Molecularly imprinted colorimetric sensor for detecting hepatitis B virus as well as preparation method and application of molecularly imprinted colorimetric sensor

Abstract

The invention discloses a molecular imprinting colorimetric sensor for detecting hepatitis B virus as well as a preparation method and application of the molecular imprinting colorimetric sensor. The sensor takes an aminated zeolite imidazate framework material (ZIF-8-NH2) with peroxidase-like activity as a catalytic core, after surface double-bond functionalization, a molecularly imprinted polymer (MIPs) layer is constructed on the surface through a free radical polymerization reaction by taking the aminated zeolite imidazate framework material as a carrier and taking HBV as a template molecule. According to the sensor, after HBV is specifically recognized and captured through MIPs, the catalytic efficiency of ZIF-8 on hydrogen peroxide is changed, then the generation amount of hydroxyl radicals is regulated and controlled, and finally visual and quantitative detection of HBV is achieved through the color fading reaction of the hydroxyl radicals and chromogenic dye crystal violet. The sensor prepared by the invention has the advantages of high sensitivity, strong specificity, low cost, simplicity and convenience in operation, rapidness in detection and the like, and can be used for rapid screening and on-site instant detection of hepatitis B viruses.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosensing and analytical detection, and particularly relates to a molecular imprinting colorimetric sensor for detecting hepatitis B virus, a preparation method and application thereof, and more particularly relates to a colorimetric sensor based on metal-organic framework nanoszyme and molecular imprinting technology, which can rapidly and visually detect hepatitis B virus. BACKGROUND

[0002] Viral infection is a major challenge to global public health. Hepatitis B virus (HBV) is a serious blood-borne pathogen that can cause chronic hepatitis, cirrhosis and liver cancer, posing a continuous threat to global public health. Therefore, developing rapid, sensitive and specific HBV detection methods is crucial for epidemic prevention and control.

[0003] Currently, a variety of detection methods have been developed for diagnosing viral infections in humans, including cell culture-based virus isolation, immunofluorescence detection, nucleic acid amplification techniques (such as PCR), and enzyme-linked immunosorbent assay (ELISA). However, these methods have problems such as time-consuming, expensive and complex instruments, and the need for professional operation, which makes it difficult to meet the needs of large-scale centralized detection and may increase the risk of viral retransmission. Therefore, in order to overcome the limitations of traditional detection methods, developing a low-cost, high-sensitivity, rapid and visual detection of virus biosensor has become an important direction of current research. At present, many different nanomaterials have been reported for biosensing, such as electrochemical sensors, piezoelectric sensors and optical sensors. These sensors improve the sensitivity and selectivity of the detection process, meeting the needs of accuracy, speed, portability and low cost. Among them, colorimetric method in optical biosensor has been widely used in the detection field based on target-induced color change due to its fast response speed, cost-effectiveness, simple operation and no need for various instruments.

[0004] Nanomaterials with peroxidase-like activity are widely used in the construction of biosensors due to their tunable catalytic activity and multifunctionality. MOF-based nanoszymes have attracted extensive attention due to their unique chemical composition and structure, high stability and easy preparation, which provide abundant catalytic sites and endow MOFs with inherent enzyme-mimic properties. ZIF-8 is a MOFs material of zeolitic imidazolate framework, which not only provides more active sites, but also promotes mass transfer in catalytic reactions, and its surface rich in metal ions makes it have superior nanoszyme catalytic activity, which is widely used as the core material of colorimetric biosensors for high-sensitivity target analysis. In order to further improve the selectivity of the sensor, molecularly imprinted polymers can be used to replace traditional biological recognition elements. Molecularly imprinted polymers are known as "artificial antibodies", which form imprinted cavities that match the template molecules in functional groups and are complementary in shape and size, and have the ability to specifically recognize targets, thereby improving the selectivity and detection performance of the sensor.

[0005] However, the combination of high-catalytic-activity ZIF-8 nanoszymes and high-selectivity MIPs technology for direct and rapid visual detection of viruses is still relatively rare. Therefore, it is of important practical application value to develop a colorimetric sensor integrating the catalytic activity of ZIF-8 nanoszymes and the specific recognition ability of MIPs to realize rapid, portable and high-sensitivity detection of HBV. SUMMARY

[0006] The present application aims to overcome the deficiencies in the prior art and provide a molecularly imprinted colorimetric sensor for hepatitis B virus, which has the advantages of fast detection speed, high sensitivity, good selectivity, low cost and visual detection.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a molecularly imprinted colorimetric sensor for detecting hepatitis B virus (HBV), characterized in that the sensor takes metal-organic framework material ZIF-8 with peroxidase-like activity as a catalytic core and an imprinted carrier, and a molecularly imprinted polymer MIPs layer with HBV as a template molecule is constructed on the surface thereof; after the sensor specifically recognizes and captures HBV through MIPs, the catalytic efficiency of ZIF-8 on hydrogen peroxide (H2O2) is changed, thereby regulating the amount of hydroxyl radicals (·OH) generated, and finally realizing the visual and quantitative detection of HBV through the fading reaction of hydroxyl radicals and color developing dye crystal violet (CV).

[0008] Preferably, the ZIF-8 is ZIF-8-NH2 modified by amino functionalization, and further grafted with C=C double bonds on the surface thereof through a silanization reaction to obtain ZIF-8@C=C as a carrier for molecular imprinting.

[0009] Preferably, the molecular imprinting polymer MIPs layer is formed by radical polymerization reaction with ZIF-8@C=C as carrier, HBV as template molecule, 4-vinylpyridine (4-VP) and acrylamide (AM) as functional monomers, and ethylene glycol dimethacrylate (EGDMA) as crosslinking agent.

[0010] In a second aspect, the present application provides a method for preparing the sensor, comprising the following steps: (1) Synthesis of ZIF-8-NH2: reacting zinc salt, 2-methylimidazole and 2-aminobenzimidazole in alkaline conditions in a water / methanol mixed solvent to obtain ZIF-8-NH2; (2) Synthesis of ZIF-8@C=C: dispersing ZIF-8-NH2 in anhydrous toluene, adding vinyltriethoxysilane for silanization reaction to graft double bonds on the surface of ZIF-8-NH2 to obtain ZIF-8@C=C; (3) Preparation of molecular imprinting polymer MIPs: dispersing ZIF-8@C=C in a solvent, sequentially adding HBV template molecule, functional monomers 4-vinylpyridine and acrylamide for pre-assembly, then adding crosslinking agent ethylene glycol dimethacrylate and initiator 2,2'-azobisisobutyronitrile (AIBN), and performing thermal initiation polymerization reaction under inert atmosphere; after the reaction is completed, the template molecule is eluted and removed to obtain MIPs.

[0011] Preferably, the molar amount ratio of the functional monomers 4-vinylpyridine, acrylamide and ethylene glycol dimethacrylate in step (3) is (1-3):(2-4):(1-3), preferably 2:3:2; Preferably, the mass molar ratio of ZIF-8@C=C4-VP and 4-vinylpyridine is 30:0.05-0.3, further preferably 30:0.1-0.2, and more preferably 30:0.2, in units of mg / mmol.

