Enzyme biological fuel cell system for detecting sugarcane pokkah boeng disease through three signals and preparation method and application of enzyme biological fuel cell system

Abstract

The invention belongs to the technical field of biological fuel cells, and particularly relates to an enzyme biological fuel cell system for detecting sugarcane pokkah boeng disease through three signals as well as a preparation method and application of the enzyme biological fuel cell system. The enzyme biological fuel cell system comprises an enzyme biological fuel cell and a photothermal conversion solution TiO2 (at) Au, the enzyme biological fuel cell comprises a biological anode, a biological cathode and an electrolyte, the biological anode is MWNT (at) ZIF-8 / AuNPs / GOD, and the biological cathode is an MWNT (at) ZIF-8 / AuNPs / DNA chain. According to the invention, false positive signals of detection results can be effectively calibrated; through combined use of three detection modes, high sensitivity, selectivity and real-time monitoring capability can be realized, and the method can be used for accurate detection of the sugarcane pokkah boeng rot.

Description

Technical Field

[0001] This invention relates to the field of biofuel cell technology, specifically to an enzyme biofuel cell system for detecting sugarcane top rot using three signals, its preparation method, and its application. Background Technology

[0002] Enzyme-catalyzed biofuel cells are gaining increasing attention in the energy conversion field due to their wide availability of fuel sources, small size, portability, and ease of assembly. The core working principle of this technology is that enzyme catalysis catalyzes the oxidation reaction between fuel and chemicals in the electrolyte at the anode, releasing electrons. These electrons flow through an external circuit to the cathode, where they undergo a reduction reaction with electron acceptors, ultimately forming a closed circuit and continuously generating electricity. Unlike traditional fuel cells, enzyme-catalyzed biofuel cells utilize biological enzymes as catalysts, offering greater flexibility in fuel selection. Enzyme-catalyzed biofuel cells can utilize organic compounds such as glucose and lactic acid as fuel. The linear relationship between the current output of a biofuel cell and the concentration change of the analyte makes it a promising candidate for quantitative detection of target analytes. Furthermore, enzyme-catalyzed biofuel cells can also serve as a highly efficient sensing tool, demonstrating unique advantages in chemical analysis, environmental monitoring, and medical testing.

[0003] Sugarcane, as a strategic agricultural product in my country, occupies an important position in sugar supply and energy conversion. Its derivatives play an irreplaceable role in the sugar industry, clean energy production, and rural heating. However, the rampant spread of sugarcane top rot poses a severe challenge to the stability of the sugarcane industry. This disease is highly insidious in its early stages, and its symptoms are easily confused with other physiological disorders, making early diagnosis extremely difficult. Current traditional detection methods, such as PCR technology, while possessing high sensitivity and specificity, have stringent requirements for experimental environments, rely on external power sources, and involve bulky instruments, severely limiting their widespread application in the fields. This makes it difficult for sugarcane farmers to conduct early detection themselves, often only intervening passively after large-scale outbreaks and significant losses. Against this backdrop, the development of miniaturized, portable, and power-free on-site molecular diagnostic technologies has become a key breakthrough for promoting precise and proactive sugarcane disease control. This has profound strategic significance and practical value in empowering sugarcane farmers to accurately identify and control diseases early, ensuring the sustainable development of the sugarcane industry. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an enzyme biofuel cell system for detecting sugarcane top rot using three signals, along with its preparation method and applications. This system achieves highly sensitive, accurate, and rapid detection of the top rot pathogen, which helps to accurately identify sugarcane plants in the initial stage of disease development.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An enzyme biofuel cell system for detecting sugarcane top rot using three signals includes an enzyme biofuel cell and a photothermal conversion solution TiO2@Au. The enzyme biofuel cell includes a bioanode, a biocathode, and an electrolyte. The bioanode is a multi-walled carbon nanotube@zeolitic imidazole framework-8 / gold nanoparticles / glucose oxidase (MWNT@ZIF-8 / AuNPs / GOD) bioanode, and the biocathode is a MWNT@ZIF-8 / AuNPs / DNA chain.

[0006] The present invention also provides a method for preparing the enzyme biofuel cell system for detecting sugarcane top rot using the three-signal method, wherein the enzyme biofuel cell is constructed by using MWNT-ZIF-8 / AuNPs, carbon cloth, DNA strands and GOD to construct the anode and cathode of the enzyme biofuel cell.

