Universal visual influenza virus fluorescent molecular imprinting sensor

Abstract

The invention provides construction of a universal influenza virus fluorescent molecular imprinting sensor and a method for visually detecting influenza viruses. In the invention, a zeolite imidazolate skeleton (ZIF-8) coated red fluorescent quantum dots (R-QDs) is used as an imprinting carrier and signal output, influenza virus H7N9 is used as a template, and the molecularly imprinted polymer (MIPs) is prepared by a sol-gel technology. Meanwhile, the surfaces of the magnetic Fe3O4 nanoparticles are modified with phenylboronic acid (Fe3O4-FPBA) to serve as a capture element, and when the H7N9 virus is specifically bound with the MIPs, Fe3O4-FPBA can be bound with glycoprotein on the surface of the virus through boric acid esterification reaction, so that a sandwich composite structure of MIPs-virus-Fe3O4-FPBA is formed. Through the formation of the structure, separation and enrichment of a target object can be realized through a magnet, and meanwhile, the change of a quantum dot fluorescence signal is caused, so that visual detection of a target virus is realized, and high-sensitivity quantitative detection can also be performed through an ultraviolet and visible spectrophotometer. The sensor prepared by the invention is a general influenza virus sensor, and the fluorescent sensor suitable for visually detecting other target objects can be prepared by selecting other subtype influenza viruses as template molecules for molecular imprinting.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry detection, and in particular relates to the construction and research of a universal visualized influenza virus fluorescent molecular imprinting sensor. Background Art

[0002] The outbreak and spread of viral infectious diseases pose a serious threat to human and animal health and have a significant impact on society and the economy. Influenza A virus (IAV) can infect a variety of hosts, including humans, pigs, and poultry, and can spread rapidly through aerosols, water, or droplets, causing infectious diseases. More than 130 subtypes of influenza viruses have been identified, based on differences in their surface hemagglutinin (HA) and neuraminidase (NA) proteins [Von Itzstein M, et al., 2011]. R, Haselhorst T. Influenzavirus, overview: structures, infection mechanisms and antivirals [M]. Springer, 2015.]. To effectively control the harm caused by such pathogen infections, it is urgent to establish simple, rapid, and highly sensitive diagnostic methods. Traditional infectious disease diagnosis relies on the detection of pathogen-specific antigens, antibodies, or nucleic acids, usually performed in clinical laboratories. This has the disadvantages of being time-consuming, complex, costly, requiring sophisticated equipment, and unable to be rapidly tested on-site. Therefore, the development of cost-effective, highly selective, sensitive, and rapidly visualized virus detection technologies has become an important research direction.

[0003] Molecularly imprinted polymers (MIPs) are a class of artificially synthesized selective recognition elements that have been widely used in analytical applications. Their preparation process primarily involves the polymerization of functional monomers in the presence of a template. After the polymerization is complete, the template molecules are washed away, leaving behind a three-dimensional cavity that is complementary to the template in shape, size, and function. These specific cavities endow MIPs with the ability to selectively recognize target molecules. With the frequent outbreaks of viral diseases in recent years, virus detection sensors based on molecular imprinting technology have gradually become a research hotspot, attracting widespread attention from a growing number of researchers. For example, Kaur et al. developed molecularly imprinted polymer nanoparticles (nanoMIPs) to detect norovirus (NoV) using a small target epitope (12 amino acids) using a solid-phase synthesis method, with a detection limit of only 6.5 pg / mL. [Kaur S, Singla P, Dann AJ, et al. Sensitive electrochemical and thermal detection of human noroviruses using molecularly imprinted polymer nanoparticles generated against a viral target[J]. ACS Applied Materials & Interfaces, 2024, 16(38): 51397-51410.] Gong et al. developed a portable, visual molecular imprinting polymer-based sensor to specifically identify H5N1. The sensor can conveniently realize self-service detection with a low detection limit [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.] These detection methods have achieved good detection results, but they have shortcomings in nonspecific adsorption, long-term stability and reusability.

[0004] Based on these significant studies, the present invention designed and constructed a universal, visual fluorescent molecular imprinting sensing platform for influenza virus detection. Specifically, a zeolite imidazolate framework (ZIF-8)-coated red fluorescent quantum dots (R-QDs) served as the imprinting carrier and signal output, with the influenza virus H7N9 as the template. Molecularly imprinted polymers (MIPs) were obtained after imprinting, and MIPs with H7N9-imprinted cavities were obtained after elution. Furthermore, phenylboronic acid (Fe3O4-FPBA) was modified on the surface of magnetic Fe3O4 nanoparticles as a capture element. After the H7N9 virus specifically binds to the MIPs, the Fe3O4-FPBA binds to glycoproteins on the virus surface through a boronate esterification reaction, forming a "MIPs-virus-Fe3O4-FPBA" sandwich structure. This structure enables the separation and enrichment of the target using a magnet, while simultaneously causing a change in the quantum dot fluorescence signal. This sensing platform offers dual detection modes: visual semi-quantitative analysis under UV light and highly sensitive quantitative analysis combined with fluorescence spectroscopy. The sensor also demonstrated excellent universal detection performance for other influenza virus subtypes, potentially addressing the challenges of on-site influenza detection during peak influenza seasons. Furthermore, the design strategy can be extended to detect other pathogens, providing new insights into the development of novel biosensors. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the purpose of the present invention is to construct a universal visual influenza virus fluorescent molecular imprinting sensor, providing a portable detection method for the specific, visual, and rapid detection of influenza viruses, thereby achieving the purpose of simple operation and rapid visual detection of influenza viruses.

