Dual-mode self-powered biosensor based on nano-enzyme fuel cell and method for detecting SRBDsrA gene

Abstract

The invention belongs to the field of biosensor preparation methods, and particularly relates to a dual-mode self-powered biosensor based on a nano-enzyme fuel cell and a method for detecting an SRB DsrA gene. A dual-mode self-powered biosensor based on a nano-enzyme fuel cell is characterized in that an anode of the biosensor is AuNPs / Cu-TCPP (Fe) / ITO, and a cathode of the biosensor is DNA nanoring / MCH / hairpin H / AuNPs / Cu-TCPP (Fe) / ITO. A dual-mode self-powered biosensor based on a nano-enzyme fuel cell is constructed, and electrochemical-colorimetric dual-mode quantitative analysis is performed on the SRB DsrA gene through the change of system open circuit potential and the change of electrolyte color (RGB Blue value). According to the dual-mode self-powered biosensor based on the nano-enzyme fuel cell and the method for detecting the SRB DsrA gene, simple, sensitive, specific, rapid and reliable dual-mode detection on the SRB DsrA gene is achieved through the method, and compared with an existing SRB detection method, the dual-mode self-powered biosensor based on the nano-enzyme fuel cell and the method for detecting the SRB DsrA gene have the advantages that the dual-mode self-powered biosensor based on the nano-enzyme fuel

Description

Technical Field

[0001] The present invention belongs to the field of biosensor preparation methods, and specifically relates to a dual-mode self-powered biosensor based on a nanozyme fuel cell and a method for detecting SRB DsrA gene. Background Art

[0002] Marine corrosion issues affect the health, service and safety of marine equipment, marine engineering platforms and facilities, and have received widespread attention. Among them, the corrosion of metal materials caused by the presence or growth metabolism of microorganisms, namely microbiologically induced corrosion (MIC), accounts for 70% to 80% of the damage to marine materials (Little B, Wagner P, Mansfeld F. An overview of microbiologically influenced corrosion [J]. Electrochimica Acta. 1992, 37: 2185-2194.). Sulfate-reducing bacteria (SRB) are the most widely studied and most corrosive microorganisms in MIC. The sensitive detection of SRB plays an important role in the mechanism research of microbial corrosion and early diagnosis of corrosion. Common SRB detection methods mainly include the maximum probable number (MPN) method, polymerase chain reaction, enzyme-linked immunosorbent assay and impedance-based immunoassay, but the above detection methods have the disadvantages of complex instruments, long time consumption and high detection cost, and may lead to poor selectivity or low sensitivity. Therefore, it is necessary to propose a more efficient, sensitive, convenient and economical SRB detection method.

[0003] Enzyme biofuel cell (EBFC) biosensors have the advantages of miniaturization, strong anti-interference ability, fast detection speed and higher sensitivity. However, the enzyme loading level significantly affects the performance of EBFC, and may cause problems such as low output power and poor stability (JL Zhang, YH Wang, K. Huang, KJ Huang, H. Jiang, XM Wang, Enzyme-based biofuel cells for biosensors and in vivo power supply, Nano Energy 84 (2021), 105853.). Nanozymes are a class of nanomaterials with enzyme-like activity, high stability, simple preparation procedures and excellent catalytic performance. Using nanozymes to replace natural enzymes, self-powered biosensors based on nanozyme fuel cells can work normally without relying on external power supplies, and effectively solve the problem of poor stability. Gold nanoparticles (AuNPs) have good conductivity and can fix biomolecules through Au-S bonds. Due to their glucose oxidase-like activity, they can catalyze glucose oxidation in the presence of O2. Cu-TCPP(Fe) can be used as a supporting material for AuNPs, which is beneficial to enhance the sensitivity and stability of biosensors.

