Self-energized sensor for detecting sugarcane smut pathogenic bacteria and preparation method of self-energized sensor

Abstract

The invention discloses a self-powered sensor for detecting sugarcane smut pathogenic bacteria and a preparation method thereof, the self-powered biosensor comprises an anode, a cathode and an electrolyte, the anode is an Au / N-GDY / GOD / BSA biological anode, the cathode is an Au / N-GDY / DNA chain biosensor, and the electrolyte comprises glucose, [Ru (NH3) 6] 3 + and a PBS buffer solution. The sensor provided by the invention is used for portable detection of pathogenic bacteria of sugarcane smut, and quantitative analysis of a target analyte is realized through change of power output of the enzyme biological fuel cell. The target object can be simply, quickly, sensitively and efficiently detected.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, and in particular to a self-powered sensor for detecting sugarcane smut pathogens and a preparation method thereof. Background Art

[0002] Traditional electrochemical sensors generally consist of four parts: identification element, signal conditioning and conversion circuit, signal amplifier, and power auxiliary circuit. As a green energy conversion device, enzyme biofuel cell (EBFC) converts biological energy into electrical energy through biocatalysts such as redox enzymes. Compared with traditional electrochemical sensors, self-powered biosensors based on EBFC do not require batteries or external power supplies. By collecting energy in EBFC, self-powered sensing is achieved. At the same time, the application of nucleic acid amplification technology integrates signal recognition and amplification functions, making the device easy to miniaturize. Relying on the principle of biological cathode and anode redox reactions, self-powered biosensors based on EBFC achieve efficient conversion of energy forms.

[0003] Sugarcane is a perennial tropical plant with perennial roots and an important economic crop in the world. The sugar produced by it is an important strategic material related to the national economy and people's livelihood. Sugarcane smut is the most common and most serious disease in the sugarcane production process. It is caused by sugarcane whip smut. When the winter spores of sugarcane whip smut infect the sugarcane buds, the hyphae parasitizes the meristem and intercellular space of the sugarcane buds, accelerating the rapid differentiation of the apical tissue. After 6-7 months, a slender black-brown whip-like structure grows from the top. Sugarcane smut absorbs nutrients from healthy cells, resulting in the stunted development of sugarcane plants and reduced reducing sugar content, which seriously threatens the stability and development of the sugarcane industry. The traditional method of identifying sugarcane smut is to judge based on the black whip at the tip of the sugarcane, but this method is very time-consuming and requires a large investment. At present, PCR-based detection technology is a relatively common method for detecting early sugarcane smut pathogens, but due to the difficulty in designing specific primers, the accuracy of PCR technology in detecting sugarcane smut pathogens is limited to a certain extent. At the same time, PCR technology requires professional laboratory operators and expensive instruments and equipment, which is not conducive to practical application and promotion. Especially in the early stage of sugarcane smut development, the pathogen content is low, the actual sample interference is more, and the traditional method is prone to low sensitivity and poor selectivity.

[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0005] The object of the present invention is to provide a self-powered sensor for detecting sugarcane smut pathogens and a preparation method thereof, so as to achieve ultra-high sensitivity and high selectivity detection of smut pathogens and help detect abnormal expression of smut pathogens.

[0006] To achieve the above-mentioned object, the present invention provides a self-powered biosensor for detecting sugarcane smut pathogens, the self-powered biosensor comprising an anode, a cathode and an electrolyte, the anode being an Au / N-GDY / GOD / BSA bioanode, the cathode being an Au / N-GDY / DNA chain biosensor, the electrolyte comprising glucose, [Ru(NH3)6] 3+ , PBS buffer solution.

[0007] A method for preparing a self-powered sensor for detecting sugarcane smut pathogens. The self-powered sensor is based on a gold nano-modified graphyne complex Au / N-GDY, carbon cloth, DNA nanostructure and glucose oxidase to construct the positive and negative poles of the self-powered biosensor.