[0006] Technical solution: The construction and research of the universal visualized influenza virus fluorescent molecular imprinting sensor of the present invention is characterized in that the method includes the following preparation steps:

[0007] (1) Preparation of a universal visualized influenza virus fluorescent molecular imprinting sensor: Zeolite imidazolate framework (ZIF-8) and red fluorescent quantum dots (R-QDs) were synthesized, and the red quantum dots were coated on ZIF-8 as an imprinting carrier and fluorescent signal output. Then, H7N9 virus was used as a template, 3-aminopropyltriethoxysilane (APTES) was used as a functional monomer, and tetraethoxysilane (TEOS) was used as a crosslinker. The synthesis was carried out by sol-gel method under the catalysis of triethylamine (TEA). After the synthesis was completed and the template was washed off, molecular imprinting polymers (MIPs) with H7N9 imprinting cavities were obtained. The silicon layer was coated on the surface of Fe3O4 nanoparticles by silanization reagents tetraethoxysilane and 3-aminopropyltriethoxysilane and amino groups were grafted. Then, 4-formylphenylboronic acid (FPBA) was grafted by Schiff base reaction to prepare Fe3O4-FPBA. In an alkaline environment, boric acid can form stable borate esters with influenza virus surface glycoproteins. When the H7N9 virus specifically binds to MIPs, Fe3O4-FPBA binds to the virus surface glycoproteins through a borate esterification reaction, forming a "MIPs-virus-Fe3O4-FPBA" sandwich structure.

[0008] (2) Synthesis of Fe3O4 magnetic nanoparticles with surface modification of 4-formylphenylboronic acid (FPBA): In order to efficiently capture and separate viruses and reduce the interference of nonspecific binding, a silicon layer was first coated on the Fe3O4 nanoparticles to increase their stability in complex environments and provide active sites for subsequent reactions; after coating with the silicon layer, highly reactive amino groups were introduced to further improve the reaction activity and biocompatibility; finally, 4-formylphenylboronic acid was modified on the surface of the material so that it could form a stable boronate ester bond with the glycoprotein on the surface of the virus in an alkaline environment, which is conducive to specific recognition and significantly improves the sensitivity and selectivity of detection.

[0009] (3) Application of a universal visualized influenza virus fluorescent molecular imprinting sensor: The prepared MIPs particles were ultrasonically dispersed and dissolved in a certain concentration of PBS solution, and then H7N9 virus of different concentrations was added and incubated for 0.5-10 hours, centrifuged and washed with PBS buffer to remove unbound H7N9; Fe3O4-FPBA particles were then ultrasonically dispersed in PBS (pH = 8.5) solution, mixed with the above-mentioned virus-bound MIPs and shaken for 10-60 minutes, and after magnetic separation, the fluorescence intensity change of the supernatant was measured by fluorescence spectroscopy for quantitative analysis. The complex after magnetic separation was washed once with PBS solution and resuspended in 0.1-10 mL PBS. The change of its fluorescence intensity was observed by naked eye under ultraviolet light to achieve visualized semi-quantitative analysis.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] (1) Using zeolite imidazolate framework (ZIF-8) as a carrier material for influenza virus molecular imprinting is low-cost and has simple synthesis steps. It not only provides a stable structure for encapsulating quantum dots, but also enhances the mechanical strength and stability of the material. Red quantum dots emit red fluorescence under ultraviolet light, providing an intuitive signal output for detection, avoiding additional fluorescent labeling steps, and simplifying the operation process;

[0012] (2) Fe3O4-FPBA, as a capture element, can bind to influenza virus surface glycoproteins through a boronate esterification reaction, forming a stable "MIPs-virus-Fe3O4-FPBA" sandwich structure. The introduction of magnetic nanoparticles enables this structure to be rapidly separated and enriched by an external magnetic field, greatly improving detection efficiency and reducing interference from complex sample matrices.

