Double-signal detection sugarcane pokkah boeng disease self-powered biosensor and preparation method thereof

Abstract

The invention discloses a self-powered biosensor for double-signal detection of sugarcane pokkah boeng disease, the self-powered biosensor comprises an anode, a cathode and an electrolyte, the anode is a CuCo-MOF / AuNPs / GOD biological anode, the cathode is a CuCo-MOF / AuNPs / DNA chain biosensor, and the electrolyte comprises glucose, MB and a PBS buffer solution. According to the self-powered biosensor, analysis is carried out through two non-interfering modes of electrochemical detection and colorimetric detection, and whether a detection result is a false positive signal or not can be effectively verified. An external power supply is not needed, and real-time outdoor detection can be realized. The material source is wide, the assembly process is simple, and accurate and rapid detection can be realized. The method has the advantages of high sensitivity, good selectivity and real-time detection, and can be used for accurately detecting the sugarcane pokkah boeng rot.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and particularly relates to a dual-signal detection self-powered biosensor for sugarcane pokkah boeng and a preparation method thereof. Background Art

[0002] As a fuel cell-type device of a green electrochemical generator, enzymatic diversity endows biofuel cells (EBFCs) with the ability to adapt to a variety of working environments. The assembly process of EBFCs is simple, with less consumables required and low cost, and it can be developed towards portable and implantable devices. It has been used in fields such as clinical medicine, sewage treatment, and food detection.

[0003] Sugarcane pokkah boeng poses a serious threat to sugarcane crops and is almost present in all sugarcane growing areas globally. In China, sugarcane pokkah boeng has also brought huge losses to sugarcane production, seriously affecting the yield and sucrose content of sugarcane stalks, and posing a severe challenge to the sustainable development of the sugarcane industry and the sugar industry. The diagnosis of sugarcane pokkah boeng is relatively difficult because its symptoms may be similar to those of other diseases or pest damages. This disease mainly infects the young leaves at the tip of sugarcane, and also infects the leaf sheaths and sugarcane stalks. At the initial stage of the disease, chlorotic yellowing appears at the base of the young leaves, the leaves are narrower than normal leaves, and are accompanied by longitudinal reddish-brown stripes. As the disease progresses, the leaves may show wrinkles, twists, or shortening. In severe cases, the heart leaves die, forming a tip rot, and ultimately leading to the death of the entire sugarcane plant. Traditional diagnostic methods, such as observing the typical symptoms of diseased plants, laboratory isolation, culture, and identification of pathogenic bacteria, can help identify sugarcane pokkah boeng, but these methods may require a long time and professional technical knowledge for farmers and agricultural workers. Therefore, timely field management is crucial for controlling the spread of the disease and reducing losses.

[0004] Currently, the PCR technique is a widely used detection method for early diagnosis of the pathogenic bacteria of sugarcane pokkah boeng. However, due to the challenge of designing specific primers, PCR has certain limitations in accurately identifying the pathogenic bacteria of sugarcane pokkah boeng. In addition, PCR detection requires professional laboratory personnel and high-cost equipment, which limits its popularization in practical applications. Especially in the early stage of sugarcane pokkah boeng, the number of pathogenic bacteria is small, and there may be various interfering factors in the samples, which makes traditional detection methods often face problems of insufficient sensitivity and poor selectivity. Therefore, there is an urgent need to develop a new method for sensitive, accurate, and rapid detection of the pathogenic bacteria of sugarcane pokkah boeng.

[0005] The information disclosed in this background art section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of implication that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0006] The object of the present invention is to provide a dual-signal detection self-powered biosensor for sugarcane pokkah boeng and its preparation method, to achieve highly sensitive, accurate and rapid detection of the pathogen of pokkah boeng, and to help accurately identify sugarcane plants at the initial stage of disease onset.

