Sugarcane smut dual-mode sensing platform and application thereof

Abstract

The invention discloses a sugarcane smut dual-mode sensing platform and application thereof, and relates to the technical field of biological detection and chemical analysis. The sensing platform comprises a three-chain DNA (Deoxyribose Nucleic Acid) and a CdTe QDs-NH2-F chain, the triplex DNA is a double strand consisting of an MB-A chain and an FAM-B chain; the MB-A chain is an A chain connected with magnetic beads; the FAM-B chain is a B chain which modifies a fluorophore and can form a hairpin structure; the CdTe QDs-NH2-F chain is an F chain which is aminated and is connected with the cadmium telluride quantum dots; a part of base sequence of the chain A is complementary to a base sequence of a sugarcane smut pathogenic bacterium gene; the base sequence of the chain F is complementary to the partial base sequence of the chain A, and the partial base sequence of the chain F is the same as the partial base sequence of the sugarcane smut pathogenic bacterium gene and the partial base sequence of the chain B. The sensing platform can sensitively amplify and efficiently amplify the genes of the pathogenic bacteria of the sugarcane smut, and can respectively obtain independent detection results of a photoelectrochemical detection mode and a fluorescence detection mode, so that the sensitivity and the accuracy of the detection results are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection and chemical analysis, and in particular to a sugarcane smut dual-mode sensing platform and application thereof. Background Art

[0002] As a fungal disease caused by sugarcane whip smut, sugarcane smut can cause serious damage such as stunted plant growth, reduced effective stems, increased fiber content, and reduced sucrose content. As one of the world's important sugar crops, the yield and quality of sugarcane directly affect the healthy and sustainable development of the sugar industry and the stable economic benefits of farmers. PCR technology is often used to detect sugarcane whip smut genes, but the equipment hardware investment and detection costs are relatively high, the operation process is relatively complicated, and highly professional analysts are required to perform experimental operations. It is not easy to promote and apply in remote areas such as towns and rural areas. Therefore, the development of a simple, fast, sensitive and accurate new method for detecting the pathogenic bacteria of sugarcane smut is crucial for early warning and precise management of sugarcane smut.

[0003] Among various detection modes, photoelectrochemical method and fluorescence method have the advantages of compact equipment and simple operation, but they have not been reported in the field of sugarcane smut detection. The above two detection modes are scientifically combined in the sensing platform to obtain independent gene content data respectively, and then the results of the two modes are mutually verified, which will effectively improve the accuracy and credibility of the detection results. Such dual-mode sensors have also not been reported in the field of sugarcane smut detection. In addition, in the early stage of sugarcane smut, the content of whip smut fungus genes is very low, and it is necessary to develop a simple and efficient amplification method to improve the sensitivity of the method. In previous reports on enzyme-free amplification technology, the reaction driving force of amplification technologies such as catalytic hairpin self-assembly and hybridization chain reaction comes from the enthalpy reduction reaction of the system, and there is also an entropy-driven strategy based on the entropy increase of the system to promote signal amplification. If the signal amplification system is rationally designed and the enthalpy reduction reaction and entropy increase reaction are integrated at the same time, the rate of the amplification reaction will be greatly accelerated, thereby improving the sensitivity of the sensing method. At present, no such amplification method that combines enthalpy reduction reaction and entropy increase reaction has been reported.

[0004] In summary, the object of the present invention is to provide a sugarcane smut dual-mode sensing platform and its application. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a dual-mode sensing platform for sugarcane smut and its application, which solves the problem that the detection accuracy and sensitivity of existing sensors are not high enough when the gene content of the pathogenic bacteria of sugarcane smut is low. The sensing platform of the present invention adopts an entropy-driven strategy with enthalpy reduction enhancement, which can sensitively and efficiently amplify the genes of the pathogenic bacteria of sugarcane smut, and can obtain independent detection results of the two detection modes of photoelectrochemical and fluorescence respectively. Through the new amplification technology and the mutual verification of the dual-mode detection results, the sensitivity and accuracy of the detection results can be made higher. The specific technical scheme is as follows:

