Construction method and application of MIL-101 (Fe)-based tobacco mosaic virus molecular imprinting sensor

Abstract

The invention belongs to the technical field of analytical chemistry detection, and particularly relates to a construction method and application of a tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe). The construction method comprises the following steps: by taking a tobacco mosaic virus TMV as a template virus, preparing a molecularly imprinted polymer MIPs with a TMV specific recognition cavity on the surface of MIL-101 (Fe) through a free radical polymerization reaction; the template virus is removed in the preparation process of the molecularly imprinted polymer MIPs, and a TMV specific recognition cavity of the molecularly imprinted polymer MIPs contains MIL-101 (Fe); the molecularly imprinted polymer MIPs is the tobacco mosaic virus molecularly imprinted sensor based on MIL-101 (Fe). Hydrochloric acid is introduced, so that the TMV specific recognition cavity structure of the molecularly imprinted polymer MIPs is cracked to release Fe < 3 + >, then potassium thiocyanate is added, and the Fe < 3 + > and KSCN are subjected to a complexation reaction to generate a red complex. And the complexation reaction of Fe < 3 + > and KSCN is used as a signal probe, the rapid semi-quantitative visual detection of the target virus TMV by naked eyes is realized, and quantitative detection and analysis are carried out through an ultraviolet spectrophotometer.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry detection, and particularly relates to a construction method and application of a tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe). Background Art

[0002] Plant virus diseases are the primary biological factor contributing to crop yield losses. Detection methods for plant viruses are primarily categorized into immunoassays and molecular methods. Immunoassays include enzyme-linked immunosorbent assays (ELISAs) and lateral flow chromatography, while molecular methods include reverse transcription loop-mediated isothermal amplification (RT-LAMP) and polymerase chain reaction. However, these methods and instruments cannot be used directly in the field, are expensive, and require complex operation. Therefore, there is an urgent need to develop highly specific, rapid, low-cost, and easy-to-use detection technologies.

[0003] Molecularly imprinted polymers (MIPs) are composite materials synthesized using the "lock and key" principle. They are polymers with specific recognition sites for template molecules. They undergo polymerization with a crosslinker and functional monomers in the presence of a molecular template (a target molecule or a structural analog). After the template molecule is removed, specific binding sites are formed in the polymer that match the template molecule in terms of functional groups and are complementary in shape and size. They exhibit high selectivity for small or large molecular structures (such as proteins, viruses, and bacteria) and offer advantages such as simple preparation, low cost, and excellent stability. For example, Bolisay et al. used polyallylamine hydrochloride (PAH) as an imprinting carrier and tobacco mosaic virus (TMV) as a template molecule. Through non-covalent interactions, they synthesized imprinted cavity hydrogels that complement TMV in size, shape, and function. [Bolisay LD, Culver JN,Kofinas P. Molecularly imprinted polymers for tobacco mosaic virus recognition[J]. Biomaterials, 2006, 27(22): 4165-4168.] Gong et al. constructed a portable dual-mode detection MIPs sensor for highly selective identification of influenza virus H5N1. [Gong H, Tang L, ChenF, et al. Self-service multimodal detection of subtype influenza A virus H5N1by visual portable molecular imprinting sensor[J]. Chemical Engineering Journal, 2024, 483: 148946.] Siavash et al. proposed a portable multi-channel molecular imprinting sensor for virus detection, which can complete the rapid and simultaneous detection of multiple viral loads such as influenza A H1N1, SARS-CoV-2 and its variants in saliva samples within 11 minutes.[Siavash Moakhar R, del Real Mata C, Jalali M, et al. Aversatile biomimic nanotemplating fluidic assay for multiplex quantitative monitoring of viral respiratory infections and immune responses in saliva and blood[J]. Advanced Science, 2022, 9(33): 2204246.]. However, although these methods have achieved good detection results, they have disadvantages such as high cost, lack of visualization, lack of portability, or long detection time. Therefore, there is an urgent need to develop a low-cost, portable, rapid, specific, and sensitive visual detection method. Summary of the Invention

[0004] In response to the above technical problems, the present invention provides a method for constructing a tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe) and its application. The method for constructing a tobacco mosaic virus molecular imprinting sensor provided by the present invention uses MIL-101 (Fe) with a rigid skeleton structure as the core, and imprints the core surface to obtain molecular imprinted polymers (MIPs). Hydrochloric acid is introduced to cleave the TMV-specific recognition cavity structure of the molecular imprinting polymer MIPs to release Fe. 3+ , followed by the addition of potassium thiocyanate, through Fe 3+ The complexation reaction with KSCN generates a red complex; the sensor realizes rapid semi-quantitative detection of tobacco mosaic virus (TMV) by naked eye visualization under visible light; and can also be quantitatively detected and analyzed by ultraviolet spectrophotometer.

[0005] The present invention is achieved through the following technical solutions: A method for constructing a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe), wherein the tobacco mosaic virus (TMV) is used as a template virus and molecular imprinting polymers (MIPs) with TMV-specific recognition cavities are prepared on the surface of MIL-101(Fe) by free radical polymerization. The template virus is removed during the preparation of the molecularly imprinted polymer MIPs, and the TMV-specific recognition cavity of the molecularly imprinted polymer MIPs contains MIL-101 (Fe); The molecular imprinting polymer MIPs is a tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe).

