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.