Universal molecular imprinting sensor based on solid phase imprinting

Abstract

The invention constructs a high-selectivity molecular imprinting sensor based on solid phase imprinting, and the high-selectivity molecular imprinting sensor is used for detecting three common viruses H5N1, H7N9 and HBV. NanoMIPs with blue fluorescence of three viruses are prepared by taking magnetic Fe3O4 as an imprinting carrier, the nanoMIPs are mixed with a universal detection bracket Fe3O4 (at) CHO, when a target virus exists, the virus can be coupled with an aldehyde group on the Fe3O4 (at) CHO and is specifically combined with the nanoMIPs to form a sandwich structure of the Fe3O4 (at) CHO-virus-nanoMIPs, and after a magnetic field is applied to enrich the sandwich structure, the fluorescence change of a sensor is observed, so that the detection accuracy of the virus-nanoMIPs is improved. The detection on the three viruses can be realized. Through a series of optimization and performance investigation, the final selectivity factors of the constructed sensor to H5N1, H7N9 and HBV are up to 12.75, 12.87 and 17.14 respectively, and the detection limits are 1.52 fM, 0.75 fM and 0.87 pM respectively, which indicates that the sensor has good selectivity and sensitivity, provides a new strategy for universal detection of various viruses, and has wide application prospects. The invention provides a low-cost and easy-to-operate detection method for virus detection and epidemic prevention and control in resource-limited areas.

Description

[0001] The present invention belongs to the technical field of analytical chemistry detection, and in particular relates to a method for preparing a universal molecular imprinting sensor based on solid phase imprinting. Background Art

[0002] Early and effective diagnosis of patients infected with the virus is a key step in controlling the epidemic. Virus detection can be divided into direct and indirect detection.

[0003] Direct detection typically requires sophisticated equipment and complex techniques. Viruses can be detected using electron microscopy, light microscopy, or conventional molecular techniques. However, advanced microscopy methods are costly. Light microscopy has poor sensitivity and specificity, and the results are sometimes difficult to interpret. Molecular techniques also exist, including nucleic acid detection and immunofluorescence assays. Nucleic acid detection utilizes the polymerase chain reaction (PCR) to detect viral genetic material, amplifying DNA or RNA chains using multiple, gradually changing temperature cycles. Amplification techniques such as PCR and LAMP offer advantages such as high specificity, high sensitivity, and ease of setup. However, the procedure is cumbersome, and the reaction behavior depends on the matrix being studied. Analysis results typically require expert evaluation. Immunofluorescence assays generally identify the entire virus. Specifically, fluorescently labeled specific antibodies are used to stain respiratory epithelial cells in human nasopharyngeal swab samples. The primary detection method is the enzyme-linked immunosorbent assay (ELISA). However, compared to nucleic acid detection, ELISA has lower accuracy and can generate false-positive signals due to cross-contamination of antibodies with other viruses.

[0004] Indirect testing primarily uses cell culture to detect viruses. This method, also known as the "gold standard" for diagnosing viral infections, relies on inoculating corresponding cell lines with clinical samples, culturing them for 7-10 days, and continuously observing the development of cytopathic effects. The presence of viral infection is ultimately confirmed by staining with red blood cells and specific antibodies or observing blood adsorption using immunofluorescence microscopy. However, the cell culture process is relatively lengthy, making rapid viral detection difficult.

[0005] Today, methods for detecting viruses are becoming increasingly diverse and sophisticated, but these methods still have limitations such as complex operation and high cost. It is urgent to develop a virus detection strategy that can effectively overcome the above shortcomings and achieve universal detection of various viruses.

[0006] Molecularly imprinted polymers (MIPs), a novel recognition material with high specificity and sensitivity, have been hailed as "artificial antibodies" since their initial proposal in 1985. MIPs were initially used primarily for metal ion detection. Subsequently, a growing number of researchers have attempted to imprint MIPs onto macromolecules, small organic molecules, proteins, viruses, and even larger bacteria. This technology has advanced to the point where, in the field of viral molecular imprinting, entire viruses can be imprinted for more accurate detection and identification. Synthesis strategies for MIPs fall into three main categories: bulk imprinting, surface imprinting, and solid-phase imprinting.

[0007] Bulk imprinting technology is one of the most classic and commonly used methods for preparing MIPs. Its core principle is to mix functional monomers, crosslinkers, and template molecules in a solution, form a three-dimensional network of imprinted polymers through polymerization, and then remove the template molecules to obtain imprinted cavities with selective binding sites that match the template molecules. For example, in 2006, Bolisay et al. used bulk imprinting technology to synthesize MIPs for tobacco mosaic virus (TMV). They conducted batch equilibrium studies using imprinted and non-imprinted polymer hydrogels and determined the virus binding capacity. At the same time, they also obtained molecularly imprinted polymers with higher specificity for viruses by using different crosslinkers [Bolisay LD, Culver JN, Kofinas P. Molecularly imprinted polymers for tobacco mosaic virus recognition [J]. Biomaterials, 2006, 27 (22): 4165-4168.]. Subsequently, in 2014, Bai et al. constructed a "double-imprinted" MIPs using bulk imprinting technology, used an aptamer-based bioimprinting strategy for protein recognition, and further prepared the apple stem pit virus molecular imprinted hydrogel into a diffraction grating sensor by imprint lithography technology, which can be read by the naked eye [Bai W, Spivak D AA double-imprinted diffraction-grating sensor based on a virus-responsive super-aptamer hydrogel derived from an impure extract [J]. Angewandte Chemie International Edition, 2014, 53 (8): 2095-2098.]. Bulk imprinting is the simplest MIP synthesis method. Its advantage is that it involves the least number of different reagents and equipment. However, bulk imprinting generally has the problem of uneven distribution of template molecules in the polymer, which may lead to the burial of some imprinted sites and the difficulty of complete removal of template molecules, affecting recognition performance.

