Preparation and application of paper-based molecularly imprinted sensor capable of visually and simultaneously detecting two subtype A influenza viruses

Abstract

The invention provides a preparation method and application of a paper-based molecularly imprinted sensor capable of visually and simultaneously detecting two subtype influenza A viruses. In the invention, an MOF (Cu) paper-based material which is low in cost, easy to obtain and convenient to carry is used as a carrier, viruses H5N1 and H7N9 are used as templates, and imprinting is carried out on the surface of the carrier to obtain the molecularly imprinted polymer (MIPs). Wherein the MOF (Cu) not only serves as a colorimetric signal output element, but also plays a metal coordination role between Cu < 2 + > in the MOF (Cu) and coat protein of the virus, so that the virus can be effectively fixed on the surface of the paper-based MOF (Cu), surface imprinting of the virus on the paper-based MOF (Cu) is facilitated, and the specific recognition capability is improved. Due to the fact that the number of imprint cavities blocked by the MIPs after the MIPs are combined with target objects with different concentrations is different, the oxidation degrees of TMB are different, visual detection of the two viruses is achieved, and high-sensitivity quantitative detection can also be conducted through an ultraviolet and visible spectrophotometer. The sensor prepared by the invention is a universal virus sensor, and the paper-based sensor suitable for visually detecting other target objects can be prepared by selecting other target objects as template molecules for imprinting.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry detection, and specifically relates to the preparation and application of a paper-based molecular imprinting sensor for visually and simultaneously detecting two subtypes of influenza A viruses. Background Art

[0002] Influenza A virus has the characteristics of rapid spread, strong destructiveness and great harm. Therefore, simple, sensitive and accurate diagnosis of influenza A virus is crucial to preventing infection, rapid spread and early treatment of the disease; it is also one of the key links in monitoring and controlling the epidemic. The conventional methods for detecting influenza A virus mainly include enzyme-linked immunosorbent assay (ELISA) [Yang F, Xiao Y, Liu F, et al. Development of a monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for detection of H7N9 subtype avianinfluenza virus[J]. Journal of Medical Virology, 2021, 93(6): 3939-3943.], viral nucleic acid amplification method (RT-PCR) [Abdel-Moneim AS, Abdel-Ghany AE, Shany SA S. Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys[J]. Journal of Biomedical Science, 2010, 17: 1-6.], and virus isolation, culture and identification [Chang P, Sadeyen JR, Bhat S, et al. Risk assessment of the newly emerged H7N9avian influenza viruses[J]. Emerging Microbes & Infections, 2023, 12(1): 2172965.]. These methods have the characteristics of high sensitivity and accuracy, strong specificity, and low false positive rate. However, there are still some shortcomings, such as high cost, long detection time, and low universality. Therefore, the development of a simple, fast, low-cost, stable and effective influenza virus detection method is highly anticipated. Molecularly imprinted polymers (MIPs), known as "artificial antibodies", have the advantages of strong specificity, good affinity, good physical and chemical stability, low cost, easy large-scale production, and reusability.Therefore, it has been successfully applied to the high-sensitivity detection of viruses. For example, Wang et al. developed a method for preparing a poliovirus imprinted potentiometric sensor [Wang Y, Zhang Z, Jain V, et al. Potentiometric sensors based on surface molecular imprinting: Detection of cancer biomarkers and viruses [J]. Sensors and Actuators B: Chemical, 2010, 146 (1): 381-387.]. Tai et al. reported a dengue virus molecular imprinting sensor [Tai DF, Lin CY, Wu TZ, et al. Recognition of dengue virus protein using epitope-mediated molecularly imprinted film [J]. Analytical Chemistry, 2005, 77 (16): 5140-5143.]. Although these virus molecular imprinting sensors have the characteristics of high sensitivity, they are inconvenient to carry and store, and the detection process is time-consuming. Therefore, the development of a portable molecular imprinting sensor that can quickly detect the target virus to achieve rapid visual detection of the virus is highly anticipated.

