Drug-resistant pathogenic bacterium detection method based on turn-on visible light photoelectrochemical sensing

Abstract

The invention discloses a detection technology based on photoelectrochemistry (PEC), a BiVO4 / MoS2 heterostructure is adopted as a photosensitive material, SiO2 is adopted as a signal inhibitor, and a trans-lysis mechanism of CRISPR / Cas12a is combined, so that turn-on type signal detection of drug-resistant pathogenic bacteria initiated by single nucleotide polymorphism (SNPs) is realized. The preparation process of the PEC sensor comprises the steps of coating the surface of a silk-screen printing carbon electrode (SPCE) with a BiVO4 / MoS2 heterojunction layer, then introducing gold nanoparticles (AuNPs) through an electrochemical deposition technology, and anchoring DNA modified SiO2 nanoparticles on the surface of the AuNPs through Au-S bonding reaction. In the presence of target drug-resistant pathogenic bacteria DNA, the trans-cleavage activity of a CRISPR / Cas12a system is activated to trigger the dissociation of a signal inhibitor SiO2, so that the originally inhibited light current signal is recovered, and the output of a turn-on signal is realized. The detection limit is 103 CFU / mL, 0.01% of drug-resistant strain proportion can be detected, and the method has the characteristics of high sensitivity, strong specificity and low background signal, and provides a new technical means for rapid and accurate diagnosis of SNPs-induced drug-resistant pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to the field of biosensor detection of foodborne pathogens, and in particular to a CRISPR / Cas12a-based turn-on photoelectrochemical detection method and its application in the detection of drug-resistant Salmonella. Background Technology

[0002] Foodborne illnesses caused by pathogenic bacteria pose a serious threat to public health and lives, resulting in an average of more than 700,000 deaths worldwide each year. The widespread contamination of Salmonella throughout the food supply chain, particularly in animal-derived foods, has made Salmonella infection one of the most important foodborne bacterial diseases.

[0003] Salmonella contamination in various foods can survive for extended periods, enabling transmission to humans and causing severe diarrhea, gastroenteritis, and typhoid fever. The prevalence of drug-resistant bacteria and the overuse and abuse of antibiotics have increased the threat to global public health. Quantitative polymerase chain reaction (PCR) for refractory mutants has been widely used in clinical witness SNP genotyping, with detection limits as low as 1%. Given the specific recognition capabilities guided by CRISPR-RNA hybridization, a CRISPR / Cas12a-based ARMS-PCR detection method for SNP resolvable detection has been developed. This Cas12a-based ARMS-PCR detection avoids the need for expensive instruments and real-time fluorescence measurements requiring pre-amplification of nucleic acids.

[0004] Photoelectrochemistry has been proven to offer high sensitivity with no amplification required and rapid analysis. It is well known that the analytical performance of photoelectrochemical chemiluminescence (PEC) largely depends on the semiconductor material used on the photoelectrode. Wide-bandgap semiconductors, such as tin oxide, zinc sulfide, and bismuth oxychloride, can only be excited by high-energy ultraviolet light, which carries the risk of damaging biomolecules, thus limiting their application in photoelectrochemistry. In contrast, semi-inductors with narrow bandgap exhibit strong responses to visible light, attracting considerable attention. However, the photocatalytic performance of semiconductors is affected by the rapid recombination of photoexcited electron-hole pairs. To address this limitation, heterojunctions are constructed by coupling with other suitable semiconductors to improve photocatalytic performance. Notably, most PEC detection strategies, especially those based on DNA, rely on a “turn-off” output, making them highly susceptible to background interference. Summary of the Invention