[0006] A dual-mode sensing platform for sugarcane smut, the sensing platform is constructed based on an entropy-driven strategy with enthalpy reduction enhancement, and the sensing platform comprises: triplex DNA and CdTe QDs-NH 2 -F chain;

[0007] The triplex DNA is a double strand composed of an MB-A strand and a FAM-B strand; the MB-A strand is an A strand connected to magnetic beads, the A strand is a single strand, and the base sequence of the A strand is shown in SEQ ID NO.1; the FAM-B strand is a B strand modified with a fluorescent group, the B strand is a DNA strand that can form a hairpin structure, and the base sequence of the B strand is shown in SEQ ID NO.2;

[0008] The CdTe QDs-NH 2 - The F chain is an amino-linked F chain connected to cadmium telluride quantum dots, the F chain is a single chain, and the base sequence of the F chain is shown in SEQ ID NO.3;

[0009] The partial base sequence of the A chain is complementary to the base sequence of a gene of a sugarcane smut pathogen, and the base sequence of the gene of the sugarcane smut pathogen is shown in SEQ ID NO.4; the base sequence of the F chain is complementary to the partial base sequence of the A chain, and the partial base sequence of the F chain is identical to the partial base sequence of the gene of the sugarcane smut pathogen and the partial base sequence of the B chain.

[0010] Furthermore, the preparation method of the triplex DNA is as follows: firstly, FAM-B is annealed in a 95°C water bath for 5 min, and then MB-A (6 μM, 20 μL) and FAM-B (6 μM, 40 μL) are mixed in TE buffer (20 mM Tris, 100 mM NaCl, 50 mM MgCl 2 , pH 7.4), and reacted at 35-37°C on a shaking platform for 9-12h, and finally hybridized to form the triplex DNA with a concentration of 1-3 μM, which was stored at 4°C for future use.

[0011] Furthermore, the preparation method of the MB-A chain is as follows: firstly, the MB (magnetic beads) are fully shaken and mixed, and then PBS (Na + The cells were washed three times with a mixture of 10 mM EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 20 mM NHs (N-hydroxysuccinimide) at 35-37 °C for 1-2 h. After the reaction, the cells were washed three times with PBS to obtain a concentration of 5 mg mL -1 The MB solution is the activated MB solution. Add 80 μL of 10 μM activated A chain to 50 μL of activated MB solution, mix, and shake at 35-37°C for 9-12 hours.

[0012] Furthermore, the CdTe QDs-NH 2 The method for preparing the -F chain comprises the following steps:

[0013] (1) Synthesis of CdTe QDs (cadmium telluride quantum dots): First, take 5 mmol·L -1 CdCl 2 2.5H 2 O and 8.5mmol·L -1 MPA (3-methylacrylic acid) solution was added to a three-necked flask and 1 mol·L -1 Adjust the pH to 11-12 with NaOH and pass N 2 Deoxygenate for 30 min, then add 120 mg NaBH 4 and 0.5mmol·L -1 NaTeO 3 , in N 2 Under the protection of , the mixture was heated under reflux at 100 °C for 6 h to obtain MPA-coated CdTe QDs, and then anhydrous ethanol was added, and the solid at the bottom was collected by centrifugation at a speed of 12000 rpm, and brown CdTe QDs powder was obtained by vacuum drying, and finally purified with acetone to obtain;

[0014] (2) Preparation of amino CdTe QDs: 2 mL of APTES (3-aminopropyltriethoxysilane) was dispersed in 20 mL of anhydrous ethanol under stirring, and 1 mol·L -1 The pH value of the HCl solution was adjusted to 5-8, and then CdTe QDs were added. After mixing and reacting for 30 minutes, the mixture was centrifuged at 10000 rpm for 5 minutes and washed to obtain CdTe QDs-NH 2 ;

[0015] (3) CdTe QDs-NH 2Preparation of -F chain: dilute the activated F chain to 2 μM with TE buffer containing TCEP (tris(2-carboxyethyl)phosphine hydrochloride), and react on a shaking table at 35-37°C for 1-2 h to obtain the treated F chain. Then, 200 μL of the treated F chain and 0.2 mg CdTe QDs-NH 2 Mix and prepare 2 mg·mL -1 The concentration was determined by shaking at 35-37°C for 9-12 hours and then stored at 4°C.

