Universal visual virus molecular imprinting sensor modified with anti-protein coating

Abstract

The invention constructs a universal visual virus molecularly imprinted sensor modified with an anti-protein coating, the sensor uses magnetic Fe3O4 as an imprinting carrier, HBV as a template and dopamine as a functional monomer for imprinting to obtain a molecularly imprinted polymer with a polydopamine coating (PDA), and before the template is removed, the molecular imprinting polymer with the polydopamine coating (PDA) is separated from the template, so that the molecular imprinting polymer with the polydopamine coating (PDA) is obtained. The surface of the PDA layer is covered with a slightly cross-linked PMEO2MA layer through a water precipitation polymerization method, and the polymer can effectively reduce non-specific protein adsorption; and finally, exposing an imprinting cavity of the PDA layer by eluting the template molecules. On the other hand, potassium permanganate (KMnO4) is used as a signal output molecule, and when no target object exists, the strong reducibility of PDA can enable KMnO4 entering the imprinting cavity to be subjected to a color fading reaction. Based on the principle, high-specificity colorimetric visual detection on the HBV is realized. The sensor can be combined with a smart phone through naked eyes to carry out visual semi-quantitative analysis and carry out quantitative detection through ultraviolet rays, and the universality of the sensor can be confirmed through a template replacement experiment. And meanwhile, the sensor shows an excellent cost advantage. The diagnosis which is high in sensitivity, low in cost and capable of achieving rapid visualization shows huge potential in practical application, and particularly has important application value in infectious disease screening and monitoring work in areas with limited resources.

Description

Technical Field

[0001] A method has been developed that belongs to the field of analytical chemistry detection technology, specifically involving the construction and research of a universal visual virus molecular imprinting sensor modified with an anti-protein coating. Background Art

[0002] Infectious diseases remain a major concern for global public health and economics. Viral diseases such as food poisoning, sepsis, pneumonia, and tuberculosis spread globally, posing a serious threat to human health. Currently, there are many methods such as enzyme-linked immunosorbent assay (ELISA) [Shang P, Xu L, Cheng T.Serological and molecular detection of citrus tristeza virus: a review [J]. Microorganisms, 2024, 12 (8): 1539.], quartz crystal microbalance [Lim HJ, Saha T, Tey BT, et al. Quartz crystal microbalance-based biosensing of hepatitis B antigen using amolecularly imprinted polydopamine film [J]. Talanta, 2022, 249: 123659.], virus-specific IgM antibodies [Nagar PK, Savargaonkar D, Anvikar A R. Detection of dengue virus-specific IgM and IgG antibodies through peptide sequences of envelope and NS1 proteins for serological identification [J]. Journal of Immunology Research, 2020, 2020(1):1820325.] and polymerase chain reaction [Fakruddin M, Mannan KSB, ChowdhuryA, et al. Nucleic acid amplification: alternative methods of polymerase chain reaction[J]. Journal of Pharmacy and Bioallied Sciences, 2013, 5(4):245-252.] and other traditional detection methods have been widely used in virus diagnosis, but these detection methods are cumbersome, time-consuming and costly, which is not conducive to rapid detection of infection [Hassanpour S, Baradaran B, de la Guardia M, et al.Diagnosis of hepatitis via nanomaterial-based electrochemical, optical or piezoelectric biosensors: a review on recent advancements[J].Microchimica Acta, 2018, 185:1-24.]. Rapid and accurate detection of pathogens is very important for epidemic control. Therefore, the development of an affordable, sensitive, portable and rapidly visual virus detection technology is of great significance.

[0003] In recent years, polydopamine, as a new type of molecularly imprinted polymer, has attracted increasing research attention. It can be synthesized in one step through the self-polymerization reaction of dopamine under weak alkaline conditions, without the participation of exogenous initiators or cross-linkers [Yin ZZ, Cheng SW, Xu LB, et al. Highly sensitive and selective sensor for sunset yellow based on molecularly imprinted polydopamine-coated multi-walled carbon nanotubes [J]. Biosensors and Bioelectronics, 2018, 100: 565-570.], [Lu CH, Zhang Y, Tang SF, et al. Sensing HIV related protein using epitope imprinted hydrophilic polymer coated quartz crystal microbalance [J]. Biosensors and Bioelectronics, 2012, 31 (1): 439-444.]. The structure of polydopamine contains many functional groups (such as amino groups, hydroxyl groups and π-π bonds), which is conducive to the formation of high-affinity imprinted cavities during the imprinting process of viruses, but it is easy to cause nonspecific adsorption problems in the non-imprinted cavity area. In order to reduce nonspecific adsorption, researchers usually modify the surface of the imprinted layer such as polyethylene glycol or introduce zwitterionic polymers. For example, Luan et al. [Luan J, Liu KK, Tadepalli S, et al. PEGylated artificial antibodies: plasmonic biosensors with improved selectivity [J]. ACS Applied Materials & Interfaces, 2016, 8 (36): 23509-23516.] PEGylated the non-imprinted cavity area of ​​the imprinted polymer to minimize nonspecific binding and significantly enhance the selectivity of the molecular imprinted polymer for the target biomolecule. The results showed that the nonspecific binding was more than 10 times lower than that of the non-PEGylated counterpart.

