Construction and application of portable rapid visual paper-based molecular imprinting sensor for tobacco mosaic virus

Abstract

The invention provides a construction method and application of a portable rapid visual tobacco mosaic virus paper-based molecular imprinting sensor. According to the sensor, tobacco mosaic virus (TMV) is taken as a template, high-specificity organic fluorescent nanogel imprinted polymer TMV-nanoMIPs obtained by solid-phase imprinting is taken as signal output, meanwhile, a cheap and portable paper base is taken as a sensing chip, TMV is imprinted on the paper base, and the paper-based imprinted polymer WP-coated MIPs with a TMV imprinted cavity is obtained after elution. After the WP-coated MIPs specifically capture the TMV, the TMV-nanoMIPs also can be combined to the TMV due to the specificity of the TMV-nanoMIPs, so that a sandwich structure of the MIPs-TMV-MIPs is formed. The TMV-nanoMIPs are used as the signal probe, the rapid semi-quantitative visual detection of the target virus TMV by naked eyes and an ultraviolet lamp is realized, the obvious change can be observed within ten minutes, and meanwhile, the TMV-nanoMIPs can be quantitatively detected and analyzed through a fluorospectro photometer. And the method has good detection capability in tobacco leaves actually infected by TMV, and the cost of the method is as low as 0.623 yuan / piece. The sensor is expected to realize on-site self-service rapid visual detection when crops are infected with viruses, and has important significance on prevention and control of plant diseases and pests.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry detection, and in particular relates to a method for preparing a portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor. Background Art

[0002] The impact of plant pathogens on global crop yields is a major societal issue. Common plant virus detection methods include reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and gel blot analysis. However, these operating methods and instruments cannot be used directly in the field and are often costly. Therefore, there is an urgent need to develop rapid, accurate, sensitive, portable, and easy-to-operate detection technologies.

[0003] Molecularly imprinted polymers (MIPs), also commonly referred to as antibody mimics or synthetic antibodies, are biomimetic materials with specific cavities to recognize and bind to target molecules (antigens). They are copolymerized by functional groups and cross-linking monomers in the presence of molecular templates (target molecules or structural analogs). They have the advantages of being easy to synthesize, cost-effective, durable, and stable over a wide pH range, various solvents, and thermal conditions. For example, Wu et al. used molecularly imprinted membrane electrospray ionization mass spectrometry to rapidly detect four types of pesticide residues in CHM, increasing the detection limit of traditional analytical methods by about 50 times. [Wu C, Li T, Li D, et al. Rapid detection of pesticide residues in Chinese herbal medicines by molecularly imprinted membrane electrospray ionization mass spectrometry[J]. Chinese Chemical Letters, 2021, 32(7): 2174-2178.]. Gong et al. developed a portable, multicolor, colorimetric molecular imprinting polymer (MIP)-based sensor that can detect viruses in three modes with a detection limit of 11.6 fM. [Gong H, Tang L, Chen F, et al. Self-service multimodal detection of subtype influenza A virus H5N1 by visual portable molecular imprinting sensor[J]. Chemical Engineering Journal, 2024, 483: 148946.]. Luo et al. developed a multifunctional molecular imprinting sensor that can be used to measure hepatitis A virus (HAV) and hepatitis B virus (HBV) at one time or simultaneously, achieving detection limits of 3.4 and 5.3 pM, respectively [Luo L, Zhang F, Chen C, et al. Visual simultaneous detection of hepatitis A and B viruses based on a multifunctional molecularly imprinted fluorescence sensor[J]. Analytical chemistry, 2019, 91(24): 15748-15756.]. However, although these methods have achieved good detection results, they have disadvantages such as high cost, 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.

[0004] As an emerging green material, paper materials do not require complex instruments for analysis and are very suitable for rapid analysis in areas with scarce medical resources and harsh environments. Compared with traditional analytical techniques, paper-based chips have low manufacturing costs, are easy to operate, convenient to transport, fast to detect, and easy to handle after testing. As a multifunctional substrate material, paper has a porous structure and contains a large number of hydroxyl functional groups, making its surface easy to modify to obtain a large surface-to-volume ratio. This makes it an excellent advantage for integrating functional nanomaterials and a flexible platform for developing complex analytical equipment. For example, Li et al. used fluorescent quantum dots combined with molecularly imprinted polymers on three-dimensional origami paper microfluidic devices to achieve specific recognition and sensitive detection of phycocyanin. [Li B, Zhang Z, Qi J, et al. Quantum dot-based molecularly imprinted polymers on three-dimensional origami paper microfluidic chip for fluorescence detection of phycocyanin[J]. ACS sensors, 2017, 2(2): 243-250.].

[0005] Based on the above background, the present invention provides a preparation method and application of a portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor. The method uses paper that is cheap, easy to obtain, has a large surface area, is easy to cut, and is easy to modify as a substrate, and obtains molecular imprinting polymers (MIPs) after imprinting on its surface. On the other hand, a highly specific organic fluorescent nanogel imprinted polymer TMV-nanoMIPs is obtained by solid phase imprinting as a signal output. The results show that the sensor achieves rapid semi-quantitative detection of tobacco mosaic virus (TMV) visualized by the naked eye under visible light and ultraviolet light, and obvious changes can be observed in ten minutes; at the same time, it can also be quantitatively detected and analyzed by a fluorescence spectrophotometer. The sensor has the advantages of rapidity, low cost, portability, high sensitivity, and high specificity. Summary of the invention

[0006] The purpose of the present invention is to construct a method for preparing a portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor, and use the sensor for specific and visualized detection of TMV virus to improve the effect of actual virus detection.

