An optoelectrochemical biosensor based on cascade amplification and its application in ultrasensitive detection of cardiac troponin

Abstract

The present invention provides a photoelectrochemical biosensor based on cascade amplification and its application in ultrasensitive detection of cardiac troponin, belonging to the field of biosensing technology. The present invention uses a nuclear pore membrane containing multiple nanopores as a substrate, and modifies the inner wall of the nanopores of the nuclear pore membrane with Z-scheme heterojunction g-C 3 N 4 @CdS quantum dots. Then, the double-stranded hybrid of aptamer Apt1 and ssDNA1 is sequentially immobilized on the inner wall of the nanopores of the nuclear pore membrane through amidation reaction. Then, ssDNA2-MB is modified on the inner wall of the pore to form a double-lock recognition interface. Then, hairpin DNAs H1 and H2 are respectively modified with Fe 3 O 4 @Pt nanozyme, pre-assembled in the nanopores, and then the non-specific sites are blocked to obtain a photoelectrochemical biosensor based on cascade amplification, realizing ultrasensitive (0.02 fmol / mL) and highly specific detection of cTnI, and at the same time simplifying the operation process and improving the anti-interference performance of the photoelectrochemical biosensor and its application in ultrasensitive detection of cardiac troponin.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing, and particularly relates to a highly sensitive electrochemiluminescence biosensor integrating CRISPR-rRNA specific recognition, hybridization chain reaction (HCR) signal amplification, methylene blue (MB) electron transfer enhancement, photoelectrochemical (PEC) signal regulation, and the synergistic catalysis of bimetallic nanozymes, which realizes the ultrasensitive and highly specific detection of cTnI, and at the same time simplifies the operation process and improves the anti-interference performance of the photoelectrochemical biosensor and its application in the ultrasensitive detection of cardiac troponin I. Background Art

[0002] In recent years, acute myocardial infarction (AMI) has become the leading cause of death globally. It is caused by acute and persistent ischemia and hypoxia of the coronary artery, resulting in myocardial injury and necrosis. Currently, the biomarkers used in clinical diagnosis of AMI include myoglobin, creatine kinase isoenzyme, cardiac troponin, etc. Among them, cardiac troponin I (cTnI) is considered the "gold standard" for detecting AMI. Currently, 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 technologies have the following bottlenecks: 1. Limited sensitivity; 2. Poor stability and insufficient anti-interference ability; 3. Slow detection time; 4. Complicated detection steps; 5. Dependence on loading equipment. Therefore, a detection method with the advantages of high sensitivity, high stability, high specificity, convenience, rapidity, easy design and construction, etc. is needed. In recent years, biosensors have attracted much attention due to their excellent detection characteristics. Compared with traditional immunosensors, biosensors exhibit the advantages of high sensitivity, high specificity, convenience, rapidity, easy design and construction, etc., and are highly favored by the majority of scientific researchers and have been widely used in the diagnosis of AMI. Based on this, the present invention combines the advantages of small size, controllable structure, simple design, and easy preparation of nucleic acid aptamers and the excellent electrochemical properties of noble metal nanomaterials to construct an electrochemiluminescence biosensor and explore its application in the highly sensitive and highly specific detection of cTnI. Summary of the Invention

[0003] The purpose of the present invention is to provide an electrochemiluminescence biosensor based on CRISPR-HCR cascade amplification, methylene blue (MB) electron transfer enhancement, and photoelectrochemical synergistic regulation within a nuclear pore membrane, which realizes the ultrasensitive and highly specific detection of cTnI, and at the same time simplifies the operation process and improves the anti-interference performance of the photoelectrochemical biosensor and its application in the ultrasensitive detection of cardiac troponin I.

[0004] To achieve the above object, the present invention adopts the following technical solutions: using a polymer film bombarded by heavy ions as the substrate, first irradiating it with ultraviolet light to sensitize it, and then chemically etching it to prepare nanochannels of different shapes, and preparing a film containing multiple nanochannels; then 3 N 4 @CdS quantum dots are modified to the inner wall of the nanopores of the nuclear pore membrane to form g-C 3 N 4 @CdS heterojunctions in the nanopores; then the nuclear pore membrane is activated with an activator, and the double-stranded hybrid of aptamer Apt1 and ssDNA1 is sequentially fixed to the inner wall of the nanopores of the nuclear pore membrane 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 hairpin DNAs H1 and H2 are respectively modified with Fe 3 O 4 @Pt nanozymes and pre-assembled in the nanopores; 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 nanochannels is columnar, conical, or irregular, and the etching solution is sodium hydroxide solution.