[0013] (3) By combining multiple detection methods, the supernatant after separation and enrichment can be quantitatively detected by fluorescence spectroscopy to provide accurate information on virus concentration; and after the separated sandwich structure is redispersed in PBS buffer, it can be semi-quantitatively visualized under ultraviolet light for naked eye detection. This combination of quantitative and semi-quantitative methods not only meets the needs of high-precision detection, but also facilitates rapid screening and preliminary judgment, and is suitable for different application scenarios;

[0014] (4) The sensor is a universal influenza virus sensor. By using other subtypes of influenza virus as template molecules for imprinting, it can be prepared into a visualized influenza virus fluorescent molecular imprinting sensor suitable for visual detection of other influenza viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] [ Figure 1 ]Schematic diagram of the preparation principle of the universal visual influenza virus fluorescent molecular imprinting sensor described in .

[0016] [ Figure 2 ](A) Feasibility principle verification of the sensor (the inset is the corresponding visual effect under 367nm UV light); (B) Fluorescence spectra of MIPs and NIPs combined with H7N9.

[0017] [ Figure 3 ]Scanning electron microscopy (SEM) images of (A) ZIF-8, (B) ZIF-8@R-QDs, (C) MIPs, and (D) NIPs.

[0018] [ Figure 4 ]Dynamic light scattering (DLS) of (A) ZIF-8, (B) ZIF-8@R-QDs, (C) MIPs, and (D) NIPs.

[0019] [ Figure 5](A) Infrared spectra of ZIF-8(a), ZIF-8@R-QDs(b), MIPs(c), and NIPs(d); (B) Infrared spectra of Fe3O4(a), Fe3O4@SiO2(b), Fe3O4@SiO2-NH2(c), and Fe3O4-FPBA(d).

[0020] [ Figure 6 ]X-ray diffraction (XRD) patterns of ZIF-8 (a) and ZIF-8@R-QDs (b) (the inset is the corresponding image under UV light).

[0021] [ Figure 7 ]Nitrogen adsorption-desorption isotherms of (A) ZIF-8, (B) ZIF-8@R-QDs; pore size distribution curves of (C) ZIF-8, (D) ZIF-8@R-QDs.

[0022] [ Figure 8 ]Zeta potential diagram of ZIF-8(a), ZIF-8@R-QDs(b), MIPs(c), NIPs(d), Fe3O4(e), Fe3O4@SiO2(f), Fe3O4@SiO2-NH2(g), Fe3O4-FPBA(h).

[0023] [ Figure 9 ](A) Linearity of MIPs in detecting different concentrations of H7N9 (0, 2.9, 5.8, 11.6, 29, 58, 87, 116, 145, 174 fM); (B) Visualization of different concentrations of H7N9; (C) Linear fitting of MIPs; (D) Linearity of NIPs in detecting different concentrations of H7N9 (0, 2.9, 5.8, 11.6, 29, 58, 87, 116, 145, 174 fM).

[0024] [ Figure 10 ](A) Selectivity of the sensor for virus H7N9; (B) Competitiveness of the sensor for virus H7N9.

[0025] [ Figure 11 ]The interference of the sensor.

[0026] [ Figure 12 ](A) Reproducibility of the sensor; (B) Temporal stability of the sensor.

[0027] [ Figure 13 ] Results of H7N9 detection in 100-fold diluted human serum.

[0028] [ Figure 14 ]Visualization results after incubation of MIPs with H7N9 virus and Fe3O4-FPBA for different times.

[0029] [ Figure 15 ](A) Linearity of H5N1-MIPs in detecting different concentrations of H5N1 (0, 5.8, 11.6, 29, 58, 87, 116, 145, 174, 203 fM); (B) Visualization of different concentrations of H5N1; (C) Linear fitting of H5N1-MIPs; (D) Linearity of NIPs in detecting different concentrations of H5N1 (0, 5.8, 11.6, 29, 58, 87, 116, 145, 174, 203 fM).

[0030] [ Figure 16 ](A) Linearity of H9N2-MIPs in detecting different concentrations of H9N2 (0, 5.8, 11.6, 29, 58, 87, 116, 145, 174, 203 fM); (B) Visualization of different concentrations of H9N2; (C) Linear fitting of H9N2-MIPs; (D) Linearity of NIPs in detecting different concentrations of H9N2 (0, 5.8, 11.6, 29, 58, 87, 116, 145, 174, 203 fM). Specific implementation plan

[0031] The following is a detailed description of the technical solution of the present invention in conjunction with the accompanying drawings and embodiments. The embodiments described below 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 universal visual influenza virus fluorescent molecular imprinting sensor

[0033] (1) Preparation of R-QDs: First, synthesize a NaHTe precursor by mixing 0.1-20 mL of ultrapure water and 0.1-20 mL of anhydrous ethanol. After deoxygenation by bubbling with argon for 10-60 min, add 10-100 mg of tellurium powder and 50-200 mg of sodium borohydride (NaBH4). Under argon protection, place the reaction system in an ice-water bath at -50-5°C and stir continuously for 1-6 h to obtain a clear NaHTe precursor solution.