[0008] The core characteristic of surface imprinting technology is that it limits the imprinting sites to the surface or near-surface region of the support material, rather than forming imprinting sites throughout the polymer bulk as in bulk imprinting technology. This imprinting method generally involves modifying the imprinting support material to allow the polymer to be synthesized on the imprinted support surface. This technology can better remove template molecules, thereby reducing mass transfer resistance. In 2003, Hayden and Dickert et al. first proposed the surface molecular imprinting method and proposed a series of virus MIPs. For example, by using a polyacrylate and polyurethane-based prepolymer mixture, under the action of a blocking agent, the virus and the prepolymer mixture could not be effectively covalently linked, thereby achieving surface imprinting and applying it to a quartz crystal microbalance sensor (QCM) [Hayden O, Lieberzeit PA, Blaas D, et al. Artificial antibodies for bioanalyte detection—Sensing viruses and proteins[J]. Advanced Functional Materials, 2006, 16(10):1269-1278.][Dickert FL, Hayden O, Bindeus R, et al. Bioimprinted QCM sensors for virus detection—screening of plant sap[J]. Analytical and Bioanalytical Chemistry, 2004, 378:1929-1934.]. Wangchareansak et al. selected five influenza A virus subtypes, namely H5N1, H5N3, H1N1, H1N3, and H6N1, and constructed a molecularly imprinted polymer (MIP) for influenza A subtypes combined with a QCM. The MIP for each influenza virus subtype exhibited unique sensor properties on the QCM. After molecular imprinting, the resulting MIP was incubated with each virus subtype to detect the selectivity of each imprinted polymer, with the subtype imprinted polymer showing excellent binding properties [Wangchareansak T, Thitithanyanont A, Chuakheaw D, et al. Influenza A virus molecularly imprinted polymers and their application in virus sub-type classification [J]. Journal of Materials Chemistry B, 2013, 1(16): 2190-2197.].

[0009] Solid-phase imprinting (SIP) involves immobilizing a template molecule on a solid support surface, followed by polymerization around the support, ultimately forming an imprinted material with specific recognition sites. This method combines the advantages of surface imprinting and solid-phase synthesis, avoiding template aggregation during pre-polymerization, significantly improving imprinting efficiency, template removal rate, and target affinity. This technique generally prioritizes solid supports with high surface area and good mechanical properties, such as silica gel, magnetic nanoparticles, glass beads, or polymer microspheres. The template molecule is then modified and immobilized on the selected support surface. Functional monomers, crosslinkers, and initiators are then added to initiate polymerization, leading to a free radical reaction. After forming the imprinted layer, the template molecule is removed using various eluents or by varying the temperature environment. Altintas et al. first used a novel solid-phase synthesis method to study surface plasmon resonance-based molecularly imprinted polymers (MIPs) using bacteriophage MS2 as a template. In the presence of the template immobilized on glass beads, they synthesized high-affinity MIP nanoparticles. Using different temperatures, they were able to separate the elution of low- and high-affinity nanoparticles. High temperature (60°C) is sufficient to successfully destroy the binding interaction between high-affinity nanoparticles and their templates, while low temperature (15°C) is sufficient to elute the low-affinity nanoparticles because the MIP binds weakly to the template molecules, thereby obtaining MIP nanoparticles with better selectivity and affinity [Altintas Z, Gittens M, Guerreiro A, et al. Detection of waterborne viruses using high affinity molecularly imprinted polymers [J]. Analytical Chemistry, 2015, 87(13): 6801-6807.] [Altintas Z, Pocock J, Thompson KA, et al. Comparative investigations for adenovirus recognition and quantification: Plastic or natural antibodies? [J]. Biosensors and Bioelectronics, 2015, 74: 996-1004.]. Summary of the Invention

[0010] In view of the shortcomings of existing methods such as sensitivity, detection time consumption, and ease of operation, the purpose of the present invention is to construct a preparation method of a fluorescent virus sensor combined with molecular imprinting technology, and use the sensor for high-specificity universal recognition and detection of similar viruses.

[0011] A method for preparing a fluorescent virus sensor combined with molecular imprinting technology, characterized in that the method has the following process steps:

[0012] (1) Molecularly imprinted polymers (MIPs) were prepared by solid-phase imprinting using magnetic nanoparticles as carrier materials, viruses (H5N1, H7N9, HBV) as template viruses, TBA, NIPAm, APMA, and AA as functional monomers, BIS as a cross-linker, and ammonia (NH3·H2O) as a catalyst. After eluting the template viruses (H5N1, H7N9, HBV) with an eluent, soluble nanomolecularly imprinted polymers (nanoMIPs) with recognition cavities identical in shape and size to the template viruses were obtained, which were used as capture and separation elements for the template viruses.

[0013] (2) Using 7-amino-4-methylcoumarin as a raw material, a blue fluorescent dye complex modified with C=C was synthesized, and the blue fluorescent dye complex was encapsulated inside nanoMIPs as a fluorescent signal output element of a universal molecular imprinting sensor for solid phase imprinting;

[0014] (3) NanoMIPs synergistically identify viruses H5N1 / H7N9 / HBV through shape, size, and hydrogen bonding between functional monomers. Viruses can also be adsorbed on the surface of magnetic nanoparticles. By using magnetic adsorption, the sandwich structure of Fe3O4@CHO-virus-MIPs is aggregated at the bottom of the tube, thereby enabling virus detection. Under handheld UV light, observing the fluorescence intensity of the supernatant enables visual detection. At the same time, the fluorescence intensity of the supernatant changes with the concentration of the virus, and the use of fluorescence spectroscopy enables highly sensitive and accurate quantitative detection of the virus.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Using magnetic nanoparticles as carrier materials for virus molecular imprinting. The small size and porous structure of magnetic nanoparticles can provide a larger specific surface area for molecular imprinting, increase the imprinting sites, and facilitate the rapid diffusion of target molecules. At the same time, they have good dispersibility in solution and are more suitable for liquid phase detection. Nanoparticles as imprinting carriers not only have the imprinting advantages of other nanomaterials, but also have the advantages of rapid separation, suitability for application under extreme conditions (such as high temperature, strong acid and strong alkali), and repeated use.