[0004] Based on these promising studies, a universal, visual, and protein-resistant viral molecular imprinting sensor was constructed. This sensor utilizes magnetic Fe₃O₄ as the imprinting carrier, HBV as the template, and dopamine as the functional monomer for imprinting, resulting in a molecularly imprinted polymer (MIP) with a polydopamine (PDA) coating. Prior to template removal, a slightly cross-linked PMEO₂MA layer was coated on the PDA surface via water precipitation polymerization. This polymer effectively reduces nonspecific protein adsorption. Finally, the template molecules were eluted to expose the imprinted cavities in the PDA layer. Potassium permanganate (KMnO₄) was used as the signal output molecule. In the absence of target, the strong reducing property of PDA causes the KMnO₄ that has entered the imprinted cavities to fade. Based on this principle, highly specific colorimetric visual detection of HBV was achieved. The sensor can be used for visual semi-quantitative analysis using both the naked eye and a smartphone, as well as for quantitative detection using UV light. Its universality was confirmed by template replacement experiments. Furthermore, the sensor exhibits excellent cost advantages. This highly sensitive, low-cost, and rapidly visualized diagnosis shows great potential in practical applications, especially in areas with limited resources for infectious disease screening and monitoring. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the purpose of the invention is to construct a universal visual virus molecular imprinting sensor modified with an anti-protein coating, providing a portable detection tool for the specific, visual and rapid detection of viruses, and achieving the purpose of simple operation and rapid visual detection of viruses.

[0006] Technical solution: A universal visual virus molecular imprinting sensor modified with an anti-protein coating was constructed and studied, characterized in that the method includes the following preparation steps:

[0007] (1) Construction of the magnetic carrier and imprinting layer: Synthesis of Fe3O4 magnetic nanoparticles: Using a solvothermal method, iron salts are used as precursors. A high-temperature reaction in the presence of a reducing agent and a surfactant yields Fe3O4 nanoparticles of uniform size. This carrier exhibits superparamagnetism, facilitating subsequent separation and manipulation.

[0008] (2) Formation of polydopamine (PDA) imprinting layer: Because the self-polymerization reaction conditions of dopamine are mild (room temperature, weak alkalinity), it avoids the damage of high temperature or organic solvents to the virus template. At the same time, the multifunctional characteristics of PDA enhance the interaction with the virus and improve the affinity of the imprinting site, which is superior to the single action mode of traditional monomers (such as acrylic acid). Fe3O4 is dispersed in a weak alkaline buffer, dopamine and template virus (such as HBV) are added, and a PDA coating is formed on the surface of the magnetic particles through the self-polymerization reaction of dopamine. PDA is rich in functional groups such as amino groups and phenolic hydroxyl groups, and can specifically bind to the virus surface through hydrogen bonds, electrostatic interactions, etc. to form an imprinting cavity.

[0009] (3) Introduction of PMEO2MA layer: Traditional molecular imprinting polymers (MIPs) often suffer from a decrease in selectivity due to nonspecific adsorption on the surface. A method was constructed to directly introduce the PMEO2MA layer after imprinting. Through its hydrophilicity and steric hindrance effect, nonspecific binding was significantly reduced, solving the bottleneck problem of MIPs in actual sample detection. Vinyl monomers were grafted onto the surface of Fe3O4@PDA@MIPs without elution of the template through metal chelation, and then the aqueous precipitation polymerization of PMEO2MA was initiated to form a cross-linked PMEO2MA anti-protein coating. PMEO2MA is a nonlinear polyethylene glycol (PEG) analogue with excellent hydrophilicity and anti-protein adsorption ability.

[0010] (4) Template elution and cavity exposure: The template virus is eluted under mild acidic conditions to retain the imprinted cavity. At the same time, the PMEO2MA layer covers nonspecific sites to reduce background interference. The virus template is eluted under low temperature acidic conditions to avoid damage to the PDA structure by strong acid or organic solvents, ensuring the integrity of the imprinted cavity.

[0011] (5) Template replacement strategy: By replacing the template virus (such as H7N9, H9N2), sensors targeting different pathogens can be quickly prepared, reflecting versatility.

[0012] (6) Colorimetric detection principle: Utilize the strong reducing property of PDA and use KMnO4 as the signal molecule. When there is no target virus, KMnO4 enters the imprint cavity and is reduced and faded by PDA; when there is a target virus, it occupies the cavity and blocks the entry of KMnO4, and the degree of fading is reduced. Semi-quantitative detection is achieved by naked eye observation or smartphone RGB analysis, and precise quantification is achieved by ultraviolet spectrophotometry. Traditional detection requires fluorescent labeling or enzyme-catalyzed amplification, but the chemical reducing property of PDA is directly utilized to construct a method that does not require complex labeling steps, reduces costs and avoids the risk of inactivation of biological molecules. At the same time, it combines naked eye visualization (suitable for on-site screening) and instrument quantification (high precision in the laboratory) to meet the needs of different scenarios.

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

[0014] (1) Existing technologies mostly use silica gel or polymer microspheres as carriers, which require complex modification steps; this paper directly utilizes the magnetic properties of Fe3O4 and the adhesive properties of PDA to simplify the preparation process. Traditional functional monomers (such as methacrylic acid) rely on a single interaction, while the multifunctional group design of PDA improves the imprinting efficiency;

[0015] (2) Existing MIPs often undergo post-modification of the surface (e.g., PEGylation) to reduce nonspecific adsorption, but the steps are cumbersome and the coverage is low. We constructed a method that simultaneously introduces a PMEO2MA layer during the imprinting process to achieve uniform coverage and better results.

