A colorimetric aptasensor based on single-atom nanoszyme and self-screening aptamer for ultrasensitive detection of tomato mosaic virus and a preparation method and application thereof

Abstract

The present application relates to a kind of based on monatomic nanozyme and self-selection aptamer for super-sensitivity detection of tomato mosaic virus colorimetric aptamer sensor and its preparation method and application, belong to electrochemistry and biological detection technical field.The present application utilizes the complex of monatomic nanozyme FeCo-NC and aptamer 1 for ToMV and the microwell plate coated with aptamer 2 for ToMV to constitute the colorimetric aptamer sensor for super-sensitivity detection of tomato mosaic virus, by combining the excellent catalytic performance of FeCo-NC with colorimetric detection system, it is first to realize that electrochemical biosensor is used in the detection of tomato mosaic virus, by verification, the sensing strategy successfully realizes the high sensitivity, high specificity visual detection of tomato mosaic virus.

Description

Technical Field

[0001] This invention relates to a colorimetric aptamer sensor based on single-atom nanozymes and self-selected aptamers for ultrasensitive detection of tomato mosaic virus, its preparation method and application, belonging to the fields of electrochemical and biological detection technology. Background Technology

[0002] Tomatoes are a widely cultivated vegetable worldwide and one of the main cultivated vegetables in my country. However, viral diseases are becoming increasingly serious in tomato production, leading to a decline in yield and quality. Therefore, breeding disease-resistant varieties is of great importance.

[0003] Tomato Mosaic Virus (ToMV) is a widespread RNA virus that severely impacts tomato yield and quality. The virus spreads through field operations or mechanical damage such as aphid feeding, and can survive on seed surfaces or in soil for over a year, remaining infectious even at high temperatures. ToMV primarily infects tobacco, tomatoes, and other Solanaceae plants, often causing mosaic streaks, twisting, wrinkling, yellowing, and even necrosis of infected leaves. Top leaf growth stagnates, and the plant is significantly stunted. It is also accompanied by flower and bud drop, and can cause internal necrosis of the fruit, resulting in substantial losses in yield and quality. Currently, breeding and promoting new varieties resistant to Tomato Mosaic Virus remains considered the most effective and environmentally friendly method. Therefore, developing effective molecular markers for detecting Tomato Mosaic Virus resistance and efficiently assisting in the breeding of new resistant varieties is crucial and urgent.

[0004] Tomato virus (ToMV) is widely distributed worldwide and can be transmitted through mechanical inoculation, grafting, and interplant contact, as well as through seeds. Seed transmission in tomatoes can reach 94%. The virus is present in the external mucus and seed coat of seeds. Sometimes the virus is also found in the endosperm, but it has not been found in the embryo. Seedlings are easily infected during transplanting. In seeds with endosperm infected with the virus, the virus can remain active for at least 9 years.

[0005] Currently, the main methods for detecting ToMV are traditional molecular biology detection techniques. For example, Chinese patent CN109762936A discloses an IC-RT-LAMP kit and detection method for detecting ToMV virus infecting Angelica sinensis, utilizing DNA loop-mediated isothermal amplification technology. Chinese patent CN119332023A discloses a CRISPR / Cas-based isothermal rapid visualization detection kit and detection method for Tomato mosaic virus. Chinese patent CN113430302A discloses RT-RAA-LFS rapid visualization detection primers, probes, and kits for Tomato mosaic virus, as well as their applications. Although traditional virus detection methods are widely used, they still have many problems, such as low efficiency and insufficient sensitivity.

[0006] Biosensors are diagnostic tools that offer high specificity, sensitivity, and rapid detection using small sample sizes. They are commonly used to detect and quantify specific compounds or bioanalytes. Currently, electrochemical biosensors, with their advantages of high efficiency, sensitivity, and low cost, have been applied in the detection of various highly pathogenic viruses. The article "Application of Electrochemical Biosensors in the Detection of Highly Pathogenic Viruses" mentions that biosensor technology is one of the most promising methods for solving the problems of low sensitivity and high cost in virus detection. For example, Chinese patent CN2018108299109 discloses a colorimetric aptamer sensor based on gold nanoparticles and a method for detecting oxytetracycline using this sensor. The colorimetric sensor designed in this invention has advantages such as simple operation, short detection time, high sensitivity, good selectivity, low detection limit, and is visible to the naked eye, making it superior to other traditional methods for detecting OTC viruses.

