Sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachine, application and detection method

Abstract

The application discloses a sugarcane disease detection biosensing platform based on an entropy-driven DNA nanomachine, application and detection method, which comprises an optical-thermal detection unit Cu2O@Au-DNA of entropy-driven DNA nanomachine signal amplification, an electrochemical / colorimetric detection unit of entropy-driven DNA nanomachine signal amplification, the electrochemical / colorimetric detection unit of entropy-driven DNA nanomachine signal amplification comprises magnetic substrate MB-S4 / S3, biological anode CoV-MOF / AuNPs / GOD, biological cathode CoV-MOF / AuNPs / DNA chain and electrolyte; the electrolyte comprises glucose, methylene blue and PBS buffer solution. The biosensing platform can analyze through three non-interfering modes of electrochemistry, colorimetry and photothermal detection, can effectively verify whether the detection result is a false positive signal, and realizes accurate detection of sugarcane top rot.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to a biosensor platform, application, and detection method for detecting sugarcane diseases based on entropy-driven DNA nanomachines. Background Technology

[0002] Biosensors, developed based on nanocomposite electrode technology, work by receiving or releasing electrons to generate a current signal when the target substance comes into contact with the electrode interface. These sensors are environmentally friendly, as the fuels and catalytic enzymes used can be directly derived from living organisms. Because they can provide the energy required for detection themselves, biosensors are easily miniaturized and portable, and can be deployed in various locations. Currently, this technology has been applied in environmental monitoring, clinical medicine, and the detection of toxic substances.

[0003] Sugarcane top rot is a significant fungal disease that severely impacts sugarcane production. It primarily infects young sugarcane shoots, causing leaf rot and even plant death, resulting in a significant decrease in yield and sugar content for sugarcane varieties. Sugarcane top rot is widespread in my country, affecting almost all sugarcane varieties. Infected varieties can have an incidence rate exceeding 38%, leading to yield reductions of 5%-20% and a decrease in sugar content of up to 3%. Sugarcane top rot is a leaf disease caused by various Fusarium species. Furthermore, top rot is one of the most significant diseases leading to reduced sugarcane yield, variety degeneration, and lower raw material quality. Early detection of the pathogen's infection process and timely intervention are crucial. Currently used detection equipment is expensive and complex to operate, making large-scale deployment in production difficult. Moreover, relying on a single signal for detection results in low accuracy and a risk of false positives. Summary of the Invention

[0004] The main objective of this invention is to provide a sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines, aiming to solve the problems of existing detection equipment using a single signal as the detection basis, resulting in low accuracy and the risk of false positive characterization.

[0005] To achieve the above objectives, the present invention proposes a sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines, comprising: a photothermal detection unit Cu2O@Au-DNA with entropy-driven DNA nanomachine signal amplification, and an electrochemical / colorimetric detection unit with entropy-driven DNA nanomachine signal amplification. The electrochemical / colorimetric detection unit with entropy-driven DNA nanomachine signal amplification includes an anode, a cathode, and an electrolyte; wherein the anode is a bioanode CoV-MOF / AuNPs / GOD, and the cathode is a biocathode CoV-MOF / AuNPs / DNA strand; the electrolyte includes glucose, methylene blue, and PBS buffer solution.

[0006] Furthermore, the photothermal detection unit Cu2O@Au-DNA includes Cu2O@Au-S1 and Cu2O@Au-S2, and the preparation method of both includes the following steps:

[0007] Cu2O@Au was assembled with a DNA strand S1 with thiol-modified surface to obtain Cu2O@Au-S1; Cu2O@Au was assembled with a DNA strand S2 with thiol-modified surface to obtain Cu2O@Au-S2.

[0008] Furthermore, the preparation method of the bioanolyte CoV-MOF / AuNPs / GOD includes the following steps:

[0009] Take 30-50µL of CoV-MOF / AuNPs and drop it onto the surface of the carbon cloth electrode. Dry it at 35-40℃ for 2-4 h. Then drop it onto 30-50µL of 3-5 mg / mL GOD and incubate for 10-14 h. After washing with water, the bioanod CoV-MOF / AuNPs / GOD is obtained.

[0010] The method for preparing the biocathode CoV-MOF / AuNPs / DNA strand includes the following steps:

[0011] 1.1) 30-50 µL of CoV-MOF / AuNPs was dropped onto the surface of a carbon cloth electrode and dried at 35-40 °C for 2-4 h. Then, 10-30 µL of gene signal receiving probe H1 was dropped onto the electrode and placed at 3-6 °C for 11-14 h. 20-50 µL of 0.5-2 mM MCH was added and reacted for 15-45 min. Excess MCH was washed away.

[0012] 1.2) 20-30µL of MB-S4 / S3 was drop-coated onto the electrode surface and incubated at 37°C to obtain the biocathode CoV-MOF / AuNPs / DNA strand.

[0013] Furthermore, the preparation method of Cu2O@Au includes the following steps:

[0014] Take 1-5 mg of dried Cu2O powder and add 10-20 mL of AuNPs into a centrifuge tube; after ultrasonic mixing for 20-30 min, obtain the Cu2O@Au complex and store it in the dark at 2-4℃.