[0008] Preferably, in the above technical scheme, the preparation of the bioanode includes: taking 30-50 μL Au / N-GDY and dropping it on the surface of the carbon cloth electrode, drying it at 35-40°C for 2-4 hours, dropping it on 20-50 μL 3-5 mg / mL glucose oxidase and incubating it for 12 to 24 hours, washing it with water, adding 0.1-2 mmol / L, 20 to 50 μL bovine serum albumin, leaving it to react and washing it with water to obtain the bioanode.

[0009] Preferably, in the above technical solution, the preparation of the biocathode comprises:

[0010] (1) 30-50 μL Au / N-GDY was drop-coated on 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 with water, and then drop-coated with 10-30 μL Truncated cone, placed at 3-6°C for 10-15 h, and 20-50 μL 0.5-2 mM hexamercaptoethanol was added to react for 0.5-1 h, and the excess hexamercaptoethanol was removed by washing;

[0011] (2) 20 to 50 μL of the hybridization chain reaction product is dropped onto the electrode surface and incubated at 35 to 40° C. for 1 to 2 h, followed by washing to obtain a biocathode.

[0012] Preferably, in the above technical solution, the preparation of the Truncated cone in step (1) comprises mixing 0.5-2 μM, 80-100 μL of each of S1, S2 and S3, heating in a water bath to 90-95° C. and maintaining for 1-30 min.

[0013] Preferably, in the above technical solution, the preparation of the hybridization chain reaction product in step (2) comprises:

[0014] 1) Preparation of DNAzyme product: 10-30 μL of LS1 and Locker were mixed, and the mixture was reacted at 35-40°C for 1-2 hours to obtain a swing chain; 40-70 μL of 5 mg / mL activated magnetic beads were mixed with 1 μM, 40-70 μL of LS2 and 10-20 μL of the swing chain, and the mixture was shaken at 35-40°C for 12-24 hours, and the mixture was adsorbed by a magnet and dispersed in 100-200 μL of PBS; 10-30 μL of pathogens of different concentrations were mixed with 40-70 μL of magnetic beads PBS, and 10-30 μL of 5 mM Mn 2+ , react at 35-40℃ for 1-2h, take the supernatant to obtain the DNAzyme product output DNA;

[0015] Step 2. Preparation of hybridization chain reaction product: Mix 0.5-2 μM, 40-70 μL each of H1 and H2 with 40-70 μL of output DNA, and incubate at 35-40°C for 1-3 hours to obtain a hybridization chain reaction product, output DNA / H1 / H2.

[0016] Preferably, in the above technical solution, the preparation of Au / N-GDY includes:

[0017] 1) Preparation of GDY: 1-2 mL of tetramethylethylenediamine (TMEDA), 5-10 mL of pyridine, and 70-120 mL of acetone were added to a round-bottom flask, and the pretreated copper plate was placed in the mixture. Then, 1-2 mL of tetrabutylammonium fluoride (TBAF) was added to the HEB-TMS dispersion (20-40 mg) in THF solution under 0°C argon atmosphere with stirring for 10-20 min. After purification, HEB was dissolved in 30-70 mL of pyridine solution as a precursor solution for the coupling reaction. Under 70°C argon atmosphere, the precursor solution was slowly added to the mixed solution in the round-bottom flask and heated in the dark for 10-15 h. After the reaction was completed, the GDY film adhered to the copper plate was extracted by ultrasonic wave and collected in the solution. Finally, it was washed with hot pyridine, DMF, HCl, ethanol, and ultrapure water in sequence, and vacuum dried at 50-70°C for 1-3 h to obtain powdered GDY.

[0018] 2) Preparation method of AuNPs: Take 400-700 μL of 1% HAuCl4 solution and mix it with 40-70 mL of ultrapure water, heat the oil bath to 120°C, and then cool the temperature to 110°C. While stirring, quickly inject 400-600 μL of 2% sodium citrate at one time. After the color of the solution changes to wine red, continue heating for 10 minutes. After stopping heating, continue stirring in the dark and cool to room temperature to obtain AuNPs.