[0007] The purpose of the present invention is achieved through the following technical solutions.

[0008] A method for preparing a portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor, characterized in that the method has the following process steps:

[0009] (1) Preparation of paper-based imprinted polymers (WP@MIPs): Inexpensive and portable filter paper was used as the imprinting material substrate. Since the main components of paper are cellulose and lignin, its surface is rich in hydroxyl groups and is easy to be modified with functional groups on the surface. By modifying double bonds on the filter paper surface, zinc acrylate (Zinc AA) and 4-vinylpyridine (4-VP) were used as functional monomers, ethylene glycol dimethyl acrylate (EGDMA) was used as a cross-linker, and N,N-azobisisobutyronitrile (AIBN) was used as an initiator. Virus molecular imprinted paper WP@MIPs with TMV-specific recognition cavities was prepared by free radical polymerization, and used as a capture and separation element for template viruses.

[0010] (2) Preparation of organic fluorescent nanogel imprinted polymer TMV-nanoMIPs: The surface of magnetic iron oxide was coated with amino groups by using silanization reagents TEOS and APTES, and then the aldehyde groups on the surface of magnetic particles were modified with glutaraldehyde by Schiff base reaction; the aldehyde groups can couple with the amino groups and then fix the virus TMV on the magnetic particles. Then, N-isopropylacrylamide (NIPAM) as a backbone monomer and N,N′-methylenebisacrylamide (BIS) as a crosslinker were combined with N-tert-butylacrylamide (TBAm), acrylic acid (AA) and aminopropylmethacrylamide (APMA), the latter three of which were used to balance the hydrophobic, cationic and anionic sites on the virus surface, respectively. The nanogel particles were fluorescently detected by adding fluorescent dye complexes, and the polymerization was initiated using ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED), and the reaction was allowed to react overnight at room temperature 25°C. After the reaction is complete, the virus template is eluted at 60°C using the temperature-sensitive properties of the functional monomer NIPAM to obtain TMV-nanoMIPs with high affinity for TMV. In addition to being able to specifically recognize viruses, this nanoparticle also has signal output capabilities. It not only fluoresces under ultraviolet light, but also displays red under visible light.

[0011] (3) Preparation of portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor: After the template virus TMV and the paper-based molecular imprinting polymer are adsorbed for a period of time under optimized experimental conditions, they are washed with ultrapure water, and then the fluorescent colorimetric probe TMV-nanoMIPs are added for adsorption for a period of time to remove the unbound TMV-nanoMIPs. Then, the paper-based MIPs and NIPs are inserted along the diagonal of the cuvette to measure their fluorescence. The excitation and emission slits are both 5nm, the voltage is 600v, and the fluorescence intensity at 588nm is collected. The color on the paper-based MIPs / NIPs under visible light and ultraviolet light is photographed using a mobile phone.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) The sensor uses paper as the sensor chip. Paper is cheap and easy to obtain, has excellent hydrophilicity, is easy to modify, easy to store, non-toxic, and easy to carry. It provides a sensitive and reliable detection method for TMV and reduces the preparation cost of the sensor, which is as low as 0.623 yuan per piece.

[0014] (2) Organic fluorescent nanogel imprinted polymer TMV-nanoMIPs is used as a signal probe. The signal probe has obvious color both under the naked eye and under ultraviolet light, and can recognize and bind to TMV with high specificity, which can significantly increase the specificity of the sensor and improve the sensitivity.

[0015] (3) The sensor can realize colorimetric semi-quantitative detection of the target object by naked eyes without the aid of any instrument, and can realize fluorescent visualization semi-quantitative detection with the aid of ultraviolet light. At the same time, it can also be used for quantitative detection and analysis by fluorescence spectrophotometer. The detection time required is short, and obvious changes can be observed in ten minutes. A rapid, specific and sensitive TMV visual detection method has been established.

[0016] (4) The sensor has good detection capability in tobacco leaves infected with TMV and can provide good visualization effect for the target virus, providing a new technical means for on-site detection of TMV.

[0017] (5) The virus molecular imprinting polymer has low preparation cost, simple sensor construction process, easy operation, sensitive and rapid detection, and low professional requirements for operators, and has broad prospects in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] [ Figure 1 ]The preparation flow chart of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor described in the present invention.

[0019] [ Figure 2 ](A) Feasibility principle verification of TMV-nanoMIPs; (B) Feasibility principle verification of WP@MIPs (the upper part of the illustration is the visual effect under visible light, and the lower part is the visual effect under 367nm UV light).

[0020] [ Figure 3 ]Dynamic light scattering patterns of (A)Fe3O4, (B)Fe3O4@SiO2, and (C)Fe3O4@SiO2-NH2.

[0021] [ Figure 4](A) Zata potential diagrams of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-NH2, Fe3O4@CHO and Fe3O4@TMV. Error bars represent the standard deviation of three replicates. (B) FTIR spectra of Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2-NH2.

[0022] [ Figure 5 ]Water contact angles of (A) WP, (B) WP@C=C, (C) WP@MIPs, and (D) WP@NIPs.