[0005] The purpose of this invention is to provide a detection method for visible light-driven photoelectrochemical analysis triggered by CRISPR / Cas12a, in order to solve the problems existing in the prior art. This invention utilizes the excellent photoactivity of the CRISPR / Cas12a cleavage and PEC signaling pathways to detect target analytes, thereby improving the detection sensitivity for pathogenic bacteria, especially drug-resistant bacteria associated with single nucleotide polymorphisms.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a detection method for CRISPR / Cas12a-triggered visible light-driven photoelectrochemical analysis, comprising the following steps: Step 1: Synthesis of BiVO4, MoS2, BiVO4 / MoS2 and DNA-SiO2: BiVO4, MoS2 and BiVO4 / MoS2 were synthesized by hydrothermal method, and thiolated DNA-SiO2 suitable for anchoring on AuNPs was synthesized. Step 2, Fabrication of PEC sensor: BiVO4 / MoS2 is coated on the surface of screen-printed carbon electrode (SPCE), dried to form a thin film, and then a layer of AuNPs is deposited. DNA-SiO2 is then anchored through Au-S bonds. Step 3, Cutting: After CRISPR / Cas12a and crRNA are incubated to form a complex, Target-DNA is added, mixed well, and then dropped onto the surface of the PEC sensor. The activated CRISPR / Cas12a will cut the DNA-SiO2 on the electrode surface. Step 4: Electrochemical testing: Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and photocurrent testing (IT) are performed on the PEC sensor using an electrochemical workstation to obtain the corresponding electrochemical signals.

[0007] Further, in step one, the method for synthesizing BiVO4 includes: taking Bi(NO3)3·5H2O NH4VO3 was added to a mixture of ethylene glycol and deionized water. After complete dissolution by ultrasonic treatment, the mixture was transferred to a 100 mL high-pressure reactor lined with polytetrafluoroethylene and reacted at 120 ~ 200 ℃ for 4 ~ 8 h. The mixture was then washed with ethanol and water and dried.

[0008] Further, in step one, the method for synthesizing MoS2 includes: transferring a mixed solution of Na2MoO4 (5~10 µM) and CH4N2S (20~40 µM) into a 100 mL polytetrafluoroethylene-lined autoclave, reacting at 200~240 °C for 10~14 h, washing with ethanol and water, and drying.

[0009] Further, in step one, the method for synthesizing BiVO4 / MoS2 includes: washing and drying the prepared BiVO4 and MoS2 to obtain solid powder, combining them in deionized water at a mass ratio of 2:1, dissolving them by sonication, transferring them to a 100 mL polytetrafluoroethylene-lined autoclave, reacting them at 200-240 °C for 10-14 h, washing them with ethanol and water, and drying them.

[0010] Further, in step one, the method for synthesizing DNA-SiO2 includes: mixing SiO2 with a particle size of 30 nm with APTES and ethanol, stirring overnight, and washing. The resulting precipitate is dispersed in deionized water to obtain NH2-SiO2 (20~50 µM). The target DNA (20~50 µM), NH2-SiO2 (20~50 µM), and glutaraldehyde solution are mixed and reacted at 37 °C for 1~2 h to obtain DNA-SiO2. Then, TCEP (25~50 mM) is added to the mixture, and reduction is carried out at 37 °C for 30~60 min to obtain thiolized DNA-SiO2 suitable for anchoring on an Au deposition electrode.

[0011] Further, in step two, the method of coating BiVO4 / MoS2 includes: taking a BiVO4 / MoS2 solution (2.5~5 mg / mL, dissolved in Nafion membrane solution) and coating it onto the surface of a screen-printed carbon electrode (SPCE), and drying it to form a thin film.

[0012] Further, in step two, the method for depositing AuNPs includes: immersing the modified electrode in a 1% HAuCl4 solution and allowing it to act at -0.2 to -0.6 V for 4 to 10 minutes.

[0013] Further, in step two, the method for immobilizing DNA-SiO2 includes: incubating the electrode with thiolized DNA-SiO2 (2.5~5 µM) overnight at room temperature to immobilize DNA-SiO2 through Au-S bonds.

[0014] Further, in step three, the method for detecting drug-resistant pathogens includes: mixing buffer (1×~10×), CRISPR / Cas12a (50 nM~1 µM), crRNA (50 nM~1 µM), and H2O, and incubating at 37 °C for 10~20 min. Then, adding different concentrations of target-DNA extracted from drug-resistant Salmonella, mixing thoroughly, and dropping onto the electrode surface. Reacting at room temperature for 30~45 min, then rinsing with ultrapure water and drying before testing.