[0004] Electrochemical biosensors have unique advantages in detecting bacteria such as SRB, showing high sensitivity and selectivity, rapid response and low cost. However, traditional biosensor methods often only use a single mode of detection, and the accuracy of the results is easily affected. Therefore, it is necessary to provide a dual-mode method for detecting bacteria such as SRB. Summary of the invention

[0005] In view of the necessity of the above-mentioned SRB detection and the limitations of traditional detection methods, the purpose of the present invention is to provide a dual-mode self-powered biosensor based on a nanozyme fuel cell and a method for detecting the SRB DsrA gene, so as to achieve high-sensitivity and high-specificity detection of the SRB DsrA gene.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A dual-mode self-powered biosensor based on a nanozyme fuel cell, characterized in that the biosensor anode is AuNPs / Cu-TCPP(Fe) / ITO, and the biosensor cathode is DNA nanoring / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO.

[0008] A method for preparing a dual-mode self-powered biosensor based on a nanozyme fuel cell: AuNPs / Cu-TCPP(Fe) preparation method, comprising the following steps:

[0009] Step 1: Synthesis of Cu-TCPP(Fe): dissolve Cu(NO3)2·3H2O, TFA and PVP in a mixed solution of anhydrous ethanol and DMF; dissolve TCPP(Fe) in a mixed solution of anhydrous ethanol and DMF, and mix the two mixed solutions; then ultrasonically treat, react at 80°C-100°C for 3h-4h, cool, centrifuge, wash and dry to obtain Cu-TCPP(Fe);

[0010] Anhydrous ethanol and DMF were mixed in a volume ratio of 1:3;

[0011] The mass ratio of Cu(NO3)2·3H2O and TCPP(Fe) is 5-12:4.4-12.

[0012] Step 2: Synthesis of AuNPs: Take HAuCl4 solution and add it into ultrapure water; then boil the solution; then add sodium citrate into the boiling HAuCl4 solution, continue to stir vigorously and boil for 10-30 minutes until the solution turns purple-red; finally cool to room temperature to obtain AuNPs;

[0013] The volume ratio of sodium citrate and HAuCl4 solution is 1-3:0.5-1.5.

[0014] Step 3: Synthesis of AuNPs / Cu-TCPP(Fe): Put the Cu-TCPP(Fe) powder obtained in step 1 and the AuNPs obtained in step 2 into a vial, mix them by ultrasonication and stir them overnight; then centrifuge and dry them to obtain AuNPs / Cu-TCPP(Fe).

[0015] The mass volume ratio of Cu-TCPP(Fe) to AuNPs is 10-20(mg):10:20(ml).

[0016] The assembly steps of the biosensor anode AuNPs / Cu-TCPP(Fe) / ITO are as follows:

[0017] AuNPs / Cu-TCPP(Fe) was dropped onto the ITO electrode and dried in vacuum; EDC / NHS was then added to react at room temperature to activate the electrode, thereby obtaining the biosensor anode AuNPs / Cu-TCPP(Fe) / ITO.

[0018] The method for preparing the DNA nanoring comprises the following steps:

[0019] Step 1: Synthesis of TWJ: Heat the L-DNA chain to 92°C-95°C and keep it for 2min-10min; then cool it to room temperature to obtain a stable TWJ;

[0020] Step 2: Synthesis of DNA nanocircles: TWJ is mixed with target DNA to obtain DNA nanocircles.

[0021] The assembly steps of the biosensor cathode DNA nanoring / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO are as follows:

[0022] AuNPs / Cu-TCPP(Fe) was dropped onto the ITO electrode and vacuum dried; EDC / NHS was then added and reacted at room temperature to activate the electrode; hairpin H was then added and reacted at 2°C-8°C, and unbound hairpin H was washed away with ultrapure water; MCH was then added and reacted at room temperature to block nonspecific binding sites, and washed with ultrapure water; finally, DNA nanorings were added and reacted to obtain the biosensor cathode DNA nanorings / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO.

[0023] A method for detecting the SRB DsrA gene by a dual-mode self-powered biosensor based on a nanozyme fuel cell, constructing the dual-mode self-powered biosensor based on a nanozyme fuel cell, and performing electrochemical-colorimetric dual-mode quantitative analysis of the SRB DsrA gene through changes in the open circuit potential of the system and changes in the color (RGB Blue value) of the electrolyte.