[0006] Furthermore, the method specifically includes: (1) Synthesis of MIL-101(Fe): FeCl3·6H2O and 2-aminoterephthalic acid were weighed and dissolved in DMF solution respectively. After ultrasonic treatment, the two solutions were mixed. After ultrasonic treatment, the mixed solution was transferred to a lined autoclave for reaction. After the reaction, the precipitate was obtained by centrifugation. The precipitate was washed alternately with DMF and methanol and dried in a vacuum drying oven to obtain the MIL-101(Fe) metal organic framework material. (2) Synthesis of MIL-101(Fe)@C=C: MIL-101(Fe) was dissolved in anhydrous toluene, ultrasonically dispersed, and then vinyltriethoxysilane was added. The mixture was stirred for reaction. After the reaction, the mixture was centrifuged to obtain a precipitate. The precipitate was washed alternately with toluene and ethanol, and vacuum dried to obtain the imprinted carrier MIL-101(Fe)@C=C. (3) Preparation of MIPs: MIL-101(Fe)@C=C was dissolved in DMF solution, tobacco mosaic virus TMV was added as a template virus, and the mixture was stirred to allow the template virus to fully combine with the imprinted carrier MIL-101(Fe)@C=C. 4-vinylpyridine and DMF containing zinc acrylate were then added. After pre-assembly, ethylene glycol dimethyl acrylate was added and deoxygenated under argon. Then, a DMF solution containing azobisisobutyronitrile was added for polymerization. The reaction was continued under argon protection. After the reaction was completed, the mixture was centrifuged to obtain a precipitate. The precipitate was repeatedly eluted with a mixed eluent of methanol and acetonitrile to remove the template virus. The precipitate was then washed with ultrapure water and dried in a vacuum drying oven to obtain molecularly imprinted polymer MIPs particles, i.e., a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) was obtained.

[0007] Furthermore, step (1) is specifically as follows: 0.5-3.0 g of FeCl3·6H2O and 0.2-2 g of 2-aminoterephthalic acid are weighed and dissolved in 5-40 mL of DMF solution respectively, and after ultrasonic treatment for 5-25 minutes, the two solutions are mixed in a volume ratio of (1-4):1, and after ultrasonic treatment for 5-30 minutes, the solution is transferred to a lined high pressure sterilizer and reacted at 80-140° C. for 6-48 hours; after the reaction is completed, the solution is centrifuged at 8000-10000 rpm / min for 1-20 minutes to obtain a precipitate, and the precipitate is washed alternately with DMF and methanol several times, and dried in a vacuum drying oven at 30-90° C. to obtain the MIL-101(Fe) metal organic framework material.

[0008] Furthermore, step (2) is specifically as follows: 0.1-2.0 g of MIL-101(Fe) is added to a dry three-necked flask, MIL-101(Fe) is dissolved in 5-40 mL of anhydrous toluene, 0.5-5 mL of vinyltriethoxysilane is added after uniform ultrasonic dispersion, and the mixture is stirred and reacted at 30-90° C. and 300-1000 rpm / min for 3-48 hours. After the reaction is completed, the mixture is centrifuged at 5000-15000 rpm / min for 1-20 minutes to obtain a precipitate, the precipitate is washed repeatedly with toluene and ethanol alternately several times, and vacuum dried at 30-90° C. to obtain the imprinted carrier MIL-101(Fe)@C=C.

[0009] Furthermore, step (3) is specifically as follows: 5-100 mg of imprinted vector MIL-101(Fe)@C=C is weighed and dissolved in 5-40 mL of DMF solution, 5-200 μL of template virus TMV is added and stirred to allow the template virus to fully combine with the imprinted vector, and then 5-200 μL of 0.05-1.0 mmol of 4-vinylpyridine and 0.2-5 mL of DMF containing 10-200 mg of 0.1-2.0 mmol of zinc acrylate are added. After pre-assembly at 20-60°C for 0.5-3.0 hours, 10-100 μL of 0.1-2.0 mmol of zinc acrylate is added. mol of ethylene glycol dimethyl acrylate was deoxygenated under argon, and 0.1-10 mL of a DMF solution containing 5-200 mg of azobisisobutyronitrile was added for polymerization. The mixture was sealed and continued to react under argon protection at 30-90°C and 400-1500 rpm / min for 1-48 hours. After the reaction, the precipitate was centrifuged and repeatedly eluted with an eluent of methanol:acetonitrile = 1:9-20:1 several times, and then washed with ultrapure water several times, each time shaking and eluting in a water bath constant temperature oscillator at 20-60°C, and then dried in a vacuum drying oven to obtain molecularly imprinted polymer MIPs particles.

[0010] The tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe) is used to perform semi-quantitative visual detection of tobacco mosaic virus (TMV), or to perform quantitative detection and analysis of tobacco mosaic virus (TMV) using an ultraviolet spectrophotometer.

[0011] Furthermore, the visual detection method includes: Hydrochloric acid was introduced to allow the TMV-specific recognition cavity structure of molecularly imprinted polymers (MIPs) to cleave and release Fe 3+ , followed by the addition of potassium thiocyanate, through Fe 3+ The complexation reaction with KSCN generates a red complex; the molecularly imprinted polymer MIPs reacts with Fe 3+The complexation reaction of KSCN and PEG-KSCN can realize the semi-quantitative visual detection of tobacco mosaic virus TMV.