[0007] There are no reports of electrochemical biosensors detecting tomato mosaic virus in the current technology. Summary of the Invention

[0008] The technical problem to be solved by this invention is to provide a colorimetric aptamer sensor based on single-atom nanozymes and self-selected aptamers for ultrasensitive detection of tomato mosaic virus, which has good repeatability, high sensitivity and specificity, as well as its preparation method and application.

[0009] The working principle of the colorimetric aptamer sensor provided by this invention for detecting tomato mosaic virus using FeCo-NC is as follows:

[0010] First, through 2-methylimidazole, Zn 2+ Fe 2+ and Co 2+A one-pot reaction was used to prepare a bimetallic zeolite imidazole framework material (FeCo-ZIF-8). This material was then thermally carbonized to transform it into a bimetallic single-atom catalyst (FeCo-NC). Subsequently, amino-modified ToMV-Apt-9 was covalently coupled to the FeCo-NC surface via bridging with APTES and glutaraldehyde, forming a FeCo-NC / ToMV-Apt-9 complex. Simultaneously, carboxyl-modified ToMV-Apt-3, after EDC / NHS activation, was fixed to the surface of a 96-well plate substrate via amide bonds, constructing a 96-well plate / ToMV-Apt-3 sensing interface. In the presence of ToMV, the specific recognition and binding of the two aptamers to different sites on the virus surface promoted the formation of a "FeCo-NC / ToMV-Apt-9-(ToMV)-96-well plate / ToMV-Apt-3" sandwich composite structure. After washing, this composite structure was effectively captured in the 96-well plate. Upon the addition of H2O2 and TMB, the FeCo-NC in the sandwich structure catalyzes the oxidation of TMB by H2O2, producing a blue TMBox with a strong absorbance signal. Conversely, in the absence of tomato mosaic virus, the catalytic reaction cannot proceed, and the absorbance signal is significantly reduced. This innovative strategy combines the superior catalytic performance of FeCo-NC with a colorimetric detection system to achieve highly sensitive and specific visual detection.

[0011] Specifically, in a first aspect, the present invention provides a colorimetric aptamer sensor for ultrasensitive detection of Tomato Mosaic Virus based on a single-atom nanozyme and a self-selected aptamer, comprising a complex of a single-atom nanozyme FeCo-NC and an aptamer 1 targeting ToMV, and a microplate coated with an aptamer 2 targeting ToMV.

[0012] Furthermore, the sequences for aptamers 1 and aptamer 2 of ToMV are SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] Furthermore, aptamer 1 for ToMV is ToMV-Apt-9, the sequence of which is shown in SEQ ID NO: 1, and aptamer 2 for ToMV is ToMV-Apt-3, the sequence of which is shown in SEQ ID NO: 2.

[0014] In a second aspect, the present invention provides a method for preparing a colorimetric aptamer sensor as described herein, comprising the following steps:

[0015] (1) Preparation of FeCo-NC;

[0016] (2) Preparation of FeCo-NC / aptamer 1;

[0017] (3) Preparation of microplates coated with aptamer 2.

[0018] Further, the preparation of FeCo-NC in step (1) is as follows:

[0019] a. Dissolve Zn salt, Fe salt and Co salt in a solvent to obtain solution A, and dissolve 2-methylimidazole in a solvent to obtain solution B;

[0020] b. Slowly pour solution A into solution B, stir at room temperature for 12-36 hours, and after the reaction is complete, centrifuge to collect the light purple product. After washing and drying, the FeCo-ZIF-8 precursor is obtained.

[0021] c. Heat the FeCo-ZIF-8 precursor at 800-1200℃ for 1-3 hours under nitrogen or inert gas protection, and collect the resulting black powder after cooling, which is FeCo-NC.

[0022] Furthermore, the Zn salt, Fe salt, and Co salt are each nitrate, sulfate, chloride salt, or hydrate of the corresponding metal.