[0034] Subsequently, 20-200 mg of cadmium chloride hydrate (CdCl2·2.5H2O) was dissolved in 10-200 mL of ultrapure water, 50-150 μL of mercaptopropionic acid (MPA) was added, and after ultrasonic dispersion, the pH was adjusted to approximately 8-12 with 0.1-10 M NaOH solution. After argon deoxygenation for 30-80 minutes, 0.1-20 mL of NaHTe precursor was injected under an argon atmosphere and refluxed at 100°C in the dark for 8-20 hours to finally produce 10-200 mL of red fluorescent quantum dot (R-QDs) solution. The resulting quantum dots were stored in a cool, dark environment until use.

[0035] (2) Preparation of ZIF-8@R-QDs: 1-20 mL of zinc nitrate hexahydrate (5-50 mg / mL) aqueous solution was mixed with 1-20 mL of R-QDs under stirring at 100-600 rpm for 1-10 min, and then 0.1-10 g of 2-methylimidazole was added. The mixture was magnetically stirred for 10-60 min and then centrifuged and collected. The mixture was washed with ultrapure water and dried to obtain 50-300 mg of ZIF-8@R-QDs.

[0036] (3) Preparation of Fe3O4-NH2: Fe3O4 magnetic nanoparticles were prepared by a solvothermal method: 0.1-10 g of FeCl3·6H2O was dissolved in 1-50 mL of ethylene glycol and ultrasonically dissolved. 0.1-10 g of NaAc and 0.01-5 g of polyethylene glycol were then added and ultrasonically dispersed. The mixture was stirred in a 10-80°C water bath for 10-60 min and then transferred to a polytetrafluoroethylene reactor for reaction at 150-250°C for 5-30 h. After the reaction, the product was washed several times with deionized water and anhydrous ethanol and dried under vacuum to obtain 200-500 mg of Fe3O4 nanoparticles.

[0037] Subsequently, 10-150 mg of Fe₃O₄ was dispersed in 1-20 mL of isopropanol, 20-500 μL of ultrapure water was added, and the mixture was sonicated for 10-60 minutes. Under mechanical stirring at 300-800 rpm, 0.1-20 mL of aqueous ammonia and 0.1-10 mL of LTEOS were added sequentially, and the mixture was allowed to react at 30-60°C for 1-10 hours. After the reaction, the product was separated using a magnet, washed with water and ethanol, and dried under vacuum at 20-80°C to yield 50-200 mg of Fe₃O₄@SiO₂.

[0038] Finally, amino modification was performed via silanization: 50-200 mg of Fe₃O₄@SiO₂ was dispersed in 1-10 mL of a 1:1 ethanol / water (v / v) mixture and sonicated for 10-50 minutes. Under nitrogen protection, 0.01-10 mL of APTES was slowly added and the reaction was allowed to proceed for 5-24 hours at 30-60°C. The product was separated using a magnet, washed several times with deionized water and ethanol, and dried under vacuum at 20-80°C to yield 20-150 mg of Fe₃O₄@SiO₂-NH₂ magnetic nanoparticles.

[0039] (4) Preparation of Fe3O4-FPBA: Dissolve 20-250 mg of 4-formylphenylboronic acid (FPBA) and 20-250 mg of NaBH3CN in 10-200 mL of ethanol. Then disperse 10-200 mg of Fe3O4@SiO2-NH2 into the above solution. Ultrasonicate for 10-60 min, stir for 2-24 h, and wash several times with ethanol and water to obtain 50-200 mg of Fe3O4-FPBA.

[0040] (5) Preparation of molecularly imprinted polymers / non-imprinted polymers (MIPs / NIPs): First, 0.1-40 mg of ZIF-8@R-QDs were dispersed in 1-20 mL of PBS. Then, 10-100 μL of H7N9 and 1-50 μL of L-APTES were added to the mixture and stirred at room temperature for 0.5-10 h. Subsequently, 20-200 μL of LTEOS and 10-200 μL of LTEA were added and stirred at room temperature for 1-15 h. Finally, the solid product was centrifuged, washed several times with 0.01-5% SDS, and finally washed with water and dried to obtain 5-20 mg of MIPs. NIPs were prepared without the addition of the template H7N9; all other procedures were the same as above.

[0041] The preparation of the universal sensor is just to change the template virus H7N9 to influenza virus H5N1 / H9N2, and the other operations are the same as above.