[0012] The mass molar ratio of ZIF-8@C=C and HBV template molecule is 30:0.17-0.5, further preferably 30:0.33-0.5, and more preferably 30:0.42, in units of mg / pmol.

[0013] Preferably, in step (3), the temperature of the polymerization reaction is 45-75°C, further preferably 55-65°C, the polymerization reaction time is 6-12 hours, further preferably 8-10h, and more preferably 10h; the eluent is a mixed solution of alcohol and acetonitrile or a 0.5% SDS solution, the alcohol is methanol or ethanol, preferably ethanol, and the volume ratio of the alcohol and acetonitrile is 4:1.

[0014] In a third aspect, the present application provides the use of the above-mentioned sensor or the sensor prepared by the above-mentioned method in detecting hepatitis B virus.

[0015] In a fourth aspect, the present application provides a method for detecting hepatitis B virus using the above-mentioned sensor or the sensor prepared by the above-mentioned method, characterized in that the method comprises the following steps: (1) mixing and incubating the sensor MIPs with a sample solution to be detected, so that the MIPs specifically capture HBV; (2) adding hydrogen peroxide and crystal violet solution to the incubated system to perform a color development reaction; (3) observing the color change of the solution by naked eye for semi-quantitative analysis, or using a spectrometer to detect the absorbance value of the solution near 580 nm for quantitative analysis; the absorbance value is positively correlated with the concentration of HBV.

[0016] Preferably, in step (1), the incubation is performed in phosphate buffer (PB), water, phosphate buffered saline (PBS), and Tris-HCl buffer, and is further preferably phosphate buffered saline with a pH of 6-8, and is further preferably 7.4. Preferably, in step (1), the incubation temperature is 25-45℃, and is further preferably 30-37℃, the incubation time is 10-90 min, and is further preferably 30-40 min, and is more further preferably 40 min; the concentration of the sensor MIPs is 0.2-1 mg·mL-1, and is further preferably 0.2-0.4 mg·mL-1, and is more further preferably 0.4 mg·mL-1. Preferably, in step (2), the mass fraction of hydrogen peroxide is 15%-30%, and is further preferably 20%. Preferably, in step (2), the concentration of crystal violet (CV) is 0.5-1.25 mM, and is further preferably 0.75-1 mM, and is more further preferably 0.75 mM. Preferably, in step (2), the color development reaction time is 1-7 min, and is further preferably 3-5 min, and is more further preferably 5 min.

[0017] Preferably, in step (3), a smart phone is used to collect the color image of the solution after the reaction, and the color channel value (RGB value) of the image is extracted, a linear relationship between the R / B value and the concentration of HBV is established, and portable quantitative detection based on a smart phone is realized.

[0018] In a fifth aspect, the present application provides the use of the above-mentioned sensor in preparing a detection kit for detecting hepatitis B virus, wherein the kit comprises the above-mentioned molecular imprinting colorimetric sensor, hydrogen peroxide solution, and crystal violet solution.

[0019] The present application has the advantages of: High sensitivity and selectivity: The present application combines the high catalytic activity of ZIF-8 nanoscale enzyme with the specific recognition ability of molecularly imprinted polymers (MIPs). The "imprinted cavity" provided by MIPs can accurately recognize HBV, effectively avoiding the interference of other substances in complex samples, and has good selectivity. The detection limit is as low as pM level, and the sensitivity is high.

[0020] Fast visual detection: Based on the principle of catalytic fading, the detection result can be directly judged by the naked eye through the color change of the solution from colorless to purple, and the whole detection process can be completed within 1 hour, realizing fast and visual detection.

[0021] Low cost and simple operation: The sensor preparation material cost is low, the detection process does not require large and expensive instruments, the operation steps are simple, non-professionals can operate after simple training, and it is very suitable for on-site rapid screening and application in resource-limited areas.

[0022] Portability and intelligence: The method can be combined with a smartphone platform, and quantitative analysis can be performed by taking photos and analyzing RGB values, greatly enhancing the portability of the detection and the accuracy of the results, providing a powerful tool for point-of-care testing (POCT).

[0023] Good stability: The prepared MIPs sensor has good reproducibility and time stability, and can maintain more than 87% of the initial detection performance after 6 weeks of storage at room temperature, which is convenient for storage and transportation. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The figure is a schematic diagram of the construction principle and process of the sensor; Figure 2 The figure is a water contact angle test result graph of ZIF-8-NH2 (A), ZIF-8@C=C (B), MIPs (C) and NIPs (D) in Example 1; Figure 3 The figure is an infrared spectrum (A) and Zeta potential graph (B) of ZIF-8-NH2 (a), ZIF-8@C=C (b), MIPs (c) and NIPs (d) in Example 1; Figure 4 The figure is a scanning electron microscope (SEM) graph of ZIF-8-NH2 (A), MIPs (B) and NIPs (C) in Example 1; Figure 5 The figure is a dynamic light scattering DLS graph of ZIF-8-NH2 (A), ZIF-8@C=C (B), MIPs (C) and NIPs (D) in Example 1; Figure 6 The XRD patterns of ZIF-8-NH2 (A), MIPs, and NIPs (B) in Example 1 are shown below. Figure 7 The N2 adsorption-desorption isotherms of ZIF-8-NH2 (A) and ZIF-8@C=C (B) in Example 1, and the pore size distribution of ZIF-8-NH2 (C) and ZIF-8@C=C (D); Figure 8 A shows the UV spectra of HBV detected by MIPs and NIPs. In the inset, a, b, c, and d are the visualization results of MIPs, MIPs+HBV, NIPs, and NIPs+HBV, respectively. Figure 8 B represents the UV absorbance of different substances with crystal violet. In the illustration, a, b, c, d, e, and f are the visualization results of ZIF-8+CV, ZIF-8@C=C+CV, H2O2+CV, ZIF-8@C=C+H2O2+CV, ZIF-8+H2O2+CV, and CV, respectively. Figure 9 The following are the UV-Vis absorption spectra (A for MIPs, C for NIPs), linear fitting curves (B), and visualization results (D) of the sensor used in Example 1 for detecting different concentrations of HBV virus. Figure 10 The graph shows the test results of the selectivity (A) and competitiveness analysis (B) of the sensor in Application Example 1; Figure 11 The results of the anti-interference capability analysis of the sensor in Application Example 1; Figure 12 The results of the investigation into the reproducibility (A) and time stability (8) of the sensor in Example 1 are presented; Figure 13 Colorimetric images of MIPs / NIPs after adding different concentrations of HBV, captured by a smartphone (A), and the linear relationship between R / B and HBV concentration in the colorimetric images (B). Figure 14 Visualization results of MIPs incubated with HBV for different times (A) and images of different groups captured by smartphones (B). Figure 15 Imprinting factor results for sensors prepared under different imprinting times (A), imprinting temperatures (B), template amounts (C), and elution solvents (D); Figure 16 The results of HBV detection under different incubation temperatures (A), incubation solvents (B), incubation pH (C), and MIPs / NIPs dosages (D); Figure 17Imprinting factor (A) and visualization results (B) for different H2O2 mass fractions in HBV detection; Figure 18 Imprinting factors (A) and visualization results (B) for different concentrations of crystal violet in HBV detection; Figure 19 Results of MIPs / NIPs and virus incubation at different times in HBV detection; Figure 20 The results are for HBV detection after adding crystal violet and reacting for different times. Detailed Implementation