[0007] In this invention, the preparation method of the bioanode further includes: taking 30-50 μL of MWNT@ZIF-8 / AuNPs and drop-coating it onto the surface of a carbon cloth electrode, drying it at 35-40 ℃ for 2-4 h, then drop-coating it with 30-50 μL of 5 mg / mL GOD and incubating it for 10-14 h, washing it with water to obtain the bioanode MWNT@ZIF-8 / AuNPs / GOD.

[0008] In this invention, the method for preparing the biocathode further includes: (1) 30-60 μL of MWNT@ZIF-8 / AuNPs was dropped onto the surface of the carbon cloth electrode and dried at 35-40 ℃ for 2-4 h. Then, 10-30 μL of gene signal receiving probe S1 was dropped onto the electrode and placed at 3-6 ℃ for 11-14 h. Then, 20-50 μL of 0.5-2 mM MCH was added and reacted for 15-45 min. Excess MCH was washed away. (2) Drop 20-50 μL of magnetic beads / A2 onto the electrode surface obtained in step (1) and incubate at 35-40 °C for 10-14 hours; (3) Add 20-30 μL of S2 to the electrode surface obtained in step (2) to obtain a biocathode.

[0009] In this invention, the preparation method of the magnetic beads / A2 and the photothermal conversion solution TiO2@Au further includes the following steps: 1) Construction of magnetic bead / A2 precursor: Mix 40-50 μL of A2 with 50-60 μL of magnetic beads and incubate in a shaker at 37 ℃ for 10-14 h; after incubation, use a magnet to separate the A2 that is not modified onto the surface of the magnetic beads under the action of an external magnetic field. 2) Composition of TiO2@Au / A3: Mix 40-50 μL of A3 with 50-60 μL of TiO2@Au and incubate at 4 ℃ for 12 h to obtain the desired result; 3) Preparation of magnetic beads / A2 / TiO2@Au / A3: Take 20 μL of magnetic beads / A2 precursor and 20 μL of TiO2@Au / A3 and incubate at 36-39℃ for 100-140 min; 4) SDA template preparation: Mix 10-15 μL of target with 10-15 μL of A1 and incubate at 37 °C for 100-140 min; 5) SDA reaction: Add 1-5 μL 10U / μL Phi29 DNA polymerase, 4-10 μL 10U / μL Phi29 Buffer, 3-8 μL DNTP, 2-4 μL 10U / μL Nt.BbvCI cleaving enzyme, 3-6 μL Nt.BbvCI Buffer and 4 μL ultrapure water to the solution obtained in step 4). Incubate at 37 °C for 120 min, then inactivate Nt.BbvCI cleaving enzyme and Phi29 DNA polymerase at 85 °C for 15 min to obtain output DNA. 6) Photothermal-Magnetic Separation: Add 40 μL of output DNA and 40 μL of magnetic beads / A2 / TiO2@Au / A3 to a centrifuge tube containing 10-15 μL of 1 / μL exonuclease III and 2 μL of 10X exonuclease III buffer. Incubate at 35-39℃ for 2 h, then inactivate exonuclease III at 75℃ for 30 min. Subsequently, use a magnet to adsorb the magnetic beads / A2 onto the inner wall of the centrifuge tube, and extract the TiO2@Au solution from the centrifuge tube. After extraction, the centrifuge tube still contains magnetic beads / A2. Add 80 μL of Na-based PBS to the centrifuge tube to obtain the magnetic bead / A2 solution. The separated solution is the TiO2@Au photothermal conversion solution.

[0010] In this invention, the preparation of the MWNT@ZIF-8 / AuNPs further includes: 1) Preparation of MWNT@ZIF-8: First, 10 mg of multi-walled carbon nanotubes (MWNT) were dissolved in 60 mL of methanol (MeOH) and sonicated for 30 min. 4 g of 2-methylimidazole was added to form a clear solution. Then, 1.68 g of Zn(NO3)2·6H2O was dissolved in 20 mL of MeOH, and the above solution was added. The mixture was stirred vigorously for 1 h. After stirring, the mixture was allowed to stand for 24 h, centrifuged and washed to obtain the product. Finally, the product was dried overnight under vacuum. 2) Preparation of AuNPs: Mix 50 μL of 2% HAuCl4 solution and 50 mL of deionized water and heat. Add 10 mg of sodium citrate while stirring. After the solution turns wine red, continue heating for 10 min. After stopping heating, stir and cool in the dark to obtain AuNPs. 3) Preparation of MWNT@ZIF-8 / AuNPs: Grind the MWNT@ZIF-8 obtained in step 1) into powder, prepare a 1 mg / mL solution, add 1 mL of acetic acid and the AuNPs obtained in step 2) to 10 mL of MWNT@ZIF-8, sonicate to obtain MWNT@ZIF-8 / AuNPs.