[0015] Furthermore, in step four, according to the method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing as described in claim 1, the CV detection in step three is performed in 100-200 µL of PBS (pH 7.0, 0.1 M), containing 5-10 mM [Fe(CN)6]. 3- / 4- The scan range was -0.5 to 0.8 V, and the scan rate was 100 mV / s; EIS tests were performed in 100–200 µL of the same PBS as CV, with a frequency range of 10. -1 ~10 5 The Hz, AC voltage amplitude (Eac) was 5 mV, and DC bias voltage (Edc) was 0.10 V; IT tests were performed in 100–200 µL of PBS (pH 7.0, 0.1M) containing 5–10 µL of H2O2.

[0016] The construction of heterojunctions enhances the separation of photogenerated carriers, thereby improving the sensitivity of photoelectrochemical detection. CV and EIS detection revealed that using a BiVO4 / MoS2 heterojunction resulted in a decrease in the peak redox current and an increase in the interfacial charge transfer resistance R2, indicating that heterojunction modification hinders electron transfer, thus reducing the conductivity of the SPCE. Modification of the Au counter electrode by electrochemical deposition significantly increased the peak current in CV, while significantly decreasing the R2 value. This is likely due to the high conductivity of Au. Subsequently, anchoring DNA-SiO2 via Au-S bonds led to a significant decrease in the peak current and a significant increase in R2. Upon detection of the target DNA, activated CRISPR / Cas12a cleaved the DNA-SiO2, resulting in an increase in the peak current. This invention utilizes the excellent photoactivity of CRISPR cleavage and the PEC signaling pathway to improve the detection sensitivity of drug-resistant and drug-sensitive pathogens, eliminating the need for nucleic acid amplification and enabling the detection of drug-resistant pathogens as low as 103 CFU / mL. The specificity of the method for detecting the target nucleic acid in this invention mainly relies on the specific recognition by CRISPR / Cas12a. We used the method of this invention to specifically detect other interfering nucleic acids that differ from the target DNA by only one or two bases, and synthesized nucleic acid sequences. Figure 4 The results show that these two mutations are key features for distinguishing between drug-resistant and drug-sensitive Salmonella, demonstrating that the method of this invention has high sensitivity for detecting both drug-resistant and drug-sensitive pathogens.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects: (1) The method of the present invention also provides the above-mentioned application of turn-on type visible light photoelectrochemical sensing in the detection of drug-resistant pathogens. The special electrode provided by the present invention is used to realize the detection of drug-resistant pathogens. The PEC sensor can detect low concentrations of drug-resistant pathogens, and has the advantages of high sensitivity, high specificity and simple operation.

[0018] (2) The BiVO4 / MoS2 heterojunction coating used in the method of the present invention greatly reduces the probability of electron-hole pair recombination and enhances light absorption in the ultraviolet-vis region, which can significantly improve photocatalytic performance.

[0019] (3) The photoelectrochemical analysis technology used in the method of the present invention has the advantages of simple operation, low cost and high sensitivity;