[0025] ZIF-8 is a metal-organic framework (MOF) material with highly efficient oxidase-like activity; therefore, this invention uses ZIF-8 as the imprinted carrier for a sensor. During the synthesis of ZIF-8, amino functional groups were introduced for -NH2 modification. These -NH2 groups can undergo amidation reactions with the -COOH groups abundant on the viral surface, effectively immobilizing the covalent bonds between the virus and the imprinted carrier during the imprinting process. This covalent bond facilitates the formation of more viral recognition sites during polymerization, thereby improving the sensor's specific recognition capability. Subsequently, functional monomers AM and 4-VP, crosslinking agent EGDMA, and initiator AIBN were added. The virus was crosslinked and embedded into the polymer network layer through the interaction between the monomers and the virus to prepare molecularly imprinted polymers (MIPs). The prepared MIPs layer contains imprinted cavities matching the template virus HBV. The ZIF-8 exposed in the cavities can catalyze the generation of hydroxyl radicals (·OH) from H2O2. Crystal violet dye was then introduced as a signal output molecule. The reaction of ·OH with crystal violet destroys its molecular structure, ultimately causing it to fade. As HBV concentration increases, the number of MIPs that specifically bind to HBV and block the blot cavity increases, preventing the reaction between ZIF-8 and H2O2, thereby inhibiting the formation of ·OH and reducing the fading of crystal violet. Ultimately, the solution exhibits a visual change from colorless to purple, achieving semi-quantitative detection. Furthermore, quantitative analysis can be achieved by detecting absorbance using UV-Vis absorption spectroscopy. Simultaneously, the MIP solution is packaged in centrifuge tubes for easy portability and on-site detection of the target virus HBV, providing a practical solution for rapid and portable virus detection. Its construction principle diagram is shown below. Figure 1 .

[0026] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0027] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0028] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0029] Information on the main reagent sources in the following examples: Zinc nitrate hexahydrate (Zn(NO3)2·6H2O), 99% purity, purchased from Xilong Chemical Co., Ltd.; 2-methylimidazole (2-MEIM), 98% purity, purchased from Shanghai Aladdin Chemical Reagent Co., Ltd.; triethylamine (TEA), ≥99.0% purity, purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; 2-aminobenzimidazole (2-ABzlm), 99.83% purity, purchased from Bide Pharmaceutical Technology Co., Ltd.; anhydrous methanol, ≥99.5% purity, purchased from Hunan Huihong Reagent Co., Ltd.; ethanol, ≥99.7% purity, purchased from Hunan Huihong Reagent Co., Ltd.; acetonitrile, ≥99.5% purity, purchased from Hunan Huihong Reagent Co., Ltd.; toluene, ≥99.5% purity, purchased from Hunan Huihong Reagent Co., Ltd.; vinyltriethoxysilane, 97% purity, purchased from [unclear - possibly a company name]. Aladdin Chemical Reagent Co., Ltd.; N,N-dimethylformamide (DMF), 99.8% purity, purchased from Hunan Huihong Reagent Co., Ltd.; Azobisisobutyronitrile (AIBN), 98% purity, purchased from Shanghai Aladdin Chemical Reagent Co., Ltd.; 4-vinylpyridine (4-VP), 95% purity, purchased from Shanghai Saen Chemical Technology Co., Ltd.; Hydrogen peroxide, 30% purity, purchased from Tianjin Damao Chemical Reagent Factory; Acrylamide (AM), ≥99.0% purity, purchased from Tianjin Kemeio Chemical Reagent Co., Ltd.; Ethylene glycol dimethacrylate (EGDMA), 98% purity, purchased from Bid Pharmaceutical Technology Co., Ltd.; Crystal violet (CV), 90% purity, purchased from Shanghai Anaiji Chemical Co., Ltd.; Hepatitis B virus (HBV), lyophilized vaccine, purchased from Beijing Minhai Biotechnology Co., Ltd.

[0030] Example 1 This embodiment provides a method for preparing a molecularly imprinted colorimetric sensor for detecting hepatitis B virus, including the following steps: (1) Synthesis of ZIF-8-NH2 In a glass bottle, (0.1 mL, 0.7 mmol) TEA was added to an aqueous solution of (0.8 mL, 0.67 mmol) Zn(NO3)2·6H2O. While stirring at 25 °C and 700 rpm / min, (396.5 mg, 4.83 mmol) 2-MEIM dissolved in 4 mL H2O and (275.6 mg, 2.07 mmol) 2-ABzlm dissolved in 2 mL methanol were added. Finally, 17.1 mL of methanol was added to bring the total solvent volume to 24 mL. After stirring for 30 min, the precipitate was collected by centrifugation at 8000 rpm / min, washed repeatedly with H2O, and dried under vacuum at 60 °C to obtain 180 mg ZIF-8-NH2.

[0031] (2) Synthesis of ZIF-8@C=C 500 mg of ZIF-8-NH2 was added to a dry three-necked flask, followed by 20 mL of anhydrous toluene. The mixture was ultrasonically dispersed until homogeneous, and then 1 mL of vinyltriethoxysilane was added. After ultrasonic dispersion for 2 min, the flask was sealed and transferred to an oil bath at 70 °C with stirring for 24 h. After the reaction was complete, the precipitate was collected by centrifugation at 10,000 rpm / min, washed three times alternately with toluene and ethanol, and dried under vacuum at 60 °C to obtain 426 mg of ZIF-8@C=C.

[0032] (3) Preparation of molecularly imprinted polymers / non-molecularly imprinted polymers Weigh 30 mg ZIF-8@C=C and dissolve it in 18 mL of DMF solution. After ultrasonic dispersion, stir at 700 rpm / min in a water bath at 37 ℃. Add 0.42 pmol of hepatitis B virus (HBV) and react for 30 min to allow HBV to fully bind with the imprinted carrier. Then add (0.2 mmol, 20 μL) 4-vinylpyridine (4-VP) and 1 mL of DMF solution containing (0.3 mmol, 14 mg) acrylamide. After pre-assembly at 37 ℃ for 1.5 h, add (0.2 mmol, 40 μL) EGDMA and deoxygenate under argon for 30 min. Then add 1 mL of DMF solution containing 20 mg AIBN and continue polymerization under argon protection at 65 ℃ for 10 h. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm / min. It was eluted four times with an ethanol:acetonitrile (v / v, 4:1) eluent, and then washed twice with ultrapure water. Each time, the precipitate was shaken for 30 min in a water bath constant temperature shaker. Finally, it was dried in a vacuum drying oven at 60℃ for 12 h to obtain 32 mg of MIPs particles.