[0011] In this invention, further, the preparation process of TiO2@Au in step 2) includes: 120 mg TiO2 was dissolved in 60 ml of aqueous solution and sonicated for 20 min. Then, 100 μL of 2% AuHCl4 solution was added and stirred for 30 min. Next, 500 μL of 2% sodium citrate was added while stirring, and the reaction was allowed to proceed for half an hour. Then, 2 mL of sodium borohydride solution (approximately 40 mg of 0.53 M sodium borohydride) was added and the reaction was allowed to proceed for 12 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 8 min and washed three times alternately with water and ethanol. The precipitate was then freeze-dried for 12 h to obtain TiO2@Au.

[0012] The present invention also provides an application of the enzyme biofuel cell system for detecting sugarcane top rot using the three-signal method in the detection of sugarcane top rot pathogens.

[0013] Furthermore, in this invention, the detection method of the enzyme biofuel cell system in detecting sugarcane top rot pathogens includes the following steps: 1) Without introducing the target top rot pathogen, measure the instantaneous current, Blue value in RGB, and photothermal temperature of the enzyme biofuel cell system; 2) When detecting different concentrations of top rot pathogens, measure the instantaneous current value, the Blue value in RGB, and the photothermal temperature value of the enzyme biofuel cell system; 3) Plot the standard curve between instantaneous current value and the concentration of specific genes of the top rot pathogen, as well as the standard curve between the Blue value in RGB and the concentration of the top rot pathogen, and the standard curve between temperature change value and the concentration of the top rot pathogen, to complete the determination of the sugarcane top rot pathogen.

[0014] By adopting the above technical solution, the beneficial effects of the present invention are as follows: (1) The enzyme biofuel cell prepared by the method of the present invention constructs a chain displacement reaction (SDA) template and uses magnetic beads / A2 / TiO2@Au / A3 and ExoⅢ exonuclease to amplify the detection signal. The amplified products are separated stepwise by magnetic beads / A2 / TiO2@Au / A3, and the magnetic beads / A2 and TiO2@Au photothermal conversion solutions are output separately. The substances in the electrolyte are used to detect three signals with the magnetically separated products. In order to improve the electron transport rate, MWNT@ZIF-8 / AuNPs are used as the substrate material of the sensor. The MWNT in MWNT@ZIF-8 / AuNPs is used to improve the point electron transfer rate. When the sugarcane top rot pathogen is present, the target substance binds to the gene chain A1 to form an SDA template. Under the action of phi29 polymerase and Nt.BbvCI nicking enzyme, Output DNA is output. Next, the output DNA undergoes base pairing with gene A2, and under the action of ExoIII exonuclease and in conjunction with an external magnetic field, the magnetic beads / A2 and TiO2@Au are separated. Subsequently, the TiO2@Au photothermal conversion solution can be used for photothermal temperature testing. The magnetic beads / A2 bind to gene probe S1 in the biocathode, and under the action of S2, replenish the remaining single strands. Under the action of the bioanode and biocathode, the resulting open-circuit voltage is positively correlated with the pathogen concentration. Furthermore, during this process, MB is reduced to colorless MBH, and the Blue value in the RGB spectrum is obtained using a mobile phone camera for colorimetric detection. This invention has the advantages of high sensitivity, good selectivity, and real-time detection capabilities, and can be used for the accurate detection of sugarcane top rot.

[0015] (2) This invention uses the SDA reaction and the interaction between magnetic beads and ExoⅢ to achieve rapid amplification. The MB redox process enables analysis in two non-interfering modes: electrochemical and colorimetric detection. Based on this, TiO2@Au is used for photothermal temperature testing to achieve a third signal, which can effectively verify and calibrate false positive signals.