[0019] 3) Preparation of N-GDY: 8-12 mg of GDY powder and 10-30 mg of melamine were mixed in 10-20 mL of water, and the mixture was subjected to hydrothermal reaction at 150°C for 10-20 h. After cooling to room temperature, the mixture was freeze-dried to obtain precursor GDY. Argon was passed through a tubular furnace, and the precursor GDY was reacted at 800°C for 1-3 h. After cooling, N-GDY was obtained.

[0020] 4) Preparation of Au / N-GDY: GDY was ground into fine powder, 1-2 mg N-GDY was added with 3-6 mL acetic acid and 6-12 mL AuNPs and ultrasonicated for 20-40 min to obtain Au / N-GDY.

[0021] An application of the self-powered sensor as described above, wherein the self-powered sensor is used to detect smut pathogens.

[0022] A method for using the self-powered sensor as described above to detect sugarcane smut pathogens, the method comprising the following steps:

[0023] (1) When the sugarcane smut pathogen is not introduced into the system, the instantaneous current of the sensor is measured;

[0024] (2) After the sugarcane smut pathogen was introduced into the system, the instantaneous current of the sensor was measured at different concentrations of the sugarcane smut pathogen;

[0025] Among them, the membraneless glucose / [Ru(NH3)6] 3+ The supporting electrolyte of the sensor contains 5mM glucose, 500μM [Ru(NH3)6] 3+ 0.01 M PBS buffer system, pH 7.4.

[0026] The working principle of the self-powered sensor prepared by the inventive method is as follows:

[0027] The self-powered biosensor of the present invention uses gold nanoparticles and nitrogen-doped graphyne two-dimensional composite nanomaterials as electrode substrate materials. Glucose in the anode glucose oxidase (GOD) catalyzes the oxidation system to efficiently generate a large number of electrons, and the cathode hexaammineruthenium receives the electrons and is reduced to form a complete electrical path. When the sugarcane smut pathogen is present, the target initiates the DNAzyme reaction, provides an activation chain for the subsequent hybridization chain reaction, and forms a large number of double-stranded structures, thereby fixing the hexaammineruthenium on the biological cathode to generate a strong electrical signal. The instantaneous current value is positively correlated with the concentration of the sugarcane smut pathogen, thereby achieving ultra-sensitive detection of the sugarcane smut pathogen.

[0028] At the same time, a small energy storage device capacitor is added to the above system, and the electrons generated by the enzyme biofuel cell can be stored in the capacitor. Then, the capacitor switch is released and switched to the discharge state, which can generate a larger instantaneous current, thereby further amplifying the detection signal. In addition, the output signal can be transmitted to a smartphone in real time using the Bluetooth device of the multimeter, allowing the system to achieve rapid and portable real-time detection of smut pathogens.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The method of the present invention constructs a self-powered biosensor based on an enzyme biofuel cell, and uses a smart phone to assist in the detection of smut pathogens. The construction of a self-powered system is the core of the sensor design. When the smut pathogen is present, the DNAzyme amplification is activated, and the hexaamineruthenium adsorbed by the biocathode increases, and the response current increases. The instantaneous current is positively correlated with the concentration of the smut pathogen, thereby achieving quantitative detection of the smut pathogen. The present invention designs a self-powered biosensor with a synergistic effect of dual nucleic acid amplification for the analysis of sugarcane smut pathogens, which can achieve simple, rapid, sensitive and efficient detection of the target object.

[0031] (2) The self-powered biosensor described in the present invention is different from the three-electrode detection system of the sensor. Only two working electrodes are needed to build the anode and cathode of the self-powered device to detect the target molecule. No external power supply is required during the test process, which effectively avoids the interference of electroactive substances that may be oxidized and reduced on the electrode surface, thereby improving the stability of the sensor. The use of instantaneous current to detect the smut pathogen not only has extremely high specificity, but also is low-cost and simple to operate, making the entire device easy to carry.

