Preparation of ECL sensor based on near-infrared alkyne functionalized Cu NSs and double-cycling waste-free Cas12a-driven signal amplifier

Abstract

The application belongs to the field of immunoassay and biosensing technology, and provides a preparation method of an ECL sensor based on near-infrared alkyne functionalized Cu NSs and a double-cycle waste-free Cas12a driven signal amplifier. Specifically, alkyne functionalized Cu NSs are used as luminophores, and the unique alkyne structure and rigid conformation of the ligand BPTA not only realize pi-pi ordered stacking, but also inhibit non-radiative transition and promote the formation of highly conjugated aggregates, so that the Cu NSs exhibit stable and efficient NIR-ECL performance at 803 nm wavelength, excellent biocompatibility and low cost advantage. In addition, based on the DNA recycling and reuse realized by the designed double-cycle strategy, and the excellent reaction kinetics and specificity of the Cas12a system, the double-cycle waste-free Cas12a driven signal amplifier not only solves the problem of DNA waste liquid in the traditional cycle, but also overcomes the defects of low cutting efficiency and poor specificity existing in the traditional enzyme cutting method. According to the difference in electrochemiluminescence signal intensity of different concentrations of the measured substance, the detection of hepatitis A virus HAV is realized.

Description

Technical Field

[0001] This invention relates to a method for fabricating an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a driving signal amplifier. Specifically, a two-dimensional sheet-like Cu NSs structure, self-assembled at 4°C, is used as the emitting element, and combined with a dual-cycle, waste-free Cas12a driving signal amplifier, to prepare an electrochemical biosensor for detecting hepatitis A virus (HAV), belonging to the field of novel functional materials and biosensing detection technology. Background Technology

[0002] Hepatitis A virus (HAV) is a major pathogen causing acute and chronic liver diseases. It is highly contagious and easily progresses to chronic hepatitis, potentially developing into liver fibrosis, cirrhosis, and even hepatocellular carcinoma, imposing a severe disease burden on the body. If not diagnosed and treated promptly, it can become life-threatening. Therefore, developing accurate, efficient, rapid, and sensitive nucleic acid detection methods is crucial for the early detection and timely treatment of HAV.

[0003] Currently, the detection of hepatitis A virus (HAV) mainly employs cytogenetics and fluorescence in situ hybridization, but these methods are still limited by factors such as low sensitivity, unsatisfactory quantitative analysis, and reliance on expensive specialized instruments, making it difficult to meet the significant demand for early HAV diagnosis. Electrochemiluminescence (ECL) has wide applications in bioimaging, medical diagnostics, and disease biomarker detection due to its advantages of high sensitivity, wide detection range, rapid response, and good controllability. Compared to traditional analytical methods, the electrochemical biosensor used in this invention has advantages such as high sensitivity, good selectivity, fast response speed, simple operation, and ease of miniaturization. Therefore, this invention prepares an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a-driven signal amplifier to achieve sensitive detection of hepatitis A virus (HAV).

[0004] This invention uses 2,5-bis(prop-2-yn-1-yloxy)-terephthalaldehyde as an organic ligand, Cu 2+Cu NSs were self-assembled at 4°C using the central ion. This material has a two-dimensional sheet-like structure, which facilitates full contact with the co-reacting reagents. Furthermore, thanks to the unique alkyne structure and rigid conformation of the ligand BPTA, it not only achieves π-π ordered stacking but also suppresses non-radiative transitions and promotes the formation of highly conjugated aggregates, thus enabling Cu NSs to exhibit stable and efficient NIR-ECL performance at 803 nm. In addition, a dual-cycle, waste-free Cas12a-driven signal amplifier was designed. Compared with traditional enzyme-induced dual-cycle methods, this approach not only solves the DNA waste problem in traditional cycles but also overcomes the shortcomings of low cutting efficiency and poor specificity in traditional enzyme digestion methods. This invention prepared an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a-driven signal amplifier, enabling quantitative detection of hepatitis A virus (HAV). It exhibits advantages such as high sensitivity, fast response speed, wide detection range, and low detection limit, as well as good stability, selectivity, and reproducibility. This strategy utilizes NIR-ECL technology to prepare novel Cu NSs, laying the foundation for early disease diagnosis and significantly expanding their application prospects in clinical diagnosis and bioanalysis. Summary of the Invention