[0015] Furthermore, the preparation method of the MB-S4 / S3 includes the following steps:

[0016] Take 20-30 µL of MB and add 30-40 µL of S3, shake on a shaker at 37℃ for 2-4 h, then add 30-40 µL of Cu2O@Au-S2 and Cu2O@Au-S1 and react for 1-2 h; add 30-40 µL of target substances of different concentrations and react at 37℃ for 1-2 h to obtain MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1; then add 30-40 µL of S4 to MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1 and react at 37℃ for 1-2 h to obtain MB-S4 / S3.

[0017] Furthermore, the preparation method of the CoV-MOF / AuNPs includes the following steps:

[0018] 2.1) Preparation of CoV-MOF: 0.6-0.8 g of polyvinylpyrrolidone was dissolved in 30-40 mL of ethylene glycol, followed by the addition of 0.25-0.27 g of VO(acac)2 and 0.6-0.8 g of Co(NO3)2·6H2O. After stirring and mixing thoroughly for 0.5-1 h, the solution was transferred to a PTFE-lined reactor and heated at 180-200 °C for 6-9 h. After the reaction was completed, the obtained brown product was centrifuged and washed repeatedly with deionized water and ethanol to remove impurities. The product was then dried at 50-70 °C for 12-24 h to obtain CoV-MOF.

[0019] 2.2) Preparation of CoV-MOF / AuNPs: Take 1-5 mg of dried CoV-MOF powder, add 10-20 mg of AuNPs and CoV-MOF powder together in a centrifuge tube, mix with sonication to obtain Au@CoV-MOF complex, and store in the dark at 2-4℃.

[0020] Furthermore, the preparation method of the AuNPs includes the following steps:

[0021] HAuCl4 solution and water were mixed and heated. Sodium citrate was added while stirring. After the solution turned wine red, heating was continued. After heating was stopped, the mixture was stirred and cooled in the dark to obtain AuNPs.

[0022] The second objective of this invention is to provide an application of a sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines, which is used to detect whether sugarcane carries top rot pathogens.

[0023] The third objective of this invention is to provide a method for detecting sugarcane top rot pathogens using a sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines, comprising the following detection steps:

[0024] The target compound and S4 were successively added to the entropy-driven nanomachine precursors MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1 obtained based on the photothermal detection unit Cu2O@Au-DNA, MB, and S3. Photothermal detection was performed in the 808 nm near-infrared environment by separating the supernatant using MB magnetic separation. The magnetic substrates MB-S4 / S3 were used to prepare a biocathode for electrochemical / colorimetric detection.

[0025] Furthermore, it also includes the following steps:

[0026] 3.1) After adding different concentrations of specific genes of the shoot rot pathogen and S4 to MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1, the supernatant of MB magnetic separation was taken for photothermal detection, and a linear regression curve was plotted. The linear equation was T = 5.38lg c + 98.05, R 2 = 0.992, detection limit is 77.02 fmol / L;

[0027] 3.2) For the sensor constructed based on MB-S4 / S3 obtained by introducing specific genes of the pathogen of shoot rot at different concentrations, the electrolyte was used for colorimetric detection, and a linear regression curve was plotted. The linear equation was Blue = 11.86 lg c + 325.28, R 2 = 0.994, detection limit is 1.66 fmol / L;

[0028] 3.3) Instantaneous current tests were performed on sensors constructed using MB-S4 / S3 obtained from specific genes of the shoot rot pathogen at different concentrations, and linear regression curves were plotted. The linear equation was Y = 9.69 lg c + 144.01, R 2 = 0.995, detection limit is 4.51 fmol / L, signal-to-noise ratio is S / N=3.

[0029] The DNA strand sequence used in this invention is as follows (synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.):

[0030]

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) The biosensing platform of this invention employs an entropy-driven DNA nanomachine, combined with an entropy-driven DNA amplification strategy and metal nanomaterials, to amplify the signal of the target substance. Through the stepwise separation and amplification of the products by the entropy-driven DNA nanomachine, the individual outputs of Cu2O@Au-S2 and Cu2O@Au-S1 were successfully achieved, and photothermal testing was performed using the magnetically separated supernatant. The magnetic substrate MB-S4 / S3 forms a double-stranded structure with the gene probe H1 on the cathode, thereby generating a more reliable signal. In the presence of sugarcane top rot pathogens, the target material initiates an entropy-driven DNA nanomachine to generate Cu2O@Au-S1. Subsequently, S4 is introduced to further generate Cu2O@Au-S2. The supernatant obtained through MB magnetic separation is then subjected to photothermal detection under 808 nm infrared and near-infrared light irradiation. The MB-S4 / S3 obtained through magnetic separation undergoes base complementary pairing with the gene probe H1 on the cathode surface, adsorbing a large amount of methylene blue onto the biocathode, thereby generating a significant electrical signal. The resulting open-circuit voltage is positively correlated with the pathogen concentration. Furthermore, during this process, methylene blue is reduced to colorless MBH, and users can obtain the blue value in the RGB spectrum using a mobile phone for colorimetric detection. This invention has the advantages of high sensitivity, excellent selectivity, and real-time detection, enabling accurate detection of sugarcane top rot.

[0033] (2) This invention combines an entropy-driven DNA amplification strategy with metal nanomaterials. Cu2O@Au can effectively absorb light and convert it into heat. The entropy drives the DNA nanomachine and utilizes the entropy-driven effect to achieve rapid amplification. Electrochemical and colorimetric detection is achieved through the methylene blue redox process. The analysis is carried out in three non-interfering modes, which helps to eliminate environmental influences and uncontrollable factors, effectively improves the reliability and accuracy of the detection results, and can be connected to a smartphone using tools such as a multimeter and Bluetooth module to achieve real-time detection.