[0016] (3) Existing colorimetric sensors mostly rely on gold nanoparticles or enzyme-catalyzed reactions, which are costly and unstable. A PDA-KMnO4 reaction was constructed, which is stable, low-cost, and does not require enzyme assistance. The principle is to use potassium permanganate (KMnO4) as a signal output molecule. In the absence of the target, the strong reducing property of PDA can cause the KMnO4 that enters the imprinted cavity to undergo a fading reaction. Based on this principle, highly specific colorimetric visual detection of HBV is achieved. The sensor can be combined with the naked eye and a smartphone for visual semi-quantitative analysis and quantitative detection by ultraviolet light, and its versatility can be confirmed by template replacement experiments. At the same time, the sensor shows excellent cost advantages;

[0017] (4) Versatility: Traditional sensors usually target a single virus and need to be redesigned and synthesized; the constructed one can adapt to multiple viruses through template replacement, significantly expanding the scope of application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] [ Figure 1 ] Schematic diagram of the preparation principle of the universal visual virus molecular imprinting sensor with modified anti-protein coating.

[0019] [ Figure 2 ](A) UV-visible absorption spectra of MIPs-PMEO2MA / NIPs-PMEO2MA before and after binding to viruses; (B) Reduction performance of different materials on KMnO4 (the inset shows the corresponding color changes); (C) Visualization of KMnO4 reduction by Fe3O4@PDA at different concentrations.

[0020] [ Figure 3]Scanning electron microscopy (SEM) images of (A)Fe3O4, (B)Fe3O4@PDA, (C)Fe3O4@PDA@C=C, (G)MIPs-PMEO2MA, (H)NIPs-PMEO2MA (8000 times magnification); Scanning electron microscopy (SEM) images of (D)Fe3O4, (E)Fe3O4@PDA, (F)Fe3O4@PDA@C=C, (I)MIPs-PMEO2MA, (J)NIPs-PMEO2MA (3000 times magnification).

[0021] [ Figure 4 ]Dynamic light scattering (DLS) of (A)Fe3O4, (B)Fe3O4@PDA, (C)Fe3O4@PDA@C=C, (D)MIPs-PMEO2MA, and (E)NIPs-PMEO2MA.

[0022] [ Figure 5 ]Infrared spectra of Fe3O4(a), Fe3O4@PDA(b), Fe3O4@PDA@C=C(c), MIPs-PMEO2MA(d), and NIPs-PMEO2MA(e).

[0023] [ Figure 6 ]Water contact angles of (A)Fe3O4; (B)Fe3O4@PDA; (C)Fe3O4@PDA@C=C; (D)MIPs-PMEO2MA; (E)NIPs-PMEO2MA.

[0024] [ Figure 7 ]Zeta potential diagrams of Fe3O4(a), Fe3O4@PDA(b), Fe3O4@PDA@C=C(c), MIPs-PMEO2MA(d), and NIPs-PMEO2MA(e).

[0025] [ Figure 8 ](A) Linearity of MIPs-PMEO2MA in detecting different concentrations of HBV (0, 4, 8, 16, 25, 42, 62.5, 83, 100, 125pM); (B) Linear fitting of MIPs-PMEO2MA; (C) Linear fitting of G / G0 and the logarithm of HBV concentration (4, 8, 16, 25, 42, 62.5, 83, 100, 125pM); (D) Visualization of different concentrations of HBV (0, 4, 8, 16, 25, 42, 62.5, 83, 100, 125pM) (the upper part is 1.25mM KMnO4 concentration, the lower part is 0.5mM KMnO4 concentration); (E) Linearity of NIPs-PMEO2MA in detecting different concentrations of HBV (0, 4, 8, 16, 25, 42, 62.5, 83, 100, 125pM).

[0026] [ Figure 9 ](A) Selectivity of the sensor for the virus HBV; (B) Competitiveness of the sensor for the virus HBV.

[0027] [ Figure 10 ]The interference of the sensor.

[0028] [ Figure 11 ](A) Reproducibility of the sensor; (B) Temporal stability of the sensor.

[0029] [ Figure 12 ]Visualization results of different incubation times of MIPs-PMEO2MA with HBV virus and its reaction with KMnO4 (colors recognized by smartphones are shown below).

[0030] [ Figure 13 ](A) Linearity of H7N9-MIPs-PMEO2MA in detecting different concentrations of H7N9 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM); (B) Linear fitting of H7N9-MIPs-PMEO2MA; (C) Linear fitting of G / G0 and the logarithm of H7N9 concentration (58, 116, 174, 232, 290, 348, 406, 464, 522 fM); (D) Visualization of different concentrations of H7N9 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM) (0.5 mM KMnO4 concentration); (E) Linearity of NIPs-PMEO2MA in detecting different concentrations of H7N9 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM).

[0031] [ Figure 14 ](A) Linearity of H9N2-MIPs-PMEO2MA in detecting different concentrations of H9N2 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM); (B) Linear fitting of H9N2-MIPs-PMEO2MA; (C) Linear fitting of G / G0 and the logarithm of H9N2 concentration (58, 116, 174, 232, 290, 348, 406, 464, 522 fM); (D) Visualization of different concentrations of H9N2 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM) (0.5 mM KMnO4 concentration); (E) Linearity of NIPs-PMEO2MA in detecting different concentrations of H9N2 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM).

[0032] [ Figure 15 ](A) Linearity of H9N2-MIPs-PMEO2MA in detecting different concentrations of H9N2 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM); (B) Linear fitting of H9N2-MIPs-PMEO2MA; (C) Linear fitting of G / G0 and the logarithm of H9N2 concentration (58, 116, 174, 232, 290, 348, 406, 464, 522 fM); (D) Visualization of different concentrations of H9N2 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM) (0.5 mM KMnO4 concentration); (E) Linearity of NIPs-PMEO2MA in detecting different concentrations of H9N2 (0, 58, 116, 174, 232, 290, 348, 406, 464, 522 fM). Specific implementation plan

[0033] 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 for illustration purposes only and do not limit the scope of application and expansion of the present invention.