[0005] One objective of this invention is to utilize 2,5-bis(prop-2-yn-1-yloxy)-terephthalaldehyde as an organic ligand, Cu 2+ Cu NSs, a dual-center ion, was self-assembled at a low temperature of 4 °C. Leveraging the unique alkyne structure and rigid conformation of the ligand BPTA, it not only achieved π-π ordered stacking but also suppressed nonradiative transitions and promoted the formation of highly conjugated aggregates, thus exhibiting stable and efficient NIR-ECL performance at a wavelength of 803 nm. Furthermore, the material's two-dimensional sheet structure facilitates sufficient contact with co-reacting reagents, thereby significantly improving ECL luminescence efficiency.

[0006] The second objective of this invention is to design a dual-cycle waste-free signal amplifier, which cleverly transfers the product from cycle one to cycle two, thus solving the problem of DNA waste in traditional cycles.

[0007] The third objective of this invention is to combine a dual-cycle waste-free signal amplifier with a CRISPR-Cas12a system, leveraging the powerful trans-cleavage activity of Cas12a to achieve rapid signal changes, thereby significantly improving the sensitivity of the biosensor.

[0008] The technical solution of the present invention is as follows: 1. A method for fabricating an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a-driven signal amplifier, comprising the following steps: (1) Polish carefully on a polishing cloth with alumina powder for 3 minutes, then rinse with ultrapure deionized water. Air dry to obtain a clean surface; (2) Add 6 μL of Cu NSs (0.8 ~ 2.4 mg / mL) and 5 μL of gold nanoparticles to GCE and let it dry; (3) Next, 10 µL of dopamine with 100 ~ 300 mmol / L was incubated at 4°C for 90 minutes. After completion, the electrode surface was cleaned with 1× phosphate buffer. (4) Block the electrode with 5 μL of 50 ~ 150 mmol / L 1-hexanethiol for 30 minutes at room temperature to reduce nonspecific binding. After completion, clean the electrode surface with 1× phosphate buffer. (5) Next, 2 μL of Cas12a diluted with 500 nM, 2 μL of crRNA and 6 μL of S2 generated by 0.00001 ~100 pmol / L HAV were mixed to form an activated CRISPR-Cas12a system. The mixture was then dropped onto the electrode and incubated at 37°C for 1 h to cleave the S3-DA bound on the electrode. The electrode surface was then washed with 1× phosphate buffer to obtain an ECL sensor based on near-infrared alkyne functionalized Cu NSs and a dual-cycle waste-free Cas12a-driven signal amplifier. The sensor was stored in a 4°C refrigerator.