[0020] (4) The specific recognition of CRISPR / Cas12a used in the method of the present invention can distinguish single nucleotide variations and has extremely high sensitivity to pathogens, especially drug-resistant pathogens. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the detection method of the present invention; Figure 2 Here are the CV electrochemical characterization diagrams; Figure 3 This is a standard curve for detecting drug-resistant bacteria using the method of the present invention; Figure 4 The method of this invention is used to detect the PEC response of different interfering DNA sequences; Figure 5 Photocurrent responses corresponding to different proportions of drug-resistant bacteria; Figure 6 Results for ARMS-qPCR detection of different proportions of drug-resistant bacteria. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The schematic diagram of the detection process of this invention is as follows: Figure 1 As shown, CRISPR / Cas12a-mediated visible light-driven photoelectrochemical analysis utilizes a BiVO4 / MoS2 heterojunction as a photosensitizer and deposits a layer of AuNPs, enhancing its photocurrent signal and endowing the PEC sensor with high sensitivity. The working principle of CRISPR / Cas12a-mediated visible light-driven photoelectrochemical analysis mainly consists of the following parts: Target-DNA extracted from pathogenic bacteria activates CRISPR / Cas12a, cleaving the single-stranded DNA at the DNA-SiO2 end, causing the SiO2, which acts as a photoelectric signal inhibitor, to detach, resulting in a turn-on photoelectric signal response. Specifically, when the Cas12a crRNA binds to the complementary sequence of the target DNA, an RNA-DNA hybrid strand is formed. This process activates the nuclease domain of CRISPR / Cas12a. CRISPR / Cas12a uses its nuclease domain to cleave the target DNA near the PAM, which is the source of the PEC sensor's specificity.

[0029] In the following examples, bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), ammonium metavanadate (NH4VO3), ethylene glycol, sodium molybdate (Na2MoO4), thiourea (CH4N2S), and 30 nm silica were obtained from Aladdin Reagents Co., Ltd. (Shanghai, China). Glutaraldehyde (C5H8O2), (3-aminopropyl)triethoxysilane (APTES), Tris(2-carboxyethyl) phosphate (TCEP), gold(III) chloride trihydrate (HAuCl4·3H2O), potassium ferricyanide (K3[Fe(CN)6]), and potassium ferrocyanide (K4[Fe(CN)6]) were purchased from Adamas-beta (Shanghai, China). 0.1 M phosphate buffer solution (PBS, pH 7.0) was prepared using 0.1 M sodium hydroxide, 0.1 M potassium dihydrogen phosphate, 0.1 M potassium chloride, and 0.1 M disodium hydrogen phosphate, all purchased from Chengdu Kelon Chemical Co., Ltd. Membrane solutions were prepared using 350 µL of deionized water, 600 µL of isopropanol ((CH3)2CHOH), and 50 µL of Nafion perfluorinated resin solution, purchased from Shanghai McLean Reagent Co., Ltd. Water was purchased from Corning (New York). All oligonucleotide sequences used in this study were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The bacterial total DNA isolation kit (#DP302-97 02) was purchased from TIANGEN (Beijing, China). CRISPR / Cas12a expression followed our previous laboratory methods. The drug-sensitive Salmonella strain (ATCC14028) was obtained from the China Industrial Culture Collection (CICC), while the drug-resistant Salmonella strain was isolated from a livestock slaughterhouse in Chengdu. The drug-resistant Salmonella used in this study had prevalent point mutations in the gyrA gene, occurring at positions 83 (Ser83Phe) and 87 (Asp87Gly).

[0030] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0031] Example 1 This example illustrates primer and probe design, and the steps are as follows: Bacterial resistance to fluoroquinolones is primarily achieved through interference with genomic DNA metabolic processes, a phenomenon often closely linked to mutations in bacterial topoisomerase genes. Specifically, variations in genes such as gyrA, gyrB, parC, and parE are key factors leading to resistance. In this study, we focused specifically on Salmonella resistance, finding that it is primarily driven by mutations in the gyrA gene caused by single nucleotide polymorphisms (SNPs). To effectively identify and detect this resistance, we carefully designed a specific crRNA based on the selected CRISPR / Cas12a system. This crRNA specifically activates CRISPR / Cas12a, thereby endowing it with the ability to recognize and bind to drug-resistant Salmonella. This strategy not only allows for precise detection of drug-resistant strains but also provides a powerful tool for further research and clinical diagnosis.

[0032] Example 2 This embodiment illustrates the step-by-step construction of a PEC sensor, and the steps are as follows: 970 mg Bi(NO3)3·5H2O and 234 mg NH4VO3 were added to a mixture of 40 mL ethylene glycol and 20 mL deionized water. After sonication to ensure complete dissolution, the solution was transferred to a 100 mL Teflon-lined autoclave and reacted at 160 °C for 6 h to obtain BiVO4.