[0034] Example 1: Preparation of a universal visual virus molecular imprinting sensor modified with an anti-protein coating

[0035] (1) Preparation of Fe3O4 magnetic nanoparticles by solvothermal method: FeCl3·6H2O was dissolved in ethylene glycol and ultrasonicated until completely dissolved. 1-5 g NaAc and 0.5-2 g polyethylene glycol were then added for ultrasonic dispersion. The mixture was stirred in a 40-80°C water bath for 30 min and then transferred to a polytetrafluoroethylene reactor for reaction at 180-220°C for 20-26 h. After the reaction, the product was washed several times with deionized water and anhydrous ethanol and then dried in vacuo to obtain Fe3O4 nanoparticles.

[0036] (2) Preparation of Fe3O4@PDA@MIPs and Fe3O4@PDA@NIPs: Fe3O4 nanoparticles were dispersed in Tris buffer (pH 7-9). 10-20 mg of DA and 40-60 μL of HBV were added, and the DA self-polymerization reaction was carried out at 20-30°C for 6-10 h. After the reaction, the imprinted polymer was quickly separated from the solution under the action of a magnet, and then the excess monomers and free HBV were removed with deionized water to obtain Fe3O4@PDA@MIPs without template removal. The preparation of Fe3O4@PDA@NIPs did not add the template virus HBV, and the other operations were consistent with the above.

[0037] (3) Modification of PMEO2MA layer: First, the coordination monomer was synthesized. A mixture of GMA (10-15 mmol), IDA (10-15 mmol) and NaOH (20-30 mmol) was stirred with 40-60 mL of distilled water at 40-60 °C for 1-3 h. Then, the synthesized monomer (denoted as GMA-IDA-2Na + ) solution was stored at 1-10°C for later use. + The solution (~26.3 μmol) was adjusted to pH 7.0 and then mixed with an equal molar amount of CuSO4·5H2O in 2-8 mL of deionized water for 1-5 h to obtain Cu 2+ Chelating monomer (denoted as GMA-IDA-Cu 2+ ).

[0038] Imprinted nanoparticles, without template removal, are incubated in this solution for 1-3 hours to introduce polymerizable vinyl groups. Next, the nanoparticles, washed with water, are redispersed in 20-30 mL of phosphate buffer (pH 7-8, 8-12 mM) containing MEO2MA (150-200 μL) and MBA (2-8 mg). After degassing with nitrogen for 10-50 minutes, SDS solution (1-3%, 350-400 μL) and APS solution (8-12%, 500-550 μL) are sequentially injected, and polymerization is continued at 35-40°C for 10-15 hours.

[0039] After polymerization, EDTA-2Na + Solution (0.1-0.3M) was used to wash the nanoparticles grafted with cross-linked PMEO2MA layer to desorb Cu 2+ The imprinted polymer was then washed with a mixture of acetic acid (1-5%, v / v) and SDS (1-5%, w / v) under ice-cold conditions to remove the embedded template. The remaining SDS and acetic acid were removed with water and a 0.2-0.8 M sodium chloride solution, and the remaining sodium chloride was removed with ultrapure water. The mixture was then vacuum-dried to obtain the PMEO2MA-modified imprinted polymer MIPs-PMEO2MA (20-25 mg) and the non-imprinted polymer NIPs-PMEO2MA (20-25 mg).

[0040] The preparation of the universal sensor only changes the template virus HBV to the influenza virus H7N9 / H9N2, and the other operations are the same as above.

[0041] (4) Colorimetric and rapid visual detection: 1-2 mg / mL of MIPs-PMEO2MA / NIPs-PMEO2MA was dispersed in PBS buffer at pH 7-8 and 8-12 mM, and different concentrations of HBV virus were added and shaken at 35-40°C for 40-60 min. After the reaction, the supernatant was separated by magnet and removed and washed three times with PBS buffer to remove unbound virus particles. The nanoparticles after magnetic separation were resuspended in 1-5 mL of ultrapure water, 300-500 μL was transferred to a centrifuge tube, and 300-500 μL of KMnO4 (1-1.5 mM) was added and shaken for 5-10 min. Subsequently, magnetic separation was performed again, and the color change of the supernatant was observed with the naked eye. At the same time, the color information (RGB value) of the supernatant was collected using a smartphone, ensuring that the sample was kept at a distance of 10 cm from the camera and a shooting angle of 45°. The RGB data was extracted and recorded using image analysis software. Finally, the absorbance was measured using UV-visible absorption spectroscopy, and the absorbance difference under the same conditions before and after the addition of the virus was recorded, that is, ΔAbs = Absi - Abs0 (Abs0 is the absorbance at 525 nm before the addition of the virus, and Absi is the absorbance at 525 nm after the addition of the virus).

[0042] Example 2: Feasibility of the universal visualized virus molecular imprinting sensor with modified anti-protein coating

[0043] Sexual Analysis

[0044] The prepared MIPs-PMEO2MA and NIPs-PMEO2MA were added with the same concentration of HBV virus to verify the feasibility of the sensor, and the changes in their absorbance values ​​were analyzed by ultraviolet-visible spectroscopy (UV-Vis). Figure 2As shown in A, the absorbance of MIPs-PMEO2MA with virus added is significantly higher than that of MIPs-PMEO2MA without virus added, while the absorbance of NIPs-PMEO2MA before and after the addition of HBV virus does not change significantly. This is because after the preparation of MIPs-PMEO2MA and the elution of the template virus, a polydopamine imprinted cavity with a specific recognition site is exposed. When HBV (10μL) virus is added for incubation, HBV specifically binds to the imprinted cavity, thereby blocking the channel for KMnO4 to enter the cavity and inhibiting the reduction reaction of KMnO4 by PDA, resulting in a significant increase in ultraviolet absorbance. MIPs-PMEO2MA without HBV exposed more imprinted cavities with a polydopamine coating, which can fully reduce KMnO4, so its absorbance is significantly reduced. However, NIPs-PMEO2MA, which lacks an HBV template during its preparation, lacks an imprinted cavity on its surface that matches the shape and size of HBV. Consequently, there is no significant change in absorbance before and after the addition of HBV. Furthermore, since the non-imprinted polymer lacks an imprinted cavity that exposes PDA, its ability to reduce KMnO4 is weaker, resulting in a consistently higher absorbance than MIPs-PMEO2MA.