[0009] 2. The near-infrared alkyne-functionalized Cu NSs is prepared by the following steps: (1) Preparation of Cu NSs: 7.1113 mg of Cu₂(NO₃)₂·3H₂O was dissolved in 5 mL of methanol and stirred vigorously at room temperature. 25 mL of dichloromethane containing an equimolar amount of the alkynyl ligand 2,5-bis(propane-2-yn-1-oxy)-terephthalaldehyde was added to this solution. Subsequently, 150 μL of triethylamine was added dropwise. The resulting mixture was allowed to stand at 4 °C in the dark for 3 days. After the reaction was complete, the formed copper nanosheets were collected by centrifugation at 10,000 rpm for 10 min. (2) Preparation of S3-DA: First, TCEP was added to S3 to activate the thiol group, and the mixture was incubated at room temperature for 1 h to ensure complete cleavage of the disulfide bonds. Then, S3 was heated to 95 °C for 5 min and slowly cooled to room temperature to form a single-chain structure. Next, the carboxyl-modified S3 was activated for 20 min using a mixed solution of 0.05 M N-hydroxysuccinimide and 0.2 M carbodiimide hydrochloride. Subsequently, a dopamine solution of 10–200 mmol / L was added to the mixture, and the reaction was carried out overnight at 4 °C. S3-DA was successfully synthesized and stored at 4 °C for subsequent use. (3) Preparation method of dual-cycle zero-waste signal amplifier: The specific operating steps are as follows: 1 ~ 10 μM MBs-H1 and 0.00001 ~ 100 pmol / L HAV are incubated at 37℃ for 2 hours to form MBs-H1-T complex; then 10 ~ 100 U / 100µL Exo III is added, and the cleavage reaction is carried out at 37℃ for 1 hour, followed by heating at 70℃ for 20 minutes to inactivate the enzyme; after magnetic separation, the cleaved hairpin structure functionalized MBs (denoted as MBs-H1') are retained and redispersed in 1 ~ 100 μL TE buffer. Meanwhile, 1-100 μL of S1 and 1-100 μL of S2 were pre-hybridized at 37°C for 2 hours to form the S1-S2 complex. This S1-S2 solution was then mixed with redispersed MBs-H1' and incubated at 37°C for 2 hours to form the MBs-H1'-S1 complex. 10-100 U / 100µL of Exo III was added again, and the mixture was cleaved at 37°C for 1 hour, followed by inactivation at 70°C for 20 minutes. Finally, after magnetic separation, the supernatant containing the released activator mimic target S2 was collected for subsequent use. (4) Preparation of activated CRISPR-Cas12: The CRISPR-Cas12a system is activated via the following steps: First, Cas12a is diluted using the provided Cas12a diluent. Then, 2 μL of Cas12a diluted at 500 nM, 2 μL of crRNA diluted at 4 μM, and 6 μL of S2 generated from 0.00001–100 pmol / L HAV are mixed. The resulting mixture is incubated at 37°C for 15 minutes to complete the activation process.

[0010] 3. For the detection of hepatitis A virus (HAV), the following steps are included: (1) An MPI-E electrochemiluminescence analysis system was used for testing in a three-electrode system. The saturated Ag / AgCl electrode was used as the reference electrode, the Pt electrode was used as the counter electrode, and the prepared near-infrared alkyne-functionalized Cu NSs ECL sensor was used as the working electrode. The voltage of the photomultiplier tube was set to 700 V. The cyclic voltammetry test voltage was -1.6 ~ 0.0 V, and the scan rate was 200 mV / s. (2) In 10 mL of phosphate buffer solution containing 0.1 mol / L potassium persulfate at pH = 7.38, the electrochemiluminescence signals generated by HAV at different concentrations of the analyte were detected and the working curves were plotted. (3) Replace the hepatitis A virus (HAV) solution with the test sample solution for detection.

[0011] Beneficial results of the present invention (1) The present invention successfully synthesized near-infrared alkyne functionalized Cu NSs. With the unique alkyne structure and rigid conformation of its ligand BPTA, the material not only achieves π-π ordered stacking, but also suppresses nonradiative transitions and promotes the formation of highly conjugated aggregates, thus enabling Cu NSs to exhibit stable and efficient NIR-ECL performance, excellent biocompatibility and low cost at a wavelength of 803 nm.

[0012] (2) In this invention, a target-induced dual-cycle waste-free Cas12a-driven signal amplifier was designed. Compared with the traditional enzyme-induced nucleic acid amplification strategy, this design can not only realize the continuous recovery and reuse of DNA and significantly reduce reagent consumption, but also achieve rapid signal change with the strong trans-cleavage activity of Cas12a, thereby greatly improving the sensitivity of the biosensor.