[0034] (3) The three-mode sensing platform can effectively overcome its limitations in accuracy, while saving the time cost required for verification results. The method is simple, easy to operate, low cost, high sensitivity, and high selectivity. The electrochemical, colorimetric and photothermal sensors described in this invention have advantages such as simple construction, high sensitivity, and high specificity, and have good applications in detecting sugarcane top rot.

[0035] (4) When the method of the present invention is used to detect sugarcane top rot, no external power supply is required, and real-time outdoor detection can be achieved. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0037] Figure 1 This is a schematic diagram of the detection process of the sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines of this invention;

[0038] Figure 2 This is a SEM image of the substrate material Au@CoV-MOF in the sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines of this invention;

[0039] Figure 3 The images shown are: CV diagram of the biocathode assembly (A), CV diagram of the presence or absence of the target on the biocathode (B), colorimetric image (C), and photothermal detection thermal imaging image (D) in the entropy-driven DNA nanomachine-based sugarcane disease detection biosensor platform of this invention.

[0040] Figure 4 This invention presents standard curves of instantaneous current, Blue value in RGB of electrolyte in colorimetric mode, and electrolyte temperature in photothermal mode, versus the concentration of specific genes of rot pathogens in a sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines. Detailed Implementation

[0041] 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, not all, of the embodiments of the present invention. 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.

[0042] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0043] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0044] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0045] To address the aforementioned problems, this invention provides a sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines, comprising: a photothermal detection unit Cu2O@Au-DNA with entropy-driven DNA nanomachine signal amplification, and an electrochemical / colorimetric detection unit with entropy-driven DNA nanomachine signal amplification. The electrochemical / colorimetric detection unit with entropy-driven DNA nanomachine signal amplification includes a magnetic substrate MB-S4 / S3, an anode, a cathode, and an electrolyte. The anode is a bioanode CoV-MOF / AuNPs / GOD, and the cathode is a biocathode CoV-MOF / AuNPs / DNA strand. The electrolyte includes glucose, methylene blue, and PBS buffer solution. The photothermal detection unit Cu2O@Au-DNA includes Cu2O@Au-S1 and Cu2O@Au-S2. The bioanode CoV-MOF / AuNPs / GOD and the biocathode CoV-MOF / AuNPs / DNA chain are prepared using CoV-MOF / AuNPs nanomaterials as the electrode substrate. Leveraging their excellent electron transfer capabilities, the electron conduction rate is accelerated, and the specific surface area is increased through CoV-MOF, increasing the amount of GOD attached. This results in a shorter response time and improved output intensity for the detection signal. Figure 2 As shown, CoV-MOF exhibits a spherical structure, and AuNPs adsorbed on the surface of CoV-MOF provide abundant binding sites for the effective adsorption of glucose oxidase (GOD) and DNA probes, thereby improving the sensor's reaction efficiency.

[0046] The principle behind this entropy-driven DNA nanomachine-based biosensor platform for detecting sugarcane disease pathogens is as follows: Figure 1 As shown:

[0047] When the pathogen of shoot rot is 0, only H1 is present on the electrode, which cannot initiate entropy-driven reactions. Cu2O@Au-DNA and MB-S4 / S3 cannot be produced, resulting in a small temperature change due to the photothermal effect. Consequently, less methylene blue is adsorbed on the biocathode, the electrical signal is weak, and the electrolyte color change is not significant. Figure 1As shown at point A, when the pathogen of shoot rot is added to the system, the target material initiates MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1 (i.e., entropy-driven DNA nanomachines). Entropy-driven action generates Cu2O@Au-S1, which is then replaced by S4 to generate Cu2O@Au-S2. The supernatant is separated to obtain the magnetic substrate MB-S4 / S3. Cu2O@Au-DNA exhibits significant temperature changes under 808 nm near-infrared light irradiation. In electrochemical detection, MB-S4 / S3 and the gene signal receiving probe H1 are complementary bases, leading to increased methylene blue adsorption at the biocathode, enhanced electrical signal, and an increase in the RGB Blue value. Figure 4 As shown, the instantaneous current, temperature value, and RGB Blue change value are positively correlated with the concentration of shoot rot pathogen, thus realizing the quantitative detection of shoot rot pathogen, and they do not interfere with each other and can mutually verify whether the experimental results are false positives.

[0048] like Figure 1 As shown, this invention provides a preparation process for a sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines, comprising the following steps:

[0049] Example 1

[0050] 1. Preparation of Cu2O@Au-DNA

[0051] ①Preparation of Cu₂O: Dissolve 1.5 g of sodium dodecyl sulfate (SDS) in 120 mL of pure water, then mix with 180 µL of 0.1 M CuCl₂·2H₂O. After stirring the mixture, add 2 mL of 0.2 M NH₂OH·HCl and 0.5 mL of 5 M NaOH. Then, gently stir the resulting mixture and let it stand for 35 min. The final product is centrifuged twice and washed with water to remove the surfactant.

[0052] ② Preparation of AuNPs: Mix 500 µL of 1% HAuCl4 solution with 50 mL of ultrapure water, heat in an oil bath to 120°C, then cool to 110°C, and quickly inject 500 µL of 2% sodium citrate solution in one go while 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.