[0016] Furthermore, the A chain, B chain and F chain are pretreated before use. The pretreatment method is: after centrifuging the A chain, B chain and F chain respectively, they are dissolved in 1×TE Buffer to prepare a solution with a concentration of 100 μM, and stored at -20°C for use.

[0017] The present invention also provides an application of the sugarcane smut dual-mode sensing platform, which is used to detect whether a target gene is a gene of a sugarcane smut pathogen.

[0018] Furthermore, the detection method comprises the following steps:

[0019] S1. Pre-process the target gene T (Target, referred to as T);

[0020] S2. Mix 200 μL triplex DNA, CdTe QDs-NH 2 -F chain (2μM, 200μL) and target gene T (50μL, 10nM) pretreated in step S1 were mixed, reacted at 35-37°C for 100-120min, and then attracted by a magnet to separate the supernatant and the precipitate. The supernatant was used for fluorescence intensity measurement, and the precipitate attracted by the lower magnet was used for photoelectric signal measurement; and the reaction time was optimized for 30min, 60min, 90min, 120min, 150min, and 180min;

[0021] S3. The supernatant was subjected to fluorescence intensity measurement: the fluorescence excitation wavelength was 430 nm, the recording wavelength range was set to 450-700 nm, the fluorescence enhancement rate was monitored every 30 s, the slits were set at 5 nm, and the changes in the fluorescence signal were detected;

[0022] S4. Detecting the photoelectric signal of the precipitate: Re-disperse the precipitate in 200 μL PBS, mix well, take 25-35 μL and drop it onto the FePc / ITO electrode, dry it, and detect the change of the photoelectric signal;

[0023] S5. Determine whether the target gene T is a gene of the pathogenic bacteria of sugarcane smut: When the fluorescence signal is enhanced in step S3 and the polarity of the photoelectric signal is reversed in step S4, it is proved that the target gene T is a gene of the pathogenic bacteria of sugarcane smut; otherwise, it is proved that the target gene T is not a gene of the pathogenic bacteria of sugarcane smut.

[0024] Furthermore, in step S1, the pretreatment method of the target gene T is the same as the pretreatment method of the A chain, B chain and F chain.

[0025] Furthermore, in step S3, the detection volume of the supernatant is 50-100 μL.

[0026] Furthermore, in step S4, the preparation method of the FePc / ITO electrode is: dissolving FePc (photoelectric material iron phthalocyanine) in PBS, ultrasonically treating it, dropping 20-30 μL onto the ITO electrode, and drying it at room temperature.

[0027] The DNA sequence used in the present invention is as follows:

[0028]

[0029]

[0030] Note: The gene sequence of the sugarcane smut pathogen is a known base sequence found in the gene bank.

[0031] The principle of the sensing platform of the present invention is as follows Figure 1 As shown, when the target gene T chain is added to the triplex DNA and CdTe QDs-NH 2 In the mixed solution of A-F chain, the reaction begins. The T chain binds to the unpaired part at the 5' end of the A chain in the triplex DNA and gradually replaces the first B chain to the right. When the reaction proceeds to a certain extent, the B chain forms a hairpin structure and quickly detaches from the A chain, exposing the bases in the middle part of the A chain. Using this as a binding site, the F chain gradually complementarily pairs with the A chain, thereby replacing the T chain and the second B chain to form a double-stranded product CdTe QDs-NH 2 -F / MB-A. The T chain separated from the A chain continues to participate in the cycle reaction, sensitively amplifying and efficiently amplifying the target gene. Finally, magnetic separation is performed in a centrifuge tube, and the reaction product CdTe QDs-NH 2-F / MB-A contains magnetic beads, which become a precipitate attracted by a magnet and are used to detect photoelectric signals, while the FAM-B chain enriched in the supernatant will be used to detect the fluorescence intensity. The driving force of the chain mixing reaction comes from the entropy increase of the system, that is, a double chain and two single chains react to generate a double chain and three single chains, and the disorder of the system increases by ΔS>0; on the other hand, it comes from the enthalpy reduction reaction caused by the formation of the B chain hairpin structure, and the bonding exothermic ΔH<0. According to the change in the Gibbs free energy of the reaction, the chain mixing reaction can proceed spontaneously and rapidly, thereby achieving sensitive amplification and efficient amplification of the target gene. Based on the above-mentioned entropy-reduced enhanced entropy-driven strategy, the sensing platform of the present invention can be successfully constructed.