[0023] [ Figure 6 ]Scanning electron micrographs of (A) WP, (B) WP@C=C, (C) WP@MIPs, (D) WP@NIPs (300 times magnification); Scanning electron micrographs of (E) WP, (F) WP@C=C, (G) WP@MIPs, (H) WP@NIPs (2000 times magnification).

[0024] [ Figure 7 ](A) Linearity of WP@MIPs in detecting different concentrations of TMV (0.0288, 0.144, 0.288, 0.411, 0.575, 1.44, 2.88, 4.11, 5.75, 14.4, 28.8 pM); (B) Visualization of different concentrations of TMV under visible light (upper) and 365 nm UV light (lower); (C) Linear fitting of WP@MIPs; (D) Linearity of WP@NIPs in detecting different concentrations of TMV (0.0288, 0.144, 0.288, 0.411, 0.575, 1.44, 2.88, 4.11, 5.75, 14.4, 28.8 pM). Error bars represent the standard deviation of three replicates.

[0025] [ Figure 8 ](A) Selectivity of WP@MIPs for virus TMV; (B) Competitiveness of WP@MIPs for virus TMV. Error bars represent the standard deviation of three replicates.

[0026] [ Fig. 9 ](A) Ion interference of WP@MIPs; (B) Reproducibility of WP@MIPs; (C) Repeatability of WP@MIPs; (D) Time stability of WP@MIPs. Error bars represent the standard deviation of three replicates.

[0027] [ Fig.10 ]Visualization of WP@MIPs detecting TMV in PBS and plant juice samples under visible and UV light.

[0028] [ Fig.11]Visualization of TMV detection time of WP@MIPs in PBS and tobacco juice samples under visible light and UV light.

[0029] [ Fig.12 ]Visualization of WP@MIPs detecting TMV in TMV-infected tobacco mosaic sap and common plant sap samples under visible and UV light.

[0030] [ Fig.13 ]Cost accounting of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor. Specific implementation plan

[0031] Here, the specific embodiments of the present invention will be further described in detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but do not limit the scope of application and extension of the present invention.

[0032] Example 1: Preparation method of a portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor

[0033] (1) Preparation of Fe3O4@SiO2: FeCl3·6H2O (5.4 g) was dissolved in ethylene glycol (160 mL) and stirred vigorously to obtain a clear solution. Then, NaAc (14.4 g) and polyethylene glycol (4 g) were added and stirred at 50°C for 30 min. The mixture was transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 24 h. The product was washed with ethanol and water several times and dried under vacuum at 50°C to obtain Fe3O4 (1.508 g). 500 mg of Fe3O4 was weighed, added with 50 mL of isopropanol and 2 mL of deionized water, and ultrasonically dispersed for 30 min. 5 mL of NH3·H2O and 2.5 mL of TEOS were added under mechanical stirring at 700 rpm and reacted at 45°C for 6 h. The obtained product was collected with a magnet and washed with ethanol and water alternately for several times. It was then placed at 50°C and dried under vacuum overnight to obtain Fe3O4@SiO2.

[0034] (2) Preparation of Fe3O4@SiO2-NH2: 500 mg of Fe3O4@SiO2 particles were taken and completely dispersed in 80 mL of anhydrous ethanol / ultrapure water (1 / 1, v / v) by ultrasonication for 30 min. The mixture was heated and stirred at 40°C for 5 min, and then 2 mL of APTES was slowly added dropwise to the flask and reacted for 12 h. The particles after the reaction were gathered at the bottom of the flask with a magnet, the supernatant was poured off, and the particles were washed alternately with ultrapure water and anhydrous ethanol for multiple times. The washed particles were vacuum dried at 60°C overnight to obtain Fe3O4@SiO2-NH2 particles.

[0035] (3) Preparation of Fe3O4@TMV: 100 mg of Fe3O4@SiO2-NH2 particles were suspended in 10 mL of glutaraldehyde (5%, v / v), deoxygenated with argon for 30 min, and stirred at 37°C for 3 h under argon protection and in the dark. Aldehyde groups were introduced on the surface of Fe3O4@SiO2-NH2 particles to obtain Fe3O4@CHO for subsequent virus fixation. The magnetic particles were collected with the help of external magnetic force and washed three times with deoxygenated PBS (0.01 M, pH = 7.4) to remove unreacted glutaraldehyde. The collected aldehyde-modified particles were then dispersed in 10 mL of deoxygenated PBS (0.01 M, pH = 7.4), 100 μL of 0.287 nM TMV tobacco mosaic virus was added, and incubated on a water bath constant temperature oscillator for 2 h. Then the magnetic particles were collected, washed to remove excess virus, and dispersed in 10 mL PBS (0.1 M, pH = 7.4) to obtain Fe3O4@TMV.

[0036] (4) Preparation of TMV-nanoMIPs: First, Rhodamine B (0.497 g, 1 mmol) was dissolved in 4 mL of ultrapure water, and then 1 mL of EDC (0.191 g, 1 mmol) and 1 mL of NHS (0.029 g, 0.25 mmol) were added, and the mixture was shaken at 37°C for 1 h to activate the carboxyl groups on Rhodamine B. 4 mL of N-(3-aminopropyl)methacrylamide hydrochloride (APMA, 0.178 g, 1 mmol) was added to the activated Rhodamine B, and the mixture was shaken at 37°C for 2 h. After the reaction was completed, the mixture was extracted twice with dichloromethane and then extracted twice with water. The organic phase was concentrated and dried by rotary evaporation. The solid was recrystallized with acetonitrile and water, and the fluorescent dye complex was obtained after filtration.