[0023] Furthermore, the molar ratio of Zn, Fe, and Co is 40:1:1.

[0024] Furthermore, the molar ratio of Zn to 2-methylimidazole is 1:5.

[0025] Furthermore, the solvent is methanol.

[0026] Further, step (2) of the preparation of FeCo-NC / aptamer 1 is as follows:

[0027] d. Disperse FeCo-NC in PBS solution containing 3-aminopropyltriethoxysilane (APTES), incubate at 25-37°C for 1-3 hours, then centrifuge to collect the precipitate, and wash to obtain FeCo-NC-NH2;

[0028] e. Disperse FeCo-NC-NH2 in PBS solution containing 20-30% (v / v) glutaraldehyde, incubate at 25-37°C for 1-3 hours, then centrifuge to collect the precipitate, and wash to obtain aldehyde-modified FeCo-NC-CHO;

[0029] f. Disperse FeCo-NC-CHO in PBS solution, add 1-10 nmol of amino-modified aptamer 1, incubate at 25-37℃ for 6-24 hours, then centrifuge to collect the precipitate, wash to obtain the complex FeCo-NC / aptamer 1, disperse it in PBS solution, and store at 0-8℃ for later use.

[0030] As used herein, amino-modified aptamers can be obtained by adding an amino group to the end of the aptamer using conventional methods well known to those skilled in the art.

[0031] Further, the preparation of the microplate coated with aptamer 2 in step (3) is as follows:

[0032] j. Add the carboxyl-modified aptamer 2 to a PBS solution containing EDC and NHS, and incubate at 25-37°C for 30-60 minutes;

[0033] h. Add the solution obtained in step j into the wells of the bottom amino-modified microplate and incubate overnight at 25-37°C;

[0034] i. After incubating the microplate in step h, wash it with PBS solution, add an appropriate amount of PBS solution to each well, and store it at -20°C for later use.

[0035] As used herein, carboxyl-modified aptamers can be obtained by adding a carboxyl group to the end of the aptamer using conventional methods well known to those skilled in the art.

[0036] Furthermore, the concentrations of EDC and NHS in the PBS solution containing dissolved EDC and NHS were each 5 mg / mL.

[0037] Furthermore, 1-20 nmol of aptamer 2 was added to a PBS solution containing EDC and NHS.

[0038] In a third aspect, the present invention provides the application of colorimetric aptamer sensors as described herein or colorimetric aptamer sensors prepared by the methods described herein in the detection of plant viruses.

[0039] Furthermore, the plant virus is tomato mosaic virus.

[0040] In a fourth aspect, the present invention provides a method for detecting tomato mosaic virus using a colorimetric aptamer sensor as described herein or a colorimetric aptamer sensor prepared by the preparation method described herein, comprising the following steps:

[0041] 1. Add the solution of the sample to be tested to the microplate coated with aptamer 2, incubate at 25-37℃ for 1-3 hours, and wash with PBS solution to remove uncaptured targets;

[0042] m. Add FeCo-NC / aptamer 1 dispersed in PBS solution to the microplate after step l treatment, incubate at 25-37°C for 1-3 hours, and wash with PBS solution to remove unbound FeCo-NC / aptamer 1;

[0043] n. Add NaAc-HAc buffer, TMB and H2O2 colorimetric combination sequentially to the microplate after step m, react at 25-37℃ for 3-5 minutes, and record the color change and ultraviolet-visible absorption spectrum of the reaction system.

[0044] The reaction system changes color when the sample contains tomato mosaic virus, but does not change color when the sample does not contain tomato mosaic virus.

[0045] Furthermore, the concentration of the NaAc-HAc buffer solution is 0.1 M, and the pH is 3.8.

[0046] Beneficial effects of the invention

[0047] (1) This invention is the first to use an electrochemical biosensor for the detection of tomato mosaic virus. Through verification, this sensing strategy has been successfully applied to the detection of tomato mosaic virus.

[0048] (2) This invention combines the excellent catalytic performance of FeCo-NC with a colorimetric detection system to achieve highly sensitive and specific visual detection. Attached Figure Description

[0049] Figure 1 The flowchart for aptamer screening is shown.