[0032] When the target gene T is the gene of the pathogenic bacteria of sugarcane smut, the T chain can replace the first B chain in the triplex DNA, thereby exposing the binding site on the A chain, creating conditions for the binding of the F chain and the subsequent replacement reaction of the T chain and the second B chain, thereby bringing about signal changes in the photoelectrochemical mode and the fluorescence mode. On the contrary, when the target gene T is not the gene of the pathogenic bacteria of sugarcane smut, the T chain cannot replace the first B chain in the triplex DNA, and the subsequent reaction cannot proceed, resulting in no change in the photoelectric signal and the fluorescence signal.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The present invention proposes for the first time an entropy-driven strategy of enthalpy reduction enhancement, which combines the advantages of enthalpy reduction reaction and entropy increase reaction, and performs sensitive and efficient amplification of genes of sugarcane smut pathogens. At the same time, the two detection modes of photoelectrochemistry and fluorescence are combined into a dual-mode sensing platform for the first time, and the two modes of independent detection results are mutually verified to be used for rapid and accurate detection of sugarcane smut. The sensing platform designed by the present invention has the technical advantages of being simple, fast, sensitive and accurate, and is suitable for early warning and precise management of sugarcane smut. In addition, the relevant signal amplification strategy and detection mode design have broad application prospects in the fields of agronomy, medicine, etc.

[0035] 2. The present invention cleverly superimposes the enthalpy reduction reaction caused by the formation of the B-chain hairpin structure into the entropy-driven strategy of increasing system disorder, thereby increasing the rate of the chain mixing reaction, and through the cyclic reaction of the target gene T, ultimately achieving sensitive amplification and efficient amplification of the target gene, solving the problem that the existing sensor has insufficient detection sensitivity when the gene content of the sugarcane smut pathogen is low.

[0036] 3. The present invention detects the precipitate and supernatant attracted by the magnet respectively, and obtains independent detection results of the two detection modes of photoelectrochemical and fluorescence. The detection results of the two modes verify each other, which can make the detection results more accurate and reliable, and solve the problem that the detection accuracy of the existing sensor is not high enough when the gene content of the pathogenic bacteria of sugarcane smut is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0038] Figure 1 This is a schematic diagram of the dual-mode sensing platform for sugarcane smut of the present invention;

[0039] Figure 2 The schematic diagram of the photoelectric detection principle of the present invention: (A) the photoelectric reaction mechanism of FePc, (B) the photoelectric reaction mechanism of FePc combined with CdTe QDs;

[0040] Figure 3 Characterization of CdTe QDs of the present invention: (A) TEM image, (B) HRTEM image, (C) XPS image;

[0041] Figure 4 The feasibility analysis of the dual-mode sensing platform for sugarcane smut of the present invention is as follows: (A) Fluorescence intensity change of triplex DNA: a is before reaction, b is after reaction; (B) Fluorescence intensity change after chain mixing reaction: a is before reaction, b is after reaction; (C) Photoelectric signal change after chain mixing reaction: a is FePc / ITO, b is CdTe QDs-NH 2 -F / MB-A / FePc / ITO;

[0042] Figure 5 The optimization of triplex DNA preparation in the present invention: (A) af is the fluorescence intensity change curve of triplex DNA reaction from 3 to 18 hours, (B) is the fluorescence intensity bar graph of triplex DNA preparation;

[0043] Figure 6 For the optimization of the reaction in the present invention: (A) af is the fluorescence intensity change curve of the reaction 30-180 min, (B) a is the FePc / ITO photoelectric signal detection, bg are CdTe QDs-NH 2 -F / MB-A / FePc / ITO photoelectric signal detection;

[0044] Figure 7 The standard curves for detecting targets of different concentrations in the present invention are: (A) fluorescence intensity detection standard curve, (B) photoelectric signal detection standard curve. DETAILED DESCRIPTION