[0007] To achieve the above object, the present invention provides a dual-signal detection self-powered biosensor for sugarcane pokkah boeng, including: the self-powered biosensor includes an anode, a cathode and an electrolyte. The anode is a CuCo-MOF / AuNPs / GOD bioanode, the cathode is a CuCo-MOF / AuNPs / DNA strand biosensor, and the electrolyte includes glucose, MB and PBS buffer solution.

[0008] A preparation method of a dual-signal detection self-powered biosensor for sugarcane pokkah boeng, the self-powered biosensor constructs the anode and cathode of the self-powered biosensor through gold nanoparticles / zinc cobalt metal-organic framework CuCo-MOF / AuNPs, carbon cloth, DNA strand and glucose oxidase.

[0009] Preferably, in the above technical solution, the preparation of the bioanode includes: taking 30-50 μL of CuCo-MOF / AuNPs and dropping it on the surface of the carbon cloth electrode, drying at 35-40 °C for 2-4 h, and then dropping 20-50 μL of glucose oxidase (GOD) with a concentration of 3-5 mg / mL and incubating for 12-24 h. After washing with water, the bioanode CuCo-MOF / AuNPs / GOD is obtained.

[0010] Preferably, in the above technical solution, the preparation of the biocathode includes:

[0011] (1) Drop 30-100 μL of CuCo-MOF / AuNPs on the surface of the carbon cloth electrode. After drying at 35-40 °C for 2-4 h, immerse it in a 5-10 mg / mL EDC / NHS solution for 0.5-1 h. After rinsing, drop 10-30 μL of rail-shaped A1 / Output DNA, place it at 3-6 °C for 10-15 h, add 20-50 μL of MCH with a concentration of 0.5-2 mM and react for 0.5-1 h, and wash to remove the excess MCH;

[0012] (2) Drop 20-50 μL of H2 and H3 on the electrode surface respectively, and incubate at 35-40 °C to obtain the biocathode.

[0013] Preferably, in the above technical solution, the preparation method of the rail-shaped A1 / Output DNA includes:

[0014] 1) Preparation of RCA precursor: Incubate 10 - 30 μL of pathogen - extracted genes with different concentrations and 20 - 40 μL of H1 at 35 - 40 °C for 1 - 2 h, then add 1 - 5 μL of 10 U / μL T4 DNA ligase and incubate at 35 - 40 °C for 1 - 2 h to obtain the RCA precursor;

[0015] 2) Preparation of interconnected hairpin - structured DNA: Add 1 - 5 μL of 10 U / μL phi29 polymerase and 1 - 10 μL of dNTP solution to the solution in step 1), and incubate at 35 - 40 °C for 1 - 2 h;

[0016] 3) Preparation of Output DNA: Add 1 - 5 μL of 10 U / μL Nt.BbvCl exonuclease to the solution in step 2), and incubate at 35 - 40 °C for 0.5 - 1.5 h to obtain the SDA product Output DNA;

[0017] 4) Preparation of planar railing - shaped DNA: Mix 15 - 30 μL of 1 μM A1 and the Output DNA prepared in step 3) and react for 1 - 2 h to obtain planar railing - shaped A1 / Output DNA.

[0018] Preferably, in the above - mentioned technical solution, the preparation of CuCo - MOF / AuNPs includes:

[0019] (1) Preparation of CuCo - MOF: Dissolve Cu(NO3)2·3H2O, Co(NO3)2·6H2O and cetyltrimethylammonium bromide in water, then inject the mixture into an aqueous solution containing 2 - methylimidazole, stir to obtain a CuCo - MOF precipitate, wash, dry and calcine;

[0020] (2) Preparation of AuNPs: Mix and heat the HAuCl4 solution and water, inject sodium citrate under stirring, continue heating after the solution color turns wine - red, stop heating and stir and cool in the dark to obtain AuNPs;

[0021] (3) Preparation of CuCo - MOF / AuNPs: Grind CuCo - MOF into powder, add acetic acid and AuNPs and sonicate to obtain CuCo - MOF / AuNPs.