[0017] (2) Using 7-amino-4-methylcoumarin as the raw material, a blue fluorescent dye complex ZIF-8-NH2 modified with C=C was synthesized to coat rhodamine B (RhB). The fluorescent dye was combined with magnetic separation and enrichment to reduce the detection limit to the fM level;

[0018] (3) Universal detection strategy: A sandwich structure (Fe3O4@CHO-virus-nanoMIPs) was designed in which the universal carrier (Fe3O4@CHO) can bind to different viruses, and multiple viruses can be detected by simply replacing nanoMIPs.

[0019] (4) The results showed that the universal molecular imprinting sensor has high specificity and satisfactory sensitivity, and is also portable and capable of rapid detection.

[0020] (5) The sensor has the ability to be applied to other virus detection, and the detection process does not require high professional skills of the operator, and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] [ Figure 1 ] Schematic diagram of the construction and detection principle of the molecular imprinting sensor described in .

[0022] [ Figure 2 ]Verification of the principle of molecular imprinting sensor for detecting three viruses.

[0023] [ Figure 3 ]DLS patterns of (A)Fe3O4, (B)Fe3O4@SiO2, (C)Fe3O4@SiO2-NH2 and (D)Fe3O4@CHO.

[0024] [ Figure 4 (A) Zeta potential diagrams of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-NH2, Fe3O4@CHO, Fe3O4-H5N1, Fe3O4-H7N9, and Fe3O4-HBV. (B) FTIR spectra of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-NH2, and Fe3O4@CHO.

[0025] [ Figure 5 ]Water contact angles of (A)Fe3O4, (B)Fe3O4@SiO2, and (C)Fe3O4@SiO2-NH2.

[0026] [ Figure 6 ]Scanning electron microscopy images of (A) H5N1-nanoMIPs, (B) H7N9-nanoMIPs, and (C) HBV-nanoMIPs.

[0027] [ Figure 7 ]Dynamic light scattering images of (A) H5N1-nanoMIPs, (B) H7N9-nanoMIPs, and (C) HBV-nanoMIPs.

[0028] [ Figure 8](A) Fluorescence intensity response of H5N1-nanoMIPs sensor after adding different concentrations of H5N1 virus (5.8, 11.6, 23.2, 34.8, 58, 69.6, 92.8, 116, 139.2, 162.4fM); (B) Linear fitting curve of H5N1; (C) Fluorescence intensity response of H7N9 sensor after adding different concentrations of H7N9 virus (46.4, 58, 69.6, 81.2, 92.8, 104.4, 116, 127.6, 139.2, 150.8, 162.4, 1 (A) Fluorescence intensity response of H7N9-nanoMIPs sensor after adding different concentrations of HBV virus (0.42, 0.84, 2.52, 4.2, 8.4, 16.8, 25.2, 33.6, 42, 50.4, 58.8, 67.2, 75.6, 84, 92.4 pM); (B) Linear fitting curve of HBV.

[0029] [ Figure 9 ]Sensor selectivity factor

[0030] [ Figure 10 ]Selectivity (A) and competitiveness (B) experiments of H5N1-nanoMIPs against H5N1; selectivity (C) and competitiveness (D) experiments of H7N9-nanoMIPs against H7N9; selectivity (E) and competitiveness (F) experiments of H7N9-nanoMIPs against H7N9.

[0031] [ Figure 11 ]Anti-interference ability of (A) H5N1-nanoMIPs, (B) H7N9-nanoMIPs and (C) HBV-nanoMIPs.

[0032] [ Figure 12 ](A) Temporal stability of H5N1-nanoMIPs, (B) H7N9-nanoMIPs, and (C) HBV-nanoMIPs.

[0033] [ Figure 13 ]Reproducibility of (A) H5N1-nanoMIPs, (B) H7N9-nanoMIPs, and (C) HBV-nanoMIPs.

[0034] [ Figure 14 ] Results of H5N1 detection in serum.

[0035] [ Figure 15 ]Results of H7N9 detection in serum.

[0036] [ Figure 16]Results of HBV detection in serum.

[0037] [ Figure 17 ]Comparison of the molecular imprinting sensor constructed in this work with other methods or biosensors for detecting viruses. Specific implementation plan

[0038] Here, the specific embodiments of the present invention will be further described in detail with reference to 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.

[0039] (1) Example 1: Preparation method of a universal molecular imprinting sensor

[0040] (2) Preparation of Fe3O4@SiO2: 2-20 g of FeCl3·6H2O was dispersed in 50-500 mL of ethylene glycol and ultrasonically dissolved to obtain an orange clear solution. 5-50 g of NaAc and 5-50 g of polyethylene glycol (6000) were then added under stirring and ultrasonically dissolved. The mixture was transferred to a 20-100°C water bath and magnetically stirred at 500-1000 r / min for half an hour to thoroughly mix the solution. The solution was then transferred to a polytetrafluoroethylene reactor and reacted in an oven at 100-1000°C for 1-48 h. After the reaction, the mixture was washed alternately with ultrapure water and anhydrous ethanol three times, and the resulting product was dried in a vacuum oven at 10-100°C for 1-48 h to obtain Fe3O4. Subsequently, 0.1-10 g of the resulting Fe₃O₄ nanoparticles are weighed and dispersed in 10-100 mL of isopropanol containing 1-10 mL of ultrapure water. 1-10 mL of NH₃·H₂O is slowly added dropwise under mechanical stirring at 500-1000 rpm. After stirring for 1-60 minutes, 1-10 mL of TEOS is slowly added dropwise. After the addition is complete, the reaction is heated at 30-100°C for 1-24 hours. After the reaction is complete, the resulting product is washed several times with ultrapure water and anhydrous ethanol until the supernatant is clear and transparent. The product is then dried in a vacuum oven at 50-200°C for 1-24 hours to obtain Fe₃O₄@SiO₂.