[0006] The Z-scheme heterojunction g-C 3 N 4 @CdS quantum dots significantly improve the visible light absorption efficiency due to their narrow bandgap characteristics, the baseline intensity of the photocurrent reaches 745 μA, and the modification of MB in the pores can accelerate the separation of photo-generated electron-hole pairs, the photocurrent response is increased by 4 times, and at the same time, the hole recombination is inhibited.

[0007] The surface of the etched nanopores 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 modified with probes, and further obtain probe-functionalized nanopores.

[0008] When cTnI is present in the technical solution, Apt1 specifically binds to cTnI, releases the single strand of ssDNA1, hybridizes with the single strand of ssDNA2-MB, activates the trans-cleavage function of Cas12a, cleaves the ssDNA2-MB strand into ssDNA2 short strands and ssDNA2 short strands-MB, and ssDNA2 short strands-MB naturally falls off, and the photocurrent signal initially rapidly decreases; triggering the HCR reaction of ssDNA2 short strands, H1, and H2 to generate a long-chain DNA structure, and the end is modified with Fe 3 O4 @Pt nanozyme catalyzes the oxidation of H 2 O 2 to oxidize TMB to form a precipitate, covering the surface of the nuclear pore membrane to inhibit the photocurrent signal, and the photocurrent signal rapidly decreases again.

[0009] A preparation method of a photoelectrochemical biosensor based on cascade amplification, which uses an electrochemical method to use a photosensitive material g-C 3 N 4 @CdS heterojunction-modified probe-functionalized nanopores as detection materials, and based on the probe hybridization reaction, the target is detected, where the photosensitive material g-C 3 N 4 @CdS heterojunction-modified probe-functionalized nanopores are prepared according to the following steps: (1) Preparation of nuclear pore membrane: Using a polymer membrane bombarded by heavy ions as the substrate, first irradiate the film with ultraviolet light for 2 hours to sensitize the film, and then immerse the film in sodium hydroxide solution for 5 minutes to chemically etch it to prepare nanopores of different shapes, and prepare a film containing multiple nanopores; (2) Design of nuclear pore membrane and photoelectrochemical coordinated amplification: Immerse the nuclear pore membrane containing nanopores in 3-5 ml of a 5-10 mg / mL solution containing Z-type heterojunction g-C 3 N 4 @CdS quantum dots for 10 minutes, then rinse and air dry; (3) MB-HCR cascade signal amplification in nanopores: Immerse the nuclear pore membrane in an activator for 1 hour, and sequentially immerse the nuclear pore membrane in the aptamer Apt1 and ssDNA1 double-stranded hybrid solution for 1 hour through amidation reaction, then modify ssDNA2-MB on the inner wall of the pore to form a double-specific recognition interface, and then modify H1 and H2 hairpin DNAs with Fe 3 O 4 @Pt nanozyme and pre-assemble it in the nanopores; (4) Blocking active sites: Wash the modified nanopores with deionized water to remove unreacted probe DNA, then use polyethylene glycol and bovine serum albumin solution to block the active sites in sequence, and then wash with deionized water to finally obtain a composite membrane containing probe DNA nanopores with blocked active sites.

[0010] Furthermore, in the step 1, the thickness of the polymer membrane is 10-20 µm, the pore diameter of the surface pores is 0.1-5 µm, 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 6 mol / L-9 mol / L.

[0011] Furthermore, in the step 2, the Z-type heterojunction g-C is prepared by a hydrothermal method3 N 4 @CdS quantum dots. Dissolve a mixture of Cd(CH 3 COO) 2 ·2H 2 O and Na 2 S·9H 2 O in deionized water, then add g-C 3 N 4 powder to the solution, then ultrasonically treat the solution, transfer the mixture to a Teflon-lined autoclave for heating, washing, drying, and grinding.