[0024] Step 1: Measure the open circuit potential (E) of the system without introducing the target DsrA gene. OCV ), and the RGBBlue value of the electrolyte;

[0025] Step 2: When the target DsrA gene is introduced, measure the E of the system containing different concentrations of DsrA gene. OCV , and the RGB Blue value of the electrolyte;

[0026] Specifically, the double-electrode system of the electrochemical workstation was used to test the open circuit potential (E OCV ) and the RGB Blue value of the electrolyte, the open circuit potential value (E OCV ) was positively correlated with the DsrA gene concentration, and the electrolyte color (RGB Blue value) was positively correlated with the DsrA gene concentration.

[0027] As the electrolyte, 5 mL of 0.01 M PBS buffer (pH=7.4) containing 5 mM glucose and methylene blue (MB) was used.

[0028] The principle of the dual-mode self-powered biosensor based on nanozyme fuel cell of the present invention for detecting SRB DsrA gene is:

[0029] AuNPs / Cu-TCPP(Fe) with glucose oxidase-like activity is modified on the sensor anode to oxidize glucose, and the reduction of methylene blue (MB) adsorbed on the sensor cathode forms a complete electrode circuit. When the DsrA gene does not exist, the DNA nanoring cannot be formed, and there are fewer double-stranded structures on the electrode. At this time, the methylene blue (MB) adsorbed on the cathode is less, the open circuit potential signal is weak, and the electrolyte color is darker (RGB Blue value is smaller); when the DsrA gene exists, the hairpin H fixed on the cathode is opened by forming a DNA nanoring to form a double-stranded structure. At this time, the methylene blue (MB) adsorbed on the cathode increases, the reduction of methylene blue (MB) increases, the open circuit potential signal is enhanced, and the electrolyte color becomes lighter (RGB Blue value increases). Therefore, the open circuit potential value (E OCV ) is positively correlated with the DsrA gene concentration, and the electrolyte color (RGB Blue value) is positively correlated with the DsrA gene concentration, thereby achieving quantitative detection of the DsrA gene. OCV The DsrA gene content was obtained by the correspondence between the RGB Blue value and the DsrA gene; the DsrA gene content was obtained by the correspondence between the RGB Blue value and the DsrA gene.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a dual-mode self-powered biosensor based on a nanozyme fuel cell and a method for detecting the SRBDsrA gene. The method realizes simple, sensitive, specific, rapid and reliable dual-mode detection of the SRB DsrA gene. Compared with the existing SRB detection method, it has the following advantages:

[0032] (1) Compared with a single-mode biosensor, the dual-mode detection system of the present invention not only possesses the inherent properties of each mode, but can also verify the results of the two modes, correcting errors caused by human operation and external conditions, thereby improving the precision and accuracy of detection;

[0033] (2) The self-powered biosensor of the present invention only requires two electrodes, namely the positive and negative electrodes of the nanozyme fuel cell, to achieve detection, and the device is simple;

[0034] (3) The self-powered biosensor described in the present invention uses AuNPs / Cu-TCPP(Fe) as the anode material, simulates the activity of glucose oxidase, can catalyze the oxidation of glucose, and has good stability and a large specific surface area, which greatly promotes the charge transfer process and effectively enhances the sensitivity, responsiveness and stability of the biosensor;

[0035] (4) The self-powered biosensor described in the present invention uses DNA nanorings for signal amplification, which increases the adsorption of the electron acceptor methylene blue (MB) on the cathode and effectively amplifies the electrical signal;

[0036] (5) The self-powered biosensor of the present invention adopts an open circuit potential (E OCV ) and color change (RGB Blue value) for electrochemical-colorimetric dual-mode detection of DsrA gene, which has the advantages of high selectivity, low cost, simple operation and reliable results;

[0037] (6) The self-powered biosensor described in the present invention does not require an additional power source, and can effectively prevent electroactive substances susceptible to redox from reacting on the electrode surface, thereby improving the anti-interference ability of the sensor and achieving integrated detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A schematic diagram of the detection principle of the dual-mode self-powered biosensor provided in an embodiment of the present invention;