[0013] (3) The electrochemical biosensor prepared by the present invention is used for quantitative detection of hepatitis A virus (HAV). It has a fast response speed, a wide linear range, a low detection limit, and good stability, selectivity and reproducibility. It can achieve simple, fast, highly sensitive and specific detection. Detailed Implementation

[0014] (The present invention will now be further described through specific embodiments, but is not limited thereto.) Example 1. A method for fabricating an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a-driven signal amplifier, comprising the following steps: (1) Polish carefully on a polishing cloth with alumina powder for 3 minutes, then rinse with ultrapure deionized water. Air dry to obtain a clean surface; (2) Add 6 μL of Cu NSs and 5 μL of gold nanoparticles to GCE and let it dry. (3) Next, 10 µL of dopamine with 100 mmol / L was incubated at 4°C for 90 minutes. After completion, the electrode surface was cleaned with 1× phosphate buffer. (4) Block the electrode with 5 μL of 50 mmol / L 1-hexanethiol for 30 minutes at room temperature to reduce nonspecific binding. After completion, clean the electrode surface with 1× phosphate buffer. (5) Next, 2 μL of Cas12a diluted with 500 nM, 2 μL of crRNA and 6 μL of S2 generated by 0.00001 ~100 pmol / L HAV were mixed to form an activated CRISPR-Cas12a system. The mixture was then dropped onto the electrode and incubated at 37°C for 1 h to cleave the S3-DA bound on the electrode. The electrode surface was then washed with 1× phosphate buffer to obtain an ECL sensor based on near-infrared alkyne functionalized Cu NSs and a dual-cycle waste-free Cas12a-driven signal amplifier. The sensor was stored in a 4°C refrigerator.

[0015] Example 2. A method for fabricating an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a-driven signal amplifier, comprising the following steps: (1) Polish carefully on a polishing cloth with alumina powder for 3 minutes, then rinse with ultrapure deionized water. Air dry to obtain a clean surface; (2) Add 6 μL of Cu NSs at a concentration of 1.5 mg / mL and 5 μL of gold nanoparticles to the GCE and let it dry. (3) Next, 10 µL of dopamine with 200 mmol / L was incubated at 4°C for 90 minutes. After completion, the electrode surface was cleaned with 1× phosphate buffer. (4) Block the electrode with 5 μL of 100 mmol / L 1-hexanethiol at room temperature for 30 minutes to reduce nonspecific binding. After completion, clean the electrode surface with 1× phosphate buffer. (5) Next, 2 μL of Cas12a diluted with 500 nM, 2 μL of crRNA and 6 μL of S2 generated by 0.00001 ~100 pmol / L HAV were mixed to form an activated CRISPR-Cas12a system. The mixture was then dropped onto the electrode and incubated at 37°C for 1 h to cleave the S3-DA bound on the electrode. The electrode surface was then washed with 1× phosphate buffer to obtain an ECL sensor based on near-infrared alkyne functionalized Cu NSs and a dual-cycle waste-free Cas12a-driven signal amplifier. The sensor was stored in a 4°C refrigerator.

[0016] Example 3. A method for fabricating an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a-driven signal amplifier, comprising the following steps: (1) Polish carefully on a polishing cloth with alumina powder for 3 minutes, then rinse with ultrapure deionized water. Air dry to obtain a clean surface; (2) Add 6 μL of Cu NSs at a concentration of 2 mg / mL and 5 μL of gold nanoparticles to the GCE and let it dry. (3) Next, 10 µL of dopamine with 300 mmol / L was incubated at 4°C for 90 minutes. After completion, the electrode surface was cleaned with 1× phosphate buffer. (4) Block the electrode with 5 μL of 150 mmol / L 1-hexanethiol for 30 minutes at room temperature to reduce nonspecific binding. After completion, clean the electrode surface with 1× phosphate buffer. (5) Next, 2 μL of Cas12a diluted with 500 nM, 2 μL of crRNA and 6 μL of S2 generated by 0.00001 ~100 pmol / L HAV were mixed to form an activated CRISPR-Cas12a system. The mixture was then dropped onto the electrode and incubated at 37°C for 1 h to cleave the S3-DA bound on the electrode. The electrode surface was then washed with 1× phosphate buffer to obtain an ECL sensor based on near-infrared alkyne functionalized Cu NSs and a dual-cycle waste-free Cas12a-driven signal amplifier. The sensor was stored in a 4°C refrigerator.