[0012] Furthermore, the quantitative detection and analysis method is specifically as follows: 0.01-20 mg of molecularly imprinted polymers (MIPs) were dispersed in 0.1-10 mL of ultrapure water by ultrasound to obtain a MIPs solution. 50-1000 μL of the MIPs solution was added to a test tube, and 10-1000 μL of 0.01-1.0 M HCl was added and reacted for 1-30 min. Then, 50-1000 μL of 0.01-2.0 M KSCN solution was added and reacted for 0.5-30 min, and the supernatant was collected. The absorbance of the supernatant at 300-775 nm was measured by UV-visible absorption spectroscopy, and the absorbance intensity at this time was recorded as Abs0; Different concentrations of TMV solution were added to the MIPs solution and oscillated for a certain period of time. Then 50-1000 μL of the solution was taken and measured in the same way. The absorbance at different concentrations was Abs. i , the linear regression equation is obtained by fitting; The linear regression equation is used to quantitatively detect and analyze the concentration of TMV in the test solution.

[0013] The present invention has the following beneficial effects: The present invention provides a method for preparing a tobacco mosaic virus molecular imprinting sensor, and uses the sensor for specific and visual detection of TMV virus, thereby improving the effect of actual virus detection.

[0014] The method for constructing a tobacco mosaic virus molecular imprinting sensor provided by the present invention uses MIL-101(Fe) as the core. MIL-101(Fe) has the advantages of a clear geometric shape, high porosity, high specific surface area, high loading rate, structural adjustability, and stability. It provides a sensitive and reliable detection method for TMV and improves the stability of the sensor.

[0015] The sensor prepared by the method provided by the present invention is Fe 3+ The complexation reaction with KSCN is used as a signal probe. The complexation reaction has a distinct color and can significantly increase the specificity of the sensor and improve the sensitivity.

[0016] The sensor prepared by the method provided by the present invention can realize colorimetric semi-quantitative detection of the target object by the naked eye without the aid of any instrument, as well as quantitative detection and analysis with the aid of an ultraviolet spectrophotometer. The required detection time is short, and obvious changes can be observed within 18 minutes. This establishes a rapid, specific, and sensitive TMV visual detection method.

[0017] The sensor prepared by the method provided by the present invention can be combined with a smart phone for detection, and can have a good visualization effect on the target virus, thereby realizing convenient and fast on-site detection of TMV.

[0018] The sensor prepared by the method provided by the present invention has a low preparation cost, with a cost as low as 0.082 yuan per piece. The sensor construction process is simple, the operation is easy, the detection is sensitive and fast, and the professional requirements for the operator are not high. The sensor is expected to achieve on-site rapid visual semi-quantitative detection during the period when tobacco leaves and other crops are infected with viruses, which is of great significance for the effective management and reduction of damage to tobacco mosaic virus crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a flow chart for preparing a tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe) metal chelation in an embodiment of the present invention; Figure 2 The UV-visible absorption spectrum absorbance diagram of MIPs / NIPs detecting TMV in an embodiment of the present invention (A) and the visualization results before and after the addition of TMV to MIPs / NIPs (B); Figure 3 Figure 1 is a graph showing dynamic light scattering (DLS) results in an embodiment of the present invention; wherein A is the dynamic light scattering of MIL-101(Fe); B is the dynamic light scattering of MIL-101(Fe)@C=C; C is the dynamic light scattering of MIPs; and D is the dynamic light scattering of NIPs. Figure 4 The infrared spectra (A) and zeta potential diagrams (B) of MIL-101(Fe), MIL-101(Fe)@C=C, MIPs, and NIPs in the embodiments of the present invention are shown. Figure 5 Graph showing water contact angles in an embodiment of the present invention, where A is the water contact angle of MIL-101(Fe); B is the water contact angle of MIL-101(Fe)@C=C; C is the water contact angle of MIPs; and D is the water contact angle of NIPs. Figure 6 The scanning electron microscopy (SEM) images of the water contact angles of MIL-101(Fe) (A), MIPs (B), and NIPs (C) in the embodiments of the present invention are shown; Figure 7Figure 3 shows the linearity graph (A) of MIPs detecting different concentrations of TMV (0.0411, 0.144, 0.288, 0.411, 0.575, 1.44, 2.88, 4.11, and 5.75 pM) in the examples of the present invention, the linear relationship graph between ∆Abs and TMV concentration (B), the linearity graph (C) of NIPs detecting different concentrations of TMV (0.0411, 0.144, 0.288, 0.411, 0.575, 1.44, 2.88, 4.11, and 5.75 pM), and the visualization results of MIPs / NIPs detecting different concentrations of TMV (D).

[0020] Figure 8 Figure 1 shows the linear fitting of the G / B values of smartphone recognition after MIPs detected different concentrations of TMV virus (0.0411, 0.144, 0.288, 0.411, 0.575, 1.44, 2.88, 4.11, and 5.75 pM) in an embodiment of the present invention (A), and images of MIPs and NIPs captured by the smartphone (B). Figure 9 The selectivity (A) of the MIPs / NIPs sensor for TMV and the competitiveness (B) of the MIPs / NIPs sensor for TMV in the embodiment of the present invention are shown.

[0021] Figure 10 1 and 2. The reproducibility (A) and time stability (B) of the MIPs sensor in the embodiment of the present invention.

[0022] Figure 11 is the anti-interference capability of the MIPs sensor in the embodiment of the present invention.