[0037] N-isopropylacrylamide (NIPAM, 7.9 mg, 70 μmol), N-tert-butylacrylamide (TBA, 3.8 mg, 30 μmol, dissolved in 500 μL ethanol), acrylic acid (AA, 1.73 μL, 1.1 μmol), N-(3-aminopropyl)methacrylamide hydrochloride (APMA, 0.9 mg, 6 μmol), and N,N-methylenebisacrylamide (BIS, 2 mg, 12.97 μmol) were mixed in 10 mL of ultrapure water, and a fluorescent composite dye (2 mg, dissolved in 500 μL ethanol) was added to the mixture. The mixture was deoxygenated with argon for 30 min and vacuumed with a water pump for 10 minutes. Before vacuuming, 4 mL of TMV-modified magnetic nanoparticles (Fe3O4@TMV) dispersed in PBS was injected into the flask using a needle, and then vacuuming was continued to deoxygenate. The flask was then sealed with a sealing film and protected with argon. The reaction solution was placed on a constant temperature oscillator and oscillated for 30 minutes. 1 mL of ammonium persulfate (APS, 6 mg, 26 μmol) and N,N,N',N'-tetramethylethylenediamine (TEMED, 6 μL) were injected into the flask under a nitrogen atmosphere, and the reaction solution was placed on a constant temperature oscillator and oscillated at 25°C for 12 hours.

[0038] After the reaction, the solid particles were collected with a magnet and washed with 25°C ultrapure water to remove unreacted functional monomers and low-affinity imprinted polymers until there was no fluorescence in the supernatant. Then, the collected magnetic particles were dispersed in 2 mL of ultrapure water and oscillated in a 60°C water bath oscillator for 60 minutes. The magnetic solid particles were separated from the supernatant by the magnetic force of an external magnet. This operation was repeated twice, and the 4 mL supernatant obtained was the soluble nanogel imprinted polymer TMV-nanoMIPs. Stored in a sealed container at 4°C.

[0039] (5) Modification of paper base: Take a 70 mm × 70 mm Whitman No. 1 qualitative filter paper, rinse it with a large amount of ethanol for activation, and vacuum dry it at 50°C. Then add 200 μL of triethylamine (TEA) and 50 mg of 4-(dimethylamino)pyridine (DMAP) to a glass culture dish containing 15 mL of dichloromethane (DCM), add 200 μL of trichlorovinylsilane (TVCs), and finally add the activated paper base to the above culture dish, shake it on a shaker at room temperature for 2 hours, and finally rinse it with ethanol three times to wash away the unreacted monomers, and vacuum dry it at 50°C to obtain the filter paper WP@C=C grafted with vinyl.

[0040] (6) Preparation of paper-based imprinted polymer WP@MIPs / WP@NIPs: 0.1 mmol 4-vinylpyridine (4-VP), 0.1 mmol zinc acrylate (Zinc AA) and 50 μL 2.87 nM TMV virus were dissolved in 20 mL DMF and prepolymerized at 37°C for two hours. Then, 0.3 mmol ethylene glycol dimethyl acrylate (EGDMA) and 4 1.3 cm × 1.3 cm sheets of WP@C=C were added, and argon was used for deoxygenation for 30 min. 20 mg initiator AIBN was added, and finally the mixture was transferred to 65°C for polymerization for 12 h.

[0041] After the polymerization is completed, the paper is taken out and washed with ultrapure water to remove the unreacted matter, and finally eluted with 1.0 mL of eluent methanol: acetic acid = 9:1 (v / v) filter paper for 4 times. Then wash with ultrapure water several times until the eluent is completely washed away, and vacuum dry at 50°C to obtain WP@MIPs. The preparation of WP@NIPs did not add the template virus TMV, and the other operations were consistent with the above.

[0042] (7) Preparation of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor: TMV virus of different concentrations dispersed in PBS (pH=7.4, 10mM) buffer was added to WP@MIPs and WP@NIPs, and the filter paper was removed and washed with ultrapure water three times after shaking incubation at 37°C for 50min. Then, 1mL of ultrasonically dispersed TMV-nanoMIPs solution was added thereto, and the shaking binding was continued at 25°C for 60min to remove the unbound TMV-nanoMIPs. Then, the paper-based MIPs and NIPs were inserted along the diagonal of the cuvette to measure their fluorescence. The excitation and emission slits were both 5nm, the voltage was 600v, and the fluorescence intensity at 588nm was collected. The color on the paper-based MIPs / NIPs under visible light and ultraviolet light was photographed using a mobile phone.