[0032] (3) The self-powered biosensor of the present invention cooperates with DNAzyme and HCR and other enzyme-free amplifications to amplify signals, specifically identifies target molecules and increases the loading amount of the cathode electron acceptor hexaamineruthenium, so that the signal of the target molecule is efficiently and accurately amplified. The substrate material has the characteristics of high electron mobility, good stability, large specific surface area, good biocompatibility, etc. It is used as a substrate material to load subsequent DNA chains and GOD in the self-powered sensor device, realizing ultra-sensitive detection of the target object, smut pathogen, and greatly improving the detection sensitivity;

[0033] (4) The self-powered biosensor of the present invention does not require an external power supply, and the actual application process does not require an expensive and complex electrochemical workstation. It is coupled with a capacitor to further amplify the detection signal, and the current signal is transmitted to a mobile phone APP in real time through the Bluetooth device of the multimeter, thereby realizing real-time monitoring of sugarcane smut pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the assembly process of the cathode and anode of the self-powered sensor for detecting sugarcane smut pathogens according to the present invention;

[0035] Figure 2 1. It is a schematic diagram of the principle of detecting sugarcane smut pathogens by the self-powered sensor for detecting sugarcane smut pathogens according to the present invention;

[0036] Figure 3 2. It is a schematic diagram of the principle of a capacitor coupled with a self-powered sensor for detecting sugarcane smut pathogens according to the present invention;

[0037] Figure 4 CV (A) of the self-powered sensor for detecting the presence of a target object at the cathode of the sugarcane smut pathogen according to the present invention, and CV (B) of each step of the assembly of the biocathode;

[0038] Figure 5 CV (A) of the self-powered sensor for detecting the presence or absence of glucose at the anode of the sugarcane smut pathogen according to the present invention, and CV (B) of each step of the assembly of the bioanode;

[0039] Figure 6 The standard curves of the open circuit voltage and the concentration of the smut pathogen of the self-powered sensor for detecting the sugarcane smut pathogen according to the present invention (A), and the standard curves of the current intensity and the concentration of the smut pathogen before charging (B) and after charging (C);

[0040] Figure 7 This is a physical picture of the self-powered sensor for detecting sugarcane smut pathogens according to the present invention. DETAILED DESCRIPTION

[0041] The specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

[0042] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0043] 1. Self-powered sensor for detecting sugarcane smut pathogens

[0044] like Figure 1-3 As shown in Figure 7, a self-powered biosensor for detecting sugarcane smut pathogens, the self-powered biosensor comprises an anode, a cathode and an electrolyte, the anode is an Au / N-GDY / GOD / BSA bioanode, the cathode is an Au / N-GDY / DNA chain biosensor, and the electrolyte comprises glucose, [Ru(NH3)6] 3+ , PBS buffer solution.

[0045] 2. Preparation of a self-powered sensor for detecting sugarcane smut pathogens

[0046] 1. Preparation of Au / N-GDY

[0047] (1) Preparation of GDY: 1 mL of tetramethylethylenediamine (TMEDA), 5 mL of pyridine, and 100 mL of acetone were added to a round-bottom flask, and then the pretreated copper plate was placed in the mixture. Then, 2 mL of tetrabutylammonium fluoride (TBAF) was added to the HEB-TMS dispersion (30 mg) in THF solution under argon atmosphere at 0 °C for 15 min. After purification, HEB was dissolved in 50 mL of pyridine solution as a precursor solution for the coupling reaction. Under argon atmosphere at 70 °C, the precursor solution was slowly added to the mixed solution in the round-bottom flask and heated in the dark for 12 h. After the reaction was completed, the GDY film adhered to the copper plate was extracted by ultrasonication and collected in the solution. Finally, it was washed with hot pyridine, DMF, HCl, ethanol, and ultrapure water in sequence and vacuum dried at 60 °C for 3 h to obtain powdered GDY.

