▎ 摘　　要

NOVELTY - A preparation method of virus molecular imprinting-aptamer sensor comprises (S1) mixing manganese nitrate and zinc nitrate hexahydrate, to obtain zwitterionic graphene oxide (ZGO) powder, ultrasonically dispersing in dimethylformamide (DMF), dripping (3-aminopropyl)triethoxysilane and adding activated H5N1 virus aptamer to the phosphate-buffered saline (PBS) buffer to obtain ZGO-H5N1 Apt, (S2) preparing magnetic iron oxide (Fe3O4) nanoparticles, adding tetraethyl orthosilicate, coating a layer of silicon dioxide (SiO2) on the surface of Fe3O4 nanoparticles, adding acrylamide, methyl acrylate, template virus H5N1, N,N'-methylenebisacrylamide and azobisisobutyronitrile, performing polymerization, eluting to obtain molecularly imprinted polymer (MIP) and (S3) dispersing the MIP in PBS buffer, adding H5N1 virus and ZGO-H5N1 Apt, incubating, performing magnetic separation, adding supernatant to a cuvette, measuring the photoluminescence intensity, recording, evaluating and constructing. USE - The method is used for preparing virus molecular imprinting-aptamer sensor based on persistent luminescence nanomaterials. The virus molecular imprinting-aptamer sensor is used for analyzing the H5N1 virus solutions with different concentrations for evaluating its selectivity and competitive adsorption capacity for H5N1 virus. The virus molecular imprinting-aptamer sensor is used for detecting different virus solutions at the same concentration and a mixed solution of the target virus and the interfering virus, and evaluating its selectivity and competitive adsorption capacity for H5N1 virus. The viral molecular imprint-aptasensor is used for detecting H5N1 virus solutions containing various interfering substances to evaluate its anti-interference ability for H5N1 virus detection, the materials stored for different times are used for constructing the virus molecular imprint-aptasensor, and analyzing the H5N1 virus solution to investigate its time stability (all claimed). ADVANTAGE - The method utilizes the unique luminescent properties of persistent luminescent nanomaterials and the high selectivity of imprinted polymers and aptamers. The constructed sensor effectively eliminates the background interference of autofluorescent substances in serum samples, and realizes highly sensitive and highly selective detection of H5N1 virus. DETAILED DESCRIPTION - A preparation method of virus molecular imprinting-aptamer sensor based on a persistent luminescent nano material comprises imprinting a magnetic iron (II, III) oxide (Fe3O4) nanoparticles as an imprinting carrier, eluting to obtain magnetically imprinted polymers with highly pathogenic avian influenza A (H5N1) imprinted cavities, synthesizing a persistent luminescent nanomaterial zinc germinate (Zn2GeO4): manganese (Mn2+)(ZGO), functionalizing using H5N1 aptamers, at the same time, using the above two materials to identify the H5N1 virus, performing magnetic separation, preparing a photoluminescence (PL) signal that varied with the concentration of H5N1 and recording the PL signal to construct the virus molecular imprint-aptamer sensor, the preparation method comprises (S1) mixing manganese nitrate (Mn(NO3)2) and zinc nitrate Hexahydrate (Zn(NO3)2.6H2O), adding 300 μL nitric acid (HNO3) (68 wt.%) and 1mL 1mol/L sodium germanate (Na2GeO3) are added successively, quickly adjusting the pH value to 9.0 using aqueous ammonia (NH3.H2O), stirring at room temperature for 1 hour, transferring to a polytetrafluoroethylene autoclave at 220℃ for 4 hours, washing, drying to obtain zwitterionic graphene oxide (ZGO) powder, dispersing the ZGO powde in sodium hydroxide (NaOH) solution, stirring overnight to obtain hydroxylated ZGO-OH, ultrasonically dispersing in dimethylformamide (DMF), dripping (3-aminopropyl)triethoxysilane (APTES), reacting at 80℃ for 24 hours to obtain ZGO-NH2, adding the activated H5N1 virus aptamer to the phosphate-buffered saline (PBS) buffer containing ZGO-NH2, shaking overnight at 37℃, and washing using PBS until the ultraviolet (UV) signal of the aptamer in the supernatant cannot be detected to obtain aptamer-functionalized ZGO material ZGO-H5N1 Apt, (S2) mixing ferric chloride hexahydrate (FeCl36H2O), sodium acetate (NaAc) and polyethylene glycol to obtain Fe3O4nanoparticles using hydrothermal reaction, adding NH3.H2O and tetraethyl orthosilicate (TEOS), coating a layer of silicon dioxide on the surface of Fe3O4nanoparticles to obtain Fe3O4loaded SiO2, which is dispersed in absolute ethanol containing maximum propylene solution (MPS), and stirred at room temperature for 24 hours to obtain Fe3O4loaded SiO2loaded C=C, ultrasonically dispersing 20 mg Fe3O4 loaded SiO2loaded C=C and in DMF solution, adding acrylamide, acrylic acid, methyl acrylate and template virus H5N1, stirring at 65℃ for 2 hours, adding N,N'-methylenebisacrylamide (MBA) as a crosslinking agent and azobisisobutyronitrile (AIBN) as an initiator, stirring under nitrogen atmosphere for 30 minutes to remove oxygen, performing polymerization at 65℃ for 6 hours, repeatedly eluting the obtained product using a mixed solution of methanol and acetic acid at a volume ratio of 9: 1 to remove the template virus H5N1 until no fluorescent signal of the H5N1 virus is observed in the supernatant to obtain molecularly imprinted polymer (MIP), similarly the preparation of non-imprinted polymer (NIP) is the same as above, except that no template virus is added and (S3) dispersing the prepared MIP in PBS buffer, adding a certain concentration of H5N1 virus, incubating with shaking under optimized adsorption conditions, adding ZGO-H5N1 Apt to the above solution, incubating with shaking under the same conditions, performing magnetic separation, collecting the supernatant, adding an appropriate amount of supernatant to a cuvette, measuring the PL intensity, recording in phosphorescence mode on a Hitachi F-4600 fluorescence spectrophotometer, evaluating the H5N1 virus concentration by calculating the PL intensity difference delta IPL of the supernatant before and after adding the H5N1 virus and constructing a viral molecular imprinting-aptamer sensor based on persistent luminescent nanomaterials to detect the H5N1 virus, where the detection conditions of the viral molecular imprinting-aptamer sensor comprises excitation wavelength is 250 nm, emission wavelength is 536 nm, excitation slit is 10 nm and emission slit is 10 nm. An INDEPENDENT CLAIM is included for use of the virus molecular imprinting-aptamer sensor for analyzing the H5N1 virus solutions with different concentrations and evaluating the detection range and detection limit of the H5N1 virus.