[0043] Example 2: Feasibility analysis of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor

[0044] In order to verify the feasibility of the present invention, this example verifies the construction principle of the sensor. Figure 2A, we incubated TMV-nanoMIPs, TMV-nanoMIPs+Fe3O4@TMV, TMV-nanoMIPs+Fe3O4@TMV+TMV and TMV-nanoMIPs+Fe3O4@TMV+H5N1 at 25℃, and then enriched them by magnets and measured the fluorescence of the supernatant. When TMV-nanoMIPs (50μL) was incubated with Fe3O4@TMV (450μL, 20mg / mL) bound to the target virus TMV, the fluorescence intensity of the supernatant decreased significantly, indicating that the magnetic Fe3O4@TMV and TMV-nanoMIPs specifically bound; when TMV (10μL, 28.75pM) was added to the above solution again, the fluorescence intensity of the supernatant rose again, because TMV competitively replaced the TMV-nanoMIPs bound to the magnetic nanoparticles, making it free in the supernatant again. In order to prove the selectivity of TMV-nanoMIPs for TMV, we added the same concentration of H5N1 virus to compete with it and found that the fluorescence only increased slightly. This shows that the prepared TMV-nanoMIPs has high selectivity for TMV.

[0045] Then we used the above TMV-nanoMIPs as signal output to further explore the feasibility of WP@MIPs on paper substrate. Figure 2 As shown in Figure B, the successfully prepared WP@MIPs / WP@NIPs were first incubated with the target virus TMV (20μL, 28.75pM), and then the WP@MIPs / WP@NIPs that captured TMV were incubated with TMV-nanoMIPs (1mL) solution. By observing the color on the paper base and measuring its fluorescence intensity, it can be found that the fluorescence intensity of WP@MIPs with TMV added is the strongest, and the visualization effect under visible light and ultraviolet light is the most obvious. However, WP@NIPs with TMV added and WP@MIPs / WP@NIPs without TMV only have a slight fluorescence intensity and are almost invisible under visible light and ultraviolet light. The above results show that the paper-based visualization sensor we constructed is highly feasible.

[0046] Example 3: Performance, morphology and structural characterization of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor and intermediates.

[0047] The prepared particles were characterized by particle size distribution, Zeta potential, infrared spectroscopy (FT-IR), water contact angle and scanning electron microscopy (SEM).

[0048] like Figure 3As shown in A, B, and C, the particle sizes of the synthesized Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2-NH2 are approximately 300nm, 425nm, and 500nm. Since TEOS is hydrolyzed and polymerized to form a silicon layer on the surface of Fe3O4, its particle size will increase significantly; after obtaining the Fe3O4 coated with the silicon layer, the aminosilane reagent APTES is used to polymerize on its surface for modification, so that its size is further increased. The above changes in particle size prove the successful synthesis of each magnetic nanoparticle.

[0049] Then, the Zata potential was used to further characterize whether the magnetic nanoparticles were successfully synthesized. Figure 4 The change in the Zata potential of A can prove that glutaraldehyde was successfully grafted onto the surface of magnetic nanoparticles and the target virus TMV was successfully immobilized on its surface. Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2-NH2 were characterized by FT-IR. Figure 4 B, 567cm -1 The Fe-O characteristic absorption peak at 803 cm -1 The Si-O symmetric stretching vibration absorption peak at 1094 cm -1 The Si-O-Si asymmetric stretching vibration indicates the successful coating of SiO2 on the Fe3O4 surface; -1 The NH bond stretching vibration absorption peak at 2930cm -1 The CH stretching vibration absorption peak at shows that the amino group was successfully modified onto the surface of the magnetic nanoparticles.

[0050] We also characterized the materials used in the construction of paper-based imprinted polymers. First, the hydrophilicity and hydrophobicity of WP, WP@C=C, WP@MIPs, and WP@NIPs were characterized by a contact angle meter. Figure 5 ), WP is a highly hydrophilic material, and its water contact angle is almost 0° ( Figure 5 A); after its surface is modified with a vinyl silane reagent and double bonds are attached, its water contact angle becomes 118° ( Figure 5 B), showing strong hydrophobicity, which indicates the successful synthesis of WP@C=C; after imprinting on the surface of WP@C=C, the WP@MIPs is about 36°( Figure 5 C), the contact angle of WP@NIPs is 40°( Figure 5D) This is because the contact angles of WP@MIPs and WP@NIPs changed after the functional monomers were successfully polymerized. Since the functional monomers and cross-linking agents used in WP@MIPs and WP@NIPs are the same, the contact angles of the two are not much different. The above results show that the paper-based imprinted polymers WP@MIPs and WP@NIPs were successfully constructed.

[0051] To further verify the imprinting effect, WP, WP@C=C, WP@MIPs and WP@NIPs were characterized by SEM. Figure 6 It can be seen that the unmodified bare paper WP appears as distinct and porous filamentous fibers, and the surface of each fiber is smooth ( Figure 6 A, E); after the double bonds are modified, the paper surface is covered with silanization reagents and the fiber surface becomes slightly rough ( Figure 6 B, F); and after imprinting, WP@MIPs ( Figure 6 C, G) and WP@NIPs( Figure 6 D, H), it can be seen that the fiber becomes very rough due to the formation of the imprinted layer, and the imprinted layer can also be clearly seen attached to the surface of a single fiber. The above results once again demonstrate the successful construction of paper-based imprinted polymers WP@MIPs and WP@NIPs.

