Enzymatic Hydrolysate-Induced Displacement Reaction with Multifunctional Silica Beads Doped with Horseradish Peroxidase-Thionine Conjugate for Ultrasensitive Electrochemical Immunoassay

A novel (invertase) enzymatic hydrolysate-triggered displacement reaction strategy with multifunctional silica beads, doped with horseradish peroxidase-thionine (HRP-Thi) conjugate, was developed for competitive-type electrochemical immunoassay of small molecular aflatoxin B₁ (AFB₁). The competitive-type displacement reaction was carried out on the basis of the affinity difference between enzymatic hydrolysate (glucose) and its analogue (dextran) for concanavalin A (Con A) binding sites. Initially, thionine-HRP conjugates were doped into nanometer-sized silica beads using the reverse micelle method. Then monoclonal anti-AFB₁ antibody and Con A were covalently conjugated to the silica beads. The immunosensor was prepared by means of immobilizing the multifunctional silica beads on a dextran-modified sensing interface via the dextran-Con A binding reaction. Gold nanoparticles functionalized with AFB₁-bovine serum albumin conjugate (AFB₁-BSA) and invertase were utilized as the trace tag. Upon target AFB₁ introduction, a competitive-type immunoreaction was implemented between the analyte and the labeled AFB₁-BSA on the nanogold particles for the immobilized anti-AFB₁ antibody on the electrode. The invertase followed by gold nanoparticles hydrolyzed sucrose into glucose and fructose. The produced glucose displaced the multifunctional silica beads from the electrode based on the classical dextran-Con A-glucose system, thus decreasing the catalytic efficiency of the immobilized HRP on the electrode relative to that of the H₂O₂-thionine system. Under optimal conditions, the detectable electrochemical signal increased with the increasing target AFB₁ in a dynamic working range from 3.0 pg mL^-1 to 20 ng mL^-1 with a detection limit of 2.7 pg mL^-1. The strong bioconjugation with two nanostructures also resulted in a good repeatability and interassay precision down to 9.3%. Finally, the methodology was further validated for analysis of naturally contaminated or spiked AFB₁ peanut samples, giving results matched well with those from a commercialized AFB₁ enzyme-linked immunosorbent assay kit. Importantly, the system provides a signal-on competitive-type immunosensing platform for ultrasensitive detection of small molecules.