[0033] A mixed solution of 12 mL Na2MoO4 (5 µM) and 48 mL CH4N2S (20 µM) was transferred to a 100 mL polytetrafluoroethylene-lined autoclave and reacted at 220 °C for 12 h to obtain MoS2.

[0034] The prepared BiVO4 and MoS2 were washed four times with ethanol and water, and then dried at 60 °C for 6 h to obtain a solid powder. The BiVO4 and MoS2 monomers were combined in 60 mL of deionized water at a mass ratio of 2:1, dissolved by ultrasonication, and transferred to a 100 mL polytetrafluoroethylene-lined autoclave, where they were reacted at 220 °C for 12 h. The resulting product was washed four times with ethanol and water, and then dried at 60 °C for 6 h to obtain a BiVO4 / MoS2 p / n heterojunction.

[0035] 30 µL of BiVO4 / MoS2 solution (2.5 mg / mL, dissolved in Nafion membrane solution) was coated onto the surface of a screen-printed carbon electrode (SPCE) and dried to form a thin film. The modified electrode was then immersed in a 1% HAuCl4 solution and incubated at -0.2 V for 6 min to deposit a layer of AuNPs. Subsequently, the electrode was incubated overnight at 4 °C with 50 µL of thiolized DNA-SiO2 (2.5 µM) to immobilize the DNA-SiO2 via Au-S bonds.

[0036] 12 mg of 30 nm diameter SiO2 was mixed with 500 µL of APTES and 10 mL of ethanol and stirred overnight. The mixture was washed four times with ethanol and water. The resulting precipitate was dispersed in 10 mL of deionized water to obtain NH2-SiO2 (20 µM). A solution containing 50 µL of Target-DNA (20 µM), 50 µL of NH2-SiO2 (20 µM), and 10 µL of glutaraldehyde was mixed and reacted at 37 °C for 1 h to obtain DNA-SiO2. 10 µL of TCEP (25 mM) was added to the mixture and reduced at 37 °C for 45 min to obtain thiolated DNA-SiO2 suitable for anchoring on an Au deposition electrode. After each modification step, the electrode was rinsed with ultrapure water to remove any unbound species.

[0037] Example 3 This embodiment is used to verify the stepwise construction and detection standard curve of the PEC sensor. The steps are as follows: In the Salmonella detection, 3 µL of buffer (10×), 3 µL of CRISPR / Cas12a (1 µM), 3 µL of crRNA (1 µM), and 18 µL of water were mixed and incubated at 37 °C for 10 min. Then, the mixture was thoroughly mixed with 3 µL of Target-DNA extracted from Salmonella and dropped onto the electrode surface. The reaction was allowed to proceed at room temperature for 30 min, followed by rinsing and drying with ultrapure water.

[0038] (1) CV test Prepare 0.1 M phosphate buffer solutions (PBS, pH 7.0) using 0.1 M NaOH, 0.1 M KH₂PO₄, 0.1 M KCl, and 0.1 M Na₂HPO₄. Then, add 5 mM [Fe(CN)₆] to 200 µl of PBS (pH 7.0, 0.1 M). 3- / 4- CV tests were performed at a scan rate of 100 mV / s between -0.5 and 0.8V.

[0039] (2) Principle verification Using SPCE as the electrode, repeat step (1) and plot its CV diagram.

[0040] Using BiVO4 / MOS2 / SPCE as electrodes, repeat step (1) and plot its CV diagram.

[0041] Using dep Au / BiVO4 / MOS2 / SPCE as electrodes, repeat step (1) and plot its CV diagram.

[0042] Using DNA-SiO2 / dep Au / BiVO4 / MOS2 / SPCE as electrodes, repeat step (1) and plot its CV diagram.

[0043] Using activated Cas12a / DNA-SiO2 / dep Au / BiVO4 / MOS2 / SPCE as electrodes, repeat step (1) and plot its CV diagram.