[0023] Figure 12 These are the visualization results of MIPs incubated for different times and reacted for different times in the examples of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] On the contrary, the present invention covers any alternatives, modifications, equivalents, and solutions that fall within the spirit and scope of the present invention as defined by the claims. Furthermore, to facilitate a better understanding of the present invention, certain specific details are described in detail below in the detailed description of the present invention. Those skilled in the art will be able to fully understand the present invention without these details.

[0026] Example 1: A method for constructing a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe), wherein the tobacco mosaic virus (TMV) is used as a template virus and molecular imprinting polymers (MIPs) with TMV-specific recognition cavities are prepared on the surface of MIL-101(Fe) by free radical polymerization. The template virus is removed during the preparation of the molecularly imprinted polymer MIPs, and the TMV-specific recognition cavity of the molecularly imprinted polymer MIPs contains MIL-101 (Fe); the molecularly imprinted polymer MIPs is a tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe).

[0027] In this embodiment, the method specifically includes: (1) Synthesis of MIL-101(Fe): Weigh 0.5-3.0 g of FeCl3·6H2O and 0.2-2 g of 2-aminoterephthalic acid and dissolve them in 5-40 mL of DMF solution. After ultrasonic treatment for 5-25 minutes, mix the two solutions in a volume ratio of (1-4):1. After ultrasonic treatment for 5-30 minutes, transfer the solution to a lined autoclave and react at 80-140°C for 6-48 hours. After the reaction, centrifuge at 8000-10000 rpm / min for 1-20 minutes to obtain a precipitate. Wash the precipitate several times with DMF and methanol alternately and dry it in a vacuum drying oven at 30-90°C to obtain the MIL-101(Fe) metal-organic framework material.

[0028] Preferably, 1.8 g of FeCl₃·6H₂O and 0.6 g of 2-aminoterephthalic acid are dissolved in 20 mL of DMF solution. After ultrasonic treatment for 15 minutes, the two solutions are mixed in a 1:1 volume ratio. After ultrasonic treatment for 15 minutes, the solution is transferred to a lined autoclave and reacted at 110°C for 24 hours. After the reaction, the precipitate is centrifuged at 10,000 rpm / min for 3 minutes to obtain a precipitate. The precipitate is washed several times alternately with DMF and methanol and dried in a vacuum oven at 60°C to obtain the MIL-101(Fe) metal-organic framework material.

[0029] (2) Synthesis of MIL-101(Fe)@C=C: 0.1~2.0g of MIL-101(Fe) was added to a dry three-necked flask, and MIL-101(Fe) was dissolved in 5~40mL of anhydrous toluene. After ultrasonic dispersion, 0.5~5mL of vinyltriethoxysilane was added. The mixture was stirred at 30~90℃ and 300~1000rpm / min for 3~48 hours. After the reaction, the mixture was centrifuged at 5000~15000rpm / min for 1~20 minutes to obtain a precipitate. The precipitate was washed repeatedly with toluene and ethanol alternately for several times, and vacuum dried at 30~90℃ to obtain the imprinted carrier MIL-101(Fe)@C=C.

[0030] Preferably, 0.5 g of MIL-101(Fe) is added to a dry three-necked flask, and MIL-101(Fe) is dissolved in 20 mL of anhydrous toluene. After ultrasonic dispersion, 1 mL of vinyltriethoxysilane is added, and the reaction is stirred at 70°C and 600 rpm / min for 24 hours. After the reaction is completed, the precipitate is centrifuged at 10,000 rpm / min for 3 minutes to obtain a precipitate. The precipitate is washed alternately with toluene and ethanol several times, and vacuum dried at 60°C to obtain the imprinted carrier MIL-101(Fe)@C=C.

[0031] (3) Preparation of MIPs: 5-100 mg of imprinted vector MIL-101(Fe)@C=C was dissolved in 5-40 mL of DMF solution, 5-200 μL of template virus TMV was added and stirred to allow the template virus to fully combine with the imprinted vector, and then 5-200 μL of 0.05-1.0 mmol 4-vinylpyridine and 0.2-5 mL of DMF containing 10-200 mg of 0.1-2.0 mmol zinc acrylate were added. After pre-assembly at 20-60 °C for 0.5-3.0 hours, 10-100 μL of 0.1-2.0 mmol ethylene glycol dimethyl acrylate was added and stirred under argon. The mixture was deoxygenated at room temperature, and 0.1-10 mL of a DMF solution containing 5-200 mg of azobisisobutyronitrile was added for polymerization. The mixture was sealed and continued to react under argon protection at 30-90° C. and 400-1500 rpm / min for 1-48 hours. After the reaction, the precipitate was centrifuged to obtain a precipitate, which was repeatedly eluted several times with an eluent of methanol:acetonitrile = 1:9-20:1, and then washed several times with ultrapure water, each time eluting in a water bath thermostat at 20-60° C., and then dried in a vacuum drying oven to obtain molecularly imprinted polymer MIPs particles, namely, a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe).

[0032] Preferably, 30 mg of the imprinted vector MIL-101(Fe)@C=C is dissolved in 18 mL of DMF solution, 50 μL of the template virus TMV is added and stirred for 30 minutes to fully combine the template virus with the imprinted vector, and then 20 μL of 0.2 mmol 4-vinylpyridine and 1 mL of DMF containing 42 mg of 0.2 mmol zinc acrylate are added. After pre-assembly at 37 ° C for 1.5 hours, 40 μL of 0.2 mmol ethylene glycol dimethyl acrylate is added, deoxygenated under argon for 30 minutes, and 1 mL of DMF solution containing 20 mg of azobisisobutyronitrile is added for polymerization. After sealing, the reaction is continued under argon protection at 65 ° C and 700 rpm / min for 12 hours; after the reaction is completed, the precipitate is centrifuged at 10,000 rpm / min for 3 minutes to obtain the precipitate, and the precipitate is repeatedly eluted 4 times with methanol: acetonitrile = 9:1 eluent, and then washed twice with ultrapure water, each time at 37 The eluted particles were shaken in a water bath thermostat at 500 °C for 30 min and then dried in a vacuum drying oven at 60 °C for 12 h to obtain 32 mg of MIPs particles.