[0003] Paper-based materials have the characteristics of good hydrophilicity, large specific surface area, low price, easy to obtain, easy to modify, good stability, easy to store and carry [Lamaoui A, Karrat A, Amine A. Molecularly imprinted polymer integrated into paper-based analytical device for smartphone-based detection: Application for sulfamethoxazole [J]. Sensors and Actuators B: Chemical, 2022, 368: 132122.], and a variety of paper-based sensors have been developed, including wearable devices [Cheng Y, Feng S, Ning Q, et al. Dual-signal readout paper-based wearable biosensor with a 3D origami structure for multiplexed analyte detection in swe at [J]. Microsystems & Nanoengineering, 2023, 9 (1): 36.], disposable test strips and biosensors [Qi J, Li B, Wang X, et al. Rotational paper-based microfluidic-chip device for multiplexed and simultaneous fluorescence detection of phenolic pollutants based on a molecular-imprinting technique[J].Analytical Chemistry,2018,90(20):11827-11834.];[Wang L,Li B,Wang J,et al.A rotary multi-positioned cloth / paper hybrid microfluidic device for simultaneous fluorescence sensing ofmer cury and lead ions by using ion imprinted technologies[J]. Journal of Hazardou s Materials, 2022,428:128165.].

[0004] Nanozymes are artificial nanomaterials that can mimic the activity of natural enzymes. Compared with natural enzymes, nanozymes have the advantages of high stability, low cost, adjustable catalytic activity, and easy modification, which makes them play an important role in environmental engineering, food safety, biosensing, and disease diagnosis and treatment. Among them, the use of the catalytic properties of nanozymes to achieve detection signal amplification has been widely used in the field of analytical chemistry. [L iu B, Zhu H, Liu J, et al. Alkali-etched imprinted Mn-based Prussian blue anal ogues with superior oxidase-mimetic activity and precise recognition for tetracy cline colorimetric sensing [J]. ACS Applied Materials & Interfaces, 2023, 15 (20): 24736-24746.]. Metal-organic frameworks (MOFs) constructed with inorganic metal ions and organic ligands as carriers are nanozymes with peroxidase-like activity. They have attracted people's attention due to their large specific surface area, good biocompatibility, rich spatial structure, and easy synthesis and functional modification.

[0005] Based on these meaningful works, this paper proposes a paper-based MOF (Cu) molecular imprinting sensor for simultaneous visual detection of H5N1 and H7N9 influenza viruses, using H5N1 and H7N9 as the analysis targets. This strategy uses paper-based MOF (Cu) as the imprinting carrier material. The MOF (Cu) structure not only provides a large specific surface area for the imprinting of the virus, but also the Cu in the MOF (Cu) 2+ It can coordinate well with the virus's coat protein, thus effectively fixing the virus on the paper-based MOF (Cu) surface, which is conducive to the imprinting of the virus on the paper-based MOF (Cu) surface and improves the specific recognition ability. In addition, MOF (Cu) has the characteristics of nanozymes, which can catalyze H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) from colorless to blue under acidic conditions, thereby realizing the visual detection of viruses. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation and application of a paper-based molecular imprinting sensor for visually detecting two subtypes of influenza A virus at the same time, and to use the sensor for specific and visual detection of the virus, aiming to provide a rapid, sensitive and simple detection method.

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

[0008] A method for preparing a paper-based molecular imprinting sensor for visually detecting two subtypes of influenza A virus simultaneously, characterized in that the method comprises the following process steps:

[0009] (1) Preparation of paper-based MIPs based on MOF (Cu): First, Whatman test paper was modified by carboxylation. Second, Cu(AcO)2 and 1,3,5-benzenetricarboxylic acid were used as raw materials to construct MOF (Cu) on the carboxyl-functionalized paper surface by an in situ growth method. Finally, MIPs were prepared by a sol-gel reaction using influenza viruses H5N1 and H7N9 as templates, 3-aminopropyltriethoxysilane (APTES) as a functional monomer, tetraethyl silicate (TEOS) as a cross-linker, and triethylamine (TEA) as a catalyst. The templates H5N1 and H7N9 were eluted with an eluent to obtain paper-based MIPs with recognition cavities of the same shape and size as the template viruses. The MOF (Cu) on the paper substrate served as a colorimetric signal output element.