[0045] Experimental results demonstrated that MIPs-PMEO2MA efficiently recognized HBV through its specific imprinted cavity, exhibiting significant changes in UV absorbance based on the reducing properties of PDA and the fading reaction of KMnO4. In contrast, NIPs-PMEO2MA exhibited a less pronounced response to HBV due to its lack of specific recognition sites. This result validated the feasibility and specificity of MIPs-PMEO2MA as a sensor for HBV detection and provided experimental evidence for its application in viral detection.

[0046] Next, the imprinted carrier Fe3O4, Fe3O4 coated with a polydopamine layer (Fe3O4@PDA), Fe3O4 with double bonds grafted on the polydopamine surface (Fe3O4@PDA@C=C), molecularly imprinted polymer MIPs-PMEO2MA, non-imprinted polymer NIPs-PMEO2MA, and a material in which PMEO2MA was directly grafted on the Fe3O4 surface without a polydopamine layer (Fe3O4@PMEO2MA) were reacted with KMnO4 (1mM) for five minutes and then magnetically separated (400μg of the above materials + 400μL KMnO4). The supernatant was then subjected to UV detection. The results are shown in Figure 2. Figure 2As shown in Figure B, Fe₃O₄ and Fe₃O₄@PMEO₂MA have no reducing effect on KMnO₄ at certain concentrations. After the reaction, the solution remains purple-red, and the absorbance does not change significantly. This indicates that the Fe₃O₄ and PMEO₂MA layers are incapable of reducing KMnO₄ under these experimental conditions. However, Fe₃O₄@PDA exhibits strong reducing activity for KMnO₄. After the reaction, the solution color changes from purple-red to light pink, and the absorbance of KMnO₄ decreases dramatically. This is because the PDA layer is rich in active functional groups such as catechol and quinone groups, enabling efficient reduction of KMnO₄. However, after double bonds are grafted onto the Fe₃O₄@PDA surface, its reducing performance decreases slightly, and its absorbance increases compared to Fe₃O₄@PDA. This is likely because the introduction of double bonds partially covers the active sites of PDA, reducing its reducing ability. The absorbance of MIPs-PMEO2MA was significantly higher than that of Fe3O4@PDA@C=C, but it still exhibited a KMnO4 reduction effect. This is due to the presence of exposed imprinted cavities on the MIPs-PMEO2MA surface, where the exposed PDA reacted with KMnO4, causing it to discolor. While NIPs-PMEO2MA theoretically cannot reduce KMnO4, the PMEO2MA layer grafted onto the polydopamine surface by the non-imprinted polymer cannot completely cover the PDA, resulting in a small number of active sites remaining. Consequently, its absorbance was slightly lower than that of the KMnO4 solution. These experimental results demonstrate that the PDA layer is crucial for reducing KMnO4.

[0047] To further verify the visualization effect of the detection system, the present invention demonstrates the visual changes of KMnO4 (0.1 mM) reduced by Fe3O4@PDA at different concentrations ( Figure 2 C). The results show that with the increase of Fe3O4@PDA concentration, the KMnO4 solution exhibits a clear color gradient from purple to pink and finally colorless. This intuitive color change confirms that the detection scheme has good visual detection potential.

[0048] Example 3: Performance, morphology and structural characterization of the universal visualized virus molecular imprinting sensor modified with the protein-resistant coating and its intermediates.

[0049] In order to verify whether the synthesized polymer is successfully prepared, the present invention uses Fourier transform infrared spectroscopy (FI-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and potentiometric particle size analyzer to characterize the properties, structure and morphology of the prepared Fe3O4, Fe3O4@PDA, Fe3O4@PDA@C=C, MIPs-PMEO2MA and NIPs-PMEO2MA.

[0050] In the infrared spectrum ( Figure 5 ) in, 590cm -1The absorption peak at 1100cm is attributed to the characteristic vibration peak of Fe-O bond in Fe3O4, indicating that Fe3O4 nanomaterials were successfully prepared. -1 The absorption peak at 1463 cm reflects the presence of CO bonds or CN bonds in polydopamine, indicating that polydopamine is successfully imprinted on the Fe3O4 surface. -1 The peak at may be related to the bending vibration of NH bond, which proves that the present invention can be used to synthesize GMA-IDA-Cu 2+ The chelating monomer successfully introduced polymerizable vinyl groups into the PDA coating. -1 The absorption peak at is attributed to the stretching vibration of the ester group (C=O), indicating that the PMEO2MA layer is successfully grafted onto the material surface.