[0033] In addition, following the above method, but without adding HBV, a non-molecularly imprinted polymer was prepared, yielding 38 mg of NIPs particles as a control.

[0034] Material characterization To verify the successful preparation of the polymers, the hydrophilicity and hydrophobicity of ZIF-8-NH2, ZIF-8@C=C, MIPs, and NIPs were first characterized using a contact angle meter. Figure 2 As can be seen from A, the water contact angle of ZIF-8-NH2 is 94°. After the surface is modified with double bonds, its hydrophobicity is enhanced, and the water contact angle is 110°, which can well prove that the double bond functionalization of ZIF-8-NH2 is successful. Figure 2 B). After imprinting on their surfaces, the water contact angles of MIPs and NIPs were 121° and 125°, respectively. Figure 2 C and Figure 2 (D) This is because the hydrophilicity of the imprinted layer formed after polymerization changes due to the alteration of the hydrophobic functional monomer 4-VP and crosslinking agent EGDMA. Furthermore, since MIPs and NIPs use the same amount of functional monomer and crosslinking agent, their hydrophilicity and hydrophobicity are similar, demonstrating that the imprinted layer was successfully coated on the surface of ZIF-8@C=C.

[0035] exist Figure 3 Figure A shows the infrared spectra (FI-IR) of ZIF-8-NH2 (a), ZIF-8@C=C (b), MIPs (c), and NIPs (d). Curve a shows the 3466 / 3371 cm⁻¹... -1 and 763 cm -1 The nearby absorption peaks belong to the vibrational absorption peaks of -NH2 and -NH in 2-aminobenzimidazole, at 2913 cm⁻¹. -1 The absorption peak is a vibrational absorption peak of CH, indicating that ZIF-8-NH2 was successfully prepared. Curve b has the same absorption peak as curve a, but at 1616 cm⁻¹... -1 The presence of a distinct C=C structure indicates successful grafting of C=C onto the ZIF-8-NH2 surface. Curves c and d are located at 1407 cm⁻¹. -1 and 1100 and 790 cm -1 The presence of new characteristic peaks corresponds to the CN bond in the AM functional monomer, the COC bond in the crosslinking agent EGDMA, and the out-of-plane bending vibration of the CH ring in the functional monomer 4-VP. These results all demonstrate the successful preparation of each particle.

[0036] To further characterize the surface charge of the material, this invention uses the Zeta potential to test the above-mentioned material, such as... Figure 3As shown in Figure B, the potential of ZIF-8-NH2(a) is approximately +24 mV. This is because 2-aminobenzimidazole was doped during synthesis, and the presence of a large number of -NH2 groups on the ZIF-8-NH2 surface contributes to the positive potential. In contrast, the potential of ZIF-8@C=C(b) drops to approximately +6 mV, indicating that C=C was successfully grafted onto the ZIF-8-NH2 surface. After the introduction of functional monomers and completion of imprinted polymerization, the potentials of MIPs(c) and NIPs(d) become negative, further demonstrating the successful polymerization of the imprinted layer. Furthermore, the difference in potential between MIPs and NIPs is due to the presence of specific recognition cavities for template molecules on the surface of MIPs, resulting in a different surface charge compared to NIPs.

[0037] The properties, structure, morphology, and size of the prepared materials ZIF-8-NH2, ZIF-8@C=C, MIPs, and NIPs were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS), X-ray diffraction (XRD), surface area measurement (BET), and thermal analysis (TGA). Figure 4 The SEM characterization results of the above materials are presented. Analysis of the SEM images shows that ZIF-8-NH2 exhibits a regular dodecahedral structure. The introduction of functional monomers and crosslinking agents leads to polymerization reactions on its surface, forming an imprinted layer, resulting in particle aggregation. MIPs and NIPs exhibit a spherical structure. Since MIPs and NIPs are synthesized from the same monomers, differing only in the presence or absence of a template, they are similar in morphology and particle size. Furthermore, the particle size distribution of DLS further reveals… Figure 5 The average particle size of ZIF-8-NH2 is approximately 300 nm; the average particle size of ZIF-8@C=C is between 250 and 350 nm, with a wider particle size distribution than ZIF-8-NH2. This is because the double bond functionalization modification on the surface weakens the dispersibility of the particles in aqueous solution, leading to particle aggregation. The particle sizes of MIPs and NIPs are approximately 400–550 nm. The SEM and DLS results of the above materials are in line with expectations, indicating that each particle was successfully prepared.

[0038] X-ray diffraction (XRD) analysis was performed on the crystallinity of each material, such as... Figure 6 As shown in Figure A, ZIF-8-NH2 exhibited multiple absorption peaks in the range of 2θ = 10~60°, and the peak intensities were relatively high, indicating that the synthesized ZIF-8-NH2 possesses high crystallinity; after the formation of MIPs and NIPs imprinted layers on its surface through polymerization, the peak intensities decreased ( Figure 6 B) indicates that the ZIF-8-NH2 surface was successfully coated with an imprinted layer, proving that the synthesis of MIPs and NIPs was successful.

[0039] Based on the N2 adsorption-desorption results of ZIF-8-NH2 and ZIF-8@C=C ( Figure 7 The specific surface areas (BET) of the two samples (AB) differ, being 941.82 m² and 941.82 m², respectively. 2 / g and 175.74 m 2 / g. Using ZIF-8-NH2, which has a large specific surface area, as an imprinting carrier can provide abundant imprinting sites, thereby improving the sensitivity of virus detection. After double bond functionalization modification on the surface, C=C occupies in the channels of ZIF-8-NH2, so the specific surface area of ​​ZIF-8@C=C decreases. According to density functional theory, ZIF-8-NH2 ( Figure 7 C) and ZIF-8@C=C ( Figure 7 The apertures of D) are 7.16 nm and 3.69 nm, respectively.

[0040] Feasibility analysis To verify the feasibility of the sensor in detecting the virus, 10 μL (0.16 nM) of HBV virus was added to both 1 mg / mL solutions of molecularly imprinted polymers (MIPs) and non-imprinted polymers (NIPs), and incubated with shaking at 37 °C for 50 minutes. Subsequently, 20 μL of 20% H₂O₂ solution was added to each solution to react with ZIF-8 exposed in the unbound HBV cavity, generating hydroxyl radicals. Then, 200 μL of 0.75 mM crystal violet solution was added, and after reacting for 5 minutes, the absorbance of the solution was detected using UV-Vis absorption spectroscopy. Figure 8 As shown in Figure A, because the template virus HBV was added to MIPs during the polymerization reaction, imprinted cavities with complementary shapes and sizes matching the functional groups of HBV were formed, enabling specific recognition of HBV. Therefore, after the addition of HBV, some cavities in the MIPs were blocked, resulting in less ·OH generated, and the UV absorbance value of the MIPs was higher than that of the MIPs without virus. In contrast, NIPs did not add HBV during the polymerization process, so they lacked specific cavities, and the absorbance value before and after the addition of HBV did not change significantly. Furthermore, the absorbance value of MIPs was significantly lower than that of NIPs, further demonstrating the successful synthesis of molecularly imprinted polymers and the ability of MIPs to specifically recognize HBV. The inset shows the visualization results of MIPs and NIPs before and after the addition of HBV. The solution after MIPs bound HBV was darker in color than that without MIPs, and the color of NIPs was darker than that of MIPs before and after the addition of the virus, which is consistent with the change in absorbance. Figure 1 These results demonstrate that the prepared sensor is highly feasible for HBV detection.