[0010] (2) Paper-based MIPs recognize and specifically bind to H5N1 and H7N9 through the specific shape and size of the imprinted cavity, as well as the hydrogen bonding between the functional monomer and the target virus. After binding to the virus, it blocks the cavity of the MIPs shell, masking the catalytic site of MOF (Cu), thereby inhibiting the color reaction of TMB oxidation. After MIPs captured H5N1 and H7N9, they were added to a mixed solution containing PBS, TMB, and H2O2, reacted for 5 minutes, and the color change of the solution was observed. As the virus concentration increased, the more the imprinted holes were filled, the more obvious the shielding effect was, the less TMB was oxidized in the solution, and the lighter the blue color displayed.

[0011] (3) Preparation and application of molecular imprinting paper-based sensors based on MOF (Cu): First, the prepared MOF (Cu) paper-based molecular imprinting polymer Paper@MOF (Cu) @MIPs / NIPs was added to a solution containing an appropriate amount of PBS (pH = 7.4) and a certain concentration of H5N1 and H7N9 viruses, and incubated at 37°C for 30 minutes under optimized experimental conditions; secondly, after the incubation was completed, the supernatant was discarded, the MIPs / NIPs paper was taken out and washed with PBS buffer, and added to a mixed solution of PBS, TMB, and H2O2, and reacted for 5 minutes to form a color change visible to the naked eye; finally, the mixed solution was placed in a quartz colorimetric dish, and its absorbance could be quantitatively detected by UV-visible absorption spectroscopy, and the UV absorbance intensity of the detection solution at a wavelength of 650nm was detected; finally, a molecular imprinting paper-based sensor based on MOF (Cu) that can be recognized by naked eye colorimetry was constructed.

[0012] At the same time, the present invention also designs a portable microfluidic chromatography device, such as Figure 1 B. The first layer is the sample loading layer; the second layer is a wax layer containing two sample inlets to prevent sample diffusion; the third layer is a sensing layer with MIPs (H5N1) and MIPs (H7N9) for colorimetric reaction; the last layer is a wax layer to prevent sample leakage. When the sample is dripped into the sample loading layer, it is transferred to the sensing layer by capillary force, which is equipped with different paper chips for colorimetric detection. The target viruses H5N1 and H7N9 can be semi-quantitatively detected by color observation. This simplified paper-based chromatography device design combined with the visual detection method for H5N1 / H7N9 viruses provides a convenient and reliable solution for on-site detection of viruses.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) Using cellulose paper with low cost, good flexibility and good biocompatibility as the carrier of the imprinting layer, it is easy to operate when in use, has low professional requirements, is low in cost, easy to carry, and has fast detection.

[0015] (2) The high catalytic ability of the peroxidase-like enzyme combined with the MOF (Cu) material causes the TMB-H2O2 system to undergo an obvious color change under natural light, thus constructing a colorimetric system for naked-eye visual detection, which has great potential for application in self-examination and screening for influenza A virus infection in the population to be tested.

[0016] (3) Experimental results show that the paper-based molecular imprinting sensor has a high specific recognition ability for H5N1 and H7N9, can quickly achieve simultaneous detection, and has good selectivity and sensitivity.

[0017] (4) The sensor is a general virus sensor, and other targets can be used as template molecules for imprinting to prepare paper-based molecular imprinting sensors suitable for visual detection of other targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] [ Figure 1 ](A) Schematic diagram of the process for preparing the paper-based MOF (Cu) virus molecular imprinting sensor; (B) microfluidic chromatography device.

[0019] [ Figure 2 ]Feasibility analysis of the paper-based MOF (Cu) virus molecular imprinting sensor.

[0020] [ Figure 3](a)Paper; (b)Paper-COOH; (c)Paper@MOF(Cu); (d)Paper@MOF(Cu)@MIPs(H5); (e)Paper@MOF(Cu)@NIPs(H5); (f)Paper@MOF(Cu)@MIPs(H7); (g)Infrared spectra of Paper@MOF(Cu)@MIPs(H7).

[0021] [ Figure 4 ]Scanning electron micrographs of (A) Paper (5000 times); (B) Paper-COOH (6000 times); (C) Paper@MOF; (D) Paper@MOF(Cu)@MIPs(H5); (E) Paper@MOF(Cu)@NIPs(H5); (F) Paper@MOF(Cu)@MIPs(H7); (G) Paper@MOF(Cu)@NIPs(H7) (10000 times).