[0041] (3) Preparation of Fe3O4@SiO2-NH2: Magnetic nanoparticles modified with amino groups, Fe3O4@SiO2-NH2, were synthesized according to literature reports: 0.02-10 g of the prepared Fe3O4@SiO2 nanoparticles were dispersed in a three-necked flask containing a mixture of 1-20 mL of ultrapure water and 1-20 mL of anhydrous ethanol. After ultrasonication, argon was used to deoxygenate for 2-200 min. The reaction apparatus was transferred to a 20-100°C water bath and mechanically stirred at 700 rpm under argon protection. While stirring, 1-10 mL of APTES was slowly injected into the flask using a needle and the reaction was continued for 1-48 h. After the reaction was completed, the product was washed alternately with anhydrous ethanol and ultrapure water several times and dried in a vacuum oven at 20-200°C for 1-48 h to obtain Fe3O4@SiO2-NH2.

[0042] (4) Aldehyde modification of Fe3O4@SiO2-NH2 and fixation of three viruses: 0.01-10 g of Fe3O4@SiO2-NH2 nanoparticles were dispersed in a mixed solution of 1-20 mL of ultrapure water and 1-10 mL of glutaraldehyde (50%), and deoxygenated with argon for 10-60 min. The reaction was then transferred to a water bath at 25-50 °C and mechanically stirred at a speed of 500-1000 r / min under argon protection for 1-24 h, thereby modifying the aldehyde group on the Fe3O4@SiO2-NH2 particles to obtain Fe3O4@CHO. After the reaction, the magnetic particles were collected with a magnet and washed several times with deoxygenated PBS (0.1 M, pH = 7.4) at 25-50 °C to wash away the unreacted glutaraldehyde. After the final wash, the Fe3O4@CHO was directly dispersed in 1–50 mL of deoxygenated PBS (0.1 M, pH 7.4). 1–100 μL of HBV (or 1–100 μL of H5N1 / H7N9) was added. The HBV concentration was 8.34 nM, and the H5N1 and H7N9 concentrations were 11.6 pM. All viral concentrations used in this analysis were the same. The mixture was incubated in a water bath at 10–100°C for 1–12 hours. After the reaction, the magnetic particles were collected using a magnet and washed four times with PBS to remove any virus not bound to the magnetic particle surface. After washing, the particles were directly dispersed in 1–10 mL of PBS to obtain Fe3O4-HBV (Fe3O4-H5N1 / Fe3O4-H7N9) particles, which were stored at 0–10°C.

[0043] (5) Preparation of blue fluorescent dye: Disperse 0.1-10 g (1.14 mmol) of 7-amino-4-methylcoumarin in 2-100 mL of dry dichloromethane (DCM), add 100-1000 μL (2.5 mmol) of triethylamine, sonicate for 1-10 min to completely dissolve, deoxygenate the solution with argon for 10-60 min, then place in an ice-water bath at 0°C with argon as the protective gas. Simultaneously, add 10-1000 μL (1.58 mmol) of acryloyl chloride to 1-10 mL of dry DCM, mix thoroughly, and slowly inject the mixed solution of acryloyl chloride into the flask with a needle while magnetically stirring under argon protection. After the addition is complete, place the reaction mixture at room temperature and continue stirring for 8 h. After the reaction, the product is purified by first washing with an alkaline solution. 1-100 mL of saturated sodium bicarbonate solution is added to the reaction solution, thoroughly mixed, and then centrifuged at 10,000 rpm for 2-10 minutes to collect the precipitate. This washing process is repeated three times. Subsequently, impurities are removed by organic phase extraction: the resulting precipitate is redispersed with 1-20 mL of DCM, thoroughly mixed, and then centrifuged at 10,000 rpm for 1-20 minutes to collect the precipitate. This washing process is repeated three times. The product is then centrifuged and dried in a vacuum oven at 20-100°C for 1-48 hours to obtain a blue fluorescent dye.

[0044] (6) Preparation of three virus molecular imprinting polymers: The functional monomers TBA, NIPAm, APMA, AA, crosslinker BIS, and fluorescent dye (1-10 mg) were added sequentially to a three-necked flask containing 1-20 mL of ultrapure water, and ultrasonicated for 10-60 min to obtain a homogeneous solution. Subsequently, the solution was deoxygenated with argon for 10-60 min, and the flask was vacuumed for 1-60 min. During this process, 0.1-10 mL of a magnetic nanoparticle suspension Fe3O4-H5N1 / H7N9 / HBV dispersed in PBS was injected into the flask. After complete injection, the solution was vacuumed and deoxygenated for another 1-60 min, and the reaction system was sealed with a sealing film. The above-mentioned mixture was placed in a water bath constant temperature oscillator at 20-100 ° C for prepolymerization for 10-60 min to promote the self-assembly between the functional monomers and the template molecules. APS (1-10 mg) and TEMED (1-10 μL) were simultaneously dispersed in 100-1000 μL of ultrapure water. After dissolution, the mixture was deoxygenated with argon for 1-10 minutes and injected into the prepolymerized mixture using a needle to initiate polymerization. The reaction solution was then shaken at 25°C for 1-48 hours. After the reaction, the magnetic nanoparticles were collected using a magnet and washed several times with 25°C ultrapure water until the supernatant showed no blue fluorescence, thereby removing weakly affinitive and unreacted substances. The magnetic particles were collected using a magnet and dispersed in 1-10 mL of ultrapure water. The mixture was then shaken and eluted in a water bath at 20-100°C for 10-60 minutes. The magnetic nanoparticles were again separated from the supernatant using a magnet. The soluble nanomolecularly imprinted polymers (nanoMIPs) were now dispersed in the supernatant. The resulting product was sealed and stored at 1-10°C.