[0050] Figure 2 The results show the binding of the screened aptamers to different viruses.

[0051] Figure 3 The affinity (KD) of the aptamer ToMV-Apt-3 was measured when the ToMV viral protein was diluted to 12 concentration gradients as coating antigens.

[0052] Figure 4 The affinity (KD) of the aptamer ToMV-Apt-9 was measured when the ToMV viral protein was diluted to 12 concentration gradients as coating antigens.

[0053] Figure 5 The affinity (KD) of the aptamer ToMV-Apt-3 was measured when the aptamer was diluted to a concentration gradient as a primary antibody.

[0054] Figure 6 The affinity (KD) of the aptamer ToMV-Apt-9 was measured when the aptamer was diluted to a concentration gradient as a primary antibody.

[0055] Figure 7 The diagram shows the synthesis and modification of FeCoNC and the detection process for tomato mosaic virus.

[0056] Figure 8 The results of the feasibility verification of Example 5 are shown in the figure: (a) blank solution; (b) low concentration ToMV; (c) UV-Vis spectrum detected by colorimetric aptamer sensor of high concentration ToMV.

[0057] Figure 9The ultraviolet-visible spectroscopy used in Example 5 reflects the relationship between the absorption signal response and the target concentration.

[0058] Figure 10 This is the linear relationship between the absorption signal change (ΔA = A - A0) and the concentration of tomato mosaic virus in Example 5. Detailed Implementation

[0059] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0060] Example 1: Selection of aptamers

[0061] Using ToMV virus protein as the positive screening target and PVY virus protein as the negative screening target, carboxyl magnetic beads were used to immobilize ToMV virus protein (denoted as MB-ToMV virus) and carboxyl magnetic beads to immobilize PVY virus protein (denoted as MB-PVY virus), respectively.

[0062] Use the lib4-76nt library (GGGACCAGCACACGCATAACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGTTATGCGTGCTACCGTG (SEQ ID NO: 3)) as Figure 1 The aptamer screening was performed as shown. In short, after dissolving and denaturing the lib4-76nt library powder, five rounds of reverse screening were performed against MB-PVY virus, followed by five rounds of forward screening against MB-ToMV. The obtained magnetic beads were washed, denatured, and eluted. The resulting eluent was then subjected to PCR amplification, and the aptamers ToMV-Apt-9 and ToMV-Apt-3 were finally screened out, with their sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0063] Example 2: Enzyme-linked oligonucleotide assay (ELONA) for detecting aptamer affinity.

[0064] Experimental methods:

[0065] 1. ToMV virus protein, PYT virus protein, and BSA protein were diluted to 20 μg / μL using pH 9.6 carbonate buffer as coating antigens and coated overnight at 4 °C. Different biotin-labeled candidate aptamers (500 nmol / L) were added separately. HRP-SA (1:5000 dilution) was used as the secondary antibody. The affinity and specificity of the aptamers for ToMV virus were detected by enzyme-linked oligonucleotide assay (ELONA).

[0066] 2. ToMV virus protein was diluted to 12 concentration gradients (40 μg / ml, 20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 μg / ml, 0.15625 μg / ml, 0.078125 μg / ml, 0.0390625 μg / ml and 0 μg / ml) using pH 9.6 carbonate buffer as coating antigen. The coating was incubated overnight at 4°C. Different biotin-labeled candidate aptamers (500 nmol / L) were added, and HRP-SA (1:5000 dilution) was used as the secondary antibody. The affinity and specificity of the aptamers for ToMV virus were detected by enzyme-linked oligonucleotide assay (ELONA).

[0067] 3. ToMV virus protein was diluted to 20 μg / μL using pH 9.6 carbonate buffer as coating antigen and coated overnight at 4°C. Candidate aptamers labeled with different concentration gradients (1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.90625 nM, and 0 nM) were added, with HRP-SA (1:5000 dilution) as the secondary antibody. The affinity and specificity of the aptamers for ToMV virus were detected using the enzyme-linked oligonucleotide assay (ELONA).

