The Development Of Novel Fluorescence Quenching Technology For Detecting Ractopamine

Objective: A fluorescence polymer dots label-free positive readout and sensitive lateral flow assay (LFA)based on fluorescence quenching has been developed for the detection of Ractopamine (Rac), a type of chemicalresidue in food harmful to human health. And It`s the first time for our lab to study the detection method of liquidFRET based system.Methods: Anti-Rac monoclonal antibody was labeled with gold nanoparticles (Au-NPs) to obtained thefluorescent quencher and then dispensed the quencher on the conjugate pad. The fluorescence polymer dots (FPDs)were closed by BSA. BSA-FNPs and a certain amount of Rac antigen were dispended on a specific area of the NCmembrane as the test line. BSA closed FPDs were prepared and dispended on a specific area of the NC membraneas a calibration line. After the fluorescence quenching lateral flow assay strip assembly was completed，a series ofprocess were carried on to optimize the performance of the developed lateral flow assay. The prepared fluorescencequenching lateral flow assay strip were used for detected Rac in water, swine urine and swine muscle tissue. Theresults were analyzed by ImageJ software to obtain quantitative results. The specificity, stability, recovery of thefluorescence quenching lateral flow assay strip were studied.Rac antigens were labeled to three different fluorescence polymer dots, Anti-Rac monoclonal antibody waslabeled with gold nanoparticles (Au-NPs) to obtained the fluorescent quencher. According to different order ofaddition, certain proportion of above FPDs and AuNPs and Rac were added to96black plates. The results wereanalyzed by safire2and detecting the variation of relative fluorescence intensity.Results: We could detect as low as0.16ng/mL Rac in PB by using the developed strips through fluorescenceLFA strip reader. The sensitivity of this LFA is15times more sensitive than commercial gold base LFA. Rac inswine urine was also tested by fluorescence quenching LFA which can be detected as low as0.16ng/mL. Theconcentration of Rac in PB could be quantitively detected within0.63ng/mL-10ng/mL by using the softwareImageJ according to the calibration curve. The LFA strips have demonstrated a high Specificity. This developednovel scheme is much suitable for small molecules rapid detection.After the study of liquid FRET based system, we got a unexpected result. Further work for theliquid FRET based system need to be done.Conclusions: A fluorescence polymer dots label-free positive readout and sensitive lateral flow assay (LFA)based on fluorescence quenching has been developed for the detection of Ractopamine (Rac), a type of chemicalresidue in food harmful to human health. Only higher concentrations of analyte produce the strong decrease insignal intensity required for users to obtain definitive results using traditional LFA strips. Compared to traditionalLFA strips, the fluorescence quenching LFA (FQLFA) strips presented here provide a positive correlation methodthat allows users to obtain results with a weak fluorescence signal. Because the characters of high sensitivity,easy to operation, low-cost, the developed fluorescence quenching lateral flow assay strips could beused for the small molecules detection.The reaction of antigen and antibody maybe not suitable for liquid FRET based system.