[0042] Example 2: Feasibility analysis of the universal visualized influenza virus fluorescent molecular imprinting sensor

[0043] Analysis

[0044] In order to verify the feasibility of the proposed scheme, we conducted a proof-of-principle test of the sensor by adding the target virus H7N9. Figure 2 As shown in (A), after incubation of MIPs with H7N9 virus and washing away unbound virus, Fe3O4-FPBA was added for binding. After magnetic separation, the fluorescence intensity of the supernatant decreased significantly. This is because the imprinted cavities on the MIPs' surfaces specifically recognize and capture the H7N9 virus. Fe3O4-FPBA, through boronate esterification, forms stable cyclic boronate bonds with cis-diol structures on the viral surface glycoprotein in a weakly alkaline environment, thereby constructing a sandwich structure. The formation of the sandwich structure and magnetic separation lead to a significant decrease in fluorescence intensity in the supernatant. The inset shows the sandwich structure redispersed in PBS buffer after separation and visualized under UV light. The virus-loaded MIPs exhibit distinct red fluorescence, visually confirming successful capture of the target virus. In contrast, since no template virus was added during the polymerization process, NIPs lack imprinted cavities for the target virus H7N9 and only bind the virus through a small amount of nonspecific adsorption. Consequently, their fluorescence intensity decreases only slightly, and no significant change is observed during visual inspection.

[0045] We then further verified that the decrease in fluorescence signal was not caused by direct quenching of quantum dots by viruses. Figure 2 As shown in (B), the fluorescence intensity of both MIPs and NIPs did not change significantly after virus binding. Therefore, the decrease in fluorescence intensity is mainly attributed to the formation of the sandwich structure and the magnetic separation process, rather than the direct quenching effect of the virus on the quantum dots.

[0046] Example 3: Performance, morphology and structural characterization of the universal visualized influenza virus fluorescent molecular imprinting sensor and its intermediates.

[0047] The structure and properties of nanomaterials were studied using a variety of characterization techniques. First, the morphology and size of the materials were studied using scanning electron microscopy (SEM) and dynamic light scattering (DLS), such as Figure 3 (A) The ZIF-8 particles are 200-250 nm in size and present a regular hexahedral structure. After the quantum dots are encapsulated in ZIF-8, the particle size is about 250 nm ( Figure 3 (B)) without changing its hexahedral structure. This is because the size of quantum dots is usually less than 5nm, so encapsulating quantum dots in it does not have a significant impact on the structure of ZIF-8. Figure 3 (B, D) are the MIPs and NIPs after imprinting, with a particle size of about 400 nm and an irregular spherical shape. This is because after the silicon imprinting on the surface of ZIF-8@R-QDs, the original regular hexahedral structure of ZIF-8 is gradually covered, causing its morphology to gradually transform into a spherical shape. In addition, by Figure 4 Dynamic light scattering further characterized the particle size of the above materials, and the results were consistent with SEM. The above results indicate that the various materials were successfully prepared.

[0048] exist Figure 5 In the infrared spectrum of (A), 2928 cm -1 The absorption peak at 1300 cm corresponds to the CH asymmetric stretching vibration of the -CH3 group in the 2-methylimidazole ligand and the stretching vibration of the CH bond on the imidazole ring. -1 The absorption peak at 2433 cm can be attributed to the stretching vibration of the CN bond in the imidazole ring, which proves the successful construction of the ZIF-8 framework; -1 The absorption peak at about 1045 cm is derived from the SH stretching vibration of mercaptopropionic acid (MPA) modified on the surface of the quantum dots, proving that the quantum dots are successfully coated by ZIF-8. -1 The absorption peak at is attributed to the asymmetric stretching vibration of Si-O-Si, proving the successful preparation of MIPs / NIPs.

[0049] exist Figure 5 (B) Middle, 584cm -1 The absorption peak at 799cm is the stretching vibration of Fe-O bond in Fe3O4, indicating that Fe3O4 was successfully prepared. -1 and 1090cm -1The absorption peaks at 2930 cm correspond to the symmetric stretching vibration of Si-O and the asymmetric stretching vibration of Si-O-Si, indicating that the Fe3O4@SiO2 core-shell structure was successfully formed. -1 The CH stretching vibration absorption peak at 1866 cm indicates that the amino group is successfully grafted onto the Fe3O4@SiO2 surface. -1 The absorption peak at corresponds to the C=O stretching vibration of the formyl group in FPBA, indicating that FPBA has been successfully grafted onto the Fe3O4@SiO2-NH2 surface.

[0050] Next, the crystal structures of ZIF-8 and ZIF-8@R-QDs composites were analyzed by X-ray diffraction (XRD). Figure 6 The XRD patterns of ZIF-8 were consistent with those reported in the literature. The ZIF-8@R-QDs composite also maintained the same diffraction peak positions as pure ZIF-8, albeit with a decrease in intensity. This indicates that the quantum dot encapsulation process did not alter the ZIF-8 crystal structure. The color change (from white to red) under UV irradiation further confirmed the successful encapsulation of the red fluorescent quantum dots (R-QDs) within the ZIF-8 framework.