[0022] [ Figure 5 ](A) Effect of different concentrations of H5N1 on the UV absorbance intensity of MIPs; (B) Effect of different concentrations of H7N9 on the UV absorbance intensity of MIPs; (C) Linear fitting curve of H5N1 concentration; (D) Linear fitting curve of H7N9 concentration; (E) Color changes corresponding to different concentrations of H5N1; (F) Color changes corresponding to different concentrations of H7N9.

[0023] [ Figure 6 ](A) Selective experiment of the sensor for H5N1; (B) Selective experiment of the sensor for H7N9; (C) Competitive experiment of the sensor for H5N1; (D) Competitive experiment of the sensor for H7N9.

[0024] [ Figure 7 ](A) Reproducibility experiment of the sensor for H5N1; (B) Reproducibility experiment of the sensor for H7N9.

[0025] [ Figure 8 ](A) Investigation of the sensor's anti-interference ability against H5N1; (B) Investigation of the sensor's anti-interference ability against H7N9. Specific implementation plan

[0026] 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.

[0027] Example 1: A method for preparing a paper-based molecular imprinting sensor for visually detecting two subtypes of influenza A virus simultaneously:

[0028] (1) Preparation of carboxylated filter paper: First, weigh NaOH (7.10 g) and CH2ClCOONa (3.50 g) in a 500 mL beaker, then add 40 mL of water to dissolve the mixture. Subsequently, immerse 4 pieces of Whatman filter paper in the solution and heat in a 40°C water bath for 2 h. Turn each piece of filter paper over every 30 min to ensure uniform reaction. After the reaction is completed, wash the filter paper with ultrapure water until neutral, then wash it twice with pH = 4-5 dilute hydrochloric acid, and dry it at 50°C for later use.

[0029] (2) Preparation of Paper@MOF(Cu): Take Cu(AcO)2 (0.78 g) and 1,3,5-trimesic acid (0.50 g) respectively, then add 10 mL each of H2O, DMF and EtOH (v:v:v=1:1:1), and sonicate for 10 min to dissolve them. Number 4 clean culture dishes 1, 2, 3, and 4, and add Cu(AcO)2 solution, H2O (30 mL), 1,3,5-trimesic acid solution, and MeOH (30 mL) in sequence. Take a piece of carboxylated filter paper moistened with ultrapure water, and place it in dish No. 1 for 10 min; in dish No. 2 for 2 min; in dish No. 3 for 10 min; in dish No. 4 for 2 min. Repeat the above steps 4 times, and dry at 50°C for later use.

[0030] (3) Preparation of Paper@MOF(Cu)@MIPs(H5) material: 10 sheets of Paper@MOF(Cu) (1×1 cm) and THF (25 mL) were added to a 50 mL flask, stirred (200 rpm / min), and then H5N1 (30 μL, 128 HUA), APTES (70 μL) were added in sequence. After prepolymerization at 25°C for 2 h, TEOS (100 μL) and TEA (100 μL) were added and stirred for 10 h. After the reaction was completed, the polymerized filter paper was rinsed with ultrapure water several times, and then repeatedly eluted with MeOH / MeCN (v:v=9:1, 5 mL) until no obvious template virus fluorescence was detected in the eluent, and dried at 50°C to obtain the paper-based imprinted polymer Paper@MOF(Cu)@MIPs(H5) with H5N1 virus imprinting sites. The non-imprinted polymer Paper@MOF(Cu)@NIPs(H5) was prepared according to the same method, but without adding the two template viruses.

[0031] (4) Preparation of Paper@MOF(Cu)@MIPs(H7) material: 10 sheets of Paper@MOF(Cu) (1×1 cm) and THF (25 mL) were added to a 50 mL flask, stirred (200 rpm / min), and then H7N9 (30 μL, 128 HUA) and APTES (100 μL) were added in sequence. After prepolymerization at 25°C for 2 h, TEOS (100 μL) and TEA (100 μL) were added and stirred for 10 h. After the reaction was completed, the polymerized filter paper was rinsed with ultrapure water several times, and then repeatedly eluted with MeOH / MeCN (v:v=9:1, 5 mL) until no obvious template virus fluorescence was detected in the eluent, and dried at 50°C to obtain the paper-based imprinted polymer Paper@MOF(Cu)@MIPs(H7) with H7N9 virus imprinting sites. The non-imprinted polymer Paper@MOF(Cu)@NIPs(H7) was prepared according to the same method, but without adding the two template viruses.