[0051] In the figure ( Figure 6 ), the hydrophilicity and hydrophobicity of Fe3O4, Fe3O4@PDA, Fe3O4@PDA@C=C, MIPs-PMEO2MA and NIPs-PMEO2MA were investigated by contact angle meter, such as Figure 6 The Fe3O4 imprinted support exhibited strong hydrophilicity, with a contact angle of 13°. Even after self-polymerization on the Fe3O4 surface to form a polydopamine imprinted coating, the Fe3O4@PDA remained highly hydrophilic. This is because the PDA structure is rich in hydrophilic functional groups such as catechol (-OH) and amino (-NH2). After the Fe3O4@PDA surface was modified with vinyl groups (C=C), the water contact angle of the Fe3O4@PDA@C=C increased to 92°, indicating hydrophobicity. This change demonstrates the successful synthesis of the Fe3O4@PDA@C=C. After PMEO2MA was grafted onto the Fe3O4@PDA@C=C surface, the water contact angles of the MIPs-PMEO2MA and NIPs-PMEO2MA increased to 38° and 41°, respectively. This is because PMEO2MA is a hydrophilic polymer, and since both utilize the same functional monomer and crosslinker, the contact angles of the two materials are similar. The above results show that molecularly imprinted polymers MIPs-PMEO2MA and NIPs-PMEO2MA were successfully constructed.

[0052] Next, the present invention also uses Zeta potential to characterize the above materials, as shown in Figure ( Figure 7 Since the surface of Fe3O4 has a small amount of hydroxyl groups, its potential is about -11mV ( Figure 7 a); after the polydopamine layer was imprinted on the surface, the potential dropped to -27mV ( Figure 7 b), this is because the PDA surface is rich in functional groups such as amino and hydroxyl groups, which are further ionized in the solution. After the double bond is grafted onto the PDA imprinting layer, the potential is further reduced to about -37mV ( Figure 7c). The potentials of MIPs-PMEO2MA and NIPs-PMEO2MA are roughly equal, about -33mV ( Figure 7 d, e). This is due to the introduction of polyethylene glycol segments into the PMEO2MA layer, which exhibit hydrophilicity in solution. Simultaneously, the ionization of some functional groups increases the surface negative charge. These changes in zeta potential demonstrate the successful synthesis of the PDA-imprinted coating, double-bond grafting, and PMEO2MA layer.

[0053] In addition, scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used to characterize the morphology and size of the material. Figure 3 ), as shown in Figures A and D, the Fe3O4 particle size is about 150nm, with typical rough texture characteristics and a regular spherical structure. After Fe3O4 self-polymerizes on its surface through dopamine to form a thin, surface-adherent PDA imprinting coating, the particle size is slightly increased (150-250nm) and the surface roughness is significantly reduced ( Figure 3 B, E). After double bonds were grafted onto the surface of Fe3O4@PDA, the material maintained a spherical morphology and the particle size distribution did not change significantly ( Figure 3 C, F). After the cross-linked PMEO2MA layer was introduced on the surface, the morphology still showed a spherical shape. MIPs-PMEO2MA ( Figure 3-3 G, I) and NIPs-PMEO2MA ( Figure 3-3 H, J) The particle size is between 200-300 nm, among which NIPs-PMEO2MA is smoother than MIPs-PMEO2MA, which may be closely related to the imprinted cavity formed after the template molecule is eluted. In addition, further DLS particle size distribution shows that ( Figure 4 The particle size of each particle is consistent with that shown in SEM. The above results indicate that each particle was successfully synthesized.

[0054] Example 4: Application of the universal visualized virus molecular imprinting sensor with modified anti-protein coating

[0055] The experimental conditions of this embodiment are as follows: 1-2 mg / mL of MIPs-PMEO2MA / NIPs-PMEO2MA is dispersed in PBS buffer of pH 7-8 and 8-12 mM, and different concentrations of HBV virus are added and shaken at 35-40 ° C for 40-60 min. After the reaction is completed, the supernatant is removed by magnetic separation and washed three times with PBS buffer to remove unbound viral particles. The nanoparticles after magnetic separation are resuspended in 1-2 mL of ultrapure water, 300-500 μL are transferred to a centrifuge tube, and 300-500 μL of KMnO4 (1-1.5 mM) are added and the reaction is shaken for 5-10 min. Subsequently, magnetic separation is performed again, and the color change of the supernatant is observed with the naked eye. At the same time, the color information (RGB value) of the supernatant is collected using a smart phone to ensure that the sample is kept at a distance of 10 cm from the camera and a shooting angle of 45 °. The RGB data is extracted and recorded by image analysis software. Finally, the absorbance was measured using UV-visible absorption spectroscopy, and the absorbance difference under the same conditions before and after the addition of the virus was recorded, that is, ΔAbs = Absi - Abs0 (Abs0 is the absorbance at 525 nm before the addition of the virus, and Absi is the absorbance at 525 nm after the addition of the virus).

[0056] (1) Linear and visual analysis of the universal visual virus molecular imprinting sensor modified with the protein-resistant coating

[0057] Under the above optimized conditions, the detection effect of the sensor on the target virus HBV in the concentration range of 4pM to 125pM was investigated. Figure 8 As shown in A, with the increase of HBV concentration, the UV absorbance of MIPs-PMEO2MA gradually increased. Figure 8 As shown in Figure 2, the absorbance at 525 nm exhibited good linearity with HBV concentration within the range of 16 to 83 pM. The linear regression equation was Abs = 0.0026CHBV + 0.66303 (R² = 0.9930), where Abs is the UV absorbance of the target HBV virus by MIPs-PMEO2MA and CHBV is the target HBV concentration. The limit of detection (LOD) was calculated to be 1.9 pM using the formula 3σ / k, where σ is the standard deviation of the blank sample signal and k is the slope of the standard curve.