[0053] ③Preparation of Cu2O@Au: Take 5 mg of dried Cu2O powder, add 10 mL of AuNPs into a centrifuge tube, and sonicate for 20 min to obtain Cu2O@Au complex, and store it in the dark at 3℃.

[0054] ④ Preparation of Cu2O@Au-DNA: Two 20 µL portions of Cu2O@Au were assembled with 40 µL of thiol-modified DNA strand S1 and 40 µL of thiol-modified DNA strand S2, respectively, and incubated at 37℃ for 4 h to obtain Cu2O@Au-S1 and Cu2O@Au-S2.

[0055] 2. Preparation of CoV-MOF / AuNPs

[0056] 2.1) Preparation of CoV-MOF: 0.8 g of polyvinylpyrrolidone (PVP) was dissolved in 40 mL of ethylene glycol (EG), followed by the addition of 0.27 g of VO(acac)2 and 0.8 g of Co(NO3)2·6H2O. After thorough mixing for 1 h, the solution was transferred to a PTFE (polytetrafluoroethylene) lined reactor and heated at 200 °C for 9 h. After the reaction was completed, the resulting brown product was centrifuged and washed repeatedly with deionized water and ethanol to remove impurities. Then, the washed product was dried at 70 °C for 12 h to obtain CoV-MOF.

[0057] 2.2) Preparation of CoV-MOF / AuNPs: Take 5 mg of dried CoV-MOF powder, add 10 mL of AuNPs (prepared by the same method as step 2 in the preparation of Cu2O@Au-DNA) and put them together with the CoV-MOF powder into a centrifuge tube. After ultrasonic mixing, Au@CoV-MOF complex is obtained and stored at 4°C in the dark.

[0058] 3. Preparation of bioanodes CoV-MOF / AuNPs / GOD

[0059] 50 µL of CoV-MOF / AuNPs was drop-coated onto the surface of a carbon cloth electrode and dried at 37 °C for 4 h. Then, 30 µL of 5 mg / mL GOD (glucose oxidase) was drop-coated onto the electrode and incubated for 12 h. After washing with water, the bioanolyte CoV-MOF / AuNPs / GOD was obtained and stored at 4 °C for later use. The assembly process is as follows: Figure 1 As shown at point B in the middle.

[0060] 4. Preparation of biocathode CoV-MOF / AuNPs / H1 / MB-S4 / S3

[0061] 1.1) Preparation of MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1: Take 30µL MB (i.e. Fe3O4) and add 40µL S3, shake on a shaker at 37℃ for 4 h, then add 40µL Cu2O@Au-S2 and 40µL Cu2O@Au-S1 and react for 1 h;

[0062] 1.1.1) Add 30 µL of different concentrations of target substances and react at 37 °C for 1 h, then collect the supernatant;

[0063] 1.1.2) Preparation of MB-S4 / S3: 40 µL of S4 was added to the supernatant obtained in step (1.1.1), and the mixture was reacted at 37 °C for 2 h to obtain the supernatant and magnetic substrate MB-S4 / S3. The supernatant was collected for photothermal detection (the supernatant contains displaced Cu2O@Au-S2 and Cu2O@Au-S1). Figure 1 The detection is shown at point C in the middle.

[0064] 1.2) Preparation of biocathode: 50 µL of 1 mg / mL CoV-MOF / AuNPs was dropped onto the surface of a 1 cm × 1 cm carbon cloth and dried at 37 °C for 3 h. Then, 20 µL of gene signal receiving probe H1 was added and reacted at 4 °C for 12 h to form Au-S chemical bonds between the SH bonds in gene signal receiving probe H1 and AuNPs in CoV-MOF / AuNPs. Subsequently, 30 µL of 1 mM MCH (i.e., hexamethylenetetramine alcohol) was added and reacted for 30 min to block the active sites (i.e., the addition of hexamethylenetetramine alcohol can occupy the active sites of AuNPs, preventing the H1 reaction from being too slow or the amount of H1 from being too small). After the reaction, excess MCH was washed away. Finally, 30 µL of MB-S4 / S3 was dropped onto the carbon cloth electrode with the gene signal receiving probe H1 introduced and incubated at 37 °C for 1.5 h to obtain the biocathode CoV-MOF / AuNPs / H1 / MB-S4 / S3. Its assembly process is as follows Figure 1 As shown at point B in the middle.

[0065] Example 2

[0066] 1. Preparation of Cu2O@Au-DNA

[0067] ①Preparation of Cu₂O: Dissolve 1.65 g of sodium dodecyl sulfate in 100 mL of pure water, then mix with 150 µL of 0.1 M CuCl₂·2H₂O. After stirring the mixture, add 3 mL of 0.2 M NH₂OH·HCl and 1 mL of 5 M NaOH. Then, gently stir the resulting mixture and let it stand for 40 min. The final product is centrifuged twice and washed with water to remove the surfactant.

[0068] ② Preparation of AuNPs: Mix 500 µL of 1% HAuCl4 solution with 50 mL of ultrapure water, heat in an oil bath to 120°C, then cool to 110°C, and quickly inject 500 µL of 2% sodium citrate solution in one go while 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.

[0069] ③Preparation of Cu2O@Au: Take 1 mg of dried Cu2O powder, add 15 mL of AuNPs to a centrifuge tube, and sonicate for 25 min to obtain Cu2O@Au complex, and store it in the dark at 2℃.