[0039] Figure 2 A schematic diagram of the anode and cathode assembly process of the dual-mode self-powered biosensor provided in an embodiment of the present invention;

[0040] Figure 3 CV (A) and LSV (B) diagrams of the dual-mode self-powered biosensor with or without glucose at the anode provided by an embodiment of the present invention;

[0041] Figure 4 CV (A) and LSV (B) diagrams of the dual-mode self-powered biosensor cathode with or without methylene blue (MB) provided by an embodiment of the present invention;

[0042] Figure 5 CV (A) and LSV (B) diagrams of the presence or absence of target fragments in the cathode of the dual-mode self-powered biosensor provided by an embodiment of the present invention;

[0043] Figure 6 The open circuit potential (E) of the dual-mode self-powered biosensor provided in the embodiment of the present invention is OCV ) and DsrA concentration (A), and the standard curve of RGB Blue value and DsrA concentration (B). DETAILED DESCRIPTION

[0044] The present invention is further described below through specific examples, which are helpful for those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0045] Based on the nanozyme fuel cell, the present invention constructs a dual-mode self-powered biosensor for efficient and sensitive detection of SRB DsrA gene. Figure 1 As shown: AuNPs / Cu-TCPP(Fe) with nanozyme activity is modified at the anode to oxidize glucose, and the reduction of methylene blue (MB) at the cathode forms a complete electrode circuit. When the DsrA gene does not exist, the DNA nanoring cannot be formed, there are fewer double-stranded structures on the electrode, less MB adsorbed at the cathode, the open circuit potential signal is weak and the electrolyte color is darker (the RGB Blue value is smaller); when the DsrA gene exists, the hairpin H fixed on the cathode is opened by forming a DNA nanoring to form a double-stranded structure. At this time, the amount of MB adsorbed at the cathode increases, the MB reduction increases, the open circuit potential signal increases, and the electrolyte color becomes lighter (the RGB Blue value increases). Open circuit potential value (E OCV ) is positively correlated with the DsrA gene concentration, and the electrolyte color (RGB Blue value) is positively correlated with the DsrA gene concentration, thus realizing the quantitative detection of the DsrA gene.

[0046] Example 1

[0047] Construction of a dual-mode self-powered biosensor based on nanozyme fuel cells: the biosensor anode is AuNPs / Cu-TCPP(Fe) / ITO, and the biosensor cathode is DNA nanoring / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO;

[0048] The preparation methods of the anode AuNPs / Cu-TCPP(Fe) / ITO and the cathode DNA nanoring / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO are as follows:

[0049] (1) Synthesis of Cu-TCPP(Fe): 7.2 mg Cu(NO3)2·3H2O, 20 μL TFA, and 20 mg PVP were dissolved in a mixed solution of 24 mL anhydrous ethanol and DMF (volume ratio = 1:3); then 8 mg TCPP(Fe) was dissolved in a mixed solution of 8 mL anhydrous ethanol and DMF (volume ratio = 1:3) and added to the mixed solution under vigorous stirring; then ultrasonic treatment was carried out for 15 min, the mixture was reacted at 80°C for 4 h, and after cooling, it was centrifuged, washed, and dried.

[0050] (2) Synthesis of AuNPs: Take 500 μL of 1% (m / V) HAuCl4 solution and add it to 47.5 mL of ultrapure water; then boil the solution in an oil bath for 20 min; then add 1 mL of 1% (m / V) sodium citrate to the boiling HAuCl4 solution, and when the color of the solution turns purple-red, continue stirring and heating for 10 min; finally cool to room temperature and store at 4°C away from light.

[0051] (3) Synthesis of AuNPs / Cu-TCPP(Fe): 10 mg of Cu-TCPP(Fe) powder and 10 mL of AuNPs were placed in a vial, ultrasonically mixed for 30 min and stirred overnight; then centrifuged and dried to obtain the AuNPs / Cu-TCPP(Fe) composite material, which was stored at 4°C in the dark.