[0012] Furthermore, in 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 a 5 - 15 mg / mL MBs solution. After the solution reacts at room temperature for 1 hour, rinse it 3 times, then add 2 - 5 µM / L of 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 polyethylene glycol aqueous solution is (8 mg / mL - 10 mg / mL, pH 7.0 - 8.0), the concentration of bovine serum albumin aqueous solution is (8 mg / mL - 10 mg / mL, pH 7.0 - 8.0), the soaking time is 1 hour, and it 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 the ultrasensitive detection of cardiac troponin. The main steps are as follows: The sealed composite membrane is placed on the ITO conductive glass as the working electrode, the silver / silver chloride electrode is used as the reference electrode, and the platinum wire is used as the counter electrode. Based on the three-electrode system, in a PBS buffer solution with a concentration of 0.1 - 0.5 mol / L and a pH of 6 - 8, using a xenon lamp as the light source and a wavelength of 350 - 380 nm, a mixed solution of troponin and Cas12a-crRNA is added, and the chronoamperometry method is used to test the electrochemical performance; according to the electrochemical test results, a linear relationship curve is plotted, and the applicable detection range of the constructed biosensor can be obtained as 0.02 fmol / mL - 2 mmol / mL, with high analytical detection ability; according to the signal test results of various interfering substances, it is shown that the constructed biosensor has high specificity; according to the results of multiple cyclic tests, it can be obtained that the constructed biosensor has good stability; according to the signal responses of biosensors from different batches, it can be obtained that 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: Using a polymer membrane bombarded by heavy ions as the substrate, first irradiate it with ultraviolet light to sensitize it, and then chemically etch it to prepare nanopores of different shapes to prepare a thin film containing multiple nanopores; Modify the Z-scheme heterojunction g-C 3 N 4 @CdS quantum dots on the inner wall of the nanopores of the nuclear pore membrane as the photosensitive material to form a g-C 3 N 4 @CdS heterojunction in the nanopores, then activate the nuclear pore membrane with an activator, and sequentially fix the double-stranded hybrid of aptamer Apt1 and ssDNA1 to the inner wall of the nanopores of the nuclear pore membrane through an amidation reaction, then modify ssDNA2-MB on the inner wall of the pores, and then modify H1 and H2 hairpin DNAs with Fe 3 O 4 @Pt nanozyme, pre-assembled in the nanopores, and then sequentially use polyethylene glycol and bovine serum albumin solution to block the active sites for 1 hour, and finally obtain a nuclear pore membrane containing probe DNA in the nanopores. When cTnI is absent, due to the Z-scheme heterojunction g-C 3 N 4@The narrow bandgap property of CdS quantum dots significantly improves the visible light absorption efficiency, with the photocurrent intensity reaching 745 μA. At the same time, due to the modification of MB in the pores, the separation of photo-generated electron-hole pairs is accelerated, the photocurrent response value reaches 1005 μA, and the hole recombination is inhibited. When cTnI is present, Apt1 specifically binds to cTnI, releasing the single-stranded ssDNA1, which hybridizes with the single-stranded ssDNA2-MB, activating the trans-cleavage function of Cas12a to cleave the ssDNA2-MB strand into ssDNA2 short strands and ssDNA2 short strands-MB. The ssDNA2 short strands-MB naturally fall off, and the photocurrent signal initially rapidly decreases. The ssDNA2 short strands initiate the HCR reaction with H1 and H2 to generate a long-chain DNA structure, with the end modified with Fe 3 O 4 @Pt nanozyme catalyzes H 2 O 2 to oxidize TMB to form a precipitate, covering the surface of the nuclear pore membrane to inhibit the photocurrent signal, and the photocurrent signal rapidly decreases again, dropping below 115 μA after the target binding.

[0017] Innovation points: 1. Nuclear pore membrane confinement and MB-PEC synergistic effect: The nuclear pore membrane concentrates the target molecules through the confinement effect, and the electron transfer of the combined MB enhances the efficient photoelectric conversion with 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 two-stage amplification and target capture: The precise cleavage of CRISPR and the HCR chain reaction cooperate, combined with the inhibition of photocurrent by nanozyme-catalyzed precipitation, and the sensitivity is increased by 150 times compared with the single CRISPR technology.

[0018] Through the CRISPR-HCR cascade amplification within the nuclear pore membrane, the enhancement of methylene blue (MB) electron transfer, and the synergistic regulation of photoelectrochemistry, the present invention realizes the ultrasensitive (0.02 fmol / mL) and highly specific detection of cTnI, overcoming the limitations of traditional sensors in sensitivity, anti-interference ability, and operation convenience. The 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 having significant clinical transformation potential and social and economic benefits. Brief Description of the Drawings

[0019] Figure 1 It is a flowchart of the preparation method of a photoelectrochemical biosensor based on cascade amplification.