[0017] Example 4. The near-infrared alkyne-functionalized Cu NSs is prepared by the following steps: (1) Preparation of Cu NSs: 7.1113 mg of Cu₂(NO₃)₂·3H₂O was dissolved in 5 mL of methanol and stirred vigorously at room temperature. 25 mL of dichloromethane containing an equimolar amount of the alkynyl ligand 2,5-bis(propane-2-yn-1-oxy)-terephthalaldehyde was added to this solution. Subsequently, 150 μL of triethylamine was added dropwise. The resulting mixture was allowed to stand at 4 °C in the dark for 3 days. After the reaction was complete, the formed copper nanosheets were collected by centrifugation at 10,000 rpm for 10 min. (2) Preparation of S3-DA: First, TCEP was added to S3 to activate the thiol group, and the mixture was incubated at room temperature for 1 h to ensure complete cleavage of the disulfide bonds. Then, S3 was heated to 95 °C for 5 min and slowly cooled to room temperature to form a single-chain structure. Next, the carboxyl-modified S3 was activated for 20 min using a mixed solution of 0.05 M N-hydroxysuccinimide and 0.2 M carbodiimide hydrochloride. Subsequently, a 50 mmol / L dopamine solution was added to the mixture, and the reaction was carried out overnight at 4 °C. S3-DA was successfully synthesized and stored at 4 °C for subsequent use. (3) Preparation method of dual-cycle zero-waste signal amplifier: The specific operation steps are as follows: 2 μM MBs-H1 was incubated with 0.00001 ~ 100 pmol / L HAV at 37℃ for 2 hours to form MBs-H1-T complex; then 20 U / 100µL Exo III was added, and the cleavage reaction was carried out at 37℃ for 1 hour, followed by heating at 70℃ for 20 minutes to inactivate the enzyme; after magnetic separation, the cleaved hairpin structure functionalized MBs (denoted as MBs-H1') were retained and redispersed in 5 μL TE buffer. Meanwhile, 2 μL of S1 and 2 μL of S2 were prehybridized at 37°C for 2 hours to form the S1-S2 complex. This S1-S2 solution was then mixed with redispersed MBs-H1' and incubated at 37°C for 2 hours to form the MBs-H1'-S1 complex. 20 U / 100µL of Exo III was added again, and the mixture was cleaved at 37°C for 1 hour, followed by inactivation at 70°C for 20 minutes. Finally, after magnetic separation, the supernatant containing the released activator mimic target S2 was collected for subsequent use. (4) Preparation of activated CRISPR-Cas12: The CRISPR-Cas12a system is activated via the following steps: First, Cas12a is diluted using the provided Cas12a diluent. Then, 2 μL of Cas12a diluted at 500 nM, 2 μL of crRNA diluted at 4 μM, and 6 μL of S2 generated from 0.00001–100 pmol / L HAV are mixed. The resulting mixture is incubated at 37°C for 15 minutes to complete the activation process.