[0048] (2) Preparation of AuNPs: 500 μL of 1% HAuCl4 solution was mixed with 50 mL of ultrapure water, heated in an oil bath to 120°C, and then cooled to 110°C. 500 μL of 2% sodium citrate was rapidly injected at one time while stirring. After the solution turned wine red, heating was continued for 10 min. After stopping heating, stirring was continued in the dark and cooled to room temperature to obtain AuNPs.

[0049] (3) Preparation of N-GDY: 10 mg of GDY powder and 20 mg of melamine were mixed in 15 mL of water, and the mixture was hydrothermally reacted at 150°C for 15 h. After cooling to room temperature, the mixture was freeze-dried to obtain precursor GDY. In a tubular furnace, argon gas was passed through the mixture, and the precursor GDY was reacted at 800°C for 2 h. After cooling, N-GDY was obtained.

[0050] (4) Preparation of Au / N-GDY: GDY was ground into fine powder. 2 mg of N-GDY was added to 6 mL of acetic acid and 12 mL of AuNPs and ultrasonicated for 30 min to obtain Au / N-GDY.

[0051] 2. Preparation of bioanode Au / N-GDY / GOD / BSA

[0052] Take 1mg / mL, 50μL Au / N-GDY and drop it on the surface of the carbon cloth electrode (1cm×1cm). After vacuum drying at 37℃ for 2h, then drop 5mg / mL, 50μL glucose oxidase (GOD) on the electrode surface, incubate at 4℃ for 12h, and wash with ultrapure water. Then drop 1mmol / L, 40μL bovine serum albumin (BSA) on the electrode, place it at room temperature for 30min, and then wash it with ultrapure water to obtain the bioanode Au / N-GDY / GOD / BSA, which is stored at 4℃ for future use. Its assembly process is as follows: Figure 1 .

[0053] 3. Preparation of biological cathode Au / N-GDY / Trucncated cone / MCH / output DNA / H1 / H2

[0054] (1) Preparation of Truncated cones: Mix 2 μM, 100 μL each of S1, S2, and S3, heat to 95°C in a water bath and keep for 5 min.

[0055] (2) Preparation of DNAzyme products: 20 μL of LS1 and Locker were mixed and reacted at 37°C for 2 h to obtain swing chain. 50 μL of 5 mg / mL activated magnetic beads (MB) were mixed with 1 μM, 40 μL of LS2 and 10 μL of swing chain, and oscillated at 37°C for 12 h. The mixture was adsorbed by a magnet and dispersed in 100 μL PBS. 10 μL of pathogens of different concentrations were mixed with 40 μL of magnetic beads PBS, and 10 μL of 5 mM Mn was added. 2+ , react at 37°C for 2h, and take the supernatant to obtain the DNAzyme product output DNA.

[0056] (3) Preparation of hybridization chain reaction (HCR) product: 2 μM, 50 μL each of H1 and H2 were mixed with 50 μL of output DNA and incubated at 37°C for 2 h to obtain the hybridization chain reaction (HCR) product output DNA / H1 / H2.

[0057] (4) Preparation of biosensor cathode: Take 1mg / ml, 50μL Au / N-GDY and drop it on the surface of the carbon cloth electrode (1cm×1cm). After vacuum drying at 37℃ for 3h, then drop 50μL of the product of step 1 on the electrode surface, incubate at 4℃ for 12h and wash with ultrapure water. Then add 1mmol / L, 25μL hexamercaptohexanol (MCH), let it stand at room temperature for 30min to block nonspecific binding sites, and wash with ultrapure water. Add 30μL of the product of step 2, incubate at 37℃ for 90min, and wash with ultrapure water. Add 50μL of the product of step 3, incubate at 37℃ for 90min, and wash with ultrapure water. The biocathode Au / N-GDY / Trucncated cone / MCH / output DNA / H1 / H2 was prepared and stored at 4℃ for future use. Its assembly process is as follows Figure 1 .