[0045] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0046] Example 1

[0047] In this embodiment, a dual-mode sensing platform for sugarcane smut includes: triplex DNA and CdTe QDs-NH 2 -F chain;

[0048] The triplex DNA is a double strand consisting of an MB-A strand and a FAM-B strand; the MB-A strand is an A strand connected to magnetic beads, the A strand is a single strand, and the base sequence of the A strand is shown in SEQ ID NO.1 (SEQ ID NO.1: NH 2 -CGC TCT GGT TCA TCA ACG AGG GGG TTC ATC AAC GAG GG); the FAM-B chain is a B chain modified with a fluorescent group, the B chain is a DNA chain that can form a hairpin structure, and the base sequence of the B chain is shown in SEQ ID NO.2 (SEQ ID NO.2: FAM-AGT TCA ATCATA CCC TCG TTG ATG AAC C);

[0049] The CdTe QDs-NH 2 -F chain is an F chain that is amino-linked and connected to cadmium telluride quantum dots, the F chain is a single chain, and the base sequence of the F chain is shown in SEQ ID NO.3 (SEQ ID NO.3: COOH-CCC TCG TTG ATG AACCCC CTC GTT GAT GAA CC);

[0050] The partial base sequence of the A chain is complementary to the base sequence of a gene of a sugarcane smut pathogen, and the base sequence of the gene of the sugarcane smut pathogen is shown in SEQ ID NO.4 (SEQ ID NO.4: CGTTGATGAACCAGAGCG); the base sequence of the F chain is complementary to the partial base sequence of the A chain, and the partial base sequence of the F chain is identical to the partial base sequence of the gene of the sugarcane smut pathogen and the partial base sequence of the B chain.

[0051] The steps for constructing the photoelectrochemical and fluorescence dual-mode sensitive detection of sugarcane smut pathogen genes based on the entropy-driven strategy of enthalpy reduction enhancement are as follows:

[0052] (1) DNA sample pretreatment: All DNA samples in the experiment (including target DNA, A chain, B chain and F chain) were centrifuged in a centrifuge for 1 min at 4000 rpm to prevent the loss of some DNA samples remaining on the wall of the centrifuge tube. Finally, the centrifuged samples were dissolved in 1×TE Buffer to a 100 μM stock solution (stored at -20°C).

[0053] (2) Preparation of MB-A chain: First, the MB was thoroughly shaken and mixed, washed three times with PBS, mixed with 10 mM EDC and 20 mM NHs, and reacted on a shaker at 37°C for 1 h. After the reaction, it was washed three times with PBS to obtain a concentration of 5 mg mL -1 The MB solution is the activated MB solution. Take 50 μL of the activated MB solution, add 80 μL of 10 μM activated A chain to it, mix, and react on a shaking table at 37°C for 12 hours.

[0054] (3) Preparation of FAM-B: It was purchased from Shanghai Zhenghe Biotechnology Co., Ltd.

[0055] (4) CdTe QDs-NH 2 - Preparation of F chain:

[0056] Synthesis of CdTe QDs: First take CdCl 2 2.5H 2 O and MPA solution were added to a three-necked flask, the pH was adjusted to 12, and N 2 Deoxygenation, followed by the addition of NaBH 4 and NaTeO 3 , in N 2 The mixture was heated under reflux under the protection of , and MPA-coated CdTe QDs were obtained. Anhydrous ethanol was added and centrifuged, and the solid at the bottom was collected and vacuum dried to obtain brown CdTe QDs powder, which was finally purified with acetone.

[0057] Preparation of amino CdTe QDs: Disperse 2 mL of APTES in 20 mL of anhydrous ethanol with 1 mol·L -1 The pH value of the HCl solution was adjusted to 7. After stirring, a certain amount of the synthesized CdTe QDs was added and reacted for 30 min. Finally, CdTe QDs-NH 2 .

[0058] CdTe QDs-NH 2 Preparation of -F chain: Dilute F chain to 2 μM with TE buffer containing TCEP and react on a shaker at 37°C for 1 h. Mix 200 μL of F chain and 0.2 mg of CdTe QDs-NH 2 Mix to make 2mg mL -1 The concentration was 100 μg / mL and the reaction was incubated at 37 °C for 12 h on a shaking platform and stored at 4 °C.