[0022] An application of a self - powered biosensor for dual - signal detection of sugarcane top rot disease, and the self - powered biosensor is used to detect sugarcane top rot disease pathogens.

[0023] A method for using a self - powered biosensor for dual - signal detection of sugarcane top rot disease to detect sugarcane top rot disease pathogens, and the method includes the following steps:

[0024] (1) Measure the E value of the sensor and the Blue value in RGB when the target sugarcane top rot pathogen is not introduced. OCV Value and the Blue value in RGB;

[0025] (2) Measure the open circuit voltage E value of the sensor and the Blue value in RGB when measuring sugarcane top rot pathogens at different concentrations. OCV Value and the Blue value in RGB;

[0026] (3) Plot the standard curve between the E value and the concentration of specific genes of the sugarcane top rot pathogen, and the standard curve between the Blue value in RGB and the concentration of the sugarcane top rot pathogen to complete the determination of the sugarcane top rot pathogen. OCV Value and the concentration of the sugarcane top rot pathogen to complete the determination of the sugarcane top rot pathogen;

[0027] Among them, the above-built membrane-free glucose / MB sensor is a 0.01M PBS buffer system containing 5 mM glucose, 5 mM MB, and pH 7.4.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The self-powered biosensor prepared by the method of the present invention constructs an interconnected hairpin structure through rolling circle amplification (RCA) of nucleic acids, and cooperates with the synergistic action of Nt.BbvCl exonuclease and Phi29 polymerase to achieve strand displacement reaction (SDA), greatly improving the detection sensitivity. The Output DNA generated by the RCA reaction binds to probe A1 to form a stable A1 / OutputDNA structure, and its planar railing-like feet bind to gold nanoparticles, enhancing the binding stability with the biocathode. In order to improve the electron transfer rate, CuCo-MOF is used as the substrate material of the sensor, and the H2O2 generated by its decomposition of glucose is used to enhance the activity of glucose oxidase on the anode. When the sugarcane top rot pathogen is present, the target binds to the hairpin structure, initiating the RCA and SDA reactions to produce A1 / Output DNA, adsorbing a large amount of MB on the biocathode, and generating a significant electrical signal. The obtained open circuit voltage is positively correlated with the pathogen concentration. In addition, in this process, MB is reduced to colorless MBH, and the Blue value in RGB is obtained by taking a mobile phone photo for colorimetric detection. The present invention has the advantages of high sensitivity, good selectivity, and real-time detection, and can be used for the accurate detection of sugarcane top rot.

[0030] (2) The present invention uses nucleic acid rolling circle amplification (RCA) reaction to drive strand displacement reaction (SDA) for the purpose of rapid amplification. Based on the planar rail-like A1 / Output DNA connected with multiple AuNPs, the stability of the device is enhanced. And based on the planar rail-like A1 / Output DNA, HCR reaction is carried out, which can adsorb more MB, increasing the electrical response signal and colorimetric signal of the sensor. Through electrochemical and colorimetric detection, two non-interfering modes are used for analysis, which can effectively verify whether the detection result is a false positive signal.

[0031] (3) When the method of the present invention is used for detecting sugarcane top rot disease, the present invention does not require an external power supply and can realize real-time outdoor detection. Using CuCo-MOF nanomaterials as the electrode substrate accelerates the electron conduction rate. Due to the addition of MOF materials, the ability to promote the decomposition of H2O2 to generate reactive oxygen species is enhanced, and the activity of glucose oxidase (GOD) is improved, so that the stability and output intensity of the detection signal are both enhanced. Using the method of the present invention to detect the pathogen of sugarcane top rot disease does not require professional operation techniques, the materials are widely sourced, the assembly process is simple, and accurate and rapid detection can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic diagram of the assembly process of the anode and cathode of the self-powered biosensor for dual-signal detection of sugarcane top rot disease according to the present invention;