[0051] like Figure 7 As shown in Figure 2, the specific surface area and pore structure of ZIF-8 and ZIF-8@R-QDs were characterized by nitrogen adsorption-desorption experiments. The specific surface area of ​​ZIF-8 is as high as 1218.96 m 2 / g, this high specific surface area provides an ideal carrier platform for efficient loading of quantum dots. The specific surface area of ​​ZIF-8@R-QDs is 771.85m 2 / g, which is slightly lower than that of ZIF-8, which proves that the quantum dots are successfully encapsulated in ZIF-8. In addition, the nitrogen adsorption results show that ZIF-8@R-QDs still maintain a large specific surface area, proving that the quantum dots are mainly encapsulated inside ZIF-8 rather than just adsorbed on the surface. The pore size distribution is determined by the Barrett-Joyner-Halenda (BJH) method. Compared with the original ZIF-8, the average pore size of ZIF-8@R-QDs is reduced from 5.21nm to 4.59nm, and the pore volume is reduced from 0.68cm 3 / g is reduced to 0.46cm 3 This decrease in pore size parameters can be attributed to the loading of quantum dots.

[0052] Finally, the prepared materials were characterized by Zeta potential. As shown in Figure (8), the potential of ZIF-8 is about +35mV (a), which is because the zinc ions exposed on the surface and the protonated ligands form a positive charge in the solution; after the red quantum dots are encapsulated in its pores, the potential is reduced to +21mV (b) due to the carboxyl groups on the surface of the quantum dots; the potentials of MIPs and NIPs obtained after imprinting on its surface are roughly the same, about +5mV (c, d), which is because after imprinting with a silanization reagent, a large number of hydroxyl groups are generated on the surface. The potential of Fe3O4@SiO2 is lowered. Due to the small amount of hydroxyl groups on its surface, its potential is -13mV (e). After coating with SiO2, the potential of Fe3O4@SiO2 is -34mV (f). After the surface of Fe3O4@SiO2 is modified with amino groups, the amino groups neutralize the negative charge on the surface, so the potential of Fe3O4@SiO2-NH2 is +30mV (g). Finally, after FPBA is grafted onto its surface, the potential of Fe3O4-FPBA is -7mV (h) because the negative charge groups of FPBA neutralize the positive charge on the material surface. The above potential changes further demonstrate the successful preparation of each material.

[0053] Example 4: Application of the universal visualized influenza virus fluorescent molecular imprinting sensor

[0054] The experimental conditions of this example are as follows: 0.01-10 mg of MIPs / NIPs particles are ultrasonically dispersed and dissolved in 0.1-10 mL of PBS solution, and then different concentrations of H7N9 virus are added and incubated for 0.5-6 h, followed by centrifugation and washing the MIPs / NIPs three times with PBS buffer to remove unbound H7N9; 0.1-10 mg of Fe3O4-FPBA particles are then ultrasonically dispersed in 0.1-20 mL of PBS solution and mixed with the virus-bound MIPs / NIPs and shaken for 10-60 min. After magnetic separation, the fluorescence intensity change of the supernatant at an excitation wavelength of 370 nm and an emission wavelength of 660 nm is measured (ΔF=Fi-F0, where F0 is the fluorescence intensity before the addition of virus and Fi is the fluorescence intensity after the addition of virus). The excitation and emission slit widths are both set to 5 nm and the voltage is 800 V; the complex after magnetic separation is washed once with PBS (pH 8.5) and resuspended in 0.1-10 mL of Visual semi-quantitative analysis was achieved by observing the changes in fluorescence intensity in PBS under UV light.

[0055] (1) Linearity and visualization analysis of a universal visualized influenza virus fluorescent molecular imprinting sensor

[0056] Under the above conditions, the prepared sensor was tested for different concentrations (2.9fM~174fM) of the target virus H7N9. Figure 9 As shown in (A), with the increase of H7N9 concentration, the fluorescence intensity of MIPs showed a regular decrease, while the visual color of the "MIPs-virus-Fe3O4-FPBA" sandwich structure obtained by magnetic separation became more and more obvious ( Figure 9 (B)), by Figure 9 (C) It can be seen that when the H7N9 concentration is in the range of 5.8fM to 116fM, a good linear relationship is shown, and its linear regression equation is ΔF = 7.96C H7N9 +51.58(R 2 =0.9963), where ΔF=F0-Fi, F0 and F i Refers to the fluorescence intensity of MIPs when H7N9 is not added and when H7N9 is added, C H7N9 is the concentration of the target virus H7N9. The limit of detection (LOD) calculated according to the formula LOD = 3σ / k is 2.2fM, where σ is the standard deviation of the blank sample signal and k is the slope of the standard curve. Figure 9 As shown in (D), NIPs lack specific imprinting cavities for H7N9 and only have a small amount of nonspecific adsorption such as electrostatic and hydrophobic interactions. Therefore, the fluorescence intensity of NIPs does not change significantly under different H7N9 concentrations.