[0016] (3) When using the method of this invention to detect sugarcane top rot, this invention does not require an external power supply and can detect three signals. Using MWNT@ZIF-8 / AuNPs nanomaterials as the electrode substrate accelerates the electron conduction rate, and the specific surface area is increased by ZIF-8, increasing the amount of GOD attached, which shortens the response time of the detection signal and improves the output intensity. Using the method of this invention to detect the pathogen of sugarcane top rot does not require professional operating skills, and its materials are widely available and the assembly process is simple, enabling accurate and rapid detection.

[0017] In summary, the enzyme biofuel cell system of the present invention analyzes the data using three non-interfering modes: electrochemical, colorimetric, and photothermal detection, which can effectively calibrate false positive signals in the detection results. Its structure is simple, the materials are widely available, and the assembly process is easy to operate. It can achieve high sensitivity, selectivity, and real-time monitoring capabilities by using the three detection modes in combination, and can be used for the accurate detection of sugarcane top rot. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the anode and cathode assembly process in the enzyme biofuel cell system for three-signal detection of sugarcane top rot according to the present invention; Figure 2 This invention presents the LSV (A) of the presence or absence of a target at the cathode in the enzyme biofuel cell system for three-signal detection of sugarcane top rot, and the CV diagram (B) of the cathode electrode assembly. Figure 3 Photograph (A) of the solution with target substance in colorimetric mode and photo (B) of the solution without target substance in colorimetric mode in the enzyme biofuel cell system for three-signal detection of sugarcane top rot of the present invention. Figure 4 The images show the target substance system solution in photothermal mode (A) and in thermal mode (B) of the enzyme biofuel cell system for detecting sugarcane top rot with three signals according to the present invention. Figure 5 The present invention provides a standard curve (A) of instantaneous current versus concentration of a specific gene of the sugarcane top rot pathogen in an enzyme biofuel cell system for three-signal detection of sugarcane top rot, a standard curve (B) of Blue value in RGB under colorimetric mode versus concentration of a specific gene of the sugarcane top rot pathogen, and a standard curve (C) of temperature change versus concentration of a specific gene of the sugarcane top rot pathogen under photothermal mode. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0020] An enzyme biofuel cell system for three-signal detection of sugarcane top rot includes an enzyme biofuel cell and a photothermal conversion solution TiO2@Au. The enzyme biofuel cell comprises a bioanode, a biocathode, and an electrolyte. The bioanode is a multi-walled carbon nanotube@zeolite imidazole framework-8 / gold nanoparticles / glucose oxidase (MWNT@ZIF-8 / AuNPs / GOD) bioanode, and the biocathode is a MWNT@ZIF-8 / AuNPs / DNA chain. The enzyme biofuel cell's anode and cathode are constructed using MWNT-ZIF-8 / AuNPs, CP, DNA chain, and GOD. The photothermal conversion solution TiO2@Au is a separate system. The electrolyte is specifically a 0.01 M PBS buffer system containing 5 mM glucose and 5 mM PBS at pH 7.4.

[0021] A method for preparing (assembly process) an enzyme biofuel cell system for three-signal detection of sugarcane top rot, wherein the enzyme biofuel cell is constructed by using MWNT-ZIF-8 / AuNPs, carbon cloth, DNA strands, and GOD to construct the anode and cathode of the enzyme biofuel cell. The specific steps are as follows: Figure 1 As shown, it includes the following steps: 1. Preparation of MWNT@ZIF-8 / AuNPs 1) Preparation of MWNT@ZIF-8: First, 10 mg of MWNT was dissolved in 60 mL of methanol (MeOH) and sonicated for 30 min. Then, 4 g of 2-methylimidazole was added to form a clear solution. Next, 1.68 g of Zn(NO3)2·6H2O was dissolved in 20 mL of MeOH, added to the above solution, and stirred vigorously for 1 h. The mixture was then allowed to stand at room temperature without stirring. After 24 h, the product was separated by centrifugation, washed repeatedly with deionized water and methanol, and finally dried overnight under vacuum. 2) Preparation of AuNPs: Mix 50 μL of 2% HAuCl4 solution and 50 mL of deionized water and heat. Add 10 mg of sodium citrate while stirring. After the solution turns wine red, continue heating for 10 min. After stopping heating, stir and cool in the dark to obtain AuNPs. 3) Preparation of MWNT@ZIF-8 / AuNPs: Grind the MWNT@ZIF-8 obtained in step 1) into powder, prepare a 1 mg / mL solution, add 1 mL of acetic acid and the AuNPs obtained in step 2) to 10 mL of MWNT@ZIF-8, sonicate to obtain MWNT@ZIF-8 / AuNPs.