[0059] (5) Preparation of triplex DNA: FAM-B was first annealed in a 95°C water bath for 5 min, and then MB-A (6 μM, 20 μL) and FAM-B (6 μM, 40 μL) were mixed in TE buffer (20 mM Tris, 100 mM NaCl, 50 mM MgCl 2 , pH 7.4), and incubated at 37°C for 12 h on a shaking platform to form triplex DNA at a concentration of 2 μM, which was then stored at 4°C for later use.

[0060] (6) Detection of target DNA: 200 μL triplex DNA, target DNA (50 μL, 10 nM) and CdTe QDs-NH 2 -F chain (2μM, 200μL) was mixed and reacted at 37°C for 120min. The supernatant and precipitate were separated by magnet attraction. The supernatant was used for fluorescence intensity measurement, and the precipitate attracted by the magnet was used for photoelectric signal measurement. The reaction time was optimized for 30min, 60min, 90min, 120min, 150min, and 180min.

[0061] When measuring the photoelectric signal, first prepare the FePc / ITO electrode, dissolve 0.5 mg FePc in 1 mL PBS, and perform ultrasonic treatment for 30 min. The obtained FePc solution is stored at 4 °C. Finally, 30 μL of FePc solution is dripped on the ITO electrode and dried at room temperature. The reaction precipitate is redispersed in 200 μL PBS, mixed evenly, and 30 μL is dripped on the FePc / ITO electrode. After drying, the photoelectric signal change is detected.

[0062] When measuring the fluorescence intensity, in the study of fluorescence spectrum, the excitation wavelength is 430nm, and the recording wavelength range is set to 450-700nm. After the components are fully mixed, 100μL of the reaction supernatant is taken for detection, and the fluorescence enhancement rate is monitored every 30s. In addition, the slit is set at 5nm.

[0063] The photoelectric detection principle of this embodiment is as follows Figure 2 As shown, the characterization of CdTe QDs in this embodiment is as follows Figure 3 As shown in the figure, the feasibility analysis of the sugarcane smut dual-mode sensing platform in this embodiment is as follows: Figure 4 shown.

[0064] Example 2

[0065] Optimize triplex DNA preparation (step (5)): first anneal FAM-B in a 95°C water bath for 5 min, then mix MB-A (6 μM, 20 μL), FAM-B (6 μM, 40 μL), in TE buffer (20 mM Tris, 100 mM NaCl, 50 mM MgCl2 , pH 7.4), the shaking reaction time was set at 37°C for 3-18 hours, and samples were taken every 3 hours and the experimental and detection process of Example 1 was repeated. The results are as follows Figure 5 shown.

[0066] Optimize the detection of target DNA (step (6)): 200 μL triplex DNA, target DNA (50 μL, 10 nM) and CdTe QDs-NH 2 -F chain (2 μM, 200 μL), the reaction time was set to 30-180 min at 37°C, and samples were taken every 30 min to repeat the experiment and detection process of Example 1. The results are shown in Figure 6 and Figure 7 The optimal reaction time for sensitive detection of the target sugarcane smut pathogen gene was obtained, and the detection result of the sugarcane smut pathogen gene was the same as that of Example 1.

[0067] Example 3

[0068] Step (6) During the photoelectric signal measurement, 20 μL of FePc solution was dripped onto the ITO electrode, and the experimental and detection process of Example 1 was repeated. The detection results of the gene of the pathogenic bacteria of sugarcane smut were the same as those of Example 1. Figure 4 (C) shown.

[0069] Example 4

[0070] In step (6) of measuring the fluorescence intensity, after the components are fully mixed, 50 μL of the reaction supernatant is taken for fluorescence intensity detection. The experimental and detection process of Example 1 is repeated, and the detection results of the gene of the pathogenic bacteria of sugarcane smut are the same as those of Example 1. The results are as follows Figure 4 (B) shown.