[0033] Figure 2 is the CV (A) of the cathode with or without the target in the self-powered biosensor for dual-signal detection of sugarcane top rot disease according to the present invention, the CV diagram (B) of the cathode electrode assembly, and the photos of the solution system with or without the target in the colorimetric mode (C);

[0034] Figure 3 is the standard curve (A) of the open circuit voltage of the self-powered biosensor for dual-signal detection of sugarcane top rot disease and the concentration of the specific gene of the top rot pathogen, and the standard curve (B) of the Blue value in RGB in the colorimetric mode and the concentration of the specific gene of the top rot pathogen. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The following will describe in detail the specific embodiments of the present invention with reference to the drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0036] Unless otherwise clearly stated, throughout the specification and claims, the term "comprising" or its variations such as "comprises" or "including" etc. will be understood to include the stated elements or components, without excluding other elements or other components.

[0037] I. Self-powered biosensor for dual-signal detection of sugarcane top rot disease

[0038] As Figure 1 shown, a self-powered biosensor for dual-signal detection of sugarcane top rot disease includes: The self-powered biosensor includes an anode, a cathode and an electrolyte. The anode is a CuCo-MOF / AuNPs / GOD biocathode, the cathode is a CuCo-MOF / AuNPs / DNA strand biosensor, and the electrolyte includes glucose, MB, and PBS buffer solution. Specifically, the electrolyte is a 0.01M PBS buffer system containing 5mM glucose, 5mM MB, and pH 7.4.

[0039] The principle of the self-powered biosensor for ultrasensitive detection of top rot pathogen is as Figure 1 shown:

[0040] When the top rot pathogen is 0, there is only H1 on the electrode, and the rolling circle amplification reaction (RCA) cannot be carried out, resulting in the difficulty of the strand displacement (SDA) reaction. Output DNA is difficult to bind to probe A1, making the hybridization chain reaction (HCR) unable to be triggered. Therefore, less MB (methylene blue) is adsorbed on the biocathode, the electrical signal is weak, and the color change of the electrolyte is not obvious. When the top rot pathogen is added to the system, the target starts the RCA reaction, forms a hairpin structure, provides a large number of binding sites for the subsequent SDA, and outputs Output DNA, triggering the HCR on the electrode surface. More MB is adsorbed on the biocathode, the electrical signal is enhanced, and the Blue value is weakened. The open circuit voltage value and the Blue change value are positively correlated with the concentration of the top rot pathogen, thereby realizing the quantitative detection of the top rot pathogen, and the electrochemical and colorimetric systems that do not interfere with each other can mutually verify whether the experimental results are false positives.

[0041] II. Preparation of a self-powered biosensor for dual-signal detection of sugarcane top rot disease

[0042] 1. Preparation of CuCo-MOF / AuNPs

[0043] (1) Preparation of CuCo-MOF: Dissolve 96.6 mg (0.4 mmol) of Cu(NO3)2·3H2O, 174.6 mg (0.6 mmol) of Co(NO3)2·6H2O, and 7.5 mg of cetyltrimethylammonium bromide (CTAB) in 10 mL of ultrapure water. Then, quickly inject it into 70 mL of aqueous solution containing 4.54 g of 2-methylimidazole and stir strongly at room temperature for 1 h. Centrifuge the precipitate (named CuCo-MOF) and wash it with ethanol three times. Finally, dry CuCo-MOF in a vacuum oven overnight and calcine it at 400 °C in air for 2 h.

[0044] (2) Preparation of AuNPs: Mix 500 μL of 1% HAuCl4 solution and 50 mL of ultrapure water, heat in an oil bath to 120 °C, then cool the temperature to 110 °C, and quickly inject 500 μL of 2% sodium citrate all at once under stirring. After the solution turns wine red, continue heating for 10 min. After stopping heating, continue stirring in the dark and cool to room temperature to obtain AuNPs.