[0041] Next, this invention verifies that the sensor is based on the principle that ZIF-8 reacts with H2O2 to generate ·OH, which then reacts with CV to cause its decolorization. Experimental results are as follows...Figure 8 As shown in Figure B, compared to pure CV, the absorbance of crystal violet did not change when ZIF-8, ZIF-8@C=C, or H2O2 were added alone. However, when ZIF-8 or ZIF-8@C=C was present simultaneously with H2O2, the absorbance of crystal violet decreased significantly. This indicates that ZIF-8, as a nanoenzyme material with oxidase-like activity, can efficiently catalyze the decomposition of H2O2 to generate ·OH, thereby destroying the molecular structure of CV and forming a fading reaction. Furthermore, the absorbance of the CV solution in the ZIF-8 and H2O2 group was lower than that in the ZIF-8@C=C and H2O2 group. This may be because the presence of the C=C group leads to poor dispersibility of ZIF-8@C=C in solution, causing aggregation and reducing the effective contact area with H2O2, thus lowering the catalytic efficiency. The inset shows that the solutions a, b, c, and f have similar colors, with d being slightly darker than e, consistent with the absorbance measurements. This indicates that the CV solution can only undergo a fading reaction when ZIF-8 and H2O2 are present simultaneously.

[0042] Application Example 1: Visual Detection and Analysis of Hepatitis B Virus The MIPspH prepared in Example 1 was prepared into a pre-concentration solution with 7.4 PBS at a concentration of 1 mg / mL. 0.4 mL of the pre-concentration solution was added to a 2 mL test tube, followed by 50 μL of 20% H2O2 solution and 200 μL of 0.75 mM crystal violet. The total volume was then brought to 1 mL with ultrapure water. After reacting for 5 min, the absorbance was measured using a UV-Vis spectrophotometer and recorded as Abs0. Then, different concentrations of HBV were added to 0.4 mL of the MIPs solution, and the mixture was incubated at 37 °C with shaking for 40 min. 50 μL of 20% H2O2 solution and 200 μL of 0.75 mM crystal violet were added, and the total volume was brought to 1 mL with ultrapure water. After reacting for 5 min, the absorbance was measured using a UV-Vis spectrophotometer and recorded as Abs0. i Therefore, the change in absorbance caused by the addition of the virus during the detection process is ∆Abs = Abs i -Abs0.

[0043] 1. Linear analysis and visualization detection 1 mL of 1 mg / mL blotted particles was added to PBS buffer solution at 37 ℃ and pH 7.4, and HBV virus of different concentrations was added and incubated with shaking for 40 min. Then, 0.4 mL of the incubation solution was added to a mixed solution containing 0.2 mL of 0.75 mM crystal violet, 0.05 mL of 20% H2O2 and 0.35 mL of H2O. The volume was adjusted to 1 mL and the reaction was carried out for 5 min. The absorbance at about 580 nm was then measured.

[0044] Experimental results are as follows Figure 9As shown, within the HBV virus concentration range of 4 pM to 150 pM, the absorbance (Abs) of MIPs is related to the viral concentration (C). HBV ) shows a linear relationship ( Figure 9 A), the linear regression equation is ∆Abs = 0.00104C HBV +0.0149 (R) 2 =0.99415)( Figure 9 B), detection limit LOD = 4.31 pM. Where ∆Abs represents the difference in absorbance between MIPs with added virus and those without virus, C HBV This represents the HBV virus concentration. In contrast, NIPs, lacking a specific HBV recognition cavity, only exhibit non-specific adsorption, therefore their absorbance curves show no significant change. Figure 9 C). From Figure 9 The visualization of D shows that as the viral concentration increases, the color of the MIPs solution gradually darkens, while the color of the NIPs solution shows no significant change, and the color of the NIPs solution is consistently darker than that of the MIPs. This phenomenon further confirms the existence of an imprinted cavity in MIPs that specifically matches HBV, allowing H2O2 to enter the cavity and react with the core ZIF-8 to generate ·OH, causing the crystal violet to fade. In contrast, because NIPs lack an imprinted cavity matching the target virus, H2O2 cannot contact the imprinted carrier and cannot generate ·OH, thus the color of the crystal violet remains unchanged. These results fully demonstrate the high specificity of MIPs in recognizing HBV.

[0045] 2. Selectivity and competition analysis To examine the specificity of the sensor, select hepatitis A virus (HAV, spherical, approximately 27 nm in diameter) which is similar in size to HBV (spherical, approximately 42 nm in diameter), influenza viruses H5N1 and H7N9 (spherical, approximately 80–120 nm in diameter) which are different in size from HBV, and enterovirus EV71 (icosahedral, approximately 20–30 nm in diameter) which has a different structure from HBV, for selective and competitive analysis.

[0046] First, the selectivity of the sensor was investigated, and the experimental results are as follows: Figure 10As shown in Figure A, after adding different viruses at the same concentration to the sensor, the sensor's ∆Abs response value for the template virus HBV was significantly higher than that for other non-template viruses, with an imprinting factor IF of 7.84. The imprinting factors for other viruses were all less than 2. This is because the MIPs have imprinting recognition sites that highly match the shape and size of HBV. Although HAV and influenza viruses (H5N1 and H7N9) are both spherical, their sizes are different, and the interactions between H5N1 and H7N9 and the functional monomers on the surface of MIPs are also different, thus making accurate identification impossible. Although EV71 virus is similar in size to HBV, its structure is very different from HBV, making specific identification impossible. These results indicate that the sensor has good selective recognition capabilities.

[0047] Similarly, a binary system of target virus and interfering virus was formed by adding them to the sensor for competitive testing. The experimental results are as follows: Figure 10 As shown in Figure B, the UV-Vis absorbance value changed significantly when the target virus HBV was added alone. However, when the interfering virus was added, the competitive virus, due to its different shape and size from the template virus, had a low matching degree with the imprint cavity and could not be specifically recognized. Therefore, compared with the HBV-only group, the absorbance change value was not significantly affected. This indicates that the prepared sensor has good competitiveness.