[0018] Example 5. The near-infrared alkyne-functionalized Cu NSs is prepared by the following steps: (1) Preparation of Cu NSs: 7.1113 mg of Cu₂(NO₃)₂·3H₂O was dissolved in 5 mL of methanol and stirred vigorously at room temperature. 25 mL of dichloromethane containing an equimolar amount of the alkynyl ligand 2,5-bis(propane-2-yn-1-oxy)-terephthalaldehyde was added to this solution. Subsequently, 150 μL of triethylamine was added dropwise. The resulting mixture was allowed to stand at 4 °C in the dark for 3 days. After the reaction was complete, the formed copper nanosheets were collected by centrifugation at 10,000 rpm for 10 min. (2) Preparation of S3-DA: First, TCEP was added to S3 to activate the thiol group, and the mixture was incubated at room temperature for 1 h to ensure complete cleavage of the disulfide bonds. Then, S3 was heated to 95 °C for 5 min and slowly cooled to room temperature to form a single-chain structure. Next, the carboxyl-modified S3 was activated for 20 min using a mixed solution of 0.05 M N-hydroxysuccinimide and 0.2 M carbodiimide hydrochloride. Subsequently, a 100 mmol / L dopamine solution was added to the mixture, and the reaction was carried out overnight at 4 °C. S3-DA was successfully synthesized and stored at 4 °C for subsequent use. (3) Preparation method of dual-cycle zero-waste signal amplifier: The specific operation steps are as follows: 5 μM MBs-H1 was incubated with 0.00001 ~ 100 pmol / L HAV at 37℃ for 2 hours to form MBs-H1-T complex; then 50 U / 100µL Exo III was added, and the cleavage reaction was carried out at 37℃ for 1 hour, followed by heating at 70℃ for 20 minutes to inactivate the enzyme; after magnetic separation, the cleaved hairpin structure functionalized MBs (denoted as MBs-H1') were retained and redispersed in 5 μL TE buffer. Meanwhile, 5 μL of S1 and 5 μL of S2 were prehybridized at 37°C for 2 hours to form the S1-S2 complex. This S1-S2 solution was then mixed with redispersed MBs-H1' and incubated at 37°C for 2 hours to form the MBs-H1'-S1 complex. 50 U / 100µL of Exo III was added again, and the mixture was cleaved at 37°C for 1 hour, followed by inactivation at 70°C for 20 minutes. Finally, after magnetic separation, the supernatant containing the released activator mimic target S2 was collected for subsequent use. (4) Preparation of activated CRISPR-Cas12: The CRISPR-Cas12a system is activated via the following steps: First, Cas12a is diluted using the provided Cas12a diluent. Then, 2 μL of Cas12a diluted at 500 nM, 2 μL of crRNA diluted at 4 μM, and 6 μL of S2 generated from 0.00001–100 pmol / L HAV are mixed. The resulting mixture is incubated at 37°C for 15 minutes to complete the activation process.

[0019] Example 6. The near-infrared alkyne-functionalized Cu NSs is prepared by the following steps: (1) Preparation of Cu NSs: 7.1113 mg of Cu₂(NO₃)₂·3H₂O was dissolved in 5 mL of methanol and stirred vigorously at room temperature. 25 mL of dichloromethane containing an equimolar amount of the alkynyl ligand 2,5-bis(propane-2-yn-1-oxy)-terephthalaldehyde was added to this solution. Subsequently, 150 μL of triethylamine was added dropwise. The resulting mixture was allowed to stand at 4 °C in the dark for 3 days. After the reaction was complete, the formed copper nanosheets were collected by centrifugation at 10,000 rpm for 10 min. (2) Preparation of S3-DA: First, TCEP was added to S3 to activate the thiol group, and the mixture was incubated at room temperature for 1 h to ensure complete cleavage of the disulfide bonds. Then, S3 was heated to 95 °C for 5 min and slowly cooled to room temperature to form a single-chain structure. Next, the carboxyl-modified S3 was activated for 20 min using a mixed solution of 0.05 M N-hydroxysuccinimide and 0.2 M carbodiimide hydrochloride. Subsequently, a 150 mmol / L dopamine solution was added to the mixture, and the reaction was carried out overnight at 4 °C. S3-DA was successfully synthesized and stored at 4 °C for subsequent use. (3) Preparation method of dual-cycle zero-waste signal amplifier: The specific operation steps are as follows: 8 μM MBs-H1 was incubated with 0.00001 ~ 100 pmol / L HAV at 37℃ for 2 hours to form MBs-H1-T complex; then 80 U / 100µL Exo III was added, and the cleavage reaction was carried out at 37℃ for 1 hour, followed by heating at 70℃ for 20 minutes to inactivate the enzyme; after magnetic separation, the cleaved hairpin structure functionalized MBs (denoted as MBs-H1') were retained and redispersed in 5 μL TE buffer. Meanwhile, 8 μL of S1 and 8 μL of S2 were prehybridized at 37°C for 2 hours to form the S1-S2 complex. This S1-S2 solution was then mixed with redispersed MBs-H1' and incubated at 37°C for 2 hours to form the MBs-H1'-S1 complex. 80 U / 100µL of Exo III was added again, and the mixture was cleaved at 37°C for 1 hour, followed by inactivation at 70°C for 20 minutes. Finally, after magnetic separation, the supernatant containing the released activator mimic target S2 was collected for subsequent use. (4) Preparation of activated CRISPR-Cas12: The CRISPR-Cas12a system is activated via the following steps: First, Cas12a is diluted using the provided Cas12a diluent. Then, 2 μL of Cas12a diluted at 500 nM, 2 μL of crRNA diluted at 4 μM, and 6 μL of S2 generated from 0.00001–100 pmol / L HAV are mixed. The resulting mixture is incubated at 37°C for 15 minutes to complete the activation process.