[0022] 2. Preparation of TiO2@Au: 120 mg TiO2 was dissolved in 60 mL of aqueous solution and sonicated for 20 min. Then, 100 μL of 2% AuHCl solution was added and stirred for 30 min. Next, 500 μL of 2% sodium citrate was added while stirring, and the reaction was allowed to proceed for half an hour. Finally, 2 mL of sodium borohydride solution (approximately 40 mg, 0.53 M) was added, and the reaction was allowed to continue for 12 h. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 8 min and washed three times alternately with water and ethanol. The precipitate was then freeze-dried for 12 h to obtain TiO2@Au.

[0023] 3. Preparation of bio-anode MWNT@ZIF-8 / AuNPs / GOD 50 μL of MWNT@ZIF-8 (1 mg / mL) was drop-coated onto the surface of a CP electrode (1 cm × 1 cm) and vacuum-dried at 37 °C for 2 h. Then, 50 µL of GOD (5 mg / mL) was drop-coated onto the electrode surface and incubated at 4 °C for 12 h. After washing with ultrapure water, the bioanode MWNT@ZIF-8 / AuNPs / GOD was obtained and stored at 4 °C for later use. The assembly process is as follows: Figure 1 .

[0024] 3. Preparation of magnetic beads / A2 and photothermal conversion solution TiO2@Au 1) Construction of the magnetic bead / A2 precursor: 40 μL of A2 was mixed with 50 μL of magnetic beads and incubated in a shaker at 37°C for 12 h. After the above process, the A2 unmodified onto the surface of the magnetic beads was separated using a magnet under the action of an external magnetic field; 2) Composition of TiO2@Au / A3: Mix 40 μL of A3 with 50 μL of TiO2@Au and incubate at 4℃ for 12 h; 3) Preparation of magnetic beads / A2 / TiO2@Au / A3: Take 20 μL of magnetic beads / A2 precursor and 20 μL of TiO2@Au / A3 and incubate at 37℃ for 100-140 min; 4) SDA template preparation: Mix 10 μL of target with 10 μL of A1 and incubate at 37°C for 100-140 min; 5) SDA reaction: Add 2 μL 10U / μL Phi29 DNA polymerase, 4 μL 10U / μL Phi29 Buffer, 5 μL DNTP, 2 μL 10U / μL Nt.BbvCI cleavage enzyme, 4 μL Nt.BbvCI Buffer, and 4 μL ultrapure water to the solution in step 4). Incubate at 37 °C for 120 min, then inactivate Nt.BbvCI cleavage enzyme and Phi29 DNA polymerase at 85 °C for 15 min to obtain output DNA.

[0025] 6) Photothermal-Magnetic Separation: 40 μL of output DNA and 40 μL of magnetic beads A2 / TiO2@Au / A3 were added to a centrifuge tube containing 10 μL of 1 / μL ExoⅢ and 2 μL of 10X ExoⅢ Buffer. The mixture was incubated at 37°C for 2 h, followed by ExoⅢ inactivation at 75°C for 30 min. Subsequently, a magnet was used to adsorb the magnetic beads / A2 onto the inner wall of the centrifuge tube, and the TiO2@Au solution was extracted from the tube. After extraction, the centrifuge tube contained magnetic beads / A2. 80 μL of Na-based PBS was then added to the centrifuge tube to obtain the magnetic beads / A2. The separated solution is the TiO2@Au photothermal conversion solution, which can be used to generate heat and produce temperature changes after irradiation with 808 nm near-infrared light.

[0026] 4. Preparation of biological cathode: (1) 50 μL of MWNT@ZIF-8 / AuNPs was dropped onto the surface of the carbon cloth electrode and dried at 37 °C for 4 h. Then, 20 μL of gene signal receiving probe S1 was dropped onto it and placed at 4 °C for 12 h. Then, 30 μL of 1 mM MCH was added and reacted for 30 min. Excess MCH was washed away. (2) 30 μL of magnetic beads / A2 was dropped onto the electrode surface obtained in step (1) and incubated at 37 °C for 12 h; (3) Add 20 μL of S2 to the electrode surface obtained in step (2) to obtain a biocathode.