[0048] 3. Analysis of anti-interference capability To examine the detection capability of the prepared MIPs sensor in real samples, some common interfering substances in serum were introduced to further investigate the sensor's anti-interference ability. Na+ was selected for the experiment. + K + Mg 2+ Ca 2+ HCO3 - HPO4 2- Glucose, L-proline, and L-alanine were used as interfering ions and substances, and the experimental results are as follows: Figure 11 As shown, the detection performance of MIPs against the target virus HBV was not affected by these interfering substances, and the value of ∆Abs fluctuated little, indicating that the influence of these interfering substances on the detection of the target virus HBV is negligible. This result shows that the sensor has excellent anti-interference ability in real complex samples.

[0049] 4. Investigation of reproducibility and time stability Five batches of MIPs sensors were prepared under the same experimental conditions as in Example 1, and the same concentration of HBV virus was detected to evaluate the reproducibility of different batches of sensors. The experimental results are as follows: Figure 12As shown in Figure A, the UV-Vis absorbance variation values ​​∆Abs of the five different batches of MIPs are relatively similar, indicating that the sensor has good reproducibility.

[0050] Next, this work investigated the temporal stability of the synthesized MIPs sensor by detecting HBV after it had been stored for different periods. The same batch of synthesized MIPs was sealed and stored at room temperature for 0, 1, 2, 4, and 6 weeks, and then the same concentration of HBV virus was detected. The experimental results are as follows: Figure 12 As shown in Figure B, the ∆Abs value decreased slightly under the same detection conditions as the storage time increased, but the MIPs after 6 weeks still retained 87.57% of the initial detection performance. This result indicates that the sensor has excellent stability and can maintain high-efficiency detection capability over a long period of time.

[0051] 5. Actual testing and smartphone-assisted testing 1 mg of MIPs particles were ultrasonically dispersed in 1 mL of human serum diluted 100 times. Different concentrations of HBV virus were added, and each concentration was tested in triplicate. The results are shown in Table 2. The recovery rate of MIPs for HBV detection ranged from 97.6% to 106.1%, indicating that the MIPs sensor has good potential for practical application.

[0052] Table 2. Results of HBV detection in human serum diluted 100-fold.

[0053]

[0054] The developed colorimetric sensor, combined with a smartphone platform, enhances its portability and further improves the accuracy of results for practical applications. Since it can distinguish color changes with the naked eye, a smartphone captures color images, which are then converted into corresponding red (R), green (G), and blue (B) data using color picker software. Figure 13 A shows the colors of images captured by a smartphone. Figure 13 B shows the linear relationship between R / B and HBV concentration. The linear equation for colorimetric detection in the 0-120 pM range is R / B = -0.00201C. HBV +0.6706 (R) 2 =0.9989), detection limit LOD=2.22 pM. The proposed sensor can successfully achieve semi-quantitative detection of HBV through visual observation, and can also provide quantitative detection with the assistance of a smartphone, demonstrating the significant potential of this sensor for HBV detection.

[0055] Furthermore, the shortest time that the sensor can achieve rapid visual detection was investigated, such as... Figure 14As shown in Figure A, the MIPs are incubated with the target virus HBV for different times, followed by different reactions with H2O2 and crystal violet for different times. The figure shows that the sensor can achieve visual detection after 10 minutes of incubation with the virus, followed by 5 minutes of reaction with H2O2 and crystal violet. Simultaneously, corresponding images are captured using a smartphone. Figure 14 B), to achieve real-time, quantitative, and accurate detection of HBV.

[0056] Example 2-10 The difference between Examples 2-10 and Example 1 is that the amounts of MAA, 4-VP, and EGDMA are different. The specific amounts are shown in Table 4 below. The rest are the same as in Example 1.

[0057] Table 3. Dosage of MAA, 4-VP, and EGDMA in Examples 2-10

[0058] Sensors were prepared according to Examples 1-10 respectively and used for HBV detection (method as in Application Example 1). The change in absorbance caused by the addition of the virus during the detection process was used to calculate the imprinting factor IF = ∆MIPs / ∆NIPs, where ∆MIPs = Abs. i -Abs0, ∆NIPs=Abs i -Abs0.

[0059] The results are shown in Table 4. 4-Vinylpyridine and acrylamide, as functional monomers, bind the template virus to the imprinted vector through electrostatic interactions and the interaction between -NH2 and -COOH on the viral surface, respectively. Therefore, insufficient amounts of 4-vinylpyridine and acrylamide reduce the number of imprinted template viruses, leading to a decrease in the number of imprinted sites; while appropriate amounts of functional monomers help form sufficient imprinted sites. The amount of cross-linking agent EGDMA affects the degree of cross-linking of the MIP shell. Insufficient cross-linking agent concentration results in a loose imprinted layer network structure, affecting the accuracy of template recognition; excessive concentration leads to over-cross-linking of the imprinted layer, making the viral template difficult to wash out, resulting in a reduction in the number of sites that can rebind the virus. The optimal amounts of MAA, 4-VP, and EGDMA are 0.2 mmol, 0.3 mmol AM, and 0.2 mmol, respectively.

[0060] Table 4 Examples 1-10 Imprinting Factors

[0061] Examples 11-14 The difference from Example 1 is that the multiples of MAA, 4-VP, and EGDMA are different, as shown in Table 5 below. The rest is the same as in Example 1.

[0062] Table 5. Dosage of MAA, 4-VP, and EGDMA in Examples 11-14

[0063] Sensors were prepared according to Examples 1-10 respectively and used for HBV detection (method as in Application Example 1). The change in absorbance caused by the addition of the virus during the detection process was used to calculate the imprinting factor IF = ∆MIPs / ∆NIPs, where ∆MIPs = Abs. i -Abs0, ∆NIPs=Abs i -Abs0.

[0064] The results are shown in Table 6. IF analysis shows that increasing the amount of functional monomer and crosslinking agent leads to a decrease in IF value. This is because the increased thickness of the imprinted layer makes virus elution more difficult and slows down the recognition rate between the template virus and the imprinted site. On the other hand, reducing the amount of functional monomer and crosslinking agent leads to an excessively thin imprinted layer, exposing the imprinted cavity on the surface and increasing the risk of non-specific adsorption. The proportion in Example 1 is the optimal proportion.

[0065] Table 6 Imprinting factors of Examples 1 and 11-14

[0066] Examples 15-17 The difference from Example 1 is that the polymerization reaction time in step 3 is different, namely 6h, 8h, and 12h, while the rest is the same as Example 1.

[0067] Examples 18-20 The difference from Example 1 is that the polymerization reaction temperature in step 3 is different, namely 45°C, 55°C, and 75°C.

[0068] Examples 21-24 The difference from Example 1 is the amount of viral template used, which is 0.17, 0.25, 0.33, and 0.5 pmol respectively. The rest is the same as Example 1.

[0069] Examples 25-26 The difference from Example 1 is in the elution solvent, which is 0.5% SDS and methanol:acetonitrile (9:1, v / v), respectively. The rest is the same as Example 1.

[0070] Sensors prepared using Examples 1 and 15-26 were used for HBV detection (method as in Application Example 1). The absorbance change caused by the addition of the virus during detection was used to calculate the imprinting factor IF = ∆MIPs / ∆NIPs, where ∆MIPs = Abs. i -Abs0, ∆NIPs=Abs i -Abs0.