[0032] (4) Preparation of the molecular imprinting sensor (Paper@MOF(Cu)@MIPs) for visual simultaneous detection of two subtypes of influenza A virus: First, the prepared Paper@MOF(Cu)@MIPs(H5), Paper@MOF(Cu)@NIP(H5), Paper@MOF(Cu)@MIPs(H7), and Paper@MOF(Cu)@NIP(H7) were placed in centrifuge tubes numbered A, B, C, and D, and then PBS (pH=7.4, 10 mM, 1 mL) buffer containing different concentrations of H5N1 or H7N9 virus was added in turn, incubated at 37°C with shaking for a period of time, the supernatant was discarded, and the sensor was washed with PBS (pH=7.4, 10 mM) buffer. Then, PBS = 800 μL (pH = 6.0), TMB = 50 μL (5 mg / mL), and H2O2 = 150 μL (15%) were added to each centrifuge tube in sequence. After 5 minutes, the absorbance of the supernatant at 650 nm was detected using a UV-2450 ultraviolet spectrophotometer.

[0033] Example 2: Feasibility analysis of the paper-based molecular imprinting sensor for visually detecting two subtypes of influenza A virus simultaneously.

[0034] In order to verify the feasibility of the present invention, this example verifies the construction principle of the paper-based molecular imprinting sensor. Figure 2As shown, the absorbance of paper-based MIPs and NIPs at around 650nm before and after the adsorption of H5N1 virus and H7N9 virus was compared. It can be seen from Figures A and B that the absorbance of paper-based MIPs after adding H5N1 and H7N9 was significantly reduced compared to before H5N1 and H7N9 were added. This is because there are specific recognition sites for H5N1 and H7N9 viruses and synergistic recognition of functional monomers in paper-based MIPs. Therefore, after the adsorption of H5N1 and H7N9 viruses, the cavity of the MIP shell will be blocked, so that the catalytic site of MOF (Cu) is masked, thereby inhibiting the color development reaction of TMB oxidation, resulting in a decrease in the absorbance at around 650nm. However, there is no obvious difference in the absorbance of paper-based NIPs before and after adding H5N1 and H7N9. This is because paper-based NIPs do not have specific recognition sites for H5N1 and H7N9, so they only have a low absorbance at around 650nm before and after adding H5N1 and H7N9 viruses. Figure 2 The two illustrations in the middle are the corresponding visualization images of H5N1 and H7N9 paper-based MIPs / NIPs before and after the addition of viruses, which are consistent with the results of the UV spectrum, and also demonstrate the good visualization effect of the paper-based sensor. The changes in the UV spectrum and the visualization color both indicate that the paper-based molecular imprinting sensor for visually detecting two subtypes of viruses simultaneously is highly feasible for the visual detection of H5N1 and H7N9 viruses.

[0035] Example 3: Performance, morphology and structural characterization of Paper@MOF(Cu)@MIPs and intermediates for simultaneous visualization of two subtypes of influenza A virus.

[0036] The structure and morphology of the prepared materials were characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Figure 3 The infrared spectra of Paper (a), Paper-COOH (b), Paper@MOF(Cu) (c), Paper@MOF(Cu)@MIPs (H5) (d), Paper@MOF(Cu)@NIPs (H5) (e), Paper@MOF(Cu)@MIPs (H7) (f), and Paper@MOF(Cu)@MIPs (H7) (g). As can be seen from the figure, 1023cm -1 The characteristic absorption peak of β-D glucosidic bond in cellulose is at 3329cm -1 The strong broad peak at 2894cm is the -OH stretching vibration peak of cellulose molecules; -1 The peak of methylene stretching vibration is at 1593cm -1 The C=O absorption peak appeared at 1642cm-1 , 1552cm -1 and 1442cm -1 The characteristic absorption peak of the benzene ring was shown, indicating that Paper@MOF(Cu) was successfully synthesized; and after imprinting, the characteristic absorption peak of O-Si-O basically overlapped with the characteristic absorption peak of the β-D glucosidic bond in cellulose, and the peak shape of its absorption peak became broader and stronger. The above results show that the imprinted layer was successfully coated on the surface of the paper-based material.