[0052] (4) Synthesis of TWJ: 10 μL of 2 μM L-DNA chain was heated to 95°C and maintained for 10 min; then cooled to room temperature for 1 h to obtain a stable TWJ.

[0053] (5) Synthesis of DNA nanocircles: 10 μL of TWJ was mixed with 10 μL of target DNA and reacted at 37°C for 2 h to obtain DNA nanocircles.

[0054] (6) Preparation of modified anode AuNPs / Cu-TCPP(Fe) / ITO: 20 μL 1 mg / mL AuNPs / Cu-TCPP(Fe) was added to the ITO electrode and dried under vacuum at 37°C for 2 h. Then 15 μL 1 mg / mL EDC / NHS was added and reacted at room temperature for 1 h to activate the electrode. The electrode was stored at 4°C. The assembly process is as follows: Figure 2 .

[0055] (7) Preparation of modified cathode DNA nanoring / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO: 20 μL 1 mg / mL AuNPs / Cu-TCPP(Fe) was added to the ITO electrode and vacuum dried at 37°C for 2 h; then 15 μL 1 mg / mL LEDC / NHS was added and reacted at room temperature for 1 h to activate the electrode; then 20 μL 2 μM hairpin H was added and reacted at 4°C for 12 h, and unbound hairpin H was washed away with ultrapure water; then 15 μL 1 M MCH was added and reacted at room temperature for 1 h to block nonspecific binding sites and washed with ultrapure water; finally 20 μL DNA nanoring was added and reacted at 37°C for 2 h and stored at 4°C. The assembly process is as follows: Figure 2 .

[0056] Electrochemical characterization of a dual-mode self-powered biosensor based on a nanozyme fuel cell

[0057] To verify the reactivity of the biosensor anode, CV and LSV tests were performed on the anode. Figure 3 As shown in A, in the electrolyte containing glucose (curve a), an obvious redox peak is observed, and the peak current is large, indicating that the material exerts nanoenzyme activity and utilizes the glucose in the electrolyte. In the electrolyte without glucose (curve b), the peak current of the redox peak is small. LSV( Figure 3The test results of B) are consistent with the CV, indicating the successful assembly of the anode.

[0058] In order to verify the reaction activity of the biosensor cathode, CV and LSV tests were performed on the cathode. Figure 4 As shown in Figure A, in the electrolyte containing MB (curve a), an obvious redox peak appears in the curve, while no redox peak is observed in the electrolyte without MB (curve b), which indicates that MB is adsorbed on the cathode, resulting in the appearance of redox signals. Figure 5 As shown in A, the redox current is larger when the target substance is present (curve a) than when the target substance is absent (curve b). LSV ( Figure 4 The test results of B and 5B) are consistent with the CV, indicating the successful assembly of the cathode.

[0059] Example 2

[0060] Nanozyme fuel cell-based dual-mode self-powered biosensor for detection of SRB DsrA gene:

[0061] In electrochemical mode, when the target DsrA gene is not introduced, the E OCV When the target DsrA gene was introduced, the system E containing different concentrations of DsrA gene was measured. OCV ; In the colorimetric mode, when the target DsrA gene is not introduced, the RGB Blue value of the electrolyte is recorded; when the target DsrA gene is introduced, the RGB Blue value of the electrolyte containing different concentrations of the DsrA gene is recorded;

[0062] As the electrolyte, 5 mL of 0.01 M PBS buffer (pH=7.4) containing 5 mM glucose and methylene blue (MB) was used.

[0063] The standard curves of open circuit potential and DsrA concentration and RGB Blue value and DsrA concentration were drawn to complete the determination of DsrA gene.

[0064] Performance characterization of dual-mode self-powered biosensor based on nanozyme fuel cell

[0065] like Figure 6 A, open circuit potential (E OCV ) test. When the DsrA gene does not exist, the DNA nanoring cannot be formed, and there are fewer double-stranded structures on the electrode. At this time, the cathode adsorbs less MB and receives fewer electrons, and E OCV The signal is weak; as the concentration of the target increases, the number of double-stranded DNA structures on the electrode increases, and E OCV In the range of 1fM to 1nM, the logarithmic scale of the target DsrA gene concentration and E OCVThere is a good linear correlation between them. The linear function is E OCV =0.01707log c+0.31668(R 2 =0.987), and the limit of detection (LOD) was calculated to be 0.33 fM (S / N=3).