[0045] (7) Example 2: Feasibility analysis of the universal molecular imprinting sensor: To verify the feasibility of the designed sensor, an exploratory experiment was conducted on the three prepared MIPs: Figure 2As shown, H5N1-nanoMIPs, H5N1-nanoMIPs+(Fe3O4@CHO+H5N1), H5N1-nanoMIPs+(Fe3O4@CHO+H7N9) were tested , H5N1-nanoMIPs+(Fe3O4@CHO+HBV) and H7N9-nanoMIPs, H7N9-nanoMIPs+(Fe3O4@CHO+H7N9), H7N9-n Fluorescence spectra of nanMIPs+(Fe3O4@CHO+H5N1), H7N9-nanoMIPs+(Fe3O4@CHO+HBV), HBV-nanoMIPs, HBV-nanoMIPs+(Fe3O4@CHO+HBV), HBV-nanoMIPs+(Fe3O4@CHO+H5N1), and HBV-nanoMIPs+(Fe3O4@CHO+H7N9). In each sample group, 100 μL of nanMIPs and 100 μL of Fe3O4@CHO were added. When viruses were added, 10 μL (11.6 pM) of H5N1 and H7N9 were added, and 5 μL (8.34 nM) of HBV was added. The total volume of each group was fixed at 1 mL. The sample composition, including the optimization of synthesis conditions described below, was the same as above. The test results demonstrate the feasibility of the proposed sensor's construction principle. For example, the supernatant fluorescence intensity of the prepared H5N1-nanoMIPs decreased most significantly only when the universal detection scaffold, Fe3O4@CHO, and the target virus, H5N1, were present. When non-target viruses (H7N9 and HBV) were added, the Fe3O4@CHO was able to bind to the viruses, but the H5N1-nanoMIPs were unable to do so. Consequently, the H5N1-nanoMIPs remained dispersed in the supernatant, resulting in minimal change in supernatant fluorescence intensity. The same principle was observed for H7N9 and HBV. Only when the target virus was present in the detection system did the Fe3O4@CHO-target virus-nanoMIP sandwich structure form, resulting in a decrease in supernatant fluorescence intensity. Otherwise, the nanoMIPs adhered to the non-target virus surface through weak nonspecific adsorption.

[0046] Example 2: Characterization of the performance, morphology and structure of the universal molecular imprinting sensor and its intermediates.

[0047] Dynamic light scattering (DLS) was used to preliminarily characterize the prepared magnetic nanoparticles. Figure 3As shown in the figure, the particle sizes of the four synthesized nanomaterials—Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-NH2, and Fe3O4@CHO—were tested. The results showed that their particle sizes were approximately 275nm, 350nm, 400nm, and 460nm, respectively. The significant increase in the Fe3O4@SiO2 particle size is due to the hydrolysis and polycondensation of TEOS under the catalysis of ammonia to form silica, which coats the Fe3O4 surface. The subsequent addition of the silanization reagent APTES modifies the amino groups on the surface, and the addition of glutaraldehyde couples the aldehyde groups. These two-step modification of the nanoparticles enhances particle adhesion, resulting in a gradual increase in particle size. The changes in particle size provide preliminary evidence of the successful preparation of the magnetic nanomaterial.

[0048] Next, the magnetic nanomaterials were further characterized using Zeta potential and Fourier transform infrared spectroscopy. Figure 4 As shown in (A), the potential of Fe3O4 is about -13mV. After the surface is coated with silicon, the introduction of a large number of negatively charged silanol (Si-OH) groups causes the potential to drop to -53mV. Subsequently, amino groups (-NH2) are modified on the surface, and amino groups are easily protonated in solution to form -NH3 + , thus increasing the positive charge on the surface of the magnetic particles and increasing the potential of Fe3O4@SiO2-NH2. After grafting the aldehyde group, the amino groups on the particle surface couple with the aldehyde group, that is, the positively charged amino groups are consumed, and the potential of the magnetic particles decreases. After adding the virus, the aldehyde group reacts with the amino groups on the virus surface to form a Schiff base reaction, and the negative charge on the virus and particle surface increases, causing the potential to further decrease. Figure 4 As shown in (B), the infrared spectrum of Fe3O4 is at 570cm -1 A strong absorption peak appears near the Fe-O stretching vibration peak, indicating that the Fe3O4 nanoparticles are successfully prepared. -1 , 800cm -1 and 470cm -1 Nearby, there are Si-O-Si bond, Si-O bond and OH stretching vibration peaks in Si-OH group, which indicate that SiO2 is successfully coated on the Fe3O4 surface. Compared with Fe3O4@SiO2, Fe3O4@SiO2-NH2 has a new peak at 2933cm -1 and 1454cm -1 The two characteristic peaks are the stretching vibration peak and bending vibration peak of the C-H bond. However, the peak around 1740 cm-1 was not observed in Fe3O4@CHO. -1 The characteristic peaks of aldehyde group and 1640 cm -1 The characteristic peaks of imine at 1635 cm -1In summary, the potential changes and infrared spectroscopy absorption peaks of each nanoparticle further prove the successful preparation of magnetic nanomaterials.

