Electrochemical proximity assay-coupled highly nonenzymatic amplifying strategy for total protein of Nosema bombycis detection

Abstract

In this study, an electrochemical proximity assay (ECPA)-coupled highly nonenzymatic amplifying strategy with electron mediator collected by a host-guest interaction was proposed for total protein of N. bombycis (TP N.b) detection. Antibody-dsDNA-gold nanoparticles functional Fe3O4 probe (termed as Ab-dsDNA-Au@Fe3O4NPs), in which the Ab-dsDNA composed by adenine rich ssDNA (A1) and Ab labeled capture ssDNA 1 (Ab-S1), was first incubated with target TP N.b and another Ab labeled capture ssDNA 2 (Ab-S2) to perform the proximity immunoreaction. Satisfyingly, the immunoreaction could not only produce sandwich immunocomplex with the proximity hybridization of S1 and S2, but also exposed the trigger ssDNA A1 to in situ form substantial methylene blue (MB) intercalated DNA concatamers via hybrid chain reaction (HCR). To further realize the nonenzymatic amplification, the MB intercalated in DNA concatemers was released into solution by duplex specific nuclease (DSN) and subsequently collected by prepared cucurbit(7)-uril (CB[7])/Au@Fe3O4NPs/GCE via host-guest interaction, which thus produced a significantly amplified signal for quantitative detection of target. Since the catalyst Fe3O4NPs on electrode surface could directly electrocatalyze the reduction of coimmobilized MB, the proposed system possessed superior advantages compared with that of traditionally enzymatic systems, which not only simplified the operation process, decreased measurement error but also improved catalytic efficiency and stability.