[0044] The results were obtained Figure 2 It can be seen that the method of the present invention improves the charge transfer capability after coating with BiVO4 / MOS2 heterojunction and gold modification.

[0045] The procedure was repeated using different concentrations of drug-sensitive Salmonella, and the photocurrent was measured.

[0046] The results were obtained Figure 3 It can be seen that the method of the present invention has a detection limit of 10³ CFU / mL for detecting SNP-related drug-resistant Salmonella, and has excellent sensitivity.

[0047] Example 4 This embodiment illustrates the specificity of the PEC sensor, and the steps are as follows: The specificity of this experimental method for detecting target nucleic acids relies primarily on the specific recognition of CRISPR / Cas12a. We used this method to detect other interfering nucleic acids that differ from the target DNA by only one or two bases. The signal response to interfering nucleic acids, particularly those with single-base mutations in the PAM region, differed only slightly from those without. Single mutations in non-PAM regions could also be identified because they produced a significant signal response.

[0048] The results were obtained Figure 4 The method of this invention has single nucleotide resolution and can accurately distinguish between drug-resistant Salmonella and drug-sensitive Salmonella associated with SNPs.

[0049] Example 5 This embodiment illustrates the ability of the PEC sensor to detect the proportion of drug-resistant bacteria and compares its performance with the ARMS-qPCR detection method. The steps are as follows:

[0050] (1) A series of mixed suspensions of drug-resistant Salmonella (0%, 0.01%, 0.1%, 1%, 10%, 50%, 100%) were prepared, and the experimental steps of Example 2 were repeated to record their photocurrent signals.

[0051] (2) A series of mixed suspensions of drug-resistant Salmonella (0%, 0.01%, 0.1%, 1%, 10%, 50%, 100%) were prepared and qPCR was performed on a QuantStudio3 real-time PCR instrument. The qPCR amplification reaction system was 20 µL, which included 2 µL of 10 µM primer set, 1 µL of 10 ng / µL target DNA template, 10 µL of qPCRmix, and 7 µL of molecular water. The cycling program was set as follows: initial denaturation at 95 °C for 10 min, followed by incubation at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 45 s, for 40 cycles.

[0052] The results were obtained Figure 5 and Figure 6 It can be seen that the identification capability of the method of the present invention is comparable to that of the standard detection method for SNP identification, ARMS-qPCR.

Claims

1. A method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing, characterized in that, Includes the following steps: Step 1: Synthesis of BiVO4, MoS2, BiVO4 / MoS2 and DNA-SiO2: BiVO4, MoS2 and BiVO4 / MoS2 were synthesized by hydrothermal method, and thiolated DNA-SiO2 suitable for anchoring on AuNPs was synthesized. Step 2, Fabrication of PEC sensor: BiVO4 / MoS2 is coated on the surface of screen-printed carbon electrode (SPCE), dried to form a thin film, and then a layer of AuNPs is deposited. DNA-SiO2 is then anchored through Au-S bonds. Step 3, Cutting: After CRISPR / Cas12a and crRNA are incubated to form a complex, Target-DNA is added, mixed well, and then dropped onto the surface of the PEC sensor. The activated CRISPR / Cas12a will cut the DNA-SiO2 on the electrode surface. Step 4: Electrochemical testing: Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and photocurrent testing (IT) are performed on the PEC sensor using an electrochemical workstation to obtain the corresponding electrochemical signals.