[0037] Figure 4 The scanning electron microscope images of Paper (A) Paper-COOH (B) Paper@MOF (C) Paper@MOF(Cu)@MIPs (H5) (D) Paper@MOF(Cu)@NIPs (H5) (E) Paper@MOF(Cu)@MIPs (H7) (F) Paper@MOF(Cu)@NIPs (H7) (G). As can be seen from the figure, there are a large number of disordered filamentous fibers on the surface of the coarse fibers on the surface of Paper. After grafting -COOH, the filamentous fibers disappear and the surface of the coarse fibers becomes smooth. After MOF (Cu) grows in situ on the paper fibers, it fills the original cavity of the modified paper. The particle size of MOF (Cu) is about 200nm-1000nm, which may be due to the presence of trimesic acid and Cu in the cavity. 2+ The low concentration is not conducive to the growth of MOF(Cu), while the outside of the cavity is conducive to the growth of MOF(Cu). After imprinting, small-sized MOF(Cu) particles are embedded, and the cavities between MOF(Cu) are filled with polymers. Among them, the imprinted polymer Paper@MOF(Cu)@MIPs presents a dense porous structure, and the surface of the imprinted layer becomes rough. This may be due to the addition of two subtypes of template viruses during the imprinting process, which are washed away, resulting in imprinted cavities of different sizes on the surface of the imprinted layer; and although the surface of Paper@MOF(Cu)@NIPs is also relatively rough, the imprinted layer covering MOF(Cu) is very smooth. The above results all indicate the successful preparation of paper-based MOF(Cu) imprinted polymers.

[0038] Example 4: Application of the paper-based MOF (Cu) molecular imprinting sensor for visually detecting two subtypes of influenza A virus simultaneously.

[0039] The experimental conditions of this embodiment are as follows: First, the prepared Paper@MOF(Cu)@MIPs(H5), Paper@MOF(Cu)@NIP(H5), Paper@MOF(Cu)@MIPs(H7), and Paper@MOF(Cu)@NIP(H7) are placed in centrifuge tubes numbered A, B, C, and D, and then PBS (pH=7.4, 10mM, 1mL) buffer containing different concentrations of H5N1 or H7N9 virus is added in sequence, incubated at 37°C for a period of time, the supernatant is discarded, and the sensor is washed with PBS (pH=7.4, 10mM) buffer. Then, PBS=800μL (pH=6.0), TMB=50μL (5mg / mL), and H2O2=150μL (15%) are added to each centrifuge tube in sequence. After 5 minutes, the absorbance of the supernatant at 650nm is detected using a UV-2450 ultraviolet spectrophotometer.

[0040] (1) Simultaneous detection and analysis of different concentrations of H5N1 and H7N9 by paper-based MOF (Cu) virus molecular imprinting sensor.

[0041] According to the above experimental steps, the paper-based MOF (Cu) virus molecular imprinting sensor of the present invention was used to detect and analyze H5N1 and H7N9 solutions of different concentrations. The results are as follows: Figure 5 As shown in the figure, the analytical concentration range of the prepared sensor for H5N1 is 6.4×10 -3 -1.28HUA / mL, detection limit is 4.2×10 -4 HUA / mL, the analytical range for H7N9 is 1.28×10 -2 -0.64HUA / mL, detection line is 11×10 -4 HUA / mL. The results showed that the sensor can perform visual detection of H5N1 and H7N9, with a wide linear range, low detection limit, and good overall effect.

[0042] (2) Selectivity and competitiveness experiments of paper-based MOF (Cu) virus molecular imprinting sensors for H5N1 and H7N9.

[0043] In this example, H5N1, H7N9, HAV, HBV, and H9N2 viruses of the same concentration were selected as targets for investigation.

[0044] According to the above experimental steps, the selectivity of the sensor was analyzed, and the experimental results are as follows Figure 6 A and Figure 6As shown in Figure B, the sensor of the present invention has the interaction between the recognition cavity and the functional monomer of the same shape and size for H5N1 and H7N9, so the adsorption capacity for H5N1 and H7N9 is significantly better than that for other interfering viruses, showing good selectivity; similarly, we investigated the competitiveness of the sensor by adding other interfering viruses to the target virus H5N1 or H7N9, and the results are shown in Figure 2. Figure 6 C and Figure 6 As shown in D, it can be seen that when the template virus H5N1 or H7N9 and other viruses (H7N9 or H5N1 or HAV or HBV or H9N2) coexist, due to the difference in shape and size of the competing viruses and the different forces acting on the functional monomers, the sensor's detection of H5N1 or H7N9 is not significantly affected, indicating that the sensor has excellent recognition ability for the target virus.