[0071] Test example (actual sample testing):

[0072] Sugarcane infected with smut was used for actual sample detection experiments. The treatment of other DNA chains was consistent with that in Example 1. 200 μL of triplex DNA and CdTe QDs-NH 2-F chain (2μM, 200μL) and freshly infected sugarcane grinding liquid were mixed and reacted at 37°C for 120min. The supernatant and precipitate were separated by magnet attraction. The magnetically separated supernatant was used for fluorescence intensity detection. In the study of fluorescence spectrum, the excitation wavelength was 430nm and the recording wavelength range was set to 450-700nm. After the components were fully mixed, 100μL of the reaction supernatant was taken for detection, and the fluorescence enhancement rate was monitored every 30s. In addition, the slits were set at 5nm. The magnetically separated precipitate was used for photoelectric signal detection. First, the FePc / ITO electrode was prepared, 0.5mgFePc was dissolved in 1mL PBS, and ultrasonic treatment was performed for 30min. Then, the obtained FePc solution was stored at 4°C. Finally, 30μL of FePc solution was dripped on the ITO electrode and dried at room temperature. The precipitate was redispersed in 200 μL PBS, mixed, and 30 μL was added dropwise to the FePc / ITO electrode. After drying, the photoelectric signal change was detected.

[0073] Test results: The test results are consistent with the test results of adding the target gene. The fluorescence intensity test results are from weak to strong (the fluorescence intensity is enhanced by 12.7%, and the fluorescence intensity is enhanced. The test results of sugarcane infected with smut are very obvious). The photoelectric signal test results are from negative current to positive current (the photocurrent is changed from -2.18μA to 1.94μA, and the polarity of the signal is reversed. The test results of sugarcane infected with smut are very obvious). The two modes have obvious test results for actual samples of sugarcane infected with smut, and the two test results verify each other to ensure the accuracy of the test results.

[0074] In summary, the present invention solves the problem that the existing sensors have low detection accuracy and sensitivity when the gene content of the pathogenic bacteria of sugarcane smut is low. The sensing platform of the present invention adopts an entropy-driven strategy with enthalpy reduction enhancement, which can sensitively and efficiently amplify the genes of the pathogenic bacteria of sugarcane smut, and can obtain independent detection results of the two detection modes of photoelectrochemical and fluorescence respectively. Through the new amplification technology and the mutual verification of the detection results of the two modes, the sensitivity and accuracy of the detection results can be made higher.

[0075] 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 dual-mode sensing platform for sugarcane smut, characterized in that: The sensing platform is constructed based on an entropy-driven strategy with enthalpy reduction enhancement, and the sensing platform includes: triplex DNA and CdTe QDs-NH2-F chains; The triplex DNA is a double strand composed of an MB-A strand and a FAM-B strand; the MB-A strand is an A strand connected to magnetic beads, the A strand is a single strand, and the base sequence of the A strand is shown in SEQ ID NO.1; the FAM-B strand is a B strand modified with a fluorescent group, the B strand is a DNA strand that can form a hairpin structure, and the base sequence of the B strand is shown in SEQ ID NO.2; The CdTe QDs-NH2-F chain is an F chain that is amino-linked and connected to cadmium telluride quantum dots. The F chain is a single chain, and the base sequence of the F chain is shown in SEQ ID NO.3; The partial base sequence of the A chain is complementary to the base sequence of a gene of a sugarcane smut pathogen, and the base sequence of the gene of the sugarcane smut pathogen is shown in SEQ ID NO.4; the base sequence of the F chain is complementary to the partial base sequence of the A chain, and the partial base sequence of the F chain is identical to the partial base sequence of the gene of the sugarcane smut pathogen and the partial base sequence of the B chain.

2. A sugarcane smut dual-mode sensing platform according to claim 1, characterized in that: The preparation method of the triplex DNA is as follows: firstly annealing the FAM-B in a 95°C water bath for 5 minutes, then mixing the MB-A and FAM-B in TE buffer, reacting on a shaking table at 35-37°C for 9-12 hours, finally hybridizing to form a triplex DNA with a concentration of 1-3 μM, and storing at 4°C for future use.

