of the target increases. At the same time, there is a certain functional relationship between the change in photocurrent and the concentration of the target. Therefore, the target can be analyzed by photoelectrochemical biosensors. By combining different photoelectrically active substances and different biorecognition elements, photoelectrochemical biosensors with different detection functions can be constructed.

[0043] Example 6, specificity experiment: In order to study the specificity of the photoelectrochemical biosensor, Figure 6 As shown, this embodiment uses four mismatches and one specific target as a control to perform a specific experiment; the results are shown in Figure 6 As shown, under the same reaction conditions, the photocurrent difference of the target object is much greater than that of the non-target object.

[0044] Example 7, stability experiment: In order to study the stability of the photoelectrochemical biosensor, Figure 7 As shown, five parallel photocurrent responses were carried out in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7. The results showed that the photoelectrochemical biosensor has excellent stability.

[0045] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A photoelectrochemical biosensor based on cascade amplification, characterized in that: Using the nuclear pore membrane containing multiple nanopores as the substrate, the Z-type heterojunction g-C3N4@CdS quantum dots were modified onto the inner wall of the nuclear pore membrane nanopore, and then the aptamer Apt1 and the ssDNA1 double-stranded hybrid were fixed to the inner wall of the nuclear pore membrane nanopore through amidation reaction. Then, ssDNA2-MB was modified on the inner wall of the pore to form a double-lock recognition interface. Then, the Fe3O4@Pt nanozyme was modified with H1 and H2 hairpin DNA respectively and preassembled in the nanopore. Finally, the nonspecific sites were blocked to obtain a photoelectrochemical biosensor based on cascade amplification.

2. A method for preparing a photoelectrochemical biosensor based on cascade amplification, characterized in that: The specific preparation steps of the photoelectrochemical biosensor are as follows: (1) Preparation of nuclear pore membrane: using a polymer membrane bombarded by heavy ions as a substrate, first sensitize it with ultraviolet light, and then chemically etching it to prepare nanopores of different shapes, and then preparing a film containing multiple nanopores; (2) Nuclear pore membrane and photoelectrochemical coordinated amplification design: modifying the Z-type heterojunction g-C3N4@CdS quantum dots to the inner wall of the nuclear pore membrane nanopore as a photosensitive material to form a g-C3N4@CdS heterojunction in the nanopore; (3) MB-HCR cascade signal amplification in the nanopore; using an activator to activate the nuclear pore membrane and reacting it through an amidation reaction The aptamer Apt1 and the ssDNA1 double-stranded hybrid are fixed to the inner wall of the nuclear pore membrane nanopore in sequence, and then ssDNA2-MB is modified on the inner wall of the pore to form a dual specific recognition interface. Then, the H1 and H2 hairpin probes are respectively modified with Fe3O4@Pt nanozymes and preassembled in the nanopore; (4) Blocking the active site: The modified nanopore is washed with deionized water to remove the unreacted probes, and then the active site is blocked with polyethylene glycol and bovine serum albumin solution for 1 hour, and then washed with deionized water to finally obtain a polyethyleneimine-modified probe-functionalized nanopore containing a blocked active site.

3. The method for preparing a photoelectrochemical biosensor based on cascade amplification according to claim 2, characterized in that: In the step (1), the polymer film is a polyethylene terephthalate (PET) film, the shape of the nanopores is columnar, conical, or irregular, and the etching solution is a sodium hydroxide solution.

4. The method for preparing a photoelectrochemical biosensor based on cascade amplification according to claim 2, characterized in that: In the step (2), the Z-type heterojunction g-C3N4@CdS quantum dots significantly improve the visible light absorption efficiency due to their narrow bandgap characteristics, and the photocurrent baseline intensity reaches 745 μA. Modified MB can accelerate the separation of photogenerated electron-hole pairs, increase the photocurrent response by 4 times, and inhibit hole recombination.

5. The method for preparing a photoelectrochemical biosensor based on cascade amplification according to claim 2, characterized in that: In step (3), the etched nanopore surface contains carboxyl functional groups, which can be activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form amine-reactive ester intermediates. These active esters are then further condensed with probes with amino groups by forming covalent bonds to obtain probe-modified nanopores, thereby obtaining probe-functionalized nanopores.

Images