Introduction of nanogold–DAB as a HRP substrate for simplifying detection in visual DNA microarrays

Abstract

Tyramine signal amplification, coupled with a gold-label silver stain (TSA–GLSS), has been demonstrated to be a sensitive visual assay for DNA microarrays. However, the procedure is laborious and time-consuming as nanogold particles do not accumulate directly in the vicinity of horseradish peroxidase (HRP). Instead, nanogold particles must accumulate through two procedures, including biotin-tyramine deposition via HRP catalysis and specific binding of biotin and streptavidin, followed by the introduction of streptavidin-nanogold. These conditions restrict this method's utility in DNA microarray detection. In the present study, nanogold was covalently linked to 3,3′-diaminobenzidine (DAB) to form a conjugate for the detection of Salmonella typhi. DAB can be oxidized by H2O2 and thus accumulate nanogold–DAB via HRP catalysis, leading to direct nanogold deposition. The results indicated that the detection limit of nanogold–DAB assay reached 103 CFU mL−1, which was comparable with that of TSA–GLSS. As one incubation step was omitted and the silver-staining time greatly shortened, this nanogold–DAB assay was more convenient than TSA–GLSS and offers great potential in detecting low concentrations of target DNA.