[0057] (2) Selectivity and competitiveness analysis of the universal visualized influenza virus fluorescent molecular imprinting sensor

[0058] The sensor's selectivity, competitiveness, and anti-interference ability are key indicators for evaluating its potential application in real-world samples. Based on the morphological and size characteristics of H7N9 (spherical, 80-120 nm), this study selected hepatitis A virus (HAV) (icosahedral, approximately 27 nm), hepatitis B virus (icosahedral, approximately 42 nm), enterovirus EV71 (icosahedral, approximately 30 nm), and influenza viruses H5N1 and H9N2 (spherical, 80-120 nm) of different subtypes as interfering viruses to evaluate the sensor's selectivity and competitiveness against H7N9.

[0059] The results of the selective experiment are as follows Figure 10 As shown in (A), analysis of fluorescence intensity and IF values ​​demonstrates that the sensor exhibits high selectivity for H7N9 (IF = 6.1). The fluorescence intensities and imprinting factors for other viruses are significantly lower. This is because the MIPs' imprinted cavity precisely matches the shape, size, and surface functional group distribution of H7N9, while other viruses are unable to adapt to the imprinted cavity, resulting in a significant reduction in their binding capacity. This demonstrates the excellent selectivity of the prepared sensor for the template virus, H7N9.

[0060] To further evaluate the competitiveness of the sensor, this study introduced the above competitive viruses into the target virus H7N9 to explore their effects on the detection signal. Figure 10 As shown in (B), the sensor's fluorescence response signal did not change significantly. This is because the competing virus does not match the imprinted cavity and cannot be specifically recognized and captured. Therefore, the sensor's fluorescence signal primarily originates from the binding of the target virus H7N9, and the presence of the competing virus does not significantly interfere with the detection results. In summary, this sensor exhibits good selectivity and competitiveness for the target virus H7N9 and has potential application value in real-world sample detection.

[0061] (3) Anti-interference ability of the universal visualized influenza virus fluorescent molecular imprinting sensor

[0062] In order to investigate the feasibility of the prepared sensor for detecting the target virus H7N9 in actual serum samples. Since the serum samples are complex and contain a variety of ions and biomolecules, these substances may interfere with the detection of the sensor. Figure 11 As shown, we added Na into the sensor based on the composition and concentration of real serum samples. + , K + , Mg 2 + , Ca 2+ , HCO 3- ,HPO4 2- , glucose, L-proline, and L-alanine were tested as interfering ions. The results showed that in a simulated serum environment containing these common interfering substances, the sensor's detection of the target virus H7N9 was not significantly interfered with. This result demonstrates that the prepared sensor has excellent anti-interference capabilities.

[0063] (4) Reproducibility and stability analysis of the universal visualized influenza virus fluorescent molecular imprinting sensor

[0064] At the same time, five batches of MIPs synthesized at different times were prepared and used to detect the same concentration of H7N9 virus to evaluate the reproducibility of the sensor. Figure 12 As shown in (A), the ΔF values ​​of the five batches of MIPs when detecting H7N9 were basically consistent, indicating that the sensor has good reproducibility.

[0065] The prepared sensors were stored for 0 weeks, 1 week, 2 weeks, 3 weeks and 4 weeks, and then the target virus H7N9 was tested to evaluate their temporal stability. Figure 12(B) Results show that the sensor's ability to identify H7N9 has a slight downward trend over time. However, after 4 weeks, its detection performance still remains at 91.0% of its initial value, demonstrating the sensor's excellent temporal stability.

[0066] (5) Analysis of the application of the universal visualized influenza virus fluorescent molecular imprinting sensor in actual detection

[0067] The prepared MIPs were tested in 100-fold diluted human serum samples for three replicates to detect H7N9 in order to evaluate the actual analytical capability of the sensor. Figure 13 The prepared sensor had a spike recovery rate of 92.5% to 106.1% for H7N9 virus, indicating that the sensor has good detection accuracy and good practical application potential.

[0068] In addition, this work also continues to explore the shortest time for rapid visual detection of the sensor, such as Figure 14 As shown, using 1 mg / mL MIPs to detect 116 fM H7N9 virus, only 10 minutes of incubation plus 10 minutes of Fe₃O₄-FPBA binding time are required to achieve visual detection under UV light. This "10+10" minute rapid detection model is highly advantageous for on-site, point-of-care testing and demonstrates significant application value in infectious disease prevention and control, public health emergency response, and other fields, particularly for rapid virus screening in resource-limited settings.