[0020] Figure 2 It is a schematic diagram of the detection principle of a photoelectrochemical biosensor based on cascade amplification for the target substance.

[0021] Figure 3 For g-C 3 N 4(A) Scanning electron micrograph and (B) nuclear pore membrane electron micrograph of CdS.

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

[0023] Figure 5 Photocurrent diagram of responses to different concentrations of the target substance.

[0024] Figure 6 Specific recognition diagram.

[0025] Figure 7 Stability exploration diagram.

[0026] Example Detailed implementation manners

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. However, the protection scope of the present invention is not limited thereto. Any changes made by professionals in the field to the technical solution of the present invention shall fall within the protection scope of the present invention.

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

[0029]

[0030] Example 1. Preparation method of a photoelectrochemical biosensor based on cascade amplification (1) Preparation of nuclear pore membrane: Using a polymer membrane bombarded by heavy ions as the substrate, first irradiate the thin film with ultraviolet light for 2 hours to sensitize it, and then immerse the thin film in a sodium hydroxide solution for 5 minutes to chemically etch it to prepare nano-pore channels of different shapes, and prepare a thin film containing multiple nano-pore channels; (2) Design of coordination amplification of nuclear pore membrane and photoelectrochemistry: Immerse the nuclear pore membrane containing nano-pore channels in 5 ml of a solution containing Z-scheme heterojunction g-C 3 N 4 @CdS quantum dots for 10 minutes, then rinse and air dry; (3) MB-HCR cascade signal amplification in nano-pore channels Immerse the nuclear pore membrane in an activator for 1 hour, and sequentially immerse the nuclear pore membrane in a double-stranded hybrid solution of aptamer Apt1 and ssDNA1 through amidation reaction for 1 hour, and then modify ssDNA2-MB on the inner wall of the pore to form a double-specific recognition interface, and then modify hairpin DNA H1 and H2 with Fe 3 O 4@Pt nanozyme, pre-assembled in nanopores; (4) Blocking active sites: Wash the modified nanopores with deionized water to remove unreacted probe DNA, then block the active sites successively with polyethylene glycol and bovine serum albumin solution, and then wash with deionized water to finally obtain a composite membrane containing probe DNA-functionalized nanopores with blocked active sites.

[0031] The polymer membrane of the technical solution is a polyethylene terephthalate (PET) membrane. The shape of the nanopores is columnar, conical, or irregular. The etching solution is sodium hydroxide solution.

[0032] The Z-scheme heterojunction g-C 3 N 4 @CdS quantum dots significantly improve the visible light absorption efficiency due to their narrow bandgap characteristics. The baseline intensity of the photocurrent reaches 745 μA. Modifying with MB can accelerate the separation of photogenerated electron-hole pairs, the photocurrent response is increased by 4 times, and at the same time, the hole recombination is inhibited.

[0033] The surface of the etched nanopores 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. Then these active esters are further condensed with the probe with amino groups through the formation of covalent bonds to obtain nanopores modified with the probe, and further obtain probe DNA-functionalized nanopores.

[0034] In the technical solution, when cTnI is present, Apt1 specifically binds to cTnI, releases the single-stranded ssDNA1, hybridizes with the single-stranded ssDNA2, activates the trans-cleavage function of Cas12a, and releases the initiating ssDNA2 strand; the initiating ssDNA2 strand and H1, H2 initiate the HCR reaction to generate a long-chain DNA structure, and the end is modified with Fe 3 O 4 @Ptt nanozyme catalyzes H 2 O 2 to oxidize TMB to generate a precipitate, covering the surface of the nuclear pore membrane to inhibit the photocurrent signal.

[0035] Furthermore, in step 1, the thickness of the polymer membrane is 15 µm, the pore diameter of the surface pores is 5 µm, the pore density is 1×10 3 per cm 2 , the ultraviolet light wavelength is 360 nm, and the concentration of the sodium hydroxide solution is 6 mol / L.

[0036] Furthermore, in step 2, the Z-scheme heterojunction g-C 3 N 4 @CdS quantum dots are prepared by a hydrothermal method. Cd(CH 3COO) 2 ·2H 2 O and Na 2 S·9H 2 The mixture of O is dissolved in deionized water, and then g-C 3 N 4 powder is added to the solution, and then the solution is ultrasonically treated. The mixture is transferred to a Teflon-lined autoclave, heated, washed, dried, and ground.