[0068] Experimental results:

[0069] 1. Experimental results on aptamer affinity and specificity are as follows: Figure 2 As shown, aptamer 15 binds well to PYT, while aptamers 2, 3, 9, 20, and 86 bind well to ToMV, but their binding to PYT is relatively weak. Monoclonal lines 1, 18, and 76 bind to both ToMV and PYT, but their specificity is poor. Therefore, the next step is to test the affinity (KD) of aptamers 3 and 9 for the ToMV virus.

[0070] 2. The ToMV viral protein was diluted to 12 concentration gradients as coating antigens, and the affinity (KD) of each aptamer was measured as follows: Figure 3 and Figure 4 As shown.

[0071] 3. The aptamers were diluted to a concentration gradient to serve as primary antibodies, and the affinities (KD) of each aptamer were measured as follows: Figure 5 and Figure 6 As shown.

[0072] It can be seen that both ToMV-Apt-3 and ToMV-Apt-9 have good affinity and specificity for ToMV.

[0073] Example 3: Fabrication of a colorimetric aptamer sensor

[0074] This embodiment provides a method for preparing a colorimetric aptamer sensor based on single-atom nanozymes and self-selected aptamers for ultrasensitive detection of tomato mosaic virus, comprising the following steps:

[0075] (1) Preparation of FeCo-ZIF-8 and FeCo-NC

[0076] The specific steps are as follows: First, 0.952 g Zn(NO3)2·6H2O (3.2 mmol), 0.023 g Co(NO3)2·6H2O (0.08 mmol), and 0.032 g Fe(NO3)3·9H2O (0.08 mmol) were dissolved in 100 mL of methanol and labeled as solution A. Separately, 1.314 g 2-methylimidazole (16 mmol) was added to 100 mL of methanol and, after complete dissolution, labeled as solution B. Then, solution A was slowly poured into solution B, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the resulting light purple product was centrifuged, thoroughly washed three times with methanol, and dried under vacuum at 60 °C overnight to obtain the FeCo-ZIF-8 precursor.

[0077] The prepared FeCo-ZIF-8 nanomaterial (250 mg) was placed in a ceramic boat in a tubular furnace under argon atmosphere protection and heated to 1000 °C at a heating rate of 2 °C·min⁻¹ and held for 1 hour. After the furnace body cooled naturally to room temperature, the resulting black powder was collected, which was the target product FeCo-NC.

[0078] (2) Preparation of FeCo-NC / ToMV-Apt-9

[0079] 2 mg of FeCo-NC was dispersed in 2 mL of PBS solution (10 mM, pH=7.4) containing 225 μL of APTES and incubated at 37 °C for 1 hour. After centrifugation, the mixture was washed three times with PBS solution to obtain FeCo-NC-NH2. Then, 2 mL of PBS solution containing 500 μL of 25% (v / v) glutaraldehyde was added, and incubation continued for 1 hour. After centrifugation and washing, aldehyde-modified FeCo-NC-CHO was obtained, which was redispersed in 2 mL of PBS solution. 1 nmol of Tomato mosaic virus aptamer ToMV-Apt-9 was added, and the mixture was incubated at 37 °C for 12 hours. Finally, the obtained FeCo-NC / ToMV-Apt-9 complex was centrifuged, washed, and dispersed in 2 mL of PBS solution, and stored at 4 °C for later use.

[0080] (3) Preparation of DNA-modified 96-well ELISA plates

[0081] The bottom-amino-modified 96-well ELISA plate was washed three times with PBS solution, and 90 μL of PBS solution was added. 5 mg EDC and 5 mg NHS were dissolved in 1 mL of PBS solution. Then, 50 μL of each solution was added to 100 μL of PBS solution containing 1 nmol ToMV-Apt-3 to form a 200 μL system, and incubated at 37 °C for 50 min. Subsequently, 10 μL of the above system was added to the wells of the 96-well plate and incubated overnight at 37 °C. Finally, the resulting 96-well plate / ToMV-Apt-3 was washed three times, 50 μL of PBS solution was added to each well, and the plate was stored at -20 °C.