[0052] Example 4: Application of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor

[0053] The experimental conditions of this embodiment are: 1 sheet of WP@MIPs (1.3cm×1.3cm), pH 7.4, virus adsorption time 50min, TMV-nanoMIPs 1mL, adsorption time 60min. The specific implementation scheme is: TMV virus of different concentrations dispersed in PBS (pH=7.4, 10mM) buffer is added to WP@MIPs and WP@NIPs, and after shaking and incubating for 50min at 37°C, the filter paper is taken out and washed with ultrapure water 3 times, and then 1mL of ultrasonically dispersed TMV-nanoMIPs solution is added thereto, and the shaking and binding is continued at 25°C for 60min to remove the unbound TMV-nanoMIPs. Then, the paper-based MIPs and NIPs are inserted along the diagonal of the cuvette to measure their fluorescence. The excitation and emission slits are both 5nm, the voltage is 600v, and the fluorescence intensity at 588nm is collected. Use a mobile phone to photograph the colors of paper-based MIPs / NIPs under visible light and ultraviolet light.

[0054] (1) Detection linearity analysis of different concentrations of TMV by the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor

[0055] According to the above experimental steps, the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor of the present invention was used to perform linear analysis on TMV of different concentrations. The results are as follows: Figure 7 As shown in A, with the increase of virus concentration, the fluorescence intensity of WP@MIPs gradually increased, and both in visible light ( Figure 7 B, above) or UV light ( Figure 7 B, below) The surface visualization color becomes more and more obvious, which indicates that the paper-based WP@MIPs we constructed have good selectivity for the target virus. Figure 7 It can be seen that TMV concentration showed good linearity in the range of 0.144-5.75 pM, and the linear regression equation was FL = 3216.98 logC TMV +5653.98, LOD = 4.80 fM, where FL is the fluorescence intensity of WP@MIPs to the target virus TMV, C TMV is the concentration of the target virus TMV. For non-imprinted WP@MIPs, there is no linear relationship within this concentration range ( Figure 7 D), this is because there is no imprinted cavity of the target virus TMV on the surface of WP@NIPs, resulting in only a small amount of nonspecific adsorption on the surface, so the fluorescence intensity of WP@NIPs has no obvious change.

[0056] (2) Experimental study on the selectivity and competitiveness of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor for TMV

[0057] Based on the shape and size of tobacco mosaic virus (TMV), H5N1, HBV, MV, and EV71 were selected as analytical targets to evaluate the selectivity and competitiveness of the sensor.

[0058] 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 8 As shown in A, through the analysis of fluorescence intensity and IF value, the sensor has a high selectivity for TMV, IF = 5.25, while the selective recognition ability for other viruses is poor.

[0059] Similarly, the competitiveness of the paper-based sensor was evaluated by adding the corresponding competitive virus to the target virus TMV. Figure 8 As shown in B, due to the shape and size of the competitive virus, the interaction force is different from that of the functional monomer and cannot be recognized by the imprinting cavity. In addition, TMV-nanoMIPs cannot specifically recognize other viruses, so the competitive virus has little effect on the detection of the target virus TMV. From the above selectivity and competition experiments, it can be seen that the sensor has good selectivity and competitiveness for the target virus TMV.

[0060] (3) Experiments on the anti-interference ability, reproducibility, reusability and time stability of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor

[0061] We chose the common Na + , K + ,Mg 2+ , Ca 2+ , HCO3 - , HPO4 2- , glucose, L-proline, and L-alanine were used as interfering ions to investigate the anti-interference ability of the sensor. Fig. 9 As shown in A, the fluorescence detection of TMV by the sensor WP@MIPs is not greatly interfered by interfering ions, indicating that the prepared MIPs have very good anti-interference ability.

[0062] Five batches of WP@MIPs were prepared to detect the target to evaluate the differences between the sensors. Fig. 9 As shown in B, the fluorescence intensity among the five different batches is not much different, indicating that the sensor has good reproducibility.

[0063] The same batch of sensors were subjected to 5 cycles of adsorption-desorption to investigate the reusability of the sensors. Fig. 9 As shown in Figure C, after 3 cycles, the fluorescence intensity of the constructed paper-based sensor for TMV detection can still be 83.24% of the original. The results show that repeated elution recognition will cause certain damage to the imprinting cavity of the imprinted polymer, but within a certain number of cycles, it still has good recognition selectivity, indicating that the sensor has good reusability.

[0064] In order to evaluate the temporal stability of the constructed paper substrate, we tested the target virus TMV after placing the same batch of WP@MIPs for 0, 1, 2, 3, 4, 5, and 6 weeks. Fig. 9 As shown in D, as the placement time increases, the fluorescence intensity of detecting the same concentration of virus decreases slightly. After 6 weeks, WP@MIPs still has 89.16% of the original, indicating that the sensor has good stability.

[0065] (4) Application analysis of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor for simulated actual detection and real sample detection

[0066] Preparation of plant juice: Grind 2 g of veinless tea leaves into powder in liquid nitrogen, then suspend the powder in 20 mL of PBS (pH=7.4, 10 mM) buffer solution and continue grinding, centrifuge at 10000 rpm for 5 min, filter the supernatant with 0.45 μm PVDF, and store at 4°C.

[0067] The prepared sensor WP@MIPs was used to detect different concentrations of TMV in the above plant juice samples. First, it was incubated with the virus in the plant juice for 50 minutes, then the WP@MIPs were taken out and oscillated with 1 mL of TMV-nanoMIPs for 60 minutes to investigate the actual sample detection ability of the sensor. The results were similar to those of the TMV detection in PBS. Fig.10 As shown in the figure, the visualization of the sensor in the actual sample is somewhat different from that in PBS due to the presence of plant juice, but it still has good detection ability. Under ultraviolet light, the fluorescence intensity decreases, probably due to the presence of plant juice, but it still has a good visualization effect on the target.