[0045] (3) Investigation on the reproducibility of paper-based MOF (Cu) virus molecular imprinting sensor for H5N1 and H7N9.

[0046] According to the above experimental steps, the reproducibility of the sensor was studied. Five different batches of synthesized sensors were prepared at the same time. The UV-visible absorbance test of H5N1 and H7N9 with the same concentration was carried out under the optimal conditions to evaluate the reproducibility of the sensor. The results are shown in Figure 7 A and Figure 7 As shown in B, there is almost no difference in the results of the five measurements, and the surface-constructed sensor has good reproducibility.

[0047] (4) Investigation of the anti-interference ability of paper-based MOF (Cu) virus molecular imprinting sensor against H5N1 and H7N9.

[0048] According to the above experimental steps, the anti-interference ability of the sensor was investigated for the same concentration of target virus. Since the detection of viruses is usually completed in complex biological samples, some interfering substances such as ions and amino acids in biological samples will affect the detection of the sensor. Therefore, we analyzed the anti-interference ability of the sensor in the presence of the interfering substance Na + , K + Mg 2+ , Ca 2+ 、HCO3 - 、HPO4 2- and glucose were used to test the anti-interference ability. Figure 8 As shown, H5N1 and H7N9 detection were almost unaffected, indicating that the proposed paper-based molecular imprinting sensor has excellent anti-interference ability.

[0049] (5) Spiked recovery of H5N1 and H7N9 by paper-based MOF (Cu) virus molecular imprinting sensor.

[0050] The spike recovery method was used to evaluate the analytical ability of the above-described method for actual samples. Six human serum samples diluted with PBS buffer (diluted 100 times, pH = 7.4) were taken, and H5N1 with concentrations of 0.128HUA / mL, 0.320HUA / mL, and 0.0640HUA / mL and H7N9 with concentrations of 50.128HUA / mL, 0.320HUA / mL, and 0.0640HUA / mL were added thereto, respectively, and the prepared WP@MIP sensor was used for detection and analysis. The results are shown in Table 1. The recovery rates for detecting H5N1 were 103.43-106.56%, and those for H7N9 were 105.46-108.18%, indicating that the sensor has great application potential in the field of simultaneous detection of H5N1 and H7N9.

[0051] Table 1 Results of detection of H5N1 and H7N9 in human serum.

[0052]