[0049] Subsequently, Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2-NH2 were characterized by contact angle meter to explore their hydrophilic and hydrophobic characteristics. Figure 5 As shown in the figure, the water contact angles of the three materials are 35±0.5°, 12±0.5°, and 20±0.5°, respectively. The Fe3O4 surface generally has a higher surface energy, resulting in a smaller contact angle, indicating hydrophilicity. After being coated with SiO2, Si-OH groups are formed on the surface of the nanoparticles, increasing their hydrophilicity and decreasing their contact angles. After amino modification, -NH2 groups are introduced on the surface of the nanoparticles, increasing their hydrophobicity and increasing their contact angles. Overall, however, all magnetic nanomaterials have good hydrophilicity.

[0050] Finally, the morphology and particle size of the synthesized H5N1-nanoMIPs, H7N9-nanoMIPs and HBV-nanoMIPs were characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). Figure 6 and Figure 7 SEM results show that the prepared nanoMIPs of each virus are spherical in shape, with a particle size of approximately 400 nm and good dispersion. DLS results show that the size of the nanoMIPs of each virus ranges from 380 to 600 nm, indicating that the nanoMIPs of each virus were successfully prepared.

[0051] Example 3: Application of the universal molecular imprinting sensor

[0052] The experimental conditions of this example are as follows: ultrasonically disperse the prepared Fe3O4@CHO nanoparticles, add 100-1000 μL (1-10 mg / mL) of Fe3O4@CHO and different concentrations of H5N1 / H7N9 / HBV virus into a centrifuge tube, and incubate the tube in a 37°C water bath thermostat for 1-12 hours to allow the virus to couple with the aldehyde groups on the magnetic nanoparticles and thus be fixed on the magnetic particles. The magnetic particles are collected with a magnet and washed four times with PBS to remove the virus not bound to the magnetic particles. The particles are then dispersed in 100-1000 μL of PBS (0.1 M, pH = 7.4). Simultaneously, three nanoMIPs (0.01-10 mg / mL) were ultrasonically dispersed. 1-1000 μL of the solution was then added to the virus-immobilized magnetic particle solution. The solution was then oscillated in a 25°C water bath on a constant-temperature oscillator for 10-100 minutes. The supernatant was separated using a magnet, and the difference in fluorescence intensity between the supernatant in the presence and absence of the target virus was expressed as F. Fluorescence spectrophotometry was used for detection with an excitation gap width of 5.0 nm and an excitation wavelength of 369 nm. Fluorescence intensity was measured at 460 nm.

[0053] (1) Detection linearity analysis and detection limit of the universal molecular imprinting sensor for different concentrations of viruses

[0054] The prepared H5N1-nanoMIPs, H7N9-nanoMIPs and HBV-nanoMIPs fluorescence sensors were added with different concentrations of H5N1, H7N9 and HBV under the above optimized conditions to investigate the detection effect of the sensors on the target viruses.

[0055] The results are as follows Figure 8 As shown in (A), (C) and (E), the changes in fluorescence intensity of H5N1 in the concentration range of 5.8-162.4fmol / L, H7N9 in the concentration range of 46.4-197.2fmol / L and HBV in the concentration range of 0.42-92.4pmol / L were investigated. It can be seen that as the concentration of the target virus increases, the fluorescence intensity of the supernatant of the detection system gradually decreases. The linear regression equation was calculated based on the relationship between the change value ΔF of the supernatant fluorescence intensity and the virus concentration. The detection limit was calculated according to the formula LOD=3δ / k, where δ is the standard deviation of the results obtained by measuring the blank sample 10 times, and k is the slope of the obtained linear equation. Figure 8As shown in (B), (D) and (F), in the detection system of H5N1, the linear range of the detection results is 11.6fmol / L to 139.2fmol / L, the linear equation is ΔF = 12.84CH5N1 + 349.06, R2 = 0.9947, and the detection limit is 1.52fmol / L; in the detection system of H7N9, the linear range of the detection results is 58fmol / L to 162.4fmol / L. The linear range for the detection of H5N1, H7N9, and HBV was 2.52 pmol / L, with a linear equation of ΔF = 17.96 CH7N9 - 190.21, R² = 0.9956, and a detection limit of 0.75 fmol / L. In the HBV detection system, the linear range of the detection results was 2.52 pmol / L to 75.6 pmol / L, with a linear equation of ΔF = 34.36 CH7N9 + 502.97, R² = 0.9962, and a detection limit of 0.87 pmol / L. The results showed that the supernatant fluorescence intensity of the constructed sensor increased with increasing virus concentration when detecting H5N1, H7N9, and HBV, with good linear relationships. All three sensors exhibited good specific recognition capabilities.

[0056] (2) Selectivity and competition experiments of the universal molecular imprinting sensor for viruses

[0057] Selective research results such as Figure 10 As shown in (A), (C), and (E), when H5N1-nanoMIPs was used to detect five different viruses at the same concentration, the sensor showed high specificity for H5N1, with the highest fluorescence response signal value. The selectivity factor of H5N1-nanoMIPs for other viruses was calculated using the formula SF = ΔFnanoMIPs, H5N1 / ΔFnanoMIPs, non-target virus. The results show that the sensor has high selectivity for H5N1 and has a selectivity factor of up to 12.21 for the non-target virus EV71. The selectivity for the target viruses H7N9 and HBV in H7N9-nanoMIPs and HBV-nanoMIPs was also significantly higher than that for other non-target viruses. Similarly, the selectivity factor for non-target viruses was calculated using the formula, as shown in the following figure. Figure 9As shown in the figure, the selectivity factor of H7N9-nanoMIPs for non-target viruses can reach up to 13.51, and the selectivity factor of HBV-nanoMIPs for non-target viruses can reach up to 18.02. This is because the prepared nanoMIPs highly match the antigenic structure on the surface of the target virus, enabling selective recognition through specific interactions (such as hydrogen bonds and electrostatic interactions). However, for non-target viruses, although there are non-target viruses with similar particle sizes, due to the significant differences in antigenic structure and surface charge between the viruses, the nanoMIPs only produce non-specific adsorption for the non-target viruses. Even different subtypes of the same virus cannot form effective specific binding with the imprinted cavity and can only be adsorbed to the nanoMIPs surface through non-specific interactions such as van der Waals forces or hydrophilic and hydrophobic interactions.

