Nanoparticle-Based Sandwich Electrochemical Immunoassay for Carbohydrate Antigen 125 with Signal Enhancement Using Enzyme-Coated Nanometer-Sized Enzyme-Doped Silica Beads

摘要

A novel nanoparticle-based electrochemical immunoassaynof carbohydrate antigen 125 (CA125) as a model wasndesigned to couple with a microfluidic strategy using anti-nCA125-functionalized magnetic beads as immunosensingnprobes.Toconstructtheimmunoassay,thionine-horseradishnperoxidase conjugation (TH-HRP) was initially doped intonnanosilica particles using the reverse micelle method, andnthen HRP-labeled anti-CA125 antibodies (HRP-anti-nCA125) were bound onto the surface of the synthesizednnanoparticles, which were used as recognition elements.nDifferent from conventional nanoparticle-based electrochemicalnimmunoassays, the recognition elements of thenimmunoassay simultaneously contained electron mediatornand enzyme labels and simplified the electrochemicalnmeasurement process. The sandwich-type immunoassaynformat was used for the online formation of the immunocomplexnin an incubation cell and captured in the detectionncell with an external magnet. The electrochemicalnsignals derived from the carried HRP toward the reductionnof H2O2 using the doped thionine as electron mediator.nUnder optimal conditions, the electrochemical immunoassaynexhibited a wide working range from 0.1 ton450 U/mL with a detection limit of 0.1 U/mL CA125.nThe precision, reproducibility, and stability of the immunoassaynwere acceptable. The assay was evaluated fornclinical serum samples, receiving in excellent accordancenwith results obtained from the standard enzyme-linkednimmunosorbent assay (ELISA) method. Concluding, thennanoparticle-based assay format provides a promisingnapproach in clinical application and thus represents anversatile detection method.