[0058] 3. Use of the self-powered biosensor of the present invention for detection of sugarcane smut pathogens

[0059] 1. Construction and measurement of a self-powered sensor for detecting sugarcane smut pathogens: When the target smut pathogen is not introduced, the sensor's E OCV After the target smut pathogen was introduced, the E of the biosensor containing different concentrations of smut pathogen was measured. OCV .

[0060] 2. Draw the standard curve of open circuit voltage and the concentration of smut pathogen, draw the standard curve of current intensity and the concentration of smut pathogen before and after charging, and complete the determination of smut pathogen.

[0061] 3. Electrochemical characterization of self-powered biosensors.

[0062] In order to verify the biocathode reaction activity of the sensor, a CV test was performed on the biocathode of the sensor. Figure 4 As shown in A, when the biocathode is not introduced with the target (curve a), the peak potential of the biocathode is small; when the biocathode is combined with the target (curve b), the peak potential of the biocathode is much greater than the peak potential without the target. The biocathode of the sensor is tested by EIS, as shown in Figure 4As shown in Figure 2, after the Au / N-GDY material was drop-coated on the surface of the bare carbon cloth (curve a) (curve b), the resistance value decreased significantly. With the subsequent loading of the DNA chain, the resistance value gradually increased (curve ce), indicating the successful assembly of the biocathode.

[0063] In order to verify the bioanode reaction activity of the sensor, a CV test was performed on the bioanode of the sensor. Figure 5 As shown in A, when the electrolyte does not contain glucose (curve a), a pair of weak redox characteristic peaks appear near -0.5V, which are characteristic peaks of glucose oxidase; when the electrolyte contains glucose (curve b), the peak potential of the bioanode is enhanced. EIS test of the bioanode of the sensor is performed, as shown in Figure 5 As shown in B, after adding GOD (curve c), the resistance value increased significantly, indicating the successful assembly of the bioanode.

[0064] 4. Performance characterization of self-powered biosensors

[0065] like Figure 6 A, open circuit voltage test of different concentrations of target (smut pathogen). When the target does not exist, there is only Truncated cone on the electrode substrate, DNAzyme amplification cannot be carried out, hybridization chain reaction amplification cannot be fixed on the electrode surface, and the biological cathode receives less electrons. At this time, the E of EBFC OCV As the concentration of the target increases, the number of DNA chains loaded on the electrode increases, and the E OCV Also gradually increased. OCV The value showed a good linear relationship with the concentration of smut pathogen in the range of 1fM-10000pM, and the linear equation was E OCV =0.0286lg c+0.625(R 2 =0.998), and the detection limit was 0.58 fmol / L (S / N=3).

[0066] like Figure 6 B. The current intensity test without capacitor was performed for different concentrations of target (smut pathogen). When the target existed, without capacitor, the multimeter (DMM) in the circuit directly recorded the current signal, and its Bluetooth device transmitted the signal to the smartphone. The current intensity increased with the increase of target concentration, and showed a good linear relationship in the range of 1fM-10000pM. The linear equation was y=1.502lg c+27.468(R 2 =0.991), the detection limit is 0.48fmol / L (S / N=3). Figure 6C, the capacitor current intensity test was performed on different concentrations of the target (smut pathogen). When the target exists, the capacitor coupled in the circuit stores the generated electrons. After turning on the discharge switch, the high-power capacitor releases a multi-fold current signal. The multimeter (DMM) in the circuit records the instantaneous current, and its Bluetooth module transmits the signal to the mobile phone. The current intensity increases with the increase of the target concentration, and shows a good linear relationship in the range of 1fM-10000pM. The linear equation is y=10.233lg c+234.628(R 2 =0.998), and the detection limit was 0.11 fmol / L (S / N=3).