[0070] ④ Preparation of Cu2O@Au-DNA: Two 20µL portions of Cu2O@Au were assembled with 40 µL S1 and 40 µL S2, respectively, to obtain Cu2O@Au-S1 and Cu2O@Au-S2;

[0071] 2. Preparation of CoV-MOF / AuNPs

[0072] 2.1) Preparation of CoV-MOF: 0.6 g of polyvinylpyrrolidone was dissolved in 30 mL of ethylene glycol, followed by the addition of 0.25 g of VO(acac)2 and 0.6 g of Co(NO3)2·6H2O. After thorough mixing for 0.5 h, the solution was transferred to a PTFE-lined reactor and heated at 180 °C for 6 h. After the reaction was completed, the resulting brown product was centrifuged and washed repeatedly with deionized water and ethanol to remove impurities. The washed product was then dried at 50 °C for 18 h to obtain CoV-MOF.

[0073] 2.2) Preparation of CoV-MOF / AuNPs: Take 1 mg of dried CoV-MOF powder, add 15 mL of AuNPs and CoV-MOF powder together in a centrifuge tube, mix by sonication to obtain Au@CoV-MOF complex, and store at 2℃ in the dark.

[0074] 3. Preparation of bioanodes CoV-MOF / AuNPs / GOD

[0075] 30 µL of CoV-MOF / AuNPs was drop-coated onto the surface of a carbon cloth electrode and dried at 35 °C for 2 h. Then, 40 µL of 3 mg / mL GOD was drop-coated onto the electrode and incubated for 10 h. After washing with water, the bioanodide CoV-MOF / AuNPs / GOD was obtained and stored at 4 °C for later use.

[0076] 4. Preparation of biocathode CoV-MOF / AuNPs / H1 / MB-S4 / S3

[0077] 1.1) Preparation of MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1: Take 20µL MB and add 30µL S3 and shake on a shaker at 37℃ for 2h. Then add 30µL Cu2O@Au-S2 and 30µL Cu2O@Au-S1 and react for 1.5h.

[0078] 1.1.1) Add 35µL of different concentrations of target substances and react at 37℃ for 1.5 h, then collect the supernatant;

[0079] 1.1.2) Preparation of MB-S4 / S3: Add 30µL of S4 to the supernatant obtained in step (1.1.1) and react at 37℃ for 1h to obtain supernatant and magnetic substrate MB-S4 / S3. Collect the supernatant for photothermal detection.

[0080] 1.2) Preparation of biocathode: 30 µL of 1 mg / mL CoV-MOF / AuNPs was dropped onto the surface of a 1 cm × 1 cm carbon cloth and dried at 35 °C for 2 h. Then, 10 µL of gene signal receiving probe H1 was added and reacted at 3 °C for 11 h. Subsequently, 20 µL of 0.5 mM MCH was added and reacted for 15 min. After the reaction, excess MCH was washed away. Finally, 20 µL of LMB-S4 / S3 was dropped onto the carbon cloth electrode with the gene signal receiving probe H1 introduced above and incubated at 37 °C for 1.5 h to obtain biocathode CoV-MOF / AuNPs / H1 / MB-S4 / S3.

[0081] The rest is the same as in Example 1.

[0082] Example 3

[0083] 1. Preparation of Cu2O@Au-DNA

[0084] ①Preparation of Cu₂O: Dissolve 1.8 g of sodium dodecyl sulfate in 140 mL of pure water, then mix with 200 µL of 0.1 M CuCl₂·2H₂O. After stirring the mixture, add 4 mL of 0.2 M NH₂OH·HCl and 2 mL of 5 M NaOH. Then, gently stir the resulting mixture and let it stand for 45 min. The final product is centrifuged twice and washed with water to remove the surfactant.

[0085] ② Preparation of AuNPs: Mix 500 µL of 1% HAuCl4 solution with 50 mL of ultrapure water, heat in an oil bath to 120°C, then cool to 110°C, and quickly inject 500 µL of 2% sodium citrate solution in one go while 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.

[0086] ③Preparation of Cu2O@Au: Take 3 mg of dried Cu2O powder, add 20 mL of AuNPs to a centrifuge tube, and sonicate for 30 min to obtain Cu2O@Au complex, and store it in the dark at 4℃.

[0087] ④ Preparation of Cu2O@Au-DNA: Two 20µL portions of Cu2O@Au were assembled with 40 µL S1 and 40 µL S2, respectively, to obtain Cu2O@Au-S1 and Cu2O@Au-S2;

[0088] 2. Preparation of CoV-MOF / AuNPs

[0089] 2.1) Preparation of CoV-MOF: 0.7 g of polyvinylpyrrolidone was dissolved in 35 mL of ethylene glycol, followed by the addition of 0.26 g of VO(acac)2 and 0.7 g of Co(NO3)2·6H2O. After thorough mixing for 1 h, the solution was transferred to a PTFE-lined reactor and heated at 190 °C for 8 h. After the reaction was completed, the resulting brown product was centrifuged and washed repeatedly with deionized water and ethanol to remove impurities. The washed product was then dried at 60 °C for 24 h to obtain CoV-MOF.

[0090] 2.2) Preparation of CoV-MOF / AuNPs: Take 2.5 mg of dried CoV-MOF powder, add 20 mL of AuNPs and CoV-MOF powder together into a centrifuge tube, mix by sonication to obtain Au@CoV-MOF complex, and store at 3℃ in the dark.