[0045] (3) Preparation of CuCo-MOF / AuNPs: Grind CuCo-MOF into fine powder, add 5 mg of CuCo-MOF to 1 mL of acetic acid and 6 mL of AuNPs, and ultrasonicate for 30 min to obtain CuCo-MOF / AuNPs.

[0046] 2. Preparation of the bioanode CuCo-MOF / AuNPs

[0047] Drop 50 μL of CuCo-MOF / AuNPs (1 mg / mL) onto the surface of a carbon cloth electrode (1 cm × 1 cm), vacuum dry at 37 °C for 2 h, then drop 50 μL of glucose oxidase (GOD) (5 mg / mL) onto the electrode surface. After incubating at 4 °C for 12 h, wash with ultrapure water to obtain the bioanode CuCo-MOF / AuNPs / GOD, and store it at 4 °C for standby. The assembly process is as Figure 1 .

[0048] 3. Preparation of the biocathode CuCo-MOF / AuNPs / A1 / Output DNA / MCH / H2 / H3

[0049] (1) Preparation of the hairpin structures of H1, H2, and H3: Anneal H1, H2, and H3 at 95 °C for 5 min to obtain the corresponding hairpin structures.

[0050] (2) Preparation of the RCA precursor: Mix 20 μL of the target with 20 μL of H1 and incubate at 37 °C for 2 h. Subsequently, add 2 μL of T4 ligase, 4 μL of 10X T4 ligase buffer, and 4 μL of ultrapure water, react at 37 °C for 4 h, and then inactivate at 65 °C for 10 min to obtain the RCA precursor.

[0051] (3) Preparation of the interconnected hairpin structure DNA: Add the mixture containing 1 μL of Phi29 DNA polymerase (10 U / μL), 5 μL of dNTP, 2 μL of 10X Phi29 polymerase buffer, and 2 μL of ultrapure water to the solution in (2), and react at 37 °C for 2 h.

[0052] (4) Preparation of Output DNA: Add 1 μL of Nt.BbvCl (10 U / μL), 2 μL of 10X Nt.BbvCl buffer, and 7 μL of ultrapure water to the solution in (3), incubate at 37 °C for 50 min, and then heat inactivate at 80 °C for 20 minutes to obtain Output DNA.

[0053] (5) Preparation of the biocathode: Drop 50 μL of 1 mg / mL CuCo-MOF / AuNPs onto the surface of a 1 cm × 1 cm carbon cloth, dry at 37 °C for 3 h, then add the EDC / NHS solution and react for 30 min, and aspirate the excess liquid; on this basis, add 20 μL of A1 / Output DNA and react at 4 °C for 12 hours to immobilize AuNPs; then add 30 μL of MCH to block the active sites; finally, add H2 and H3 participating in the HCR reaction and incubate for 1.5 h to obtain the biocathode.

[0054] III. Detection of the pathogen of sugarcane top rot using the dual-signal self-powered biosensor of the present invention

[0055] 1. Construction and detection of the dual-signal self-powered top rot pathogen biosensor: When the target top rot pathogen is not introduced, measure the E OCV value and the Blue value in RGB; after introducing the target top rot pathogen, measure the E OCV value and the Blue value in RGB of the system containing different concentrations of the top rot pathogen.

[0056] 2. Plot the standard curve between the E OCV value and the concentration of the specific gene of the top rot pathogen, and the standard curve between the Blue value in RGB and the concentration of the top rot pathogen.

