Establishment Of Folic Acid Rapid Detection Based On Time-resolved Fluorescence Microspheres

Lateral flow immunoassay(LFIA)is a kind of in vitro diagnostic experiment,which is widely used because of its advantages of rapidity,convenience,stability,low cost,and easy to read results.Among them,spherical gold nanoparticles are the most widely used.However,the detection method based on conventional spherical gold nanoparticles has insufficient sensitivity,which hinders the further application of lateral flow immunoassay test strip.Time resolved fluoroimmunoassay(TRFIA)is a nonisotope immunoassay developed in the last decade,and is also the most sensitive microanalysis technology.TRFIA uses lanthanide to label antigen or antibody.According to the luminescence characteristics of lanthanide chelates,time-resolved technique is used to measure fluorescence,and two parameters,wavelength and time,are simultaneously detected for signal resolution,which can effectively eliminate the interference of non-specific fluorescence.In order to improve the sensitivity of lateral flow immunoassay and realize high sensitivity detection of folic acid content,a timeresolved fluorescence lateral flow immunoassay for quantitative detection of folic acid was developed by combining time-resolved fluorescence with lateral flow immunoassay.The main results of this paper are as follows:Polystyrene microspheres with different colors,uniform particle size and good dispersibility were synthesized by using styrene as raw material,soap-free emulsion polymerization method,ammonium persulfate as initiator to adjust microsphere synthesis,methyl methacrylate and sodium methacrylate as copolymers by changing dyes.The microstructures,morphologies,optical properties and particle size distribution of latex microspheres were characterized by field emission scanning electron microscopy,ultraviolet spectroscopy and particle size analyzer.The labeling conditions of the microspheres were explored.Based on two different color polystyrene microspheres labeled with streptococcal protein G,a brucellosis lateral flow immunoassay test strip was constructed,which can be used for qualitative detection of Brucellosis antibodies.The lower limit of qualitative detection of the immunoassay strip is 40 IU/ml,which can detect positive samples on the strip within 10 minutes and display the results.The sensitivity,specificity,stability and repeatability of the lateral flow immunoassay test strip for brucellosis were evaluated experimentally,and the detection effect was compared with that of conventional spherical gold nanoparticle lateral flow immunoassay test strips.This microsphere-based strip was a field test for brucellosis and a high sensitivity for brucellosis was achieved compared with other traditional biological detection methods.It also lays the foundation for the preparation and application of time-resolved fluorescent carboxy polystyrene microspheres.Time-resolved fluorescent carboxyl polystyrene microspheres were prepared from styrene by dispersive polymerization.The microspheres were characterized by field emission scanning electron microscopy,ultraviolet spectroscopy,particle size analyzer,Fourier transform infrared absorption spectrometer and fluorescence spectrometer for their microstructure,morphology,infrared spectra,fluorescence intensity,quantum yield,fluorescence lifetime,particle size distribution and surface groups.Established the time-resolved fluorescence intensity and concentration of fluorescent microspheres relation curve,the labeling conditions of time-resolved fluorescent carboxy polystyrene microspheres were explored,and time-resolved fluorescent carboxy polystyrene microspheres were used as labeling materials to label folic acid monoclonal antibodies.A lateral flow immunoassay with time-resolved fluorescence was developed,which can be used for quantitative detection of folic acid.The sensitivity,specificity,stability and repeatability of folic acid immunoassay test strips were evaluated experimentally.The LOD value of folic acid immunoassay was determined to be 0.78 ng/ml by sensitivity curve fitting,and the test results could be stable and read within 10 minutes.Compared with conventional folic acid detection methods,time-resolved fluorescence lateral flow immunoassay achieves rapid,sensitive and quantitative detection of folic acid molecules.The folic acid lateral flow immunoassay test strip constructed in this paper has high sensitivity and can be used for rapid quantitative detection of folic acid.In addition,the ordinary polystyrene latex microspheres and time-resolved polystyrene latex microspheres synthesized in this paper can label one or multiple protein or nucleic acid probes and other markers because of their carboxyl groups on the surface,which have broad application prospects in the field of biological applications.