[0033] In this example, the preparation method of NIPs was the same as above, except that TMV was not added, and 38 mg of NIPs particles were obtained.

[0034] Feasibility analysis of tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe) metal chelation: In order to verify the feasibility of the present invention, this example verifies the constructed tobacco mosaic virus molecular imprinting sensor. Figure 2 Figure A compares the absorbance at around 475 nm of MIPs and NIPs before and after 60 minutes of adsorption of the same concentration of TMV virus. As can be seen from the figure, the absorbance of MIPs is significantly reduced after the addition of TMV compared to when no target virus is added. This is because there are synergistic recognition sites for TMV and functional monomers in MIPs. Therefore, after the adsorption of TMV, the MIPs will block the cavity of the MIPs shell, reducing the exposed MIL-101 (Fe). Correspondingly, the Fe released after adding a certain concentration of hydrochloric acid (HCl) is reduced. 3+ The concentration of MIL-101(Fe) in NIPs is also reduced, thereby inhibiting the complexation reaction with potassium thiocyanate (KSCN). The resulting red solution also becomes lighter, resulting in a decrease in absorbance around 475 nm. In contrast, there is no significant difference in the absorbance of NIPs before and after the addition of TMV. This is because NIPs do not have a specific recognition site for TMV and no exposed MIL-101(Fe). Therefore, the absorbance at around 475 nm is only low before and after the addition of TMV virus. The experimental results were then photographed, as shown in the figure below. Figure 2As shown in B, the visualization results of MIPs / NIPs polymer before and after adding TMV virus are shown, which are consistent with the UV spectrum. Figure 1 The results are consistent, further demonstrating the sensor's excellent visualization. Both the UV spectrum and the visualization results indicate that the proposed metal-organic framework (MOF)-based visualized molecular imprinting sensor is highly feasible for detecting tobacco mosaic virus. These results demonstrate the high feasibility of the constructed visualized sensor.

[0035] Performance, morphology and structural characterization of tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) metal chelation and its intermediates: The prepared particles were characterized by particle size distribution, zeta potential, infrared spectroscopy (FT-IR), water contact angle and scanning electron microscopy (SEM); Figure 3 As shown in Figures A, B, C, and D, the average particle size of the synthesized MIL-101(Fe) is about 400 nm; the average particle size of MIL-101(Fe)@C=C is about 450 nm. This may be because the hydrophobicity becomes stronger after the double bond is grafted, so the dispersibility in water is poor, resulting in the agglomeration of MIL-101(Fe)@C=C, which increases the particle size. The average particle size of MIPs and NIPs is about 500~600 nm. The difference in particle size between the two is because template viruses are added to MIPs during the synthesis process, while template viruses are not added to NIPs.

[0036] The individual particles were then further characterized by Fourier transform infrared spectroscopy and Zeta potential, such as Figure 4 As shown in A, curve a at 3368 cm -1 and 1578 cm -1 The characteristic peaks appearing at 1656 cm-1 belong to the stretching vibrations of -NH2 and C=N in 2-aminoterephthalic acid, which indicates that MIL-101(Fe) was successfully prepared. Curve b has similar characteristic absorption peaks to curve a, but at 1656 cm-1 -1 A new characteristic absorption peak appeared, which was derived from the stretching vibration of C=C on vinyltriethoxysilane, indicating that C=C was successfully modified on MIL-101(Fe); the absorption peaks of MIPs and NIPs were consistent, and compared with the material before imprinting, the absorption peak at 1718 cm -1 and 838 cm -1 New characteristic peaks appeared on the left and right, belonging to the C=O on EGDMA or Zinc AA and the CH bending vibration outside the 4-VP pyridine ring, indicating that the functional monomers and cross-linkers were successfully polymerized to form MIPs and NIPs.

[0037] like Figure 4As shown in B, MIL-101(Fe) contains a large amount of Fe 3+ , resulting in a positive potential of approximately +27 mV. After C=C grafting, the potential dropped to approximately +5 mV. Following the introduction of functional monomers and crosslinkers, the potentials of both MIPs and NIPs became negative. Both infrared spectroscopy and zeta potential results demonstrate relevant changes in the surface properties of the polymers during synthesis, demonstrating the successful preparation of each material.

[0038] The chemical properties of the surfaces of various materials were characterized. Figure 5 The water contact angles of MIL-101(Fe), MIL-101(Fe)@C=C, MIPs, and NIPs show that MIL-101(Fe) is hydrophilic and becomes hydrophobic after surface vinyl functionalization. After imprinting, the contact angles of MIPs and NIPs change due to the hydrophobicity of the functional monomers, cross-linkers, and initiators, proving that the polymerization of MIPs and NIPs is successful.