[0027] Detection of sugarcane top rot pathogen using the three-signal enzyme biofuel cell system of this invention 1. Without introducing the target shoot rot pathogen, measure the instantaneous current value, the Blue value in RGB, and the temperature value of the enzyme biofuel cell system; after introducing the target shoot rot pathogen, measure the instantaneous current value, the Blue value in RGB, and the temperature change value of the system containing different concentrations of shoot rot pathogen.

[0028] 2. Plot the standard curves between instantaneous current values ​​and the concentration of specific genes of the shoot rot pathogen, the standard curves between the Blue value in RGB and the concentration of the shoot rot pathogen, and the standard curves between temperature changes and the concentration of specific genes of the shoot rot pathogen under photothermal mode.

[0029] like Figure 1When the concentration of *S. truncatella* pathogen is zero, only S1 is present on the electrode. Since an SDA template cannot be formed, Output DNA cannot be output, preventing the generation of magnetic beads / A2. Therefore, less MB is adsorbed on the biocathode, resulting in a weak electrical signal and insignificant color change in the electrolyte. When *S. truncatella* pathogen is added to the system, the target analyte initiates the SDA reaction and outputs Output DNA. Subsequently, the Output DNA binds to A3 in the magnetic beads / A2 / TiO2@Au / A3, and TiO2@Au is separated under the action of ExoⅢ. The photothermal temperature change is measured under 808 nm infrared light. The complementary base pairing between magnetic beads / A2 and S1 increases the adsorption of MB on the biocathode. With the addition of S2, the electrical signal strengthens again, and the Blue value increases. The instantaneous current, Blue value change, and temperature change are positively correlated with the concentration of *S. truncatella* pathogen, thus enabling quantitative detection of the pathogen. Furthermore, the non-interfering electrochemical and colorimetric systems and photothermal system can mutually calibrate the experimental results to prevent false positive signals.

[0030] Electrochemical characterization of a three-signal enzyme biofuel cell system To verify the biocathode reaction activity of the enzyme biofuel cell system, LSV testing was performed on the biocathode of the enzyme biofuel cell system. For example... Figure 2 As shown in Figure A, when the biocathode does not contain the target material (curve b), the peak current of the biocathode is relatively small; when the biocathode is combined with the target material (curve a), the peak current of the biocathode is much larger than the peak current without the target material. The CV test diagram of the biocathode assembly process is shown below. Figure 2 As 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 MWNT@ZIF-8 / AuNPs. Compared with a, the peak current of b is significantly increased. When probe S1 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 continues to increase after the addition of magnetic beads / A2 (curve d). When S2 is added, the peak current value reaches the maximum. In summary, the electrode indicates the successful assembly of the biocathode.

[0031] Figure 3 A is a colorimetric diagram of the target object in colorimetric mode; Figure 3 B is a colorimetric diagram without a target object in colorimetric mode. It can be seen that the color with a target object is much lighter than the color without a target object. Figure 4 A is the temperature diagram of the target object under photothermal mode; Figure 4 B is the temperature map without a target object under the photothermal mode, from Figure 4 It can be seen that the nucleic acid signal amplification part can successfully separate TiO2@Au photothermal conversion solution and magnetic beads / A2 in the presence of the specific gene for top rot, indicating that the temperature signal can also be used to determine whether sugarcane has top rot.

[0032] 4. Performance characterization of enzyme biofuel cells like Figure 5 As shown in Figure A, transient current measurements were performed on specific genes of the twig rot pathogen at different concentrations. In the absence of the target analyte, only the capture probe S1 was present on the electrode substrate. At this time, because the target analyte could synthesize an SDA template, the subsequent ExoIII reaction was inhibited, resulting in the inability to output the magnetic bead / A2. Consequently, the biocathode received fewer electrons, leading to a lower transient current value in the enzyme biofuel cell system. With increasing target analyte concentration, the number of DNA strands loaded on the electrode also increased, and the transient current value of the enzyme biofuel cell system gradually increased. The transient current value showed a good linear relationship with the twig rot pathogen concentration in the range of 0.1 fM–10000 pM, with the linear equation Y = 3.359 lg c + 59.229 (R² = 0.997), and a detection limit of 31.82 amol / L (S / N = 3).

[0033] exist Figure 5 In step B, after detecting specific genes of the pathogen of shoot rot at different concentrations, the electrolyte was used for colorimetric detection. The Blue value in RGB showed a good linear relationship with the concentration of the pathogen of shoot rot in the range of 0.1 fM-10000 pM, and the linear equation was Blue = 8.117 lg c+295.237 (R2 = 0.996), with a detection limit of 10.977 amol / L.