[0067] The membraneless glucose / [Ru(NH3)6] 3+ The supporting electrolyte of the sensor contains 5mM glucose, 500μM [Ru(NH3)6] 3+ 0.01 M PBS buffer system, pH 7.4.

[0068] Target: 5'-CGT TGATGA ACC AGA GCG-3'

[0069] S1 sequence: 5'-SH-(CH2)6-TGAATT GAG TAT TGT TTC TTG TGT T GC TTA TCT GA-3'

[0070] S2 sequence: 5'-AAACAA TAT TTA CTC CAC TTT GCA CTG CAA CAC AAG-3'

[0071] S3 sequence: 5'-AAA CAA TAG CAG TGC AAA GTG GAG TAA AAACAC AAG-3'

[0072] LS1 sequence: 5'-NH2-(CH2)6-CGC ATT AGC CAG GT GCT TAT TAACAT T / rA / TCT GATAAG CTC TGA TAA GC-3'

[0073] LS2 sequence: 5'-NH2-(CH2)6-TTT CAA TGC AAC TTT TTT TTT TTT TTT TTT TTT TTTTAT CAG CTT ATC ATG AGG TCT CCC CAG CAT GTT A-3'

[0074] Locker sequence: 5'-CGC TCT GGT TCA TCA ACG-3'

[0075] H1 sequence: 5'-TCA GAC TTC TTT TTT TTT CTT CAG ACT-3'

[0076] H2 sequence: 5'-TTT GCT TAT CTG AAG TCT GAA GGC TTA TCT GA -3'

[0077] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A self-powered sensor for detecting sugarcane smut pathogens, characterized in that: The self-powered biosensor comprises an anode, a cathode and an electrolyte, wherein the anode is an Au / N-GDY / GOD / BSA bioanode, the cathode is an Au / N-GDY / DNA chain biosensor, and the electrolyte comprises glucose, [Ru(NH3)6] 3+ , PBS buffer solution.

2. A method for preparing a self-powered sensor for detecting sugarcane smut pathogens as claimed in claim 1, characterized in that: The self-powered sensor is based on a gold nano-modified graphene composite Au / N-GDY, carbon cloth, DNA nanostructure and glucose oxidase to construct the positive and negative poles of the self-powered biosensor.

3. The method according to claim 2, characterized in that The preparation of the bioanode comprises: taking 30-50 μL Au / N-GDY and dropping it on the surface of the carbon cloth electrode, drying it at 35-40° C. for 2-4 hours, dropping it on 20-50 μL 3-5 mg / mL glucose oxidase and incubating it for 12-24 hours, washing it with water, and then dropping 0.1-2 mmol / L, 20-50 μL bovine serum albumin, leaving it to react and washing it with water to obtain the bioanode.

4. The method according to claim 2, characterized in that: The preparation of the biocathode comprises: (1) 30-50 μL Au / N-GDY was drop-coated on 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 with water, and then drop-coated with 10-30 μL Truncated cone, placed at 3-6°C for 10-15 h, and 20-50 μL 0.5-2 mM hexamercaptoethanol was added to react for 0.5-1 h, and the excess hexamercaptoethanol was removed by washing; (2) 20 to 50 μL of the hybridization chain reaction product is dropped onto the electrode surface and incubated at 35 to 40° C. for 1 to 2 hours, followed by washing to obtain a biocathode.

5. The method according to claim 4, characterized in that The preparation of the Truncated cone in step (1) includes mixing 0.5-2 μM, 80-100 μL of each of S1, S2 and S3, heating in a water bath to 90-95° C. and maintaining the mixture for 1-30 minutes.