[0071] See results Figure 15 As the reaction time increases, the imprinting factor gradually increases, and then shows a decreasing trend after 10 hours. Figure 15 A). This is because when the imprinting time is too short, the imprinted layer is not fully formed, resulting in a large area of ​​the imprinted carrier being exposed, and the change in UV-Vis absorbance gradually decreases. Furthermore, excessive distribution of functional monomers on the surface hinders precise virus recognition, increasing non-specific adsorption; conversely, excessively long imprinting times may lead to overly high polymer cross-linking, causing the virus to be deeply embedded within the imprinted layer, increasing elution difficulty and reducing the effective capture capacity of the imprinted cavity for the target virus. Compared to imprinting time, polymerization temperature has a more significant impact on the performance of MIPs. Experimental results are as follows: Figure 15 As shown in Figure B, the imprinting factor (IF) of MIPs for detecting viruses reaches its maximum value at a polymerization temperature of 65 °C. Under low-temperature conditions, the polymerization reaction rate on the surface of the imprinting carrier is slow, making it difficult to form an imprint layer of sufficient thickness, resulting in an imprint cavity that is too thin and cannot effectively identify the target virus. Under high-temperature conditions, the polymerization rate is too fast and the activity of the viral template is reduced, both of which lead to a decrease in the number of imprinting sites.

[0072] The amount of template used directly determines the number of imprinting sites during the imprinting process, playing a crucial role in achieving highly sensitive virus detection. Experimental results are as follows... Figure 15 As shown in Figure C, the blotting factor (IF) of MIPs gradually increases with the increase of template virus dosage; however, the blotting factor begins to decrease when the template dosage exceeds 0.42 pmol. This is because, during incubation, when the same concentration of virus is added, excessive blotting sites reduce the binding efficiency of the virus per unit site, and adsorption reaches saturation, leading to a decrease in detection performance.

[0073] After the target analyte is embedded in the imprinted polymer layer, effective template removal is crucial for the specific recognition of the imprinted polymer. The eluent removes the template by disrupting the electrostatic interactions, hydrogen bonds, and ionic bonds between the template virus and the functional monomers. The degree of template removal determines the number of imprinted cavities: the more thorough the template removal, the more imprinted cavities are formed, resulting in stronger target analyte recognition and better sensor specificity; conversely, insufficient template removal or even template residue in the polymer reduces the number of imprinted cavities and lowers its recognition ability. Figure 15As shown in Figure D, the lowest IF value was obtained when eluting with 0.5% SDS containing 0.01 M acetic acid. SDS, as a surfactant, effectively disrupts the ionic bonds between the template and functional monomers, thereby promoting template release. Simultaneously, the presence of acetic acid affects the pH of the eluent, weakening the binding force between the virus and MIPs, thus enhancing the elution effect. However, since the imprinted vector is a metal-organic framework material, its internal structure collapses in an acidic environment. This not only reduces specific recognition sites but also lowers the overall ΔAbs. The highest IF value was obtained when eluting with ethanol:acetonitrile (4:1, v / v). The ethanol-acetonitrile mixed solvent provides a milder environment, which can weaken the interaction force between the template and functional monomers through polarity and solvation effects to efficiently elute the template without damaging the MIP structure.

[0074] Application Example 2-6: Visual Detection and Analysis of Hepatitis B Virus HBV virus (10 μL, 0.16 nM) was incubated with shaking at different temperatures (25, 30, 37, 40, 45 °C), and the detection performance of the sensor was evaluated by measuring the UV-Vis absorbance and comparing the IF values. All other conditions were the same as in Application Example 1.

[0075] The results are as follows Figure 16 A. The results showed that the optimal incubation temperature was 37 ℃. When the temperature was below 37 ℃, the effect on the detection performance of MIPs was not significant, indicating that the structure of MIPs and HBV does not change at low temperatures. When the temperature was above 37 ℃, the ∆Abs and IF of MIPs decreased significantly, because the activity of the virus was affected under high temperature conditions.

[0076] Application Example 7-10: Visual Detection and Analysis of Hepatitis B Virus Four solvents with a pH of 7.4 were selected: phosphate buffer (PB), water, phosphate buffered saline (PBS), and Tris-HCl buffer. MIPs / NIPs were dispersed in each of the four solvents, and the same concentration of virus was added. The mixtures were then incubated at 37 °C with shaking for 2 hours. All other conditions were the same as in Application Example 1.

[0077] The results are as follows Figure 16 The results showed that the ΔAbs and IF of MIPs initially increased and then decreased, with the ΔAbs and IF values ​​reaching their maximum when PBS was used as the incubation solvent. This may be because PBS provides a favorable physiological environment for the virus, maintaining its activity and thus promoting its effective binding to the blot cavity.

[0078] Application Examples 11-14: Visual Detection and Analysis of Hepatitis B Virus Virus detection was performed using PBS solutions with pH values ​​of 6, 7, 7.4, and 8, with all other conditions being the same as in Example 1.

[0079] The results are as follows Figure 16 As shown in Figure C, when the pH of the PBS solution is below 7.4, the values ​​of ∆Abs and IF gradually increase, reaching their maximum at pH 7.4. This may be because acidic conditions destroy the surface structure of the virus and cause the metal-ligand coordination bonds in the blot vector to break, leading to structural collapse and weakening the binding ability of the virus to the blot cavity. Under neutral conditions at pH 7.4, the environment is closest to the physiological state, which can effectively maintain the activity of the virus and thus improve the binding efficiency of the virus to MIPs. When the pH of the system exceeds 7.4, the surface structure of the virus will denature under alkaline conditions, which is not conducive to the effective binding of the virus to the blot cavity.

[0080] Application Examples 15-19: Visual Detection and Analysis of Hepatitis B Virus Different concentrations (0.2, 0.4, 0.6, 0.8, 1 mg·mL⁻¹) of blotted particles were added to the same concentration of virus for detection, and the remaining detection conditions were the same as in Application Example 1.

[0081] The results are as follows Figure 16 As shown in Figure D, ∆Abs and IF reach their maximum values ​​when the blot particle concentration is 0.4 mg / mL. This is because when the blot particle concentration is too low, the number of particles containing viral blot cavities in the system is insufficient, limiting the ability to re-recognize and bind to the virus; when the blot particle concentration is too high, the number of viral templates in the system is relatively insufficient, and the amount of virus adsorbed by the blot cavities reaches saturation, resulting in a decrease in overall absorbance. Therefore, a blot particle concentration of 0.4 mg / mL was determined to be the optimal detection concentration.

[0082] Application Examples 20-23: Visual Detection and Analysis of Hepatitis B Virus Different concentrations (15%, 20%, 25%, 30%) of H2O2 were added during the test, and the other conditions were the same as in Application Example 1.

[0083] See results Figure 17 When the mass fraction of H2O2 is 20%, the IF value is the highest. Although the absorbance change value of 25% H2O2 is very high, NIPs also exhibit this phenomenon, and the IF value eventually decreases (17A). Figure 17 B is its corresponding visualization.