[0066] like Figure 6 B, colorimetric test of different concentrations of DsrA gene. When the target is not present, DNA nanorings cannot be formed, and there are fewer double-stranded structures on the electrode. At this time, less MB is adsorbed on the cathode, so less MB is reduced, and the electrolyte color is darker (RGB Blue value is smaller). As the concentration of DsrA gene increases, more MB is adsorbed on the cathode, more MB is reduced, and the electrolyte color becomes lighter (RGB Blue value increases). The RGB Blue value shows a good linear correlation with the logarithmic concentration of the DsrA gene in the range of 1fM-10nM, and the linear regression equation is RGB Blue=14.90194log c+308.22775(R 2 =0.993), and the LOD was calculated to be 0.41 fM (S / N=3).

[0067] The supporting electrolyte of the biosensor prepared above was 5 mL 0.01 M PBS buffer (pH=7.4), which contained 5 mM glucose and methylene blue (MB).

[0068] DsrA fragment sequence: 5'-CGTAAGCTGATCGACCTGTG-3'

[0069] Hairpin H sequence: 5'-AGACTCCCCAACGAGTATTCCTC-3'

[0070] L-DNA sequence: 5'-SH C6-ACT CAC ACTGAG GAA GAC TC TCC ACA GGT CGA TCA GCTTAC CT AGA GGA AGA CTC TCT ATG GTT TTT TCT GCC TAC TTG AGG AAG ACT CTC AAGTGT GAG T-3'.

[0071] Application Examples

[0072] Verify the actual detection capability of the dual-mode self-powered biosensor based on the nanozyme fuel cell, specifically: use the actual cultured SRB bacterial solution and PCR technology to extract and amplify the DsrA gene. Add the SRB strain to the culture medium, culture at 30℃ for 72h, take 1mL of the bacterial solution to extract DNA, and use it as a template for PCR amplification. Place the diluted PCR product in a centrifuge tube, heat at 95℃ for 2min, and then cool it quickly to obtain denatured ssDNA (single-stranded DNA).

[0073] The ssDNA of the DsrA gene at different concentrations was hybridized with TWJ at 37°C to obtain DNA nanorings. The DNA nanorings were modified at the cathode and subjected to E OCV And colorimetric test. At the same time, qPCR test was performed on the actual samples to be tested, and the results of the dual mode method were compared with the qPCR method to verify its reliability. All tests were repeated three times.

[0074] The dual-mode sensor was used to detect the DsrA gene and the obtained E OCV , RGB Blue value, and the concentration of the target DNA was calculated by the standard curve, as shown in Table 1, E OCV The RSD (relative standard deviation) of the mode was 1.45-3.06%, and the recovery rate was 96.3-103.15%; the RSD of the colorimetric mode was 1.29-2.17%, and the recovery rate was 95.38-107.04%.

[0075] As can be seen from Table 1, the results of the dual-mode detection method of the present invention are close to those of the qPCR method, thus proving the reliability of the dual-mode detection method of the present invention. At the same time, the method of the present invention is more compact and has lower cost than the qPCR technology, so the dual-mode detection method of the present invention can be used for quantitative analysis of the DsrA gene in actual samples.

[0076] Table 1 Comparison of dual-mode sensor and qPCR in DsrA gene determination

[0077]

[0078] As can be seen from the above, the present invention aims at the problems existing in the existing detection methods, constructs a dual-mode self-powered biosensor based on a nanozyme fuel cell, and performs electrochemical-colorimetric dual-mode quantitative analysis of the SRB DsrA gene through changes in the open circuit potential of the system and changes in the color (RGB Blue value) of the electrolyte. Compared with a single-mode biosensor, the dual-mode self-powered biosensor of the present invention not only has the inherent properties of each mode, but also can verify the results of the two modes with each other, correct errors caused by human operation and external conditions, thereby improving the precision and accuracy of the detection, and can achieve simple, sensitive, specific, rapid and reliable detection of the DsrA gene.