[0058] Competitive research results such as Figure 10 As shown in (B), (D), and (F), the competitiveness of each viral nanoMIP was investigated by adding the target virus and interfering viruses at the same concentration as the target virus to the detection system. When the target virus H5N1 was added to the H5N1-nanoMIPs, the fluorescence intensity changed significantly. Similarly, when other non-target competing viruses were added simultaneously, the fluorescence intensity change of the sensor was not much different from that when the target virus was added alone, indicating that the interfering viruses had little competitive ability against H5N1. Similar results were obtained for the competitive experiments investigating the H7N9-nanoMIPs and HBV-nanoMIPs. This demonstrates that the prepared sensor exhibits good selectivity and competitiveness for the target virus.

[0059] (3) The universal molecular imprinting sensor recovers H5N1 serum spiked with

[0060] The prepared nanoMIPs and the universal detection scaffold Fe3O4@CHO were used to detect the target viruses H5N1, H7N9, and HBV in 100-fold diluted human serum samples to evaluate the practical application value of the constructed sensor. The serum was obtained from Xiangtan University Hospital and stored at 4°C. Each test was performed in triplicate, and the results were averaged to obtain the recovery rate. The test results are shown in Figure 2. Figure 14 、 Figure 15 and Figure 16 The results show that the recovery rates of the prepared H5N1-nanoMIPs for H5N1 detection were 97.47-102.86%, the recovery rates of H7N9-nanoMIPs for H7N9 detection were 96.26-108.79%, and the recovery rates of HBV-nanoMIPs for HBV detection were 96.03-102.81%. The good recovery rates indicate that the prepared nanoMIPs can provide a better detection method for the actual detection of viruses.

[0061] (4) Comparison of the universal molecular imprinting sensor with other methods or biosensors for detecting viruses

[0062] The molecular imprinting sensor constructed in this work was compared with other methods or biosensors for detecting viruses. Figure 17 As shown in the figure, there are currently a variety of detection methods for influenza A virus and hepatitis B virus. Among them, electrochemical sensors have high sensitivity, but generally require antibodies for detection, which not only increases costs but also makes antibody screening complex and time-consuming. Detection methods similar to quartz crystal microbalance sensors are relatively insensitive and also require the use of aptamers. Methods related to molecular imprinting technology for virus detection have improved linear range and detection limit compared to other methods, but their selectivity is poor. The detection method proposed in this work combines a wide linear detection range and a low detection limit, and has good selective recognition effect for similar viruses, providing a universal strategy for virus detection.