2. The method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing according to claim 1, characterized in that, In step one, the method for synthesizing BiVO4 includes: Bi(NO3)3·5H2O and NH4VO3 were added to a mixture of ethylene glycol and deionized water. After being completely dissolved by ultrasonic treatment, the mixture was transferred to a 100 mL high-pressure reactor lined with polytetrafluoroethylene and reacted at 120 ~ 200 ℃ for 4 ~ 8 h. The mixture was then washed with ethanol and water and dried. In step one, the method for synthesizing MoS2 includes: transferring a mixed solution of Na2MoO4 (5~10 µM) and CH4N2S (20~40 µM) into a 100 mL polytetrafluoroethylene-lined autoclave, reacting at 200~240 °C for 10~14 h, washing with ethanol and water and drying; In step one, the method for synthesizing BiVO4 / MoS2 includes: washing and drying the prepared BiVO4 and MoS2 to obtain solid powder, combining them in deionized water at a mass ratio of 2:1, dissolving them by sonication, transferring them to a 100 mL polytetrafluoroethylene-lined autoclave, reacting them at 200-240 °C for 10-14 h, washing them with ethanol and water, and drying them.

3. The method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing according to claim 1, characterized in that, In step one, the method for synthesizing DNA-SiO2 includes: mixing SiO2 with a particle size of 30 nm with APTES and ethanol, stirring overnight, washing, and dispersing the resulting precipitate in deionized water to obtain NH2-SiO2 (20~50 µM); mixing Target-DNA (20~50 µM), NH2-SiO2 (20~50 µM), and glutaraldehyde solution, and reacting at 37 °C for 1~2 h to obtain DNA-SiO2. 2， Then TCEP (25~50 mM) was added to the mixture, and the mixture was reduced at 37 °C for 30~60 min to obtain thiolated DNA-SiO2 suitable for anchoring on the electrode via Au-S bonds.

4. The method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing according to claim 1, characterized in that, In step two, the method of coating BiVO4 / MoS2 includes: taking a BiVO4 / MoS2 solution (2.5~5 mg / mL, dissolved in Nafion membrane solution) and coating it on the SPCE surface, then drying it to form a thin film.

5. The method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing according to claim 1, characterized in that, In step two, the method for depositing AuNPs includes immersing the modified electrode in a 1% HAuCl4 solution and allowing it to react at -0.2 to -0.6 V for 4 to 10 minutes.

6. The method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing according to claim 1, characterized in that, In step two, the method for anchoring DNA-SiO2 includes: incubating the modified electrode with thiolized DNA-SiO2 (2.5~5 µM) overnight at room temperature to anchor DNA-SiO2 through Au-S bonds.

7. The method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing according to claim 1, characterized in that, In step three, for the detection of drug-resistant pathogens, buffer (1×~10×), CRISPR / Cas12a (50 nM~1 µM), crRNA (50 nM~1 µM) and H2O are mixed and incubated at 37 ℃ for 10~20 min. Then, different concentrations of Target-DNA extracted from drug-resistant Salmonella are added, mixed thoroughly, and dropped onto the electrode surface. The mixture is reacted at room temperature for 30~45 min, rinsed with ultrapure water and dried before the experiment.

8. The method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing according to claim 1, characterized in that, In step three, CV detection was performed in 100–200 µL of PBS (pH 7.0, 0.1 M) containing 5–10 mM [Fe(CN)6]. 3- / 4- The scan range was -0.5 to 0.8 V, and the scan rate was 100 mV / s; EIS tests were performed in 100–200 µL of the same PBS as CV, with a frequency range of 10. -1 ~10 5 The frequency was Hz, the AC voltage amplitude (Eac) was 5~10 mV, and the DC bias voltage (Edc) was 0.1~0.2 V; the IT test was performed in 100~200 µL of PBS (pH 7.0, 0.1 M) containing 5~10 µL of H2O2.

9. The method for detecting drug-resistant pathogens based on turn-on visible light photoelectrochemical sensing according to claim 1, characterized in that, The NH-T13-SH sequence of synthesized DNA-SiO2 (5' modified with NH2, 3' modified with HS-SH), crRNA sequence, Target-DNA sequence, single-base mutant sequence TS-G>A, single-base mutant sequence TS-T>C, double-base mutant sequence TS-G>A, T>C, random sequence Random-DNA, ARMS-qPCR front primer arms-F, and ARMS-qPCR back primer arms-Q are shown in SEQ ID NO.1~SEQ ID NO.9 of the sequence listing, respectively.

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