[0057] 3. Electrochemical characterization of the dual-signal self-powered biosensor

[0058] To verify the biocathode reaction activity of the sensor, perform CV testing on the biocathode of the sensor. As Figure 2 shown in A, when no target is introduced to the biocathode (curve b), the peak current of the biocathode is small at this time; when the biocathode binds to the target (curve a), the peak current of the biocathode is much larger than that without the target. The CV test diagram during the biocathode assembly process is as Figure 2As shown in Figure B, curve a is the CV curve of the bare electrode; curve b is the CV curve of the bare electrode modified with CuCo-MOF. Compared with a, the peak current of b increases significantly. When the probe A1 is further immobilized on the electrode, the peak current continues to increase, indicating that the electron probe MB has been adsorbed into the DNA molecular backbone (curve c). Subsequently, the peak current increases to the maximum after adding H2 / H3, indicating that the HCR reaction has been successfully carried out. In summary, it shows the successful assembly of the bio-cathode on the electrode surface. Figure 2 Figure C shows the colorimetric pictures with and without the target in the colorimetric mode. a is the color of the system solution with the target, and b is the color of the system solution without the target. It can be seen that the color with the target is much lighter than that without the target.

[0059] 4. Performance Characterization of the Self-Powered Biosensor

[0060] As Figure 3 shown in Figure A, the open-circuit voltage tests were carried out on specific genes of the Dieback pathogen at different concentrations. When the target is absent, only the capture probe A1 is on the electrode substrate. At this time, since the target does not trigger the RCA reaction, the subsequent SDA reaction is inhibited, resulting in the inability to output Output DNA, making the HCR reaction generated by H2 / H3 unable to be immobilized on the electrode surface, thus reducing the electrons received by the bio-cathode. Therefore, the open-circuit voltage value (E OCV ) of the biosensor is relatively low. As the concentration of the target increases, the number of DNA strands loaded on the electrode also increases, and the EOCV value of the biosensor gradually rises. The EOCV value has a good linear relationship with the concentration of the Dieback pathogen in the range of 0.1 fM - 10000 pM. The linear equation is E OCV = 0.011lgc + 0.333 (R 2 = 0.988), and the detection limit is 10.2 amol / L (S / N = 3).

[0061] In Figure 3 Figure B, after detecting specific genes of the Dieback pathogen at different concentrations, the electrolyte was taken for colorimetric detection. The Blue value in RGB has a good linear relationship with the concentration of the Dieback pathogen in the range of 0.1 fM - 10000 pM. The linear equation is Blue = 11.8lg c + 336 (R 2 = 0.996), and the detection limit is 17.6 amol / L.

[0062] The supporting electrolyte of the constructed dual-signal self-powered biosensor described above is a 0.01 M PBS buffer system containing 5 mM glucose, 5 mM MB, and pH 7.4.

[0063] Target (target pathogen): 5’-GTT GTAAAC TCG GTAATGATC CCT-3’

[0064] H1 sequence: 5'-CGA GTT TACAACGAGCTGA GGC TTGACT TGT GGT CCT CAG CTC GTTAGGGAT CATTAC-3'

[0065] A1 sequence: 5'-SH-GGG GGGAGC CTCACAAGT CA-3'

[0066] H2 sequence: 5'-GTC CTCATGA CAT TGA GGA CCA-3'

[0067] H3 sequence: 5'-ATC TCATGA GGA CGCATG GTC CTCATG-3'

[0068] The foregoing description of the specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. A dual-signal self-powered biosensor for detecting sugarcane tip rot, characterized in that: include: The self-powered biosensor includes an anode, a cathode and an electrolyte, wherein the anode is a CuCo-MOF / AuNPs / GOD bioanode, the cathode is a CuCo-MOF / AuNPs / DNA chain biosensor, and the electrolyte includes glucose, MB, and PBS buffer solution.

2. A method for preparing a dual-signal self-powered biosensor for detecting sugarcane tip rot as claimed in claim 1, characterized in that: The self-powered biosensor is a yin and yang electrode of the self-powered biosensor constructed by gold nanoparticles / copper cobalt metal organic material framework CuCo-MOF / AuNPs, carbon cloth, DNA chain and glucose oxidase.

3. The preparation method according to claim 2, characterized in that: The preparation of the bioanode includes: taking 30-50 μL CuCo-MOF / AuNPs and dropping it on the surface of the carbon cloth electrode, drying it at 35-40° C. for 2-4 hours, then dropping it on 20-50 μL 3-5 mg / mL glucose oxidase (GOD) and incubating it for 12-24 hours, and washing it with water to obtain the bioanode CuCo-MOF / AuNPs / GOD.