Claims

1. A method for preparing a universal molecularly imprinted sensor, using inexpensive and readily available Fe3O4 magnetic nanoparticles as a carrier material, viruses (H5N1, H7N9, HBV) as template molecules, epitope imprinting to prepare molecularly imprinted polymers, and 7-amino-4-methylcoumarin as a raw material to synthesize a C=C-modified blue fluorescent dye complex. The blue fluorescent dye complex is used as a fluorescent signal output element, specifically comprising the following steps: Step 1) 2-20 g of FeCl3·6H2O is dispersed in 50-500 mL of ethylene glycol and ultrasonically dissolved to obtain an orange clear solution. 5-50 g of NaAc and 5-50 g of polyethylene glycol (6000) are then added under stirring and ultrasonically dissolved. The mixture is then transferred to a 20-100° C. water bath and magnetically stirred at a speed of 500-1000 r / min for half an hour to fully mix the solution. The solution is then transferred to a polytetrafluoroethylene reactor and reacted in a 100-1000° C. oven for 1-48 hours. After the reaction is completed, the mixture is washed alternately with ultrapure water and anhydrous ethanol three times, and the resulting product is dried in a 10-100° C. vacuum oven for 1-48 hours to obtain Fe3O4. Subsequently, 0.1-10g of the obtained Fe3O4 nanoparticles are weighed and dispersed in 10-100mL of isopropanol containing 1-10mL of ultrapure water. 1-10mL of NH3·H2O is slowly added dropwise under mechanical stirring at 500-1000r / min. After stirring for 1-60min, 1-10mL of LTEOS is slowly added dropwise. After the addition is complete, the reaction is heated at 30-100°C for 1-24h. After the reaction is completed, the resulting product is washed several times with ultrapure water and anhydrous ethanol until the supernatant is clear and transparent, and then dried in a vacuum oven at 50-200°C for 1-24h to obtain Fe3O4@SiO2. Step 2) Synthesize amino-modified magnetic nanoparticles Fe3O4@SiO2-NH2 according to literature reports: Disperse 0.02-10g of the prepared Fe3O4@SiO2 nanoparticles in a three-necked flask containing a mixture of 1-20mL ultrapure water and 1-20mL anhydrous ethanol, and use argon bubbling to deoxygenate for 2-200min after ultrasonication. Transfer the reaction apparatus to a 20-100℃ water bath, and mechanically stir the reaction at a speed of 700r / min under argon protection. While stirring, slowly inject 1-10mL APTES into the flask with a needle, and continue the reaction for 1-48h. After the reaction is completed, the obtained product is washed alternately with anhydrous ethanol and ultrapure water several times, and placed in a vacuum oven at 20-200℃ to dry for 1-48h to obtain Fe3O4@SiO2-NH2; Step 3) Disperse 0.01-10 g of Fe3O4@SiO2-NH2 nanoparticles in a mixed solution of 1-20 mL of ultrapure water and 1-10 mL of glutaraldehyde (50%), deoxygenate with argon for 10-60 min, then transfer the reaction to a water bath at 25-50°C and mechanically stir at 500-1000 rpm under argon protection for 1-24 h, thereby modifying the Fe3O4@SiO2-NH2 particles with aldehyde groups to obtain Fe3O4@CHO. After the reaction, collect the magnetic particles with a magnet and wash them several times with deoxygenated PBS (0.1 M, pH = 7.4) at 25-50°C to remove unreacted glutaraldehyde. After the last wash, Fe3O4@CHO was directly dispersed in 1-50 mL of deoxygenated PBS (0.1 M, pH = 7.4), and 1-100 μL of HBV (or 1-100 μL of H5N1 / H7N9) was added. The HBV concentration was 8.34 nM, and the H5N1 and H7N9 concentrations were 11.6 pM. All viral concentrations mentioned hereafter are the same. The mixture was incubated in a water bath on a constant-temperature oscillator at 10-100°C for 1-12 hours. After the reaction, the magnetic particles were collected using a magnet and washed four times with PBS to remove any virus not immobilized on the surface of the magnetic particles. After washing, the particles were directly dispersed in 1-10 mL of PBS to obtain Fe3O4-HBV (Fe3O4-H5N1 / Fe3O4-H7N9) particles, which were then stored at 0-10°C. Step 4) Disperse 0.1-10 g (1.14 mmol) of 7-amino-4-methylcoumarin in 2-100 mL of dry dichloromethane (DCM). Add 100-1000 μL (2.5 mmol) of triethylamine and sonicate for 1-10 minutes to completely dissolve the solution. Deoxygenate the solution with argon for 10-60 minutes and then place it in a 0°C ice-water bath with argon as the shielding gas. Simultaneously, add 10-1000 μL (1.58 mmol) of acryloyl chloride to 1-10 mL of dry DCM. After thorough mixing, slowly inject the acryloyl chloride mixture into the flask with a needle while magnetically stirring under argon. After the addition is complete, the reaction mixture is stirred continuously at room temperature for 8 hours. After the reaction is completed, the product is purified: first, it is washed with an alkaline solution. 1-100 mL of saturated sodium bicarbonate solution is added to the reaction solution, mixed thoroughly, and then centrifuged at 10,000 rpm for 2-10 minutes to collect the precipitate. The washing is repeated three times. Then, an organic phase extraction method is used to remove impurities: the resulting precipitate is redispersed with 1-20 mL of DCM, mixed thoroughly, and centrifuged at 10,000 rpm for 1-20 minutes to collect the precipitate. The washing is repeated three times, and the product is collected by centrifugation. The resulting product is dried in a vacuum oven at 20-100°C for 1-48 hours to obtain a blue fluorescent dye. Step 5) Each functional monomer, TBA, NIPAm, APMA, AA, a crosslinker, BIS, and a fluorescent dye (1-10 mg) are sequentially added to a three-necked flask containing 1-20 mL of ultrapure water. The mixture is ultrasonicated for 10-60 minutes to obtain a homogeneous solution. Subsequently, the mixture is deoxygenated with argon for 10-60 minutes and the flask is vacuumed for 1-60 minutes. During this process, 0.1-10 mL of a suspension of magnetic nanoparticles, Fe3O4-H5N1 / H7N9 / HBV, dispersed in PBS is injected into the flask. After complete injection, vacuum deoxygenation is continued for 1-60 minutes, and the reaction system is sealed with a sealing film. The above mixture is placed in a water bath thermostat oscillator at 20-100°C for prepolymerization for 10-60 minutes to promote self-assembly between the functional monomers and the template molecules. APS (1-10 mg) and TEMED (1-10 μL) were simultaneously dispersed in 100-1000 μL of ultrapure water. After dissolution, the mixture was deoxygenated with argon for 1-10 minutes and injected into the prepolymerized mixture using a needle to initiate polymerization. The reaction solution was then shaken at 25°C for 1-48 hours. After the reaction, the magnetic nanoparticles were collected using a magnet and washed several times with 25°C ultrapure water until the supernatant showed no blue fluorescence, thereby removing weakly affinitive and unreacted substances. The magnetic particles were collected using a magnet and dispersed in 1-10 mL of ultrapure water. The mixture was then shaken and eluted in a water bath at 20-100°C for 10-60 minutes. The magnetic nanoparticles were again separated from the supernatant using a magnet. The soluble nanomolecularly imprinted polymers (nanoMIPs) were now dispersed in the supernatant. The resulting product was sealed and stored at 1-10°C.

2. The method for preparing a universal molecular imprinted sensor according to claim 1, wherein: Step 4) When using a fluorescence spectrophotometer for detection, set the excitation gap width to 5.0 nm, the excitation wavelength to 369 nm, and observe the fluorescence intensity at 460 nm.

3. The use of the universal molecular imprinted sensor prepared by the method for preparing a universal molecular imprinted fluorescent sensor according to claim 1, characterized in that: The prepared Fe3O4@CHO nanoparticles were ultrasonically dispersed. 100–1000 μL (1–10 mg / mL) of Fe3O4@CHO and varying concentrations of H5N1 / H7N9 / HBV viruses were added to a centrifuge tube and incubated in a 37°C waterbath on a shaker for 1–12 hours to allow the viruses to couple with the aldehyde groups on the magnetic nanoparticles and become immobilized on the magnetic particles. The magnetic particles were collected with a magnet and washed four times with PBS to remove unbound viruses. The particles were then dispersed in 100–1000 μL of PBS (0.1 M, pH 7.4). Simultaneously, three nanoMIPs (0.01–10 mg / mL) were ultrasonically dispersed. 1–1000 μL of the solution was then added to the virus-immobilized magnetic particle solution. The solution was then shaken in a 25°C waterbath on a shaker for 10–100 minutes. The supernatant was separated with a magnet, and F represents the difference in fluorescence intensity between the presence and absence of the target virus.

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