[0082] Example 4: Detection method for colorimetric aptamer sensors

[0083] 50 μL of tomato mosaic virus solutions of different concentrations were added to each well of a 96-well plate containing ToMV-Apt-3 and incubated at 37 °C for 1 hour. After washing three times, 70 μL of PBS solution and 30 μL of FeCo-NC / ToMV-Apt-9 were added, and the plates were incubated again at 37 °C for 1 hour. Subsequently, the resulting sandwich structure was formed, and 192 μL of HAc-NaAc buffer solution (0.1 M, pH = 3.8) was added to each well of a 96-well plate. Then, 4 μL of TMB and 4 μL of H2O2 solution were added to the HAc-NaAc buffer solution, and the plates were incubated at 37 °C for 5 minutes. Finally, the absorbance signal after the reaction was measured using a full-wavelength microplate reader.

[0084] Example 5: Detection of tomato samples using the colorimetric aptamer sensor prepared in Example 1

[0085] To verify the practicality of the sensor prepared in Example 3, tomato leaf extract was used as the actual sample, and a standard addition method was employed. First, 10 grams of finely chopped tomato leaves were soaked in sterile PBS solution. After 12 hours, the filtrate was removed, resulting in a turbid solution containing leaf tissue. Subsequently, tomato mosaic virus solutions of different standard concentrations were added to this mixture. After thorough mixing, solid precipitates were filtered off, preparing a practical sample suitable for detection. Then, the prepared sample was colorimetrically detected using the sensor detection strategy of Example 4. Finally, the obtained detection data were compared with the initially added standard concentrations to evaluate the sensor's performance.

[0086] (1) Feasibility verification

[0087] like Figure 8As shown, in the absence of the target protein, no sandwich structure is formed, resulting in minimal absorption signal. Conversely, even the introduction of trace amounts of tomato mosaic virus into the detection system resulted in a significant increase in the absorption signal. Furthermore, the absorption signal increased accordingly with increasing tomato mosaic virus concentration. These findings confirm the successful application of this sensing strategy in the detection of tomato mosaic virus.

[0088] (2) Linear detection

[0089] Under optimal experimental conditions, ultraviolet-visible spectroscopy is used to reflect the relationship between the absorption signal response and the target concentration. For example... Figure 9 As shown, increasing the target concentration resulted in a corresponding enhancement of the absorption signal at 653 nm. This phenomenon is attributed to an increase in the formation of sandwich structures in the system, which is promoted by the presence of tomato mosaic virus. Therefore, the increased ·OH content in the detection leads to the oxidation of more TMB, thereby enhancing the spectral signal.

[0090] Figure 10 The linear relationship between the absorption signal change (ΔA = A - A0) and the concentration of tomato mosaic virus is shown. Here, A and A0 represent the absorption peak at 653 nm in the presence and absence of tomato mosaic virus, respectively. The linear equation is ΔA = 0.08434logC + 0.18678 (R0). 2 =0.9994), with a detection limit of 1.4 pg / mL. Furthermore, the sensitivity and small sample size requirements of this sensing strategy make it suitable for the detection of trace samples. Notably, the system exhibits a significant color change before and after the reaction, enabling direct visual detection and semi-quantitative assessment.

[0091] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A colorimetric aptamer sensor based on single-atom nanozymes and self-selected aptamers for ultrasensitive detection of tomato mosaic virus, characterized in that, The colorimetric aptamer sensor comprises a complex of a single-atom nanozyme FeCo-NC and an aptamer 1 for ToMV, and a microplate coated with an aptamer 2 for ToMV. The aptamer 1 for ToMV is ToMV-Apt-9, the sequence of which is shown in SEQ ID NO: 1, and the aptamer 2 for ToMV is ToMV-Apt-3, the sequence of which is shown in SEQ ID NO:

2.

2. A method for preparing a colorimetric aptamer sensor as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of FeCo-NC; (2) Preparation of FeCo-NC / aptamer 1; (3) Preparation of microplates coated with aptamer 2.