[0068] Next, this work also explored the shortest time for the sensor WP@MIPs to detect TMV virus (0.5 mL, 14.38 pM) in healthy tobacco leaf juice (0.5 mL, 1 g / mL). Fig.11 As shown in the figure, it can be found that the sensor can detect TMV in 10 minutes, indicating that the prepared WP@MIPs has good practical application potential.

[0069] Subsequently, the ability of the sensor WP@MIPs to detect TMV virus in TMV-infected tobacco leaf sap was investigated. First, WP@MIPs were incubated with TMV-infected tobacco leaf sap and ordinary plant sap for 10 minutes, then WP@MIPs were taken out and oscillated with 1 mL of TMV-nanoMIPs for 10 minutes. Fig.12 As shown in the figure, the surface of WP@MIPs incubated with TMV-infected tobacco leaf juice has obvious pink color; at the same time, under ultraviolet light, WP@MIPs incubated with TMV-infected tobacco leaf juice has obvious orange fluorescence, while WP@MIPs incubated with ordinary plant juice has no obvious orange fluorescence. This shows that the sensor has the ability to detect actual samples.

[0070] Finally, the preparation cost of the sensor was calculated ( Fig.13 ), and its preparation cost is as low as 0.623 yuan / piece, indicating that the sensor has excellent application potential and good market prospects.