[0058] Figure 8 The visualization graph above D is the corresponding visualization graph under linear conditions (1-1.5mM KMnO4). As the HBV concentration increases, its color gradually changes from light purple to dark purple. In order to achieve a more obvious visualization effect, the present invention also performs visualization detection under low concentration KMnO4. Figure 8As can be seen below D, under the condition of 0.5mM KMnO4, as the virus concentration increases, the color gradually changes from light pink to light purple, providing a more significant color change, suitable for rapid visual detection. Therefore, in practical applications, the appropriate KMnO4 concentration can be selected according to the detection requirements: in quantitative analysis requiring high sensitivity and accuracy, a 1.25mM KMnO4 concentration is used, which can provide a larger UV absorbance difference, thereby improving the sensitivity and accuracy of quantitative analysis; in application scenarios requiring rapid visual detection and semi-quantitative analysis, a 0.5mM KMnO4 concentration is used to fully utilize its significant color change and excellent visualization effect. This differentiated concentration strategy can leverage the advantages of different KMnO4 concentrations and improve the flexibility and practicality of detection.

[0059] When users are not sensitive to color changes, the present invention can also use smart phones to achieve more sensitive visual quantitative detection, such as Figure 15 C. The present invention uses a smartphone color recognition program to collect the three primary color light intensity parameters (R: red, G: green, B: blue) of a sample (0.5mM KMnO4). Within the range of 8 to 83pM, G / G0 and the logarithm of HBV concentration show a good linear relationship. The linear regression equation is Y = 1.1937-0.23505X (R2 = 0.996), where Y is the ratio of G to G0, G is the green light intensity parameter of the sample collected by the smartphone, G0 is the green light intensity parameter collected without the addition of the target virus, and X is the logarithm of the HBV concentration. These results demonstrate the successful construction of a new HBV detection method based on a smartphone sensing platform that combines portability, visualization, and high sensitivity.

[0060] like Figure 8 E. Since there is no specific imprinting cavity for HBV on the surface of NIPs-PMEO2MA, there is only a small amount of nonspecific adsorption, so its absorbance value has no significant change.

[0061] (2) Selectivity and competitiveness analysis of the universal visualized virus molecular imprinting sensor with modified anti-protein coating

[0062] To evaluate the sensor's selectivity and competitiveness against HBV, we selected different pathogens with similar morphology and size to the HBV (icosahedral, approximately 42 nm in diameter) as interfering viruses for analysis. Specifically, the interfering viruses selected were hepatitis A virus (HAV), influenza viruses (H5N1, H7N9, and H9N2), and enterovirus 71 (EV71).

[0063] The results are as follows Figure 9As shown in A, the ΔAbs and IF values ​​caused by the binding of HBV virus to MIPs-PMEO2MA are the largest, indicating that the developed sensor exhibits high selectivity for HBV (IF=12.7). This is because there is only a specific imprinted cavity for HBV on the surface of MIPs-PMEO2MA, while other viruses differ from HBV in morphology and size. For example, although HAV is also icosahedral, its size is 27-32nm, while H5N1 and H7N9 are spherical structures with a particle size of 80-120nm. Due to the differences in their surface functional groups, shapes and sizes, other viruses cannot adapt to the imprinted cavity of HBV, so the sensor exhibits excellent selectivity. Next, the above-mentioned non-target virus was also used for competitive testing. As shown Figure 9 As shown in Figure B, the competitive virus has little effect on the detection of the target virus HBV because its shape and size cannot be recognized by the imprinted cavity. This indicates that the prepared sensor has excellent competitiveness against the target virus HBV.

[0064] (3) Analysis of the anti-interference ability and stability of the universal visual virus molecular imprinting sensor modified with the anti-protein coating

[0065] In order to investigate the detection and analysis capabilities of the prepared sensor in serum samples, the present invention conducted an anti-interference performance test on various substances that may exist in serum. Figure 10 As shown, interfering ions (Na + , K + , Mg 2+ , Ca 2+ ,HCO3 - ,HPO4 2- , glucose, L-proline, L-alanine) were tested and the results were as follows Figure 10 As shown in the figure, the sensor was not significantly interfered with the detection of the target virus HBV. This result shows that the prepared sensor has excellent anti-interference ability.

[0066] In order to evaluate the temporal stability of the constructed sensor, the present invention tested HBV after storing the same batch of synthesized sensors for 0, 1, 2, 3, and 4 weeks. Figure 11 As shown in Figure B, as the storage time goes by, the ΔAbs for detecting the same concentration of HBV decreases slightly, but after 4 weeks, the detection performance of the sensor can still be maintained at 90.1% of the initial performance, indicating that the sensor has good time stability.

[0067] (4) Reproducibility of the universal visualized virus molecular imprinting sensor modified with the protein-resistant coating

[0068] To further evaluate the reproducibility of the sensor, five batches of the same sensor were prepared and used to detect the target virus at the same concentration. The results are as follows: Figure 11 As shown in A, the ΔAbs values ​​obtained from 5 different batches of sensors are basically consistent, which shows that the sensor has excellent reproducibility.

[0069] (5) Analysis of the application of the universal visual virus molecular imprinting sensor modified with the anti-protein coating in actual detection

[0070] To investigate the actual analytical performance of the sensor, 1 mL of MIPs-PMEO2MA at a concentration of 1-2 mg / mL was placed in a 100-fold diluted human serum sample and three parallel tests were performed for HBV. Figure 12 As shown in the figure, the recovery rate of HBV detection by the prepared sensor ranged from 94.11% to 110.1%. This indicates that the constructed sensor can still accurately measure HBV content in complex biological sample detection scenarios, indicating that the sensor has good practical application potential.