[0091] 3. Preparation of bioanodes CoV-MOF / AuNPs / GOD

[0092] 40 µL of CoV-MOF / AuNPs was dropped onto the surface of a carbon cloth electrode and dried at 40 °C for 3 h. Then, 50 µL of 4 mg / mL GOD was dropped onto the electrode and incubated for 14 h. After washing with water, the bioanodide CoV-MOF / AuNPs / GOD was obtained and stored at 4 °C for later use.

[0093] 4. Preparation of biocathode CoV-MOF / AuNPs / H1 / MB-S4 / S3

[0094] 1.1) Preparation of MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1: Take 25µL MB and add 35µL S3 and shake on a shaker at 37℃ for 3h. Then add 35µL Cu2O@Au-S2 and 35µL Cu2O@Au-S1 and react for 2h.

[0095] 1.1.1) Add 40 µL of different concentrations of target substances and react at 37 °C for 2 h, then collect the supernatant;

[0096] 1.1.2) Preparation of MB-S4 / S3: Add 35µL of S4 to the supernatant obtained in step (1.1.1) and react at 37℃ for 1.5h to obtain supernatant and magnetic substrate MB-S4 / S3. Collect the supernatant for photothermal detection.

[0097] 1.2) Preparation of biocathode: 40 µL of 1 mg / mL CoV-MOF / AuNPs was dropped onto the surface of a 1 cm × 1 cm carbon cloth and dried at 40 °C for 4 h. Then, 30 µL of gene signal receiving probe H1 was added and reacted at 4 °C for 14 h. Subsequently, 50 µL of 2 mM MCH was added and reacted for 45 min. After the reaction, excess MCH was washed away. Finally, 25 µL of MB-S4 / S3 was dropped onto the carbon cloth electrode with the gene signal receiving probe H1 introduced above and incubated at 37 °C for 1.5 h to obtain the biocathode CoV-MOF / AuNPs / H1 / MB-S4 / S3.

[0098] The supernatant obtained in step 3 of the preparation of the biocathode in Examples 1 to 3 was placed in a 1.5 mL glass bottle and tested with 808 nm near-infrared light (power density of 1.5 W / cm²). 2 Irradiation was performed for 1 min, with a distance of 10 cm between the light source and the sample. Temperature signals were simultaneously acquired using a near-infrared imager, and sensors were constructed using the biocathodes and bioanodes obtained in Examples 1 and 3 to detect instantaneous current values. As shown in Table 1 below, the color of the sample showed a significant trend of lightening compared to the bare electrode (i.e., the blank item), and the electrical signal increased with rising temperature.

[0099] Table 1 shows the signal characterization data of the biosensors constructed using the examples and bare electrodes.

[0100]

[0101] This invention provides a sugarcane disease detection biosensor platform based on entropy-driven DNA nanomachines, applied to the detection and method of sugarcane top rot pathogens, comprising the following steps:

[0102] The target compound and S4 were successively added to the entropy-driven nanomachine precursors MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1 obtained based on the photothermal detection units Cu2O@Au-DNA, MB, and S3. Photothermal detection was performed in an 808 nm near-infrared environment by magnetic separation of the supernatant using MB. The magnetic substrates MB-S4 / S3 were used to prepare a biocathode for electrochemical / colorimetric detection, i.e., measuring the instantaneous current value, electrolyte temperature, and RGB Blue value, and comparing them with the corresponding values ​​of the sensing system without the introduction of the target shoot rot pathogen. By analyzing these signal changes, the correlation between pathogen concentration and signal was further analyzed.

[0103] Furthermore, the electrochemical, colorimetric, and photothermal characterization of this entropy-driven DNA nanomachine-based sugarcane disease detection biosensor platform is as follows: Cyclic voltammetry (CV) tests were performed to verify the reactivity of the sensor's biocathode. Figure 3 In section A, the CV curves of the biocathode assembly process are shown. Curve a is the CV curve of CoV-MOF / AuNPs; curve b is the CV curve of CoV-MOF / AuNPs modified with gene signal receiving probe H1. Compared with curve a, the peak current of curve b is significantly increased, indicating that the methylene blue electron probe has been successfully adsorbed onto the DNA molecular backbone; curve c is the CV curve of CoV-MOF / AuNPs after adding MB-S4 / S3 and H1 base complementary pairing. Its peak current is further increased compared with curve b, reaching the maximum value. Figure 3 The middle B area shows the CV test results with and without the target object. Curve b indicates that the current is smaller when the target object is not introduced, while curve a indicates that the current increases significantly after the target object is introduced, indicating that the combination of the target object enhances the current signal. Figure 3 The results in the middle C area are the test results in colorimetric mode. a is the color of the solution containing the target substance, and b is the color of the solution without the target substance. It is obvious that the solution containing the target substance is lighter in color (i.e., methylene blue is reduced to colorless MBH). Figure 3 The D region shows thermal images under photothermal mode. a is a thermal image of the solution containing the target, and b is a thermal image of the solution without the target. The results show that the temperature of the solution containing the target is higher than that of the solution without the target.