[0034] exist Figure 5 In step C, the TiO2@Au photothermal conversion solution obtained after amplifying the nucleic acid signal was subjected to photothermal testing. The photothermal heating temperature showed a good linear relationship with the concentration of the shoot rot pathogen in the range of 10 fM-10000 pM, and the linear equation was Temperature = 4.233lg c + 92.559 (R2 = 0.996), with a detection limit of 1.617 fmol / L.

[0035] Therefore, through Figure 5 It can be seen that the combination of sugarcane top rot gene molecules with biofuel cells exhibits high sensitivity and good linearity with changes in target concentration (R0 for the three modes). 2 >0.99).

[0036] The sequences used in this invention are as follows: Target pathogen: 5'-GTT GTA AAC TCG GTA ATG ATC CCT-3' A1 sequence: 5'- CCG TTC ATC CTG AGG GAT CAT CAT TAC CGA GTT TAC AAC -3' A2 sequence: 5'- NH2-CGG CTA TCG CTA GCA GTG CGC GAT ACA GCT ATC GAG TATGGA -3' A3 sequence: 5'- SH-CGG CTC GAC TGT ATC GCG CAC TGC TTC TAG CTG AGG -3' S2 sequence: 5'- GAT AGC TGT ATC GCG CAC TGC TAC CGA TAG CCG -SH -3' S2 sequence: 5'- GCC ATA CTAC GAT AGC TGT-3' The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. An enzyme-based biofuel cell system for detecting sugarcane top rot using three signals, characterized in that, The invention includes an enzyme biofuel cell and a photothermal conversion solution TiO2@Au. The enzyme biofuel cell includes a bioanode, a biocathode, and an electrolyte. The bioanode is a MWNT@ZIF-8 / AuNPs / GOD bioanode, and the biocathode is a MWNT@ZIF-8 / AuNPs / DNA chain.

2. A method for preparing an enzyme biofuel cell system for three-signal detection of sugarcane top rot according to claim 1, characterized in that, The enzyme biofuel cell is constructed by using MWNT-ZIF-8 / AuNPs, carbon cloth, DNA strands, and GOD to construct the anode and cathode of the enzyme biofuel cell.

3. The preparation method according to claim 2, characterized in that, The preparation method of the bioanode includes: taking 30-50 μL of MWNT@ZIF-8 / AuNPs and drop-coating it onto the surface of a carbon cloth electrode, drying it at 35-40 ℃ for 2-4 h, then drop-coating it with 30-50 μL of 5 mg / mL GOD and incubating it for 10-14 h, washing it with water to obtain the bioanode MWNT@ZIF-8 / AuNPs / GOD.

4. The preparation method according to claim 2, characterized in that, The method for preparing the biocathode includes: (1) 30-60 μL of MWNT@ZIF-8 / AuNPs was dropped onto the surface of the carbon cloth electrode and dried at 35-40 ℃ for 2-4 h. Then, 10-30 μL of gene signal receiving probe S1 was dropped onto it and placed at 3-6 ℃ for 11-14 h. Then, 20-50 μL of 0.5-2 mM MCH was added and reacted for 15-45 min. Excess MCH was washed away. (2) Drop 20-50 μL of magnetic beads / A2 onto the electrode surface obtained in step (1) and incubate at 35-40 °C for 2 h; (3) Add 20-30 μL of S2 to the electrode surface obtained in step (2) to obtain a biocathode.