[0020] Example 7: Detection of Hepatitis A Virus (HAV), comprising the following steps: (1) An MPI-E electrochemiluminescence analysis system was used for testing in a three-electrode system. The saturated Ag / AgCl electrode was used as the reference electrode, the Pt electrode was used as the counter electrode, and the prepared near-infrared alkyne-functionalized Cu NSs ECL sensor was used as the working electrode. The voltage of the photomultiplier tube was set to 700 V. The cyclic voltammetry test voltage was -1.6 ~ 0.0 V, and the scan rate was 200 mV / s. (2) In 10 mL of phosphate buffer solution containing 0.1 mol / L potassium persulfate at pH = 7.38, the electrochemiluminescence signals generated by HAV at different concentrations of the analyte were detected and the working curves were plotted. (3) Replace the hepatitis A virus (HAV) solution with the test sample solution for detection.

Claims

1. A method for fabricating an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a-driven signal amplifier, comprising the following steps: (1) Polish carefully on a polishing cloth with alumina powder for 3 minutes, then rinse with ultrapure deionized water. Air dry to obtain a clean surface; (2) Add 6 μL of Cu NSs (0.8 ~ 2.4 mg / mL) and 5 μL of gold nanoparticles to GCE and let it dry; (3) Next, 10 µL of dopamine with 100 ~ 300 mmol / L was incubated at 4°C for 90 minutes. After completion, the electrode surface was cleaned with 1× phosphate buffer. (4) Block the electrode with 5 μL of 50 ~ 150 mmol / L 1-hexanethiol for 30 minutes at room temperature to reduce nonspecific binding. After completion, clean the electrode surface with 1× phosphate buffer. (5) Next, 2 μL of Cas12a diluted with 500 nM, 2 μL of crRNA and 6 μL of S2 generated by 0.00001 ~ 100 pmol / L HAV were mixed to form an activated CRISPR-Cas12a system. The mixture was then dropped onto the electrode and incubated at 37°C for 1 h to cleave the S3-DA bound on the electrode. The electrode surface was then washed with 1× phosphate buffer to obtain an ECL sensor based on near-infrared alkyne functionalized CuNSs and a dual-cycle waste-free Cas12a-driven signal amplifier. The sensor was stored in a 4°C refrigerator.