[0039] In order to further verify the imprinting effect, MIL-101(Fe), MIL-101(Fe)@C=C, MIPs, and NIPs were characterized by SEM. Figure 6 As can be seen in Figure A, the surface of MIL-101(Fe) is a smooth octahedral morphology with an average particle size of about 400 nm. The MIPs ( Figure 6 (shown in B), NIPs ( Figure 6 The morphology of the particles (shown in Figure C) changed to a spherical shape and agglomerated. The DLS size was similar to that in the SEM image, confirming the successful preparation of each material.

[0040] Example 2: Application of tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) metal chelation; The tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) constructed in Example 1 was used to perform semi-quantitative visual detection of tobacco mosaic virus TMV, or to perform quantitative detection and analysis of tobacco mosaic virus TMV using an ultraviolet spectrophotometer.

[0041] The visual detection method includes: Hydrochloric acid was introduced to allow the TMV-specific recognition cavity structure of molecularly imprinted polymers (MIPs) to cleave and release Fe 3+ , followed by the addition of potassium thiocyanate, through Fe 3+ The complexation reaction with KSCN generates a red complex; the molecularly imprinted polymer MIPs reacts with Fe 3+ The complexation reaction of KSCN and PEG-KSCN can realize the semi-quantitative visual detection of tobacco mosaic virus TMV.

[0042] The quantitative detection and analysis method is specifically as follows: 0.01-20 mg of molecularly imprinted polymers (MIPs) were dispersed in 0.1-10 mL of ultrapure water by ultrasound to obtain a MIPs solution. 50-1000 μL of the MIPs solution was added to a test tube, and 10-1000 μL of 0.01-1.0 M HCl was added and reacted for 1-30 min. Then, 50-1000 μL of 0.01-2.0 M KSCN solution was added and reacted for 0.5-30 min, and the supernatant was collected. The absorbance of the supernatant at 300-775 nm was measured by UV-visible absorption spectroscopy, and the absorbance intensity at this time was recorded as Abs0; Different concentrations of TMV solution were added to the MIPs solution and oscillated for a certain period of time. Then 50-1000 μL of the solution was taken and measured in the same way. The absorbance at different concentrations was Abs. i , the linear regression equation is obtained by fitting; The linear regression equation is used to quantitatively detect and analyze the concentration of TMV in the test solution.

[0043] Specifically, the experimental conditions in this example are: the dosage of MIPs is 0.4 mg / mL, the virus adsorption time is 60 min, the reaction time of HCl is 1 min, and the optimal reaction time of KSCN is 3 min.

[0044] MIPs solution was obtained by dispersing 1 mg of molecularly imprinted polymers (MIPs) in 1 mL of ultrapure water by ultrasound; 400 μL of MIPs solution was added to the test tube, and 100 μL of 0.1 M HCl was added and reacted for 1 min. Then 600 μL of 0.2 M KSCN solution was added and reacted for 3 min. The supernatant was collected. The absorbance of the supernatant at 475 nm was measured by UV-visible absorption spectroscopy, and the absorbance intensity at this time was recorded as Abs0; TMV solutions of different concentrations were added to the MIPs solution and oscillated for a certain period of time. Then 400 μL of the solution was taken and measured in the same way. The absorbance at different concentrations was Abs. i , the linear regression equation is obtained by fitting; The linear regression equation is used to quantitatively detect and analyze the concentration of TMV in the test solution.

[0045] Detection linearity analysis of different concentrations of TMV by the tobacco mosaic virus molecular imprinting sensor based on metal chelation MIL-101 (Fe): According to the above experimental steps, the tobacco mosaic virus molecular imprinting sensor based on metal chelation MIL-101 (Fe) of the present invention was used to perform detection linearity analysis of different concentrations of TMV. The results are as follows: Figure 7 As shown in Figure A, the absorbance change value (∆Abs) of MIPs showed a linear correlation with the increase of TMV concentration. The linear regression equation obtained by fitting was ∆Abs=0.04412 logC TMV +0.0794, (R 2 =0.99447, Figure 7 The results are shown in Figure B), indicating that MIPs have excellent quantitative analysis capabilities for TMV detection. Based on the standard deviation of the blank response value, the detection limit of the sensor was calculated to be 0.0132 pM. ∆Abs is defined as the difference in absorbance before and after the addition of TMV virus to the MIPs (∆Abs = Abs0 - Abs i ), reflecting the specific recognition ability of MIPs for TMV. In contrast, NIPs, due to the lack of TMV-specific recognition cavity, only adsorbed the virus through nonspecific interactions, and their absorbance values did not change significantly at different TMV concentrations ( Figure 7 The specific recognition performance of MIPs was further verified. Figure 7 (D) intuitively demonstrates the concentration dependence of the MIPs detection system: with the increase of TMV concentration, the color of the solution shows an obvious gradient change, while the NIPs solution always remains light in color with no obvious color difference.

[0046] At the same time, the RGB value of each colorimetric image was calculated using the image acquisition system of the smartphone. In the range of 0-5.75 pM, there was a good linear relationship between the concentration of TMV and G / B, and the linear equation was G / B=-0.28834+1.48228 ( Figure 8 (as shown in A in the figure). Figure 8 Middle B shows an image of MIPs captured by a smartphone. As the TMV concentration increases, the color of the MIPs gradually changes from reddish brown to light orange, while the color of the NIPs does not change significantly and all appear lighter.

[0047] The selectivity and competition experiment of the tobacco mosaic virus molecular imprinting sensor based on metal chelation MIL-101 (Fe) to TMV Influenza virus (H5N1), enterovirus 71 (EV71), hepatitis A virus (HAV), and Japanese encephalitis (JEV) were selected as analytical targets for selectivity and competition experiments.