[0069] (6) Verification of the universality of the universal visualized influenza virus fluorescent molecular imprinting sensor

[0070] To verify the universal application capability of the constructed molecular imprinting sensor, the present invention replaced the template virus H7N9 with H5N1 and H9N2 by replacing the template molecule, keeping the other synthesis steps unchanged, and evaluated the sensor's detection ability for the new target virus. Figure 15 As shown in (A) and 16(A), with the increase of H5N1 and H9N2 concentrations, the fluorescence intensity in the supernatant of H5N1-MIPs and H9N2-MIPs decreased regularly (the excitation and emission slit widths were both set to 5 nm, and the voltage was 700 V). The "MIPs-virus-Fe3O4-FPBA" sandwich structure obtained by magnetic separation gradually changed from colorless to red under ultraviolet light, and the color became more and more obvious ( Figure 15 (B), 16(B)). Figure 15 (C) When the H5N1 concentration is in the range of 29fM to 174fM, a good linear relationship is shown, and the linear regression equation is ΔF = 3.2987C H5N1 +102.85(R 2=0.97367), and according to the formula LOD=3σ / k, LOD=6.8fM, and the imprinting factor is 3.6. Figure 16 (C) H9N2 showed a good linear relationship in the range of 29 to 145 fM, and the linear regression equation was ΔF = 3.9031C H9N2 +56.92(R 2 =0.99778), according to the formula LOD = 3σ / k, LOD = 6.0fM, and the imprinting factor is 4.2. Figure 15 (D) and 16(D), since NIPs do not have H5N1 and H9N2 imprinting cavities and only a small amount of nonspecific adsorption occurs, there is no obvious change in fluorescence intensity at different H5N1 and H9N2 virus concentrations.

[0071] The detection performance demonstrated by the sensor developed in this study during universal validation was based on optimal detection conditions for the H7N9 virus, and no systematic optimization of conditions was performed for H5N1 and H9N2 viruses. However, due to significant differences in surface charge distribution, hemagglutinin (HA) protein conformation, and receptor binding properties among other influenza virus subtypes, such as H5N1 and H9N2, the preparation conditions suitable for H7N9 (including functional monomer ratios, polymerization time, and elution parameters) may not be fully compatible with other subtypes. Although experimental data confirm that the sensor has certain universal detection potential for different influenza virus subtypes (such as H5N1 and H9N2), further improvements in imprinting factors and selectivity require systematic optimization of conditions for different viruses.

Claims

1. A universal, visual influenza virus fluorescent molecular imprinting sensor. This sensor combines molecularly imprinted nanoparticles with magnetic nanoparticles modified with phenylboronic acid in the presence of the target virus, H7N9, to form a sandwich structure. This structure allows for the separation and enrichment of the target virus using a magnet, while simultaneously causing changes in the quantum dot fluorescence signal for visual detection. The same detection results can be achieved by replacing the target virus with other influenza virus subtypes.

2. A universal visualized influenza virus fluorescent molecular imprinting sensor according to claim 1, characterized in that: The preparation process includes the following steps: (1) Zeolite imidazolate framework (ZIF-8) and red fluorescent quantum dots (R-QDs) were synthesized, and the red quantum dots were coated on the zeolite imidazolate framework (ZIF-8) as the imprinting medium and the fluorescence signal output source; (2) Using zeolite imidazolate framework coated with red quantum dots (ZIF-8@R-QDs) as a carrier, H7N9 as a template, 3-aminopropyltriethoxysilane (APTES) as a functional monomer, and tetraethoxysilane (TEOS) as a cross-linker, the synthesis was carried out by a sol-gel method under the catalysis of triethylamine (TEA). After the synthesis was completed and the template was washed off, molecularly imprinted polymers (MIPs) with H7N9 imprinted cavities were obtained; (3) Fe3O4 magnetic nanoparticles were synthesized, and a silicon layer was coated on their surface and amino groups were grafted using silanization reagents tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES). Then, 4-formylphenylboronic acid (FPBA) was grafted onto the magnetic nanoparticles using a Schiff base reaction to obtain Fe3O4-FPBA as a capture element.

3. A universal visualized influenza virus fluorescent molecular imprinting sensor, characterized in that: The prepared MIPs particles were ultrasonically dispersed and dissolved in a PBS solution of a certain concentration. H7N9 virus of different concentrations was added to the solution and incubated for 0.5-10 hours, followed by centrifugation and washing with PBS buffer to remove unbound H7N9. Fe3O4-FPBA particles were then ultrasonically dispersed in a PBS solution and mixed with the above-mentioned virus-bound MIPs and shaken for 10-60 minutes. After magnetic separation, the fluorescence intensity change of the supernatant was measured by fluorescence spectroscopy for quantitative analysis. The magnetically separated complex was washed once with PBS solution and resuspended in 0.1-10 mL PBS. The change in fluorescence intensity was observed by the naked eye under ultraviolet light to achieve visual semi-quantitative analysis. The particle dosage used for the sensor to detect the virus was 1 mg / mL, and the shortest incubation time required for visualization was 10 minutes.

4. A universal visualized influenza virus fluorescent molecular imprinting sensor, characterized in that: The molecularly imprinted sensor has dual detection modes: semi-quantitative analysis visualized by the naked eye under UV light and highly sensitive quantitative analysis combined with fluorescence spectroscopy. The sensor also demonstrates excellent universal detection performance for other influenza virus subtypes, potentially solving the challenge of on-site detection during peak influenza seasons.

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