[0084] Application Examples 20-23: Visual Detection and Analysis of Hepatitis B Virus Different concentrations (0.5 mM, 0.75 mM, 1 mM, 1.25 nM) of crystal violet were added to the test, and the other conditions were the same as in Application Example 1.

[0085] See results Figure 18 A. The highest IF value was observed when the concentration of crystal violet was 0.75 mM, as seen from visualization. Figure 18 As can be seen from B, the distinction between MIPs and NIPs is highest when the crystal violet concentration is 0.75 mM.

[0086] Application Examples 23-32: Visual Detection and Analysis of Hepatitis B Virus After adding the virus, the cells were incubated for different times (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 min), with the remaining conditions being the same as in Application Example 1.

[0087] The results are as follows Figure 19 As shown, the ΔAbs of both MIPs and NIPs gradually increased with increasing incubation time before 40 min, and then tended to reach equilibrium after 40 min. This is because before 40 min, MIPs contain a large number of virus recognition sites, which efficiently recognize HBV through electrostatic interactions and hydrogen bonding, resulting in a rapid increase in ΔAbs. After 40 min, the virus capture by MIPs in the system reaches saturation, and the system enters a dynamic adsorption equilibrium state, where ΔAbs no longer changes significantly. Therefore, 40 min was ultimately chosen as the incubation time for subsequent virus detection.

[0088] Application Examples 32-35: Visual Detection and Analysis of Hepatitis B Virus During the detection process, crystal violet was added and reacted for different times (1 min, 3 min, 5 min, 7 min), while the other conditions were the same as in Application Example 1.

[0089] The results are as follows Figure 20 As shown, the IF value reaches its maximum (20A) when the crystal violet fading reaction takes 5 minutes. If the reaction time is too short, the ∆Abs values ​​of MIPs before and after virus addition are too small; if the reaction time is too long, the crystal violet fades to almost transparency, resulting in a small ∆Abs value. As can be seen from the visualization results (20B), when the ∆Abs is small, it is not visually possible to distinguish the MIPs before and after virus addition. Therefore, 5 minutes is the optimal time for crystal violet fading.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A molecularly imprinted colorimetric sensor for detecting hepatitis B virus, characterized in that, The sensor uses ZIF-8, a metal-organic framework material with peroxidase-like activity, as the catalytic core and imprinted carrier. A molecularly imprinted polymer (MIP) layer with HBV as the template molecule is constructed on its surface. After the sensor specifically recognizes and captures HBV through MIPs, it changes the catalytic efficiency of ZIF-8 for hydrogen peroxide, thereby regulating the amount of hydroxyl radicals generated. Finally, the sensor achieves visualization and quantitative detection of HBV through the fading reaction of hydroxyl radicals with the colorimetric dye crystal violet.

2. The sensor according to claim 1, characterized in that, The ZIF-8 is an amino-functionalized ZIF-8-NH2, and further C=C double bonds are grafted onto its surface through a silanization reaction to obtain ZIF-8@C=C as a carrier for molecular imprinting.

3. The sensor according to claim 2, characterized in that, The molecularly imprinted polymer (MIPs) layer is formed by free radical polymerization using ZIF-8@C=C as a carrier, HBV as a template molecule, 4-vinylpyridine and acrylamide as functional monomers, and ethylene glycol dimethacrylate as a crosslinking agent.

4. A method for preparing the sensor as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Synthesis of ZIF-8-NH2: Zinc salt, 2-methylimidazole and 2-aminobenzimidazole were reacted in a water / methanol mixed solvent under alkaline conditions to obtain ZIF-8-NH2; (2) Synthesis of ZIF-8@C=C: ZIF-8-NH2 was dispersed in anhydrous toluene, and vinyltriethoxysilane was added to carry out silanization reaction. Double bonds were grafted onto the surface of ZIF-8-NH2 to obtain ZIF-8@C=C. (3) Preparation of molecularly imprinted polymers (MIPs): ZIF-8@C=C was dispersed in a solvent, and HBV template molecules, functional monomers 4-vinylpyridine and acrylamide were added sequentially for pre-assembly. Then, crosslinking agent ethylene glycol dimethacrylate and initiator 2,2'-azobisisobutyronitrile were added, and thermally initiated polymerization was carried out under an inert atmosphere. After the reaction was completed, the template molecules were eluted to remove them and MIPs were obtained.

5. The method according to claim 4, characterized in that, The molar ratio of the functional monomer 4-vinylpyridine, acrylamide and ethylene glycol dimethacrylate in step (3) is (1-3):(2-4):(1-3); the molar ratio of ZIF-8@C=C4-VP to 4-vinylpyridine is 30:0.05-0.3, in mg / mmol; the molar ratio of ZIF-8@C=C to HBV template molecule is 30:0.17-0.5, in mg / pmol. Preferably, in step (3), the polymerization reaction is carried out at a temperature of 45-75°C for 6-12 hours; the eluent is a mixed solution of alcohol and acetonitrile or a 0.5% SDS solution, wherein the alcohol is methanol or ethanol.

6. The use of the sensor as described in any one of claims 1-3 or the sensor prepared by the method as described in any one of claims 4-5 in the detection of hepatitis B virus.

7. A method for detecting hepatitis B virus using a sensor as described in any one of claims 1-3 or a sensor prepared by the method as described in any one of claims 4-5, characterized in that, Includes the following steps: (1) The sensor MIPs are mixed and incubated with the sample solution to allow the MIPs to specifically capture HBV; (2) Add hydrogen peroxide and crystal violet solution to the incubated system to carry out a colorimetric reaction; (3) Semi-quantitative analysis can be performed by observing the color change of the solution with the naked eye, or quantitative analysis can be performed by using a spectrometer to detect the absorbance value of the solution near 580 nm; the absorbance value is positively correlated with the HBV concentration.

8. The method according to claim 7, characterized in that, In step (1), the incubation is carried out in phosphate buffer, water, phosphate-buffered saline (PBS) or Tris-HCl buffer, with a pH of 6 to 8, an incubation temperature of 25 to 45°C, an incubation time of 10 to 90 minutes, and the concentration of the sensor MIPs is 0.2 to 1 mg·mL⁻¹; in step (2), the mass fraction of hydrogen peroxide is 15% to 30%, the concentration of crystal violet is 0.5 to 1.25 mM, and the colorimetric reaction time is 1 to 7 minutes.

9. The method according to claim 7 or 8, characterized in that, In step (3), a smartphone is used to collect a color image of the solution after the reaction, and the color channel values ​​of the image are extracted. The R / B value is then used to establish a linear relationship with the HBV concentration, thereby realizing portable quantitative detection based on a smartphone.

10. The use of the sensor according to any one of claims 1-3 or the sensor prepared by the method according to any one of claims 4-5 in the preparation of a detection kit for detecting hepatitis B virus, the kit comprising the molecularly imprinted colorimetric sensor, hydrogen peroxide solution, and crystal violet solution.

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