[0048] The experiment was carried out according to the above steps, and the selectivity of the sensor was analyzed. The experimental results are as follows Figure 9 As shown in Figure A, the results show that the ∆Abs of MIPs to the target virus TMV is significantly higher than that of other control viruses. Due to differences in size, shape, and surface functional groups between other viruses and TMV, they cannot effectively match the recognition sites on the surface of MIPs, resulting in low selectivity of MIPs to them. In contrast, due to the lack of specific recognition sites, NIPs have lower absorbance values for TMV and other viruses, which indicates that the sensor has excellent selective recognition ability for TMV. Similarly, the competitiveness of the paper-based sensor was evaluated by adding the corresponding competitive virus to the target virus TMV. The results are shown in Figure 2. Figure 9 As shown in Figure B, in the presence of the competing virus, the absorbance change of the MIPs for TMV was not significantly different from that when TMV alone was added. This indicates that the presence of the competing virus does not significantly affect the MIPs' ability to specifically recognize TMV. In the competition experiment, the absorbance changes of the NIPs for TMV and other viruses were low and non-significant, further demonstrating that the selective recognition ability of the MIPs stems from the specific recognition cavities on their surfaces, rather than nonspecific adsorption. This demonstrates that the prepared sensor has good competitive properties for TMV.

[0049] Experimental study on the reproducibility, time stability and anti-interference ability of the tobacco mosaic virus molecular imprinting sensor based on metal chelation MIL-101 (Fe) The MIPs synthesized from five different batches were tested under the same conditions against the target virus TMV at the same concentration. Figure 10 As shown in Figure A, the absorbance change ∆Abs of 5 different batches of MIPs when detecting the same concentration of TMV is basically unchanged, indicating that the sensor has good reproducibility.

[0050] In order to investigate the effect of the prepared sensor on the detection performance after being placed for a period of time, the target virus TMV was detected after the MIPs synthesized in the same batch were placed for 0 weeks, 1 week, 2 weeks, 4 weeks, and 6 weeks. Figure 10 As shown in Figure B, with the extension of the storage time, the ∆Abs of TMV detection under the same conditions showed a slight downward trend. However, after 6 weeks, the ∆Abs of MIPs was 84.85% of the initial detection performance, a decrease of only 15.15%, indicating that the sensor has good stability.

[0051] Some common interfering substances Na + , K + Mg 2+ , Ca 2+ 、HCO3 - 、HPO42- , glucose, L-proline, and L-alanine were added to the detection solution to evaluate the anti-interference ability of the sensor. Figure 11 As shown in the figure, in the presence of the above-mentioned interfering substances, the change in the UV absorbance of TMV by MIPs is not significantly different from the absorbance value when no interfering substances are added. This shows that the prepared sensor has good anti-interference ability and is highly feasible for detecting TMV in actual samples.

[0052] (4) In addition, we also explored the time it takes to achieve rapid detection after incubating MIPs with TMV virus for different times and then reacting with HCl and KSCN for different times. The experimental results are as follows: Figure 12 As shown in the figure, the MIPs sensor can detect tobacco mosaic virus with the naked eye after incubation with TMV for 15 minutes and then reacting for 3 minutes. This result shows that this sensor is conducive to the immediate visual detection of target viruses on site.

[0053] Finally, the preparation cost of the sensor was calculated as shown in Table 1. Its preparation cost is as low as 0.082 yuan per piece, indicating that the sensor has excellent application potential and good market prospects.

[0054] Table 1 Cost accounting table of MIPs sensors Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe), characterized in that: The construction method uses tobacco mosaic virus (TMV) as a template virus and prepares molecularly imprinted polymers (MIPs) with TMV-specific recognition cavities on the surface of MIL-101 (Fe) through free radical polymerization. The template virus is removed during the preparation of the molecularly imprinted polymer MIPs, and the TMV-specific recognition cavity of the molecularly imprinted polymer MIPs contains MIL-101 (Fe); The molecular imprinting polymer MIPs is a tobacco mosaic virus molecular imprinting sensor based on MIL-101 (Fe).

2. The method for constructing a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) according to claim 1, characterized in that: The method specifically includes: (1) Synthesis of MIL-101(Fe): FeCl3·6H2O and 2-aminoterephthalic acid were weighed and dissolved in DMF solution respectively. After ultrasonic treatment, the two solutions were mixed. After ultrasonic treatment, the mixed solution was transferred to a lined autoclave for reaction. After the reaction, the precipitate was obtained by centrifugation. The precipitate was washed alternately with DMF and methanol and dried in a vacuum drying oven to obtain the MIL-101(Fe) metal organic framework material. (2) Synthesis of MIL-101(Fe)@C=C: MIL-101(Fe) was dissolved in anhydrous toluene, ultrasonically dispersed, and then vinyltriethoxysilane was added. The mixture was stirred for reaction. After the reaction, the mixture was centrifuged to obtain a precipitate. The precipitate was washed alternately with toluene and ethanol, and vacuum dried to obtain the imprinted carrier MIL-101(Fe)@C=C. (3) Preparation of MIPs: MIL-101(Fe)@C=C was dissolved in DMF solution, tobacco mosaic virus TMV was added as a template virus, and the mixture was stirred to allow the template virus to fully combine with the imprinted carrier MIL-101(Fe)@C=C. 4-vinylpyridine and DMF containing zinc acrylate were then added. After pre-assembly, ethylene glycol dimethyl acrylate was added and deoxygenated under argon. Then, a DMF solution containing azobisisobutyronitrile was added for polymerization. The reaction was continued under argon protection. After the reaction was completed, the mixture was centrifuged to obtain a precipitate. The precipitate was repeatedly eluted with a mixed eluent of methanol and acetonitrile to remove the template virus. The precipitate was then washed with ultrapure water and dried in a vacuum drying oven to obtain molecularly imprinted polymer MIPs particles, i.e., a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) was obtained.