Claims

1. Construction and application of a portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor. First, using tobacco mosaic virus (TMV) as a template, artificial antibody nanogel TMV-nanoMIPs with composite organic fluorescent dye signals is obtained by solid phase imprinting as a signal probe; secondly, using paper as a carrier material, imprinting TMV on the paper, and eluting to obtain a paper-based imprinted polymer, and constructing a paper-based molecular imprinting sensor that can be used for visual detection of TMV by using the paper-based imprinted polymer and the fluorescent colorimetric signal probe TMV-nanoMIPs, which specifically includes the following steps: Step 1) Preparation of Fe3O4@SiO2: FeCl3·6H2O (5.4 g) was dissolved in ethylene glycol (160 mL), stirred vigorously to obtain a clear solution, then NaAc (14.4 g) and polyethylene glycol (4 g) were added and stirred at 50°C for 30 min, transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 24 h. The product was washed with ethanol and water several times, and dried under vacuum at 50°C to obtain Fe3O4 (1.508 g). 500 mg of Fe3O4 was weighed, added to 50 mL of isopropanol and 2 mL of deionized water, ultrasonically dispersed for 30 min, 5 mL of NH3·H2O and 2.5 mL of TEOS were added under mechanical stirring at 700 rpm and reacted at 45°C for 6 h. The obtained product was collected with a magnet, and washed with ethanol and water alternately for several times, and then placed at 50°C and dried overnight in vacuum to obtain Fe3O4@SiO2; Step 2) Preparation of Fe3O4@SiO2-NH2: Take 500mg of Fe3O4@SiO2 particles, and completely disperse them in 80mL of anhydrous ethanol / ultrapure water (1 / 1, v / v) by ultrasonication for 30min. Heat and stir at 40°C for 5min, then slowly drop 2mL of APTES into the flask and react for 12h. The particles after the reaction are gathered at the bottom of the flask with a magnet, the supernatant is poured off, and the particles are alternately washed with ultrapure water and anhydrous ethanol for multiple times. The washed particles are vacuum dried at 60°C overnight to obtain Fe3O4@SiO2-NH2 particles; Step 3) Preparation of Fe3O4@TMV: 100 mg of Fe3O4@SiO2-NH2 particles were suspended in 10 mL of glutaraldehyde (5%, v / v), deoxygenated with argon for 30 min, and stirred at 37°C for 3 h in the dark under argon protection. Aldehyde groups were introduced on the surface of Fe3O4@SiO2-NH2 particles to obtain Fe3O4@CHO for subsequent virus fixation. The magnetic particles were collected with the help of external magnetic force and washed three times with deoxygenated PBS (0.01 M, pH = 7.4) to remove unreacted glutaraldehyde. The collected aldehyde-modified particles were then dispersed in 10 mL of deoxygenated PBS (0.01 M, pH = 7.4). M, pH = 7.4), add 100 μL 0.287nM TMV tobacco mosaic virus, and incubate on a water bath constant temperature oscillator for 2 hours. Then collect the magnetic particles, wash to remove excess virus, and disperse in 10mL PBS (0.1M, pH = 7.4) to obtain Fe3O4@TMV; Step 4) Preparation of TMV-nanoMIPs: First, Rhodamine B (0.497 g, 1 mmol) was dissolved in 4 mL of ultrapure water, and then 1 mL of EDC (0.191 g, 1 mmol) and 1 mL of NHS (0.029 g, 0.25 mmol) were added, and the mixture was shaken at 37°C for 1 h to activate the carboxyl groups on Rhodamine B. 4 mL of N-(3-aminopropyl) methacrylamide hydrochloride (APMA, 0.178 g, 1 mmol) was added to the activated Rhodamine B, and the mixture was shaken at 37°C for 2 h. After the reaction was completed, the mixture was extracted twice with dichloromethane, and then extracted twice with water, leaving the organic phase, which was concentrated and dried by rotary evaporator, and the solid was recrystallized with acetonitrile and water, and the fluorescent dye complex was obtained after filtration. N-isopropylacrylamide (NIPAM, 7.9 mg, 70 μmol), N-tert-butylacrylamide (TBA, 3.8 mg, 30 μmol, dissolved in 500 μL ethanol), acrylic acid (AA, 1.73 μL, 1.1 μmol), N-(3-aminopropyl)methacrylamide hydrochloride (APMA, 0.9 mg, 6 μmol), and N,N-methylenebisacrylamide (BIS, 2 mg, 12.97 μmol) were mixed in 10 mL of ultrapure water, and a fluorescent composite dye (2 mg, dissolved in 500 μL ethanol) was added to the mixture. The mixture was deoxygenated with argon for 30 min and vacuumed with a water pump for 10 minutes. Before vacuuming, 4 mL of TMV-modified magnetic nanoparticles (Fe3O4@TMV) dispersed in PBS was injected into the flask using a needle, and then vacuuming was continued to deoxygenate. The flask was then sealed with a sealing film and protected with argon. The reaction solution was placed on a constant temperature oscillator and oscillated for 30 minutes. 1 mL of ammonium persulfate (APS, 6 mg, 26 μmol) and N,N,N',N'-tetramethylethylenediamine (TEMED, 6 μL) were injected into the flask under a nitrogen atmosphere, and the reaction solution was placed on a constant temperature oscillator and oscillated at 25°C for 12 hours. After the reaction, the solid particles were collected with a magnet and washed with 25°C ultrapure water to remove unreacted functional monomers and low-affinity imprinted polymers until there was no fluorescence in the supernatant. Then, the collected magnetic particles were dispersed in 2 mL of ultrapure water and oscillated in a 60°C water bath oscillator for 60 minutes. The magnetic solid particles were separated from the supernatant by the magnetic force of an external magnet. This operation was repeated twice, and the 4 mL supernatant obtained was the soluble nanogel imprinted polymer TMV-nanoMIPs. Stored in a sealed container at 4°C. Step 5) Modification of paper base: Take a 70mm×70mm Whitman No. 1 qualitative filter paper, rinse it with a large amount of ethanol for activation, and vacuum dry it at 50°C. Then add 200μL triethylamine (TEA) and 50mg 4-(dimethylamino)pyridine (DMAP) to a glass culture dish containing 15mL dichloromethane (DCM), add 200μL trichlorovinylsilane (TVCs), and finally add the activated paper base to the above culture dish, shake it on a shaker at room temperature for 2 hours, and finally rinse it with ethanol 3 times to wash away the unreacted monomers, and vacuum dry it at 50°C to obtain the filter paper WP@C=C grafted with vinyl. Step 6) Preparation of paper-based imprinted polymer WP@MIPs / WP@NIPs: 0.1mmol 4-vinylpyridine (4-VP), 0.1mmol zinc acrylate (Zinc AA) and 50μL 2.87nM TMV virus were dissolved in 20mL DMF and prepolymerized at 37°C for two hours. Then 0.3mmol ethylene glycol dimethyl acrylate (EGDMA) and 4 1.3cm×1.3cm WP@C=C were added, argon was deoxygenated for 30min, 20mg initiator AIBN was added, and finally transferred to 65°C for polymerization for 12h. After the polymerization is completed, the paper is taken out and washed with ultrapure water to remove the unreacted matter, and finally eluted with 1.0 mL of eluent methanol: acetic acid = 9:1 (v / v) filter paper for 4 times. Then wash with ultrapure water several times until the eluent is completely washed away, and vacuum dry at 50°C to obtain WP@MIPs. The preparation of WP@NIPs did not add the template virus TMV, and the other operations were consistent with the above.

2. The detection conditions of a portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor according to claim 1: TMV virus of different concentrations dispersed in PBS (pH=7.4, 10mM) buffer was added to WP@MIPs and WP@NIPs, and the filter paper was taken out and washed with ultrapure water 3 times after shaking incubation at 37°C for 50min, and then 1mL of ultrasonically dispersed TMV-nanoMIPs solution was added thereto, and the shaking binding was continued at 25°C for 60min to remove the unbound TMV-nanoMIPs. Then, the paper-based MIPs and NIPs were inserted along the diagonal of the cuvette to measure their fluorescence. The excitation and emission slits were both 5nm, the voltage was 600v, and the fluorescence intensity at 588nm was collected. The colors on the paper-based MIPs / NIPs under visible light and ultraviolet light were photographed using a mobile phone.

3. The application of a portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor according to claim 1, characterized in that: The portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor is used to analyze TMV solutions of different concentrations to evaluate its detection range and detection limit for TMV; the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor is used to detect interfering virus solutions of the same concentration and mixed solutions in which target viruses and interfering viruses coexist to evaluate its selective recognition and competitive adsorption capabilities for TMV; the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor is applied to the detection of tobacco leaf juice infected with TMV to evaluate its actual sample detection capability for TMV; the preparation cost of the portable rapid visualization tobacco mosaic virus paper-based molecular imprinting sensor is calculated to evaluate its application potential and market prospects.

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