An Immunosensing System Using Stilbene Glycoside as a Fluorogenic Substrate for an Enzymatic Reaction Model

A natural product, stilbene glycoside (2,3,5,4′-tetrahydroxy-diphenylethylene-2-O-glucoside, TBG), has been evaluated for the first time as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reactions. The properties of TBG as a fluorogenic substrate for HRP and its application in a fluorometric enzyme-linked immunosensing system were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red using Brucella melitensis antibody (BrAb) as a model analyte. The immunosensing body based on HRP-BrAb was constructed by dispersing graphite, BrAg and paraffin wax at room temperature. In a competitive immunoassay procedure, the BrAb competed with HRP-BrAb to react with the immobilized BrAg. In the enzymatic reaction, the binding HRP-BrAb on the sensing body surface can catalyze the polymerization reaction of TBG by H(2)O(2) forming fluorescent dimers and causing an increase in fluorescence intensity. TBG showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 3.5×10(−8)∼7.6×10(−6)g/L and with a detection limit of 1.7×10(−9) g/L. The immobilized biocomposite surface could be regenerated with excellent reproducibility (RSD=3.8%) by simply polishing with an alumina paper. The proposed immunosensing system has been used to determine the BrAb in rabbit serum samples with satisfactory results.