Ultrasensitive electrochemical detection of Bacillus thuringiensis transgenic sequence based on in situ Ag nanoparticles aggregates induced by biotin-streptavidin system

A novel electrochemical biosensor was developed for detecting short DNA oligonucleotide of Bacillus thuringiensis (Bt) transgenic sequence based on Ag nanoparticle aggregates. To fabricate this DNA biosensor, the thiol-modified capture DNA (cDNA) was first anchored on gold (Au) electrode, and then the target DNA (tDNA) was hybridized with the immobilized cDNA. Subsequently, the probe DNA (pDNA) functionalized by biotinylated Ag nanoparticle was associated with the fixed tDNA, and the single Ag nanoparticle label was obtained (cited as SAg label). Finally, dissociative biotinylated Ag nanoparticle was bound to the resultant biotinylated SAg label assembled on Au electrode by virtue of bridge molecule streptavidin (SA) through biotin-SA specific interaction, which could lead to in situ aggregate of Ag nanoparticles on Au electrode and induce a novel tag including multiple Ag nanoparticles (cited as MAg tag). The novel tag exhibited excellent electroactive property in the solid-state Ag/AgCl process and was successfully applied to Bt transgenic sequence assay. A detection limit of 10 fM was achieved, which was improved by three orders of magnitude as compared to the SAg label. Furthermore, this novel DNA biosensor demonstrated a good selectivity towards tDNA.