[0037] 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).

[0038] 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 soaking time is 1 hour, and it is carried out at room temperature.

[0039] Example 2. In addition, the present invention also provides an application of a photoelectrochemical biosensor based on cascade amplification in the ultrasensitive detection of cardiac troponin. The main steps are as follows: The sealed composite membrane is placed on an ITO conductive glass as a working electrode, a silver / silver chloride electrode is used as a reference electrode, and a platinum wire is used 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, using a xenon lamp as a light source and a wavelength of 360 nm, a troponin and Cas12a-crRNA mixed solution is added, and chronoamperometry is used to test the electrochemical performance; According to the electrochemical test results, a linear relationship curve is drawn, and the applicable detection limit of the constructed biosensor can be obtained as 0.02 fmol / mL, with high-efficiency analysis and detection capabilities; According to the signal test results of various interfering substances, it shows that the constructed biosensor has high specificity; According to the results of multiple cyclic tests, the constructed biosensor has good stability; According to the signal responses of biosensors of different batches, the constructed biosensor has good reproducibility.

[0040] 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.

[0041] Example 3. In this example, scanning electron microscopy (SEM) is used as a powerful technique to help observe the morphology and structure of the prepared materials. As Figure 3 shown in Figure a, the structure of the g-C 3 N 4 @CdS composite material can be clearly seen. The g-C 3N 4 Successfully coated on the surface of spherical CdS. Although the combination of the two monomers makes the surface of CdS rough, the spherical structure of individual CdS remains almost unchanged, and good contact is established at the interface. This unique hierarchical structure enables g-C 3 N 4 @CdS to have a large specific surface area and provide more active sites for the reaction. As shown in Figure b of Figure 3 , a uniform pore distribution exists on the nuclear pore membrane.

[0042] Example 4. Experimental verification of the principle: To prove the feasibility of this technical solution, this example uses a method of preparing a photoelectrochemical biosensor with different modified electrodes. The results are as shown in Figure 4 . After coating the ITO conductive glass with a nuclear pore membrane with Z-scheme heterojunction g-C 3 N 4 @CdS, a photocurrent response is 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 is observed, indicating that the nuclear pore membrane with Z-scheme heterojunction g-C 3 N 4 @CdS has good photocurrent performance; after activation with EDC and NHS and then carrying out a photocurrent response in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, a photocurrent value of 730 µA is observed; after modification with apt1-ssDNA1 and then carrying out a photocurrent response in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, a photocurrent value of 715 µA is observed; after modification with ssDNA2-MB and then carrying out a photocurrent response in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, a photocurrent value of 1005 µA is observed; after modification with H1-H2-Fe 3 O 4 @Pt and then carrying out a photocurrent response in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, a photocurrent value of 985 µA is observed; after blocking and then carrying out a photocurrent response in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, a photocurrent value of 965 µA is observed; after incubation with the target and then carrying out a photocurrent response in a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7, a photocurrent value of 115 µA is observed, which is much smaller than the photocurrent value before incubation. This indicates that when the target is present, specific recognition occurs with the probe, thereby changing the transfer state of photo-generated electron-hole pairs on the electrode surface and causing a change in photocurrent.

[0043] Example 5, Sensitivity Experiment: To evaluate the sensitivity of the detection target of this technical solution, under the optimal experimental conditions, photocurrents of targets with different concentrations were measured. As Figure 5 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 magnitude of the target concentration. Therefore, the target can be analyzed by the optoelectrochemical biosensor. By combining different optoelectroactive substances with different biorecognition elements, optoelectrochemical biosensors with different detection functions can be constructed.

[0044] Example 6, Specificity Experiment: To study the specificity of the optoelectrochemical biosensor, as Figure 6 shown, in this example, a specificity experiment was carried out with four mismatches and one specific target as controls; the results are as Figure 6 shown. Under the same reaction conditions for different types of analytes, the photocurrent difference of the target is much larger than that of non-targets.

[0045] Example 7, Stability Experiment: To study the stability of the optoelectrochemical biosensor, as Figure 7 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 show that the optoelectrochemical biosensor has excellent stability.

[0046] It should be noted that the above examples are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently 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.

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