[0104] The performance characterization of this entropy-driven DNA nanomachine-based biosensor platform for sugarcane disease detection is shown below:

[0105] like Figure 4As shown at point A, the instantaneous current of the sensor constructed based on MB-S4 / S3 obtained by introducing specific genes of *Trichoderma truncatum* at different concentrations was measured. When the target substance was absent, only the gene signal receiving probe H1 was present on the electrode substrate. Since the target substance did not trigger the entropy-driven DNA nanomachine, the subsequent entropy-driven reaction was suppressed, resulting in no output of MB-S4 / S3. Consequently, the biocathode received fewer electrons, leading to a lower instantaneous current value for the biosensor. As the target substance concentration increased, the number of DNA strands loaded on the electrode substrate also increased, and the instantaneous current value of the biosensor gradually increased. The instantaneous current value showed a good linear relationship with the *Trichoderma truncatum* concentration in the range of 10 fM–10000 pM, with the linear equation Y = 9.69 lg c + 144.01 (R0)2. 2 = 0.995), and the detection limit is 4.51 fmol / L (S / N=3).

[0106] exist Figure 4 As shown at point B, the electrolyte of the MB-S4 / S3 sensor, constructed based on the introduction of specific genes from the shoot rot pathogen at different concentrations, was used for colorimetric detection. The Blue value in the RGB values ​​showed a good linear relationship with the shoot rot pathogen concentration in the range of 10 fM-10000 pM; that is, the higher the shoot rot pathogen concentration, the lighter the electrolyte color. The linear equation was Blue = 11.86 lg c + 325.28 (R0)2. 2 = 0.994), and the detection limit is 1.66 fmol / L.

[0107] exist Figure 4 In step C, different concentrations of specific genes of the shoot rot pathogen and S4 were added to MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1. The supernatant from the MB magnetic separation was then subjected to photothermal detection. A good linear relationship was observed between the temperature and the concentration of the shoot rot pathogen in the range of 100 fM to 10000 pM; that is, the higher the concentration of the shoot rot pathogen, the higher the electrolyte temperature. The linear equation was T = 5.38 lg c + 98.05 (R 2 = 0.992), and the detection limit is 77.02 fmol / L.

[0108] In summary, by combining an entropy-driven DNA amplification strategy with metal nanomaterials, Cu2O@Au can effectively absorb light and convert it into heat, thereby driving the DNA nanomachines and utilizing entropy-driven action to achieve rapid amplification. Furthermore, electrochemical and colorimetric detection are achieved through the methylene blue redox process. The analysis using three non-interfering modes helps eliminate environmental influences and uncontrollable factors, effectively improving the reliability and accuracy of the detection results. It can also be connected to smartphones using tools such as multimeters and Bluetooth modules for real-time detection. The three-mode sensing platform effectively overcomes its limitations in accuracy while saving time costs required for result verification. This method is simple, easy to operate, low-cost, highly sensitive, and highly selective. The electrochemical, colorimetric, and photothermal sensors described in this invention have advantages such as simple construction, high sensitivity, and high specificity, making them well-suited for detecting sugarcane top rot. When detecting sugarcane top rot using this method, no external power supply is required, enabling real-time outdoor detection. No specialized operating techniques are needed, the materials are widely available, the assembly process is simple, and accurate and rapid detection can be achieved.

[0109] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. An entropy-driven DNA nanomachine-based sugarcane disease detection biosensing platform, characterized in that, The application relates to an entropy-driven DNA nanomachine signal amplification photo-thermal detection unit Cu2O@Au-DNA, an entropy-driven DNA nanomachine signal amplification electrochemical / colorimetric detection unit, and a preparation method of the entropy-driven DNA nanomachine signal amplification electrochemical / colorimetric detection unit. The photo-thermal detection unit Cu2O@Au-DNA comprises Cu2O@Au-S1 and Cu2O@Au-S2, and the preparation method of the two comprises the following steps: Cu2O@Au is assembled with a thiol-modified DNA chain S1 to obtain Cu2O@Au-S1, and Cu2O@Au is assembled with a thiol-modified DNA chain S2 to obtain Cu2O@Au-S2; The preparation method of the biological anode CoV-MOF / AuNPs / GOD comprises the following steps: 30-50 muL of CoV-MOF / AuNPs is dropped on the surface of a carbon cloth electrode, dried at 35-40 DEG C for 2-4 h, 30-50 muL of 3-5 mg / mL GOD is then dropped and incubated for 10-14 h, and then the biological anode CoV-MOF / AuNPs / GOD is obtained after washing with water; The preparation method of the biological cathode CoV-MOF / AuNPs / DNA chain comprises the following steps: 1.1) 30-50 muL of CoV-MOF / AuNPs is dropped on the surface of a carbon cloth electrode, dried at 35-40 DEG C for 2-4 h, 10-30 muL of a gene signal receiving probe H1 is then dropped, placed at 3-6 DEG C for 11-14 h, 20-50 muL of hexamercaptohexanol with a concentration of 0.5-2 mM is added and reacted for 15-45 min, and then the excess hexamercaptohexanol is removed by washing; 1.2) 20-30 muL of a magnetic substrate MB-S4 / S3 is dropped on the surface of the electrode, incubated at 37 DEG C, and then the biological cathode CoV-MOF / AuNPs / DNA chain is obtained. ​ The gene sequence of S1 in the Cu2O@Au-S1 is 5'-SH-CGAAGAAGGAGGTTTAAAGAGGAGGTCACTG-3'; the gene sequence of S2 in the Cu2O@Au-S2 is 5'-CCAGGGATCATTACCGAGTTTACAACCC-3'; the gene sequence of H1 is 5'-SH-AATTGAATAAGCTGGTAAAA-3'; the gene sequence of S3 is 5'-NH2-ATCATTACCGAGTTTACAACAAGGGCAGTGACCTCCTCAGAAAAAATTTTT-3'; and the gene sequence of S4 is 5'-TCTGAGGAGGTCACTGCCCTTGTTGTAAACTCGGTAATGTACCCTTTTACC-3'.

