Enhanced Fluorescence ELISA Based on HAT Triggering Fluorescence Turn-on with Enzyme-Antibody Dual Labeled AuNP Probes for Ultrasensitive Detection of AFP and HBsAg

At present, enzyme-linked immunosorbent assay (ELISA) is considered to be the most appropriate approach in clinical biomarker detection, with good specificity, low cost, arid straightforward readout. However, unsatisfactory sensitivity severely hampers its wide application in clinical diagnosis. Herein, we designed a new kind of enhanced fluorescence enzyme-Inked immunosorbent assay (FELISA) based on the human alpha-thrombin (HAT) triggering fluorescence turn-on signals. In this system, detection antibodies (Ab(2)) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamineno(110) with fluorescence quenched served as the substrate of HAT. After the sandwich immunoreaction, HAT on the sandwich structure could catalyze the cleavage of the fluorescence-quenched substrate, leading to a strong fluorescence signal for sensing ultralow levels of alpha fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg). Under the optimized reaction conditions, AFP and HBsAg were detected at the ultralow concentrations of 10(-8) ng ml(-1) and 5 x 10(-4) IU mL(-I), respectively, which were at least 10(4) times lower than those of the conventional fluorescence assay and 106 times lower than those of the conventional ELISA. In addition, we further discussed the efficiency of the sensitive FELISA in clinical serum samples, showing great potential in practical applications.