Detection Method Of Coronavirus Based On Upconversion Fluorescence Signal

In recent years,a variety of coronaviruses have frequently attacked the normal life of human society,and the outbreak of SARS-Co V-2 at the end of 2019 has swept the globe.In view of the serious harm of coronavirus,it is necessary to establish a rapid and effective detection method.Rapid test strip can accurately detect whether an individual is a positive infected person in a short time.Due to the excellent optical properties of up-conversion fluorescent materials,they have broad application prospects,it had attracted much attention of many scholars.As a novel biofluorescent marker,up-conversion fluorescent materials have the advantages of stable photochemical properties,low toxicity,low fluorescence background,and no interference from samples or carriers.In this thesis,up-conversion fluorescent materials and gold nanorods（AuNRs）are used as markers.Utilizing the fluorescence resonance energy transfer effect（FRET）between AuNRs and upconversion nanoparticles（UCNPs）in combination with immunochromatography,an immunochromatographic test strip for quantitative detection of coronavirus nucleocapsid protein（N protein）was developed.The specific research contents are as follows:We prepared AuNRs of different length-diameter ratio by using improved seed induction method.The synthesized particles were characterized for their phase and morphology using X-ray diffraction（XRD）and transmission electron microscopy（TEM）.Biotin and antibody were modified on the surface of AuNRs respectively,and their absorption properties were investigated by UV-visible spectrophotometer.Na Gd F₄:Yb,Er and Na Gd F₄:Yb,Tm of hexagonal phase were prepared by high temperature pyrolysis,under the excitation of 980 nm near-infrared light（IR-980 nm）,they emit yellow light and blue-violet light respectively.Using ultraviolet-induced sulfhydryl alkenyl click reaction,the surface of UCNPs was hydrophilic modified with HS-PEG-NH₂ and HS-PEG-COOH.In this way,UCNPs will carry amino or carboxyl groups which can be further modified with biotin and antibodies.The luminescent properties of two UCNPs were characterized by fluorescence spectrometer.Two types of UCNPs were excited by IR-980 nm,and strong emission peaks were observed at 650nm and 800 nm,respectively.The particle luminescence intensity decreased after hydrophilic modification and modification with biotin and Ab.After AuNRs and UCNPs were modified with biotin,under the action of avidin,FRET effect occurred between them,resulting in fluorescence quenching.Use fluorescence spectrometer to explore the degree of fluorescence quenching of AuNRs-UCNPs FRET system in solution with the increase of the amount of avidin;Antibody modified AuNRs and UCNPs cause the FRET process of the two particles to cause fluorescence quenching under the antigen-antibody immune recognition.With the increase of the concentration of N protein in the sample,the FRET effect increases and the fluorescence intensity ratio（I₂/I₁）at the T line of the test strip decrease.On this base,the ICA test strip was established to quantitatively detect the content of coronavirus N protein in the sample.A standard curve with a linear regression equation of I₂/I₁=-11.65lnc _Ag+75.74（R²=0.9820）and I₂/I₁=-8.52lnc _Ag+72.59（R²=0.9822）was established with a linear range of 10 ng·mL^-1-1280 ng·mL^-1.The detection limit of blank were 0.199 ng·mL^-1 and 0.154 ng·mL^-1 respectively.The limit of detection of test strips were 1.15 ng·mL^-1 and 0.93 ng·mL^-1,and have good detection specificity.