2. The entropy-driven DNA nanomachine-based bio-sensing platform for sugarcane disease detection according to claim 1, wherein, The preparation method of the Cu2O@Au comprises the following steps: 1-5 mg of Cu2O powder dried is taken, 10-20 mL of AuNPs is added into a centrifuge tube, ultrasonic mixing is performed for 20-30 min, and then Cu2O@Au composite is obtained and stored in dark conditions at 2-4 DEG C.

3. The entropy-driven DNA nanomachine-based bio-sensing platform for sugarcane disease detection according to claim 1, wherein, The preparation method of the magnetic substrate MB-S4 / S3 comprises the following steps: 20-30 μL of magnetic beads (MB) is taken, 30-40 μL of S3 is added, shaking is performed at 37 DEG C for 2-4 h, 30-40 μL of Cu2O@Au-S2 and Cu2O@Au-S1 is added, and reaction is performed for 1-2 h; 30-40 μL of target objects of different concentrations is added, reaction is performed at 37 DEG C for 1-2 h, and then MB-S3 / Cu2O@Au-S2 / target objects and Cu2O@Au-S1 are obtained; then 30-40 μL of S4 is added to the MB-S3 / Cu2O@Au-S2 / target objects, reaction is performed at 37 DEG C for 1-2 h, and then the magnetic substrate MB-S4 / S3 is obtained.

4. The entropy-driven DNA nanomachine-based bio-sensing platform for sugarcane disease detection according to claim 1, wherein, The preparation method of the CoV-MOF / AuNPs comprises the following steps: 2.1) Preparation of CoV-MOF: 0.6-0.8 g of polyvinylpyrrolidone is dissolved in 30-40 mL of ethylene glycol, then 0.25-0.27 g of VO(acac)2 and 0.6-0.8 g of Co(NO3)2.6H2O are added, the mixture is fully stirred and mixed for 0.5-1 h, then the solution is transferred into a PTFE-lined reaction kettle, and heating is performed at 180-200 DEG C for 6-9 h; after the reaction is completed, the brown product obtained is subjected to centrifugal separation, and is washed with deionized water and ethanol for multiple times to remove impurities; and then the product is dried at 50-70 DEG C for 12-24 h, and CoV-MOF is obtained; 2.2) Preparation of CoV-MOF / AuNPs: Take 1-5 mg of dried CoV-MOF powder, add 10-20 mL of AuNPs to the CoV-MOF powder in a centrifuge tube, mix by ultrasonic, obtain Au@CoV-MOF composite, and store in the dark at 2-4°C.

5. The entropy-driven DNA nanomachine-based bio-sensing platform for sugarcane disease detection according to claim 2 or 4, wherein, The preparation method of the AuNPs comprises the following steps: Mix the HAuCl4 solution and water and heat, inject the sodium citrate solution under stirring, continue to heat after the solution color changes to wine red, stop heating, avoid light, stir and cool, and obtain the AuNPs.

6. Use of the sugarcane disease detection biosensing platform based on entropy-driven DNA nanomachines according to any one of claims 1 to 5, characterized in that, The application is for detecting whether sugarcane carries the pathogen of top rot disease.

7. A method for detecting the pathogen of sugarcane top wilt by using the sugarcane disease detection biosensor platform based on the entropy-driven DNA nanomachine according to any one of claims 1 to 5, characterized in that, The detection steps comprise: The target and S4 are added to the entropy-driven nanomachine precursor MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1 based on the photothermal detection unit Cu2O@Au-DNA and magnetic beads (MB) and S3, the supernatant is separated by magnetic beads (MB), and photothermal detection is carried out under 808 nm near-infrared light, and the magnetic substrate MB-S4 / S3 is used to prepare a biological cathode for electrochemical / colorimetric detection.

8. The method of detecting the sugarcane smudge pathogen by the entropy-driven DNA nanomachine-based sugarcane disease detection biosensor platform according to claim 7, characterized in that, The steps further comprise: 3.1) After adding different concentrations of the specific genes of the tip blight pathogen and S4 to MB-S3 / Cu2O@Au-S2 / Cu2O@Au-S1, the magnetic beads (MB) were magnetically separated from the supernatant for photothermal detection, and a linear regression curve was drawn, with a linear equation of T = 5.38lg c + 98.05, R 2 = 0.992, and a detection limit of 77.02 fmol / L; 3.2) The MB-S4 / S3 sensor based on the introduction of different concentrations of specific genes of the branch canker pathogen was detected by colorimetric detection of the electrolyte, and the linear regression curve was drawn, and the linear equation was Blue = 11.86 lg c+325.28, R 2 = 0.994, and the detection limit was 1.66 fmol / L; 3.3) The transient current test was carried out on the sensor constructed based on the MB-S4 / S3 obtained by introducing different concentrations of specific genes of the branch canker pathogen, and the linear regression curve was drawn, the linear equation was Y = 9.69 lg c + 144.01, R 2 = 0.995, the detection limit was 4.51 fmol / L, and the signal-to-noise ratio was S / N = 3.

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