4. The preparation method according to claim 2, characterized in that: The preparation of the biocathode comprises: (1) 30-100 μL of CuCo-MOF / AuNPs was dropped onto the surface of the carbon cloth electrode, dried at 35-40°C for 2-4 h, immersed in 5-10 mg / mL EDC / NHS solution for 0.5-1 h, rinsed, and then dropped with 10-30 μL of railing A1 / Output DNA, placed at 3-6°C for 10-15 h, and 20-50 μL of 0.5-2 mM MCH was added for reaction for 0.5-1 h, and the excess MCH was removed by washing; (2) 20-50 μL of H2 and H3 were respectively applied to the electrode surface and incubated at 35-40°C to obtain a biocathode.

5. The preparation method according to claim 4, characterized in that: The preparation method of the railing A1 / Output DNA comprises: 1) Preparation of RCA precursor: 10-30 μL of pathogenic bacteria extract genes of different concentrations were incubated with 20-40 μL of H1 at 35-40°C for 1-2 h, and then 1-5 μL of 10 U / μL T4 DNA ligase was added and incubated at 35-40°C for 1-2 h to obtain RCA precursor; 2) Preparation of interconnected hairpin structure DNA: Add 1-5 μL 10U / μL phi29 polymerase and 1-10 μL dNTP solution to the solution in step 1), and incubate at 35-40°C for 1-2h; 3) Preparation of Output DNA: Add 1-5 μL 10U / μL Nt.BbvCl exonuclease to the solution in step 2), incubate at 35-40°C for 0.5-1.5h to obtain the SDA product Output DNA; 4) Preparation of planar railing DNA: Mix 15-30 μL of 1 μM A1 and the Output DNA prepared in step 3) for 1-2 hours to obtain planar railing A1 / Output DNA.

6. The preparation method according to any one of claims 2 to 5, characterized in that: The preparation of the CuCo-MOF / AuNPs includes: (1) Preparation of CuCo-MOF: Dissolve Cu(NO3)2·3H2O, Co(NO3)2·6H2O and hexadecyltrimethylammonium bromide in water, inject the mixture into an aqueous solution containing 2-methylimidazole, stir to obtain a CuCo-MOF precipitate, wash, dry and calcine; (2) Preparation of AuNPs: Mix HAuCl4 solution and water and heat them, then add sodium citrate while stirring. Continue heating after the solution changes to wine red. Stop heating and cool with stirring in the dark to obtain AuNPs. (3) Preparation of CuCo-MOF / AuNPs: CuCo-MOF was ground into powder, acetic acid and AuNPs were added and ultrasonicated to obtain CuCo-MOF / AuNPs.

7. An application of a dual-signal self-powered biosensor for detecting sugarcane tip rot according to any one of claims 1 to 6, characterized in that: The self-powered biosensor is used to detect sugarcane tip rot pathogens.

8. A method for detecting the pathogenic bacteria of sugarcane tip rot using the dual-signal self-powered biosensor for detecting sugarcane tip rot as claimed in any one of claims 1 to 7, characterized in that: The method comprises the following steps: (1) When the target shoot rot pathogen is not introduced, the E of the sensor is measured. OCV value and the Blue value in RGB; (2) The open circuit voltage E of the sensor when measuring different concentrations of shoot rot pathogens OCV value and the Blue value in RGB; (3) Draw E OCV The standard curve between the values ​​and the specific gene concentration of the shoot rot pathogen, and The standard curve of Blue value in RGB and the concentration of the pathogen of sugarcane tip rot was used to complete the determination of the pathogen of sugarcane tip rot. Among them, the membraneless glucose / MB sensor constructed above contains glucose, MB, and PBS buffer system.

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