Abstract
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 H2O2 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∼7.6×10−6g/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.
Highlights
Enzyme-linked immunosorbent assays (ELISA), introduced in the 20th century in early seventies and coupling the amplification effect of enzymatic reactions with selective antigen-antibody binding can provide sensitivity comparable with that of radioimmunoassay (RIA) [1, 2]
When the substrate solution containing TBG and H2O2 swept over the incubated biocomposite surface, the horseradish peroxidase (HRP) of the Brucella melitensis antigens (BrAg)-Brucella melitensis antibody (BrAb)-HRP complexes bound on the support body surface catalyze the reaction to convert a part of TBG into a fluorescent dimmer product
The results showed that the fluorescence decrease rate of TBG was 8 % at 80°C, while the fluorescence decrease values for p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red were 11.2%, 47% and 26%, respectively
Summary
Enzyme-linked immunosorbent assays (ELISA), introduced in the 20th century in early seventies and coupling the amplification effect of enzymatic reactions with selective antigen-antibody binding can provide sensitivity comparable with that of radioimmunoassay (RIA) [1, 2]. Several enzymes, such as alkaline phosphatase (AP), β-D-galactosidse and horseradish peroxidase (HRP) may be used as labels. Work in our laboratory has demonstrated that the use of a suitable fluorogenic substrate for HRP makes possible the detection of enzyme reaction products at much lower levels than can be detected using the aforementioned substrates with conventional determination techniques. BrAg-graphite-paraffin based immunosensing system has been applied to determination of BrAb in rabbit serum samples using a competitive binding assay with the aid of HRP-BrAb
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