Afterglow-colorimetric Dual-mode Lateral Flow Immunochromatographic Strip For Estradiol

Estradiol(E2)is the endocrine disruptor chemical with the strongest estrogen effect.When the amount of E2 accumulated through food and drinking water exceeds the threshold in human body,it can cause potential harm,such as affecting the endocrine system.At present,the commonly detection methods for E2 could be divided into analytical methods and on-site methods in which lateral flow assay has the advantages of low-cost and easy operation.Traditional lateral flow assay with colloidal gold(CG)as labeling material performs semi-quantitative analysis based on colorimetric intensity by naked eyes.However,this method usually has insufficient sensitivity and possibility of false negative or false positive.The fluorescence-based lateral flow assay has low signal-to-noise ratio due to the luminescent interferences from strip materials under the ultraviolet(UV)lamp.Therefore,the afterglow-colorimetric dual-mode lateral flow immunochromatographic strip(DM-ICS)based on the principle of antigen-antibody specific recognition and F(?)ster resonance energy transfer was established to visually screen and highly-sensitive detection of E2 in food samples.The main research contents and results of the paper are as follows:(1)Preparation and characterization of afterglow donor and acceptor: estradiol carboxymethyl ether(E2-CME),also called E2-hapten,was synthesized by nucleophilic substitution reaction;bovine serum albumin(BSA)was used as the carrier protein and the coating antigen(E2-BSA)was prepared through coupling BSA with E2-CME by carbodiimide method.The products were successfully synthesized through characterization of liquid chromatography,ESI-QQQ mass spectrometer,NMR spectrometer and MALDI-TOF mass spectrometer.The afterglow particles(APs)were modified by silica encapsulation and carboxyl modification,and then conjugated with E2-BSA by the carbodiimide method to prepared the coupling product(E2-BSA-APs)which was used as afterglow donor.The products were successfully synthesized through characterization of Fourier transform infrared spectrometer and Zeta potential.CG was prepared by sodium citrate reduction method and CG-labeled anti-E2 monoclonal antibody(CG-m Ab)was prepared by adsorption interaction which was used as afterglow acceptor and colorimetric label.The products were successfully prepared through characterization of transmission electron microscope and ultraviolet-visible absorption spectrophotometer.(2)Construction and performance verification of ICS: The DM-ICS based on the principle of antigen-antibody specific recognition and F(?)ster resonance energy transfer was established by using E2-BSA-APs as the afterglow donor and CG-m Ab as the afterglow acceptor to provide colorimetric signals at the same time.The optimal experimental conditions were obtained by optimizing the parameters of DM-ICS mainly based on the sensitivity of the afterglow mode where excitation time was 5 s and capture time was 0.5 s after turning off UV lamp.Under optimal conditions,the detection limit(LOD)of DM-ICS was 10 ng/mL in the colorimetric mode and 0.5 ng/mL in the afterglow mode,with good specificity.In order to compare and evaluate the analytical performance of the established DM-ICS,the traditional colloidal gold colorimetric lateral flow immunochromatographic strip(CG-ICS)was constructed and its parameters were optimized based on the colorimetric sensitivity.The LOD of CG-ICS was 10 ng/mL under the optimal conditions,which was equivalent to the LOD of DM-ICS in the colorimetric mode,indicating that the optimal conditions for the afterglow mode were also better conditions for the colorimetric mode in DM-ICS.The LOD of the established DM-ICS was 1/20 of that of CG-ICS by comparison.(3)Detection of actual samples: The water samples and milk samples after simple pretreatment were detected and spiked experiments of samples were also performed.These experiments showed that the DM-ICS detection results were stable and the LOD(1 ng/mL)was lower than that of CG-ICS in the complex samples,which was also consistent with the results of HPLC-FLD.This DM-ICS could effectively avoid the fluorescence background of strip and the fluorescence interference from the complex matrix,and be applied to the detection of E2 in water samples and milk samples.