3. The preparation method according to claim 2, characterized in that, Step (1) Preparation of FeCo-NC is as follows: a. Dissolve Zn salt, Fe salt and Co salt in a solvent to obtain solution A, and dissolve 2-methylimidazole in a solvent to obtain solution B; b. Slowly pour solution A into solution B, stir at room temperature for 12-36 hours, and after the reaction is complete, centrifuge to collect the light purple product. After washing and drying, the FeCo-ZIF-8 precursor is obtained. c. Heat the FeCo-ZIF-8 precursor at 800-1200℃ for 1-3 hours under inert gas protection, and collect the resulting black powder after cooling, which is FeCo-NC.

4. The preparation method according to claim 3, characterized in that, The Zn salt, Fe salt, and Co salt are each a nitrate, sulfate, chloride salt, or hydrate of the corresponding metal.

5. The preparation method according to claim 3, characterized in that, The molar ratio of Zn, Fe and Co is 40:1:

1.

6. The preparation method according to claim 3, characterized in that, The molar ratio of Zn to 2-methylimidazole is 1:

5.

7. The preparation method according to claim 3, characterized in that, The solvent is methanol.

8. The preparation method according to claim 2, characterized in that, Step (2) Preparation of FeCo-NC / aptamer 1 is as follows: d. Disperse FeCo-NC in PBS solution containing 3-aminopropyltriethoxysilane (APTES), incubate at 25-37°C for 1-3 hours, then centrifuge to collect the precipitate, and wash to obtain FeCo-NC-NH2; e. Disperse FeCo-NC-NH2 in PBS solution containing 20-30% (v / v) glutaraldehyde, incubate at 25-37°C for 1-3 hours, then centrifuge to collect the precipitate, and wash to obtain aldehyde-modified FeCo-NC-CHO; f. Disperse FeCo-NC-CHO in PBS solution, add 1-10 nmol of amino-modified aptamer 1, incubate at 25-37℃ for 6-24 hours, then centrifuge to collect the precipitate, wash to obtain the complex FeCo-NC / aptamer 1, disperse it in PBS solution, and store at 0-8℃ for later use.

9. The preparation method according to claim 2, characterized in that, Step (3) Preparation of the microplate coated with aptamer 2 is as follows: g. Add the carboxyl-modified aptamer 2 to a PBS solution containing EDC and NHS, and incubate at 25-37°C for 30-60 minutes; h. Add the solution obtained in step g to the wells of the bottom amino-modified microplate and incubate overnight at 25-37°C; i. After incubating the microplate in step h, wash it with PBS solution, add an appropriate amount of PBS solution to each well, and store it at -20°C for later use.

10. The preparation method according to claim 9, characterized in that, The concentrations of EDC and NHS in the PBS solution containing EDC and NHS were each 5 mg / mL.

11. The preparation method according to claim 9, characterized in that, Add 1-20 nmol of aptamer 2 to a PBS solution containing EDC and NHS.

12. The application of the colorimetric aptamer sensor as described in claim 1 or the colorimetric aptamer sensor prepared by any one of claims 2-11 in the detection of plant viruses.

13. The application according to claim 12, characterized in that, The plant virus in question is the tomato mosaic virus.

14. A method for detecting tomato mosaic virus using a colorimetric aptamer sensor as described in claim 1 or a colorimetric aptamer sensor prepared by the preparation method according to any one of claims 2-11, characterized in that, Includes the following steps:

1. Add the solution of the sample to be tested to the microplate coated with aptamer 2, incubate at 25-37℃ for 1-3 hours, and wash with PBS solution to remove uncaptured targets; m. Add FeCo-NC / aptamer 1 dispersed in PBS solution to the microplate after step l treatment, incubate at 25-37°C for 1-3 hours, and wash with PBS solution to remove unbound FeCo-NC / aptamer 1; n. Add NaAc-HAc buffer, TMB and H2O2 colorimetric combination sequentially to the microplate after step m, react at 25-37℃ for 3-5 minutes, and record the color change and ultraviolet-visible absorption spectrum of the reaction system. The reaction system changes color when the sample contains tomato mosaic virus, but does not change color when the sample does not contain tomato mosaic virus.

15. The method according to claim 14, characterized in that, The concentration of the NaAc-HAc buffer solution is 0.1 M, and the pH is 3.8.

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