Claims

1. Preparation and application of a paper-based molecular imprinting sensor for visually detecting two subtypes of influenza A virus simultaneously, characterized in that: First, the filter paper is carboxylated and modified, and a MOF (Cu) with peroxidase catalytic activity is constructed on the paper surface by an in-situ growth method; secondly, the MOF (Cu) paper-based material is used as a carrier, and the influenza virus H5N1 and H7N9 are used as template molecules. The molecular imprinting technology is used to synthesize a cavity with specific recognition of H5N1 and H7N9 on the paper surface, and hydrogen peroxide enters the cavity and oxidizes the colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue under the catalysis of MOF (Cu). When H5N1 and H7N9 are present, they are specifically captured by the imprinted cavity of the imprinted polymer, which will block the cavity of the MIPs shell layer, so that the catalytic site of MOF (Cu) is masked, thereby inhibiting the color development reaction of TMB oxidation, thereby realizing the colorimetric detection of influenza virus H5N1 and H7N9. The preparation method comprises the following steps: Step 1) Preparation of carboxylated filter paper: First, weigh NaOH (7.10 g) and CH2ClCOONa (3.50 g) in a 500 mL beaker, then add 40 mL of water to dissolve the mixture. Subsequently, immerse 4 Whatman filter papers in the solution, heat in a 40°C water bath for 2 h, and turn each filter paper over every 30 min to ensure uniform reaction of the filter paper. After the reaction is completed, wash the filter paper with ultrapure water until neutral, then wash it twice with pH=4-5 dilute hydrochloric acid, and dry it at 50°C for use. Step 2) Preparation of Paper@MOF(Cu): Take Cu(AcO)2 (0.78g) and 1,3,5-trimesic acid (0.50g) respectively, then add 10mL each of H2O, DMF and EtOH (v:v:v=1:1:1), and sonicate for 10min to dissolve. Number 4 clean culture dishes as 1, 2, 3, and 4, and add Cu(AcO)2 solution, H2O (30mL), 1,3,5-trimesic acid solution, and MeOH (30mL) in sequence according to the numbers. Take a piece of carboxylated filter paper moistened with ultrapure water, and place it in dish No. 1 for 10min; in dish No. 2 for 2min; in dish No. 3 for 10min; in dish No. 4 for 2min, repeat the above steps 4 times, and dry at 50℃ for use. Step 3) Preparation of Paper@MOF(Cu)@MIPs(H5) material: 10 sheets of Paper@MOF(Cu) (1×1cm) and THF (25mL) were added to a 50mL flask, stirred (200rpm / min), and then H5N1 (30μL, 128HUA), APTES (70μL) were added in sequence, and prepolymerized at 25°C for 2h, and then TEOS (100μL) and TEA (100μL) were added, and stirring continued for 10h. After the reaction was completed, the polymerized filter paper was rinsed with ultrapure water several times, and then repeatedly eluted with MeOH / MeCN (v:v=9:1, 5mL) until no obvious template virus fluorescence was detected in the eluent, and dried at 50°C to obtain the paper-based imprinted polymer Paper@MOF(Cu)@MIPs(H5) with H5N1 virus imprinting sites. The non-imprinted polymer Paper@MOF(Cu)@NIPs(H5) was prepared according to the same method, but without adding the two template viruses. Step 4) Preparation of Paper@MOF(Cu)@MIPs(H7) material: 10 sheets of Paper@MOF(Cu) (1×1cm) and THF (25mL) were added to a 50mL flask, stirred (200rpm / min), and then H7N9 (30μL, 128HUA), APTES (100μL) were added in sequence, and prepolymerized at 25°C for 2h, and then TEOS (100μL) and TEA (100μL) were added, and stirring continued for 10h. After the reaction was completed, the polymerized filter paper was rinsed with ultrapure water several times, and then repeatedly eluted with MeOH / MeCN (v:v=9:1, 5mL) until no obvious template virus fluorescence was detected in the eluent, and dried at 50°C to obtain the paper-based imprinted polymer Paper@MOF(Cu)@MIPs(H7) with H7N9 virus imprinting sites. The non-imprinted polymer Paper@MOF(Cu)@NIPs(H7) was prepared according to the same method, but without adding the two template viruses.

2. The method for preparing a paper-based molecular imprinting sensor for visually detecting two subtypes of influenza A virus simultaneously according to claim 1, characterized in that: The prepared MIP / NIP was placed in a centrifuge tube, and then PBS (pH = 7.4, 10mM, 1mL) buffer containing different concentrations of H5N1 or H7N9 virus was added in turn, incubated at 37°C for a period of time, the supernatant was discarded, and the sensor was washed with PBS (pH = 7.4, 10mM) buffer. Then PBS = 800μL (pH = 6.0), TMB = 50μL (5mg / mL), H2O2 = 150μL (15%) were added to each centrifuge tube in turn. After 5 minutes, the colorimetric effect of the solution was observed with the naked eye and the absorbance of the supernatant at 650nm was detected using a UV-2450 ultraviolet spectrophotometer, which can not only realize portable detection, but also improve the accuracy and sensitivity of real-time virus detection.

3. The use of a paper-based molecular imprinting sensor for visually detecting two subtypes of influenza A virus simultaneously according to claim 1, characterized in that: The paper-based MOF (Cu) virus molecular imprinting sensor was used to analyze H5N1 and H7N9 solutions of different concentrations, respectively, to evaluate its detection range and detection limit for H5N1 and H7N9; the paper-based MOF (Cu) virus molecular imprinting sensor was used to detect interfering virus solutions of the same concentration and mixed solutions containing target viruses and interfering viruses, to evaluate its selective recognition and competitive adsorption capabilities for H5N1 and H7N9; the paper-based MOF (Cu) virus molecular imprinting sensor was applied to the spike recovery of H5N1 and H7N9 in human serum, to evaluate its actual analytical capabilities for H5N1 and H7N9.

Images