[0079] The above are only exemplary embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dual-mode self-powered biosensor based on a nanozyme fuel cell, characterized in that: The anode of the biosensor was AuNPs / Cu-TCPP(Fe) / ITO, and the cathode of the biosensor was DNA nanoring / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO.

2. A method for detecting SRBDsrA gene using a dual-mode self-powered biosensor based on a nanozyme fuel cell according to claim 1, characterized in that: Construct the dual-mode self-powered biosensor based on the nanozyme fuel cell as described in claim 1, and perform electrochemical-colorimetric dual-mode quantitative analysis of the SRB DsrA gene through changes in the open circuit potential of the system and changes in the color of the electrolyte (RGB Blue value).

3. The method according to claim 2, characterized in that: The double-electrode system of the electrochemical workstation was used to test the open circuit potential (E OCV ) and the RGB Blue value of the electrolyte, the open circuit potential value (E OCV ) was positively correlated with the DsrA gene concentration, and the electrolyte color (RGB Blue value) was positively correlated with the DsrA gene concentration.

4. The method according to claim 2 or 3, characterized in that: As the electrolyte, 5 mL of 0.01 M PBS buffer (pH=7.4) containing 5 mM glucose and methylene blue (MB) was used.

5. A method for preparing a dual-mode self-powered biosensor based on a nanozyme fuel cell according to claim 1, characterized in that: The preparation method of AuNPs / Cu-TCPP(Fe) comprises the following steps: Step 1: Synthesis of Cu-TCPP(Fe): dissolve Cu(NO3)2·3H2O, TFA and PVP in a mixed solution of anhydrous ethanol and DMF; dissolve TCPP(Fe) in a mixed solution of anhydrous ethanol and DMF, and mix the two mixed solutions; then ultrasonically treat, react at 80°C-100°C for 3h-4h, cool, centrifuge, wash and dry to obtain Cu-TCPP(Fe); Step 2: Synthesis of AuNPs: Take HAuCl4 solution and add it into ultrapure water; then boil the solution; then add sodium citrate into the boiling HAuCl4 solution, continue to stir vigorously and boil for 10-30 minutes until the solution turns purple-red; finally cool to room temperature to obtain AuNPs; Step 3: Synthesis of AuNPs / Cu-TCPP(Fe): The Cu-TCPP(Fe) powder obtained in step 1 and the AuNPs obtained in step 2 were ultrasonically mixed and stirred overnight; then centrifuged and dried to obtain AuNPs / Cu-TCPP(Fe).

6. The preparation method according to claim 5, characterized in that: The steps of assembling the biosensor anode are as follows: AuNPs / Cu-TCPP(Fe) was dropped onto the ITO electrode and dried in vacuum; EDC / NHS was then added to react at room temperature to activate the electrode, thereby obtaining the biosensor anode AuNPs / Cu-TCPP(Fe) / ITO.

7. The preparation method according to claim 5, characterized in that: The method for preparing the DNA nanoring comprises the following steps: Step 1: Synthesis of TWJ: Heat the L-DNA chain to 92°C-95°C and keep it for 2min-10min; then cool it to room temperature to obtain a stable TWJ; Step 2: Synthesis of DNA nanocircles: TWJ is mixed with target DNA to obtain DNA nanocircles.

8. The preparation method according to claim 5, characterized in that: The assembly steps of the biosensor cathode DNA nanoring / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO are as follows: AuNPs / Cu-TCPP(Fe) was dropped onto the ITO electrode and vacuum dried; EDC / NHS was then added and reacted at room temperature to activate the electrode; hairpin H was then added and reacted at 2°C-8°C, and unbound hairpin H was washed away with ultrapure water; MCH was then added and reacted at room temperature to block nonspecific binding sites, and washed with ultrapure water; finally, DNA nanorings were added and reacted to obtain the biosensor cathode DNA nanorings / MCH / hairpin H / AuNPs / Cu-TCPP(Fe) / ITO.

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