title: Enzymatic hydrolysate-induced displacement reaction with multifunctional silica beads doped with horseradish peroxidase-thionine conjugate for ultrasensitive electrochemical immunoassay.
abstract: 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.
authors.affiliation: †Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education and Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China.
authors.forename: Youxiu
authors.initials: Y
authors.lastname: Lin
authors.affiliation: †Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education and Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: †Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education and Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China.
authors.forename: Yuping
authors.initials: Y
authors.lastname: Lin
authors.affiliation: †Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education and Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China.
authors.forename: Dianping
authors.initials: D
authors.lastname: Tang
authors.affiliation: ‡Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Niessner
authors.affiliation: ‡Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Knopp
mesh_terms: D016604:Aflatoxin B1 / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D010367:Arachis / Q000378:metabolism; D002417:Cattle; D003208:Concanavalin A / Q000737:chemistry; D055664:Electrochemical Techniques; D004566:Electrodes; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D053768:Metal Nanoparticles / Q000737:chemistry; D010640:Phenothiazines / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry / Q000276:immunology; D012822:Silicon Dioxide / Q000737:chemistry*; D013395:Sucrose / Q000737:chemistry; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000378:metabolism
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D004800:Enzymes, Immobilized; D010640:Phenothiazines; D012709:Serum Albumin; C052669:aflatoxin B1-bovine serum albumin; D003208:Concanavalin A; D013395:Sucrose; D006046:Gold; D012822:Silicon Dioxide; D016604:Aflatoxin B1; D006735:Horseradish Peroxidase; D043324:beta-Fructofuranosidase; C009469:thionine