2. The method for fabricating an ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle waste-free Cas12a-driven signal amplifier as described in claim 1, wherein the method for fabricating S3-DA, the method for fabricating the dual-cycle waste-free signal amplifier, and the method for fabricating activated CRISPR-Cas12 are characterized in that, The steps are as follows: (1) Preparation of Cu NSs: 7.1113 mg of Cu₂(NO₃)₂·3H₂O was dissolved in 5 mL of methanol and stirred vigorously at room temperature. 25 mL of dichloromethane containing an equimolar amount of the alkynyl ligand 2,5-bis(propane-2-yn-1-oxy)-terephthalaldehyde was added to this solution. Subsequently, 150 μL of triethylamine was added dropwise. The resulting mixture was allowed to stand at 4 °C in the dark for 3 days. After the reaction was complete, the formed copper nanosheets were collected by centrifugation at 10,000 rpm for 10 min. (2) Preparation of S3-DA: First, TCEP was added to S3 to activate the thiol group, and the mixture was incubated at room temperature for 1 h to ensure complete cleavage of the disulfide bonds. Then, S3 was heated to 95 °C for 5 min, followed by slow cooling to room temperature to form a single-chain structure. Next, the carboxyl-modified S3 was activated for 20 min using a mixed solution of 0.05 M N-hydroxysuccinimide and 0.2 M carbodiimide hydrochloride. Subsequently, a dopamine solution of 10–200 mmol / L was added to the mixture, and the reaction was carried out overnight at 4 °C. S3-DA was successfully synthesized and stored at 4 °C for subsequent use. (3) Preparation method of dual-cycle zero-waste signal amplifier: The specific operation steps are as follows: 1 ~ 10 μM MBs-H1 and 0.00001 ~ 100 pmol / L HAV are incubated at 37℃ for 2 hours to form MBs-H1-T complex; then 10 ~ 100 U / 100µL Exo III is added, and the cleavage reaction is carried out at 37℃ for 1 hour, followed by heating at 70℃ for 20 minutes to inactivate the enzyme; after magnetic separation, the cleaved hairpin structure functionalized MBs (denoted as MBs-H1') are retained and redispersed in 5 μL TE buffer. Meanwhile, 1-10 μL of S1 and 1-10 μL of S2 were prehybridized at 37°C for 2 hours to form the S1-S2 complex. This S1-S2 solution was then mixed with redispersed MBs-H1' and incubated at 37°C for 2 hours to form the MBs-H1'-S1 complex. 10-100 U / 100µL of Exo III was added again, and the mixture was cleaved at 37°C for 1 hour, followed by inactivation at 70°C for 20 minutes. Finally, after magnetic separation, the supernatant containing the released activator mimic target S2 was collected for subsequent use. (4) Preparation of activated CRISPR-Cas12: The CRISPR-Cas12a system is activated via the following steps: First, Cas12a is diluted using the provided Cas12a diluent. Then, 2 μL of Cas12a diluted at 500 nM, 2 μL of crRNA diluted at 4 μM, and 6 μL of S2 generated from 0.00001–100 pmol / L HAV are mixed. The resulting mixture is incubated at 37°C for 15 minutes to complete the activation process.

3. The ECL sensor based on near-infrared alkyne-functionalized Cu NSs and a dual-cycle, waste-free Cas12a-driven signal amplifier, prepared by the method described in claim 1, is used for the detection of hepatitis A virus (HAV), characterized in that... The steps are as follows: (1) An MPI-E electrochemiluminescence analysis system was used for testing in a three-electrode system. The saturated Ag / AgCl electrode was used as the reference electrode, the Pt electrode as the counter electrode, and the prepared near-infrared alkyne-functionalized Cu NSs ECL sensor was used as the working electrode. The voltage of the photomultiplier tube was set to 700 V. The cyclic voltammetry test voltage was -1.6 ~ 0.0 V, and the scan rate was 200 mV / s. (2) In 10 mL of phosphate buffer solution containing 0.1 mol / L potassium persulfate at pH = 7.38, the electrochemiluminescence signals generated by HAV at different concentrations of the analyte were detected and the working curves were plotted. (3) Replace the hepatitis A virus (HAV) solution with the test sample solution for detection.