3. The sugarcane smut dual-mode sensing platform according to claim 1, characterized in that: The preparation method of the MB-A chain is as follows: first, the MB is fully shaken and washed, then mixed with EDC and NHs, and reacted on a shaker at 35-37°C for 1-2 hours, and washed with PBS to obtain a concentration of 5 mg·mL -1 MB solution was prepared to a concentration of 5 mg mL -1 Add the pretreated A chain to the MB solution and react on a shaking table at 35-37°C for 9-12 hours to obtain the product.

4. The sugarcane smut dual-mode sensing platform according to claim 1, characterized in that: The preparation method of the CdTeQDs-NH2-F chain comprises the following steps: (1) Synthesis of CdTe QDs: First, CdCl2·2.5H2O and MPA solution were added to a three-necked flask, the pH was adjusted to 11-12, and N2 was passed for deoxidation. Then, NaBH4 and NaTeO3 were added in sequence. The mixture was heated under reflux under the protection of N2 to obtain MPA-coated CdTeQDs. Anhydrous ethanol was added and centrifuged. The solid at the bottom was collected and vacuum dried to obtain brown CdTe QDs powder. Finally, it was purified with acetone to obtain the obtained product. (2) Preparation of amino CdTe QDs: APTES was dispersed in anhydrous ethanol, the pH value was adjusted to 5-8, and then the CdTe QDs were added, mixed and reacted, and centrifuged and washed to obtain CdTe QDs-NH2; (3) Preparation of CdTe QDs-NH2-F chain: dilute the F chain with TE containing TCEP, and then mix it with the CdTe QDs-NH2 to make the required concentration. After shaking, store it at 4°C to obtain the product.

5. The sugarcane smut dual-mode sensing platform according to claim 1, characterized in that: The A chain, B chain and F chain were pretreated before use. The pretreatment method was as follows: the A chain, B chain and F chain were centrifuged respectively, dissolved in 1×TE Buffer to prepare a solution with a concentration of 100 μM, and stored at -20°C for later use.

6. An application of the sugarcane smut dual-mode sensing platform according to any one of claims 1 to 5, characterized in that: The application is used to detect whether the target gene is a gene of the pathogenic bacteria of sugarcane smut.

7. The application of a dual-mode sensing platform for sugarcane smut according to claim 6, characterized in that: The detection method comprises the following steps: S1. Pre-process the target gene T; S2. The triplex DNA, CdTe QDs-NH2-F chain and the target gene T pretreated in step S1 are mixed, reacted at 35-37°C for 100-120 minutes, and then attracted by a magnet to separate the supernatant and the precipitate. The supernatant is used for fluorescence intensity measurement, and the precipitate attracted by the lower magnet is used for photoelectric signal measurement; S3. The supernatant was subjected to fluorescence intensity measurement: the fluorescence excitation wavelength was 430 nm, the recording wavelength range was set to 450-700 nm, the fluorescence enhancement rate was monitored every 30 s, the slits were set at 5 nm, and the changes in the fluorescence signal were detected; S4. Detecting the photoelectric signal of the precipitate attracted by the magnet: redispersing the precipitate in PBS and mixing it, then dropping it onto the FePc / ITO electrode, drying it, and detecting the change of the photoelectric signal; S5. Determine whether the target gene T is a gene of the pathogenic bacteria of sugarcane smut: When the fluorescence signal is enhanced in step S3 and the polarity of the photoelectric signal is reversed in step S4, it is proved that the target gene T is a gene of the pathogenic bacteria of sugarcane smut; otherwise, it is proved that the target gene T is not a gene of the pathogenic bacteria of sugarcane smut.

8. The application of a dual-mode sensing platform for sugarcane smut according to claim 7, characterized in that: In step S1, the pretreatment method of the target gene T is the same as the pretreatment method of the A chain, B chain and F chain.

9. The application of a dual-mode sensing platform for sugarcane smut according to claim 7, characterized in that: In step S3, the detection volume of the supernatant is 50-100 μL.

10. The use of a dual-mode sensing platform for sugarcane smut according to claim 7, characterized in that: In step S4, the preparation method of the FePc / ITO electrode is as follows: dissolving FePc in PBS, ultrasonically treating it, dropping 20-30 μL onto the ITO electrode, and drying it at room temperature.

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