Manganese(II)-Enhanced Graphene Oxide Fluorescence Switch-Based Hybridization Chain Reaction for the Enzyme-Free and Sensitive Determination of Bacillus thuringiensis (Bt) in Transgenic Crops

Abstract

Bacillus thuringiensis, as a special DNA sequence, has been introduced into crops for insect control. At the same time, the safety risk of transgenic crops has received wide concern. Herein, a simple, enzyme-free and sensitive novel nanosensor for the determination of Bacillus thuringiensis by manganese(II)-enhanced graphene oxide (GO) fluorescence switch (Mn(II)-GO-FS)-based hybridization chain reaction (HCR) signal amplification was developed. The high affinity of Mn(II) to oxygen-containing groups in double-stranded DNA and on the surface of GO was able to overcome the steric effect of rigid double chains and drive the efficient adsorption of HCR hairpin probes with sticky ends on GO. The efficient quenching of 6-carboxyfluorescein (FAM) labeled on the HCR hairpin probes guaranteed a low background. The Bacillus thuringiensis acted as a trigger to initiate a cascade of hybridization events between the two metastable HCR hairpin probes (H1 and H2) to polymerize into a nicked long double-helix structure (dsDNA). The products desorbed from the surface of GO and produced strong fluorescence. The results included a wide linear range (0.1 pM-50 nM) and a low detection limit (0.05 pM). This nanosensor was successfully applied to determine Bacillus thuringiensis in maize seeds with spiked recoveries between 97.5% and 103%. The Mn(II)-GO-FS had a simple design and was conveniently used for the determination of other special genes by changing the corresponding HCR hairpin probe sequence. Moreover, this novel nanosensor did not require harsh experimental conditions, operating in a neutral environment.