6. The method according to claim 4, characterized in that The preparation of the hybridization chain reaction product in step (2) comprises: 1) Preparation of DNAzyme product: 10-30 μL of LS1 and Locker were mixed, and the mixture was reacted at 35-40°C for 1-2 hours to obtain a swing chain; 40-70 μL of 5 mg / mL activated magnetic beads were mixed with 1 μM, 40-70 μL of LS2 and 10-20 μL of the swing chain, and the mixture was shaken at 35-40°C for 12-24 hours, and the mixture was adsorbed by a magnet and dispersed in 100-200 μL of PBS; 10-30 μL of pathogens of different concentrations were mixed with 40-70 μL of magnetic beads PBS, and 10-30 μL of 5 mM Mn 2+ , react at 35-40℃ for 1-2h, take the supernatant to obtain the DNAzyme product output DNA; Step 2. Preparation of hybridization chain reaction product: Mix 0.5-2 μM, 40-70 μL each of H1 and H2 with 40-70 μL of output DNA, and incubate at 35-40°C for 1-3 hours to obtain a hybridization chain reaction product, output DNA / H1 / H2.

7. The method according to any one of claims 2 to 6, characterized in that: The preparation of Au / N-GDY includes: 1) Preparation of GDY: tetramethylethylenediamine, pyridine and acetone were mixed and added to a copper plate. Under an argon atmosphere at -5-5°C, tetrabutylammonium fluoride was added to a HEB-TMS dispersion in a THF solution, and after purification, HEB was dissolved in a pyridine solution as a precursor solution for a coupling reaction; under an argon atmosphere at 60-80°C, the precursor solution was added to a mixed solution of tetramethylethylenediamine, pyridine and acetone, and heated in the dark for reaction; after the reaction was completed, the GDY film adhered to the copper plate was extracted, washed, and dried to obtain powdered GDY; 2) Preparation method of AuNPs: HAuCl4 solution and water are mixed and heated for reaction, sodium citrate is added under stirring, and heating is continued after the solution color changes to wine red. After stopping heating, stirring is continued in the dark and cooled to room temperature to obtain AuNPs; 3) Preparation of N-GDY: GDY powder and melamine are mixed in water for hydrothermal reaction, and then freeze-dried after cooling to room temperature to obtain precursor GDY. Argon gas is passed through a tube furnace, and the precursor GDY reacts at 700-1000°C, and N-GDY is obtained after cooling; 4) Preparation of Au / N-GDY: Grind N-GDY into fine powder, add acetic acid and AuNPs to N-GDY for ultrasonic reaction to obtain Au / N-GDY.

8. The method according to any one of claim 7, characterized in that: In step 1), the volume ratio of tetramethylethylenediamine, pyridine and acetone is 1-2:5-10:70-120; the washing is washing with hot pyridine, DMF, HCl, ethanol and water in sequence; Step 2) The mass concentration of HAuCl4 solution is 1%, and the volume ratio of the mixture with water is 0.4-0.7:40-70; the mass concentration of sodium citrate is 2%, and the volume ratio of the mixture with HAuCl4 solution is 4-6:4-7; Step 3) The mass ratio of GDY powder to melamine is 8-12:10-30; Step 4) The mass and volume ratio of N-GDY, acetic acid and AuNPs mixed is 1-2 mg: 3-6 mL: 6-12 mL.

9. An application of a self-powered sensor as claimed in any one of claims 1 to 8, characterized in that: The self-powered sensor is used to detect smut pathogens.

10. A method for detecting sugarcane smut pathogens using a self-powered sensor as claimed in any one of claims 1 to 9, characterized in that: The method comprises the following steps: (1) When the sugarcane smut pathogen is not introduced into the system, the instantaneous current of the sensor is measured; (2) After the sugarcane smut pathogen was introduced into the system, the instantaneous current of the sensor was measured at different concentrations of the sugarcane smut pathogen; Among them, the membraneless glucose / [Ru(NH3)6] 3+ The supporting electrolyte of the sensor contains 5mM glucose, 500μM [Ru(NH3)6] 3+ 0.01 M PBS buffer system, pH 7.4.

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