Highly Selective and Sensitive Electrochemical Immunoassay of Cry1C Using Nanobody and π-π Stacked Graphene Oxide/Thionine Assembly.

Abstract

Cry1C is one of the emerging toxin proteins produced by the Bacillus thuringiensis in the genetically modified crops for pest control in agriculture; thus, it is vital to measure the Cry1C level in crops for the healthy and environmental concerns. Current detections of Cry1C mainly rely on instrumental analysis such as high-performance liquid chromatography, which are time-consuming and are generally cost-prohibitive. Herein, a simple nanobodies (Nbs)-based electrochemical immunosensor has been first proposed for highly selective and sensitive detection of Cry1C. The Nbs pair, i.e., Nb51 and Nb54, which bind to different epitopes on Cry1C, was screened out from an immunized Bactrian camel, with an extra benefit of higher stability compared with conventional antibodies. Further, by using a π-π stacked graphene oxide/thionine assembly that had fast electron transfer kinetics as an electroactive label, the immunoreaction that occurred between the two Nbs and Cry1C can be highly sensitively quantified by square wave voltammetry. The linear detection range was from 0.01 to 100 ng·mL-1, and the low detection limit was 3.2 pg·mL-1. This method was further successfully applied for sensing Cry 1C in spiked samples with recoveries ranging from 100.17% to 106.69% and relative standard deviation less than 4.62%. This proposed assay would provide a simple highly sensitive and selective approach for the Cry1C toxin detection and be applicable to be extended to other toxin proteins sensing in foods.