[0071] In addition, this work also explores the rapid detection capability of the sensor. Figure 13 As shown, when using 1.5mg / mL MIPs-PMEO2MA to detect 83pM HBV virus, a color change can be observed with the naked eye after only 10 minutes of incubation combined with a 5-minute KMnO4 color development reaction. Notably, even more pronounced color changes can be observed via smartphone analysis. This "10+5" minute rapid detection mode is highly advantageous for on-site, immediate testing. Furthermore, the sensor's combination of visual visualization and smartphone detection technology provides an efficient and convenient solution for on-site virus screening, demonstrating promising application prospects in virus prevention and control.

[0072] (6) Verification and analysis of the universality of the universal visual virus molecular imprinting sensor modified with the anti-protein coating

[0073] To verify the universal application capability of the constructed molecular imprinting sensor, this study replaced the template molecule, the template virus HBV with influenza viruses H7N9 and H9N2, and kept the other synthesis steps unchanged, and evaluated the sensor's ability to detect new target viruses. Figure 14 As shown in A and 15A, with the increase of H7N9 and H9N2 concentrations, the UV absorbance of H7N9-MIPs-PMEO2MA and H9N2-MIPs-PMEO2MA gradually increased, and the visual color gradually changed from light yellow to purple ( Figure 14 B, 15B). Figure 14C, H7N9 showed a good linear relationship with the absorbance at 525nm in the range of 116-406fM, the linear regression equation was Abs=4.9383CH7N9+0.60972(R2=0.99798), LOD=58fM, and the imprinting factor for H7N9 was 6.7. Figure 14 D. Analysis by smartphone software showed a good linear relationship between G / G0 and the logarithm of H7N9 concentration in the range of 116 to 464 fM, and the linear regression equation was Y = 1.39338-0.19338X (R2 = 0.98241), LOD = 116 fM. Figure 15 C, H9N2 showed a good linear relationship with the absorbance at 525nm in the range of 58 to 464fM, and the linear regression equation was Abs = 3.5482CH9N2 + 0.69394 (R2 = 0.99613), LOD = 58fM, and the imprinting factor for H9N2 was 7.6. The analysis of the smartphone software showed that G / G0 and the logarithm of the H9N2 concentration showed a good linear relationship in the range of 116 to 406fM of the target concentration, and the linear regression equation was Y = 1.65235-0.32277X (R2 = 0.98189), LOD = 116fM ( Figure 15 D). Figure 14 E, 15E, Since NIPs-PMEO2MA has no imprinting cavity for H7N9 and H9N2, there is only a small amount of nonspecific adsorption, so the absorbance values ​​do not change significantly at different H7N9 and H9N2 virus concentrations.

[0074] The detection performance demonstrated in this sensor's universal validation was based on optimal detection conditions for HBV, and no systematic optimization of conditions was performed for influenza viruses H7N9 and H9N2. However, due to differences in size (80-120nm), surface charge, and molecular conformation between influenza viruses and HBV (42nm), the preparation conditions suitable for HBV may not be fully applicable to other viruses. This work only validates the sensor's significant universal detection performance, but further optimization is required to achieve higher imprinting factors and more satisfactory selectivity.

Claims

1. A universal visual virus molecular imprinting sensor modified with an anti-protein coating, characterized in that: include: Using magnetic Fe3O4 as the imprinting carrier and viral molecules (HBV, H7N9 or H9N2) as the template, and dopamine (DA) as the functional monomer, a polydopamine (PDA) imprinting coating is formed on the Fe3O4 surface through self-polymerization. Before removing the template, a slightly cross-linked PMEO2MA layer was covered on the surface of the PDA coating by water precipitation polymerization. The template molecules were eluted to expose the specific imprinted cavities of the PDA layer. Potassium permanganate (KMnO4) was used as the signal output molecule, and the reducing property of PDA was utilized to achieve visual detection of the target virus. The sensor can be semi-quantitatively detected by visual observation of color changes or smartphone RGB analysis, or quantitatively detected by measuring the absorbance change (ΔAbs) at 525nm using a UV-visible spectrophotometer.

2. The universal visual virus molecular imprinting sensor according to claim 1, characterized in that: The method for introducing the PMEO2MA layer comprises the following steps: Synthesis of GMA-IDA-Cu 2+ Chelating monomers; imprinted nanoparticles without template removal were mixed with GMA-IDA-Cu 2+ Incubate to introduce polymerizable vinyl groups; in the presence of MEO2MA and crosslinker MBA, a PMEO2MA layer is formed on the surface of the imprinted nanoparticles by water precipitation polymerization; EDTA-2Na + Solution washing to desorb Cu 2+ , and then the template molecules are eluted to obtain the imprinted polymer modified with the PMEO2MA layer.

3. The universal visual virus molecular imprinting sensor according to claim 1 or 2, characterized in that: The detection method comprises the following steps: MIPs-PMEO2MA was dispersed in PBS buffer, the target virus was added, and the mixture was incubated with shaking. After magnetic separation, the mixture was washed and resuspended in ultrapure water. KMnO4 solution was added, and after shaking, the color change of the supernatant was observed or the absorbance change was measured. Semi-quantitative or quantitative detection of the target virus was achieved through color change or ΔAbs value.

4. The universal visual virus molecular imprinting sensor according to claim 1, characterized in that: The sensor has specific recognition capabilities for HBV, H7N9 and H9N2 viruses, and universal detection is achieved by replacing the template virus.

5. The universal visual virus molecular imprinting sensor according to claim 1, characterized in that: The PMEO2MA layer can significantly reduce nonspecific protein adsorption and improve the specificity of imprinting factors.

6. A universal visual virus molecular imprinting sensor integrated with any one of claims 1 to 5, capable of simultaneously semi-quantitatively detecting two viruses and achieving highly sensitive and specific quantitative detection through fluorescence spectroscopy.

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