3. The method for constructing a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) according to claim 1, characterized in that: Step (1) is specifically as follows: 0.5-3.0 g of FeCl3·6H2O and 0.2-2 g of 2-aminoterephthalic acid are weighed and dissolved in 5-40 mL of DMF solution respectively, and after ultrasonic treatment for 5-25 minutes, the two solutions are mixed in a volume ratio of (1-4):1, and after ultrasonic treatment for 5-30 minutes, the solution is transferred to a lined high-pressure sterilizer and reacted at 80-140° C. for 6-48 hours; after the reaction is completed, the solution is centrifuged at 8000-10000 rpm / min for 1-20 minutes to obtain a precipitate, and the precipitate is washed alternately with DMF and methanol several times, and dried in a vacuum drying oven at 30-90° C. to obtain the MIL-101 (Fe) metal organic framework material.

4. The method for constructing a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) according to claim 1, characterized in that: Step (2) is specifically as follows: 0.1~2.0g MIL-101(Fe) is added to a dry three-necked flask, MIL-101(Fe) is dissolved in 5~40mL anhydrous toluene, 0.5~5mL vinyltriethoxysilane is added after ultrasonic dispersion, and the mixture is stirred and reacted at 30~90°C and 300~1000rpm / min for 3~48 hours. After the reaction is completed, the mixture is centrifuged at 5000~15000rpm / min for 1~20 minutes to obtain a precipitate, the precipitate is washed repeatedly with toluene and ethanol alternately several times, and vacuum dried at 30~90°C to obtain the imprinted carrier MIL-101(Fe)@C=C.

5. The method for constructing a tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) according to claim 1, characterized in that: Step (3) is specifically as follows: 5~100 mg of imprinted vector MIL-101(Fe)@C=C is dissolved in 5~40 mL of DMF solution, 5~200 μL of template virus TMV is added and stirred to allow the template virus to fully combine with the imprinted vector, and then 5~200 μL of 0.05~1.0 mmol of 4-vinylpyridine and 0.2~5 mL of DMF containing 10~200 mg of 0.1~2.0 mmol of zinc acrylate are added. After preassembly at 20~60°C for 0.5~3.0 hours, 10~100 μL of 0.1~2.0 mmol of zinc acrylate is added. 1 ethylene glycol dimethyl acrylate was deoxygenated under argon, and 0.1-10 mL of a DMF solution containing 5-200 mg of azobisisobutyronitrile was added for polymerization. The mixture was sealed and continued to react under argon protection at 30-90°C and 400-1500 rpm / min for 1-48 hours. After the reaction, the precipitate was centrifuged and repeatedly eluted with an eluent of methanol:acetonitrile = 1:9-20:1 several times, and then washed with ultrapure water several times, each time eluting in a water bath constant temperature oscillator at 20-60°C, and then dried in a vacuum drying oven to obtain molecularly imprinted polymer MIPs particles.

6. Application of the tobacco mosaic virus molecular imprinting sensor based on MIL-101(Fe) constructed by the construction method according to any one of claims 1 to 5, characterized in that: The MIL-101(Fe)-based tobacco mosaic virus molecular imprinting sensor is used to perform semi-quantitative visual detection of tobacco mosaic virus TMV, or a UV spectrophotometer is used to perform quantitative detection and analysis of tobacco mosaic virus TMV.

7. The use according to claim 6, characterized in that The visual detection method includes: Hydrochloric acid was introduced to allow the TMV-specific recognition cavity structure of molecularly imprinted polymers (MIPs) to cleave and release Fe 3+ , followed by the addition of potassium thiocyanate, through Fe 3+ The complexation reaction with KSCN generates a red complex; the molecularly imprinted polymer MIPs reacts with Fe 3+ The complexation reaction of KSCN and PEG-KSCN can realize the semi-quantitative visual detection of tobacco mosaic virus TMV.

8. The use according to claim 7, characterized in that The quantitative detection and analysis method is specifically as follows: 0.01-20 mg of molecularly imprinted polymers (MIPs) were dispersed in 0.1-10 mL of ultrapure water by ultrasound to obtain a MIPs solution. 50-1000 μL of the MIPs solution was added to a test tube, and 10-1000 μL of 0.01-1.0 M HCl was added and reacted for 1-30 min. Then, 50-1000 μL of 0.01-2.0 M KSCN solution was added and reacted for 0.5-30 min, and the supernatant was collected. The absorbance of the supernatant at 300-775 nm was measured by UV-visible absorption spectroscopy, and the absorbance intensity at this time was recorded as Abs0; Different concentrations of TMV solution were added to the MIPs solution and oscillated for a certain period of time. Then 50-1000 μL of the solution was taken and measured in the same way. The absorbance at different concentrations was Abs. i , the linear regression equation is obtained by fitting; The linear regression equation is used to quantitatively detect and analyze the concentration of TMV in the test solution.

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