5. The preparation method according to claim 4, characterized in that, The preparation method of the magnetic beads / A2 and the photothermal conversion solution TiO2@Au includes the following steps: 1) Construction of magnetic bead / A2 precursor: Mix 40-50 μL of A2 with 50-60 μL of magnetic beads and incubate in a shaker at 37 ℃ for 10-14 h; after incubation, use a magnet to separate the A2 that is not modified onto the surface of the magnetic beads under the action of an external magnetic field. 2) Composition of TiO2@Au / A3: Mix 40-50 μL of A3 with 50-60 μL of TiO2@Au and incubate at 4 ℃ for 12 h to obtain the desired result; 3) Preparation of magnetic beads / A2 / TiO2@Au / A3: Take 20 μL of magnetic beads / A2 precursor and 20 μL of TiO2@Au / A3 and incubate at 36-39℃ for 100-140 min; 4) SDA template preparation: Mix 10-15 μL of target with 10-15 μL of A1 and incubate at 37 °C for 100-140 min; 5) SDA reaction: Add 1-5 μL 10U / μL Phi29 DNA polymerase, 4-10 μL 10U / μL Phi29 Buffer, 3-8 μL DNTP, 2-4 μL 10U / μL Nt.BbvCI cleaving enzyme, 3-6 μL Nt.BbvCI Buffer and 4 μL ultrapure water to the solution obtained in step 4). Incubate at 37 °C for 120 min, then inactivate Nt.BbvCI cleaving enzyme and Phi29 DNA polymerase at 85 °C for 15 min to obtain output DNA. 6) Photothermal-Magnetic Separation: Add 40 μL of output DNA and 40 μL of magnetic beads / A2 / TiO2@Au / A3 to a centrifuge tube containing 10-15 μL of 1 / μL exonuclease III and 2 μL of 10X exonuclease III buffer. Incubate at 35-39℃ for 2 h, then inactivate exonuclease III at 75℃ for 30 min. Subsequently, use a magnet to adsorb the magnetic beads / A2 onto the inner wall of the centrifuge tube, and extract the TiO2@Au solution from the centrifuge tube. After extraction, the centrifuge tube still contains magnetic beads / A2. Add 80 μL of Na-based PBS to the centrifuge tube to obtain the magnetic beads / A2 solution. The separated solution is the TiO2@Au photothermal conversion solution.

6. The preparation method according to claim 5, characterized in that, The preparation of the MWNT@ZIF-8 / AuNPs includes: 1) Preparation of MWNT@ZIF-8: First, 10 mg of multi-walled carbon nanotubes were dissolved in 60 mL of methanol and sonicated for 30 min. Then, 4 g of 2-methylimidazole was added to form a clear solution. Next, 1.68 g of Zn(NO3)2·6H2O was dissolved in 20 mL of methanol and added to the above solution. The mixture was stirred vigorously for 1 h. After stirring, the mixture was allowed to stand for 24 h, centrifuged and washed to obtain the product. Finally, the product was dried overnight under vacuum. 2) Preparation of AuNPs: Mix HAuCl4 solution and deionized water and heat. Add sodium citrate while stirring. After the solution turns wine red, continue heating for 10 min. After stopping heating, stir and cool in the dark to obtain AuNPs. 3) Preparation of MWNT@ZIF-8 / AuNPs: Grind the MWNT@ZIF-8 obtained in step 1) into powder, prepare a 1 mg / mL solution, add 1 mL of acetic acid and the AuNPs obtained in step 2) to 10 mL of MWNT@ZIF-8, sonicate to obtain MWNT@ZIF-8 / AuNPs.

7. The preparation method according to claim 5, characterized in that, Step 2) The preparation process of TiO2@Au includes: 120 mg TiO2 was dissolved in 60 ml of aqueous solution and sonicated for 20 min. Then, 100 μL of 2% AuHCl solution was added and stirred for 30 min. Next, 500 μL of 2% sodium citrate was added while stirring, and the reaction was allowed to proceed for half an hour. Then, 2 mL of sodium borohydride solution was added and the reaction was allowed to proceed for 12 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 8 min and washed three times alternately with water and ethanol. The precipitate was then freeze-dried for 12 h to obtain TiO2@Au.

8. The application of the enzyme biofuel cell system for detecting sugarcane top rot according to claim 1 in the detection of sugarcane top rot pathogens.

9. The application according to claim 8, characterized in that, The detection method of the enzyme biofuel cell system for detecting sugarcane top rot pathogens includes the following steps: 1) Without introducing the target top rot pathogen, measure the instantaneous current, Blue value in RGB, and photothermal temperature of the enzyme biofuel cell system; 2) When detecting different concentrations of top rot pathogens, measure the instantaneous current value, the Blue value in RGB, and the photothermal temperature value of the enzyme biofuel cell system; 3) Plot the standard curve between instantaneous current value and the concentration of specific genes of the top rot pathogen, as well as the standard curve between the Blue value in RGB and the concentration of the top rot pathogen, and the standard curve between temperature change value and the concentration of the top rot pathogen, to complete the determination of the sugarcane top rot pathogen.

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