SERS-based cascade amplification bioassay protocol of miRNA-21 by using sandwich structure with biotin-streptavidin system.

In our bioassay protocol, the Ag@4MBA@DNA-biotin probes were synthesized by linking biotin-modified DNA and 4-mercaptobenzoic acid-covered Ag nanoparticles, and the Si@Ag@anti-digoxin/digoxin-DNA substrate was fabricated by immune linking of digoxin-DNA and anti-digoxin immobilized on a Ag-coated wafer. Then, the probes, miRNA-21 and the substrate were constructed into a "sandwich structure" to detect the variation in the SERS signals with respect to miRNA-21 concentrations. Next, streptavidin and extra probes were alternately introduced to implement the cascade amplification of the SERS signal to increase the detection sensitivity. The results show that the limit of detection (LOD) with cascade amplification is remarkably improved from 97.81 pM to 38.02 fM, which is three orders of magnitude higher than the original data without cascade amplification. Furthermore, the SERS-based cascade amplification mechanism was analyzed and is attributed to the "hot spots effect" of the noble metal nanostructure. The biotin-streptavidin (B-S) system was introduced into the SERS detection platform, and the novel SERS-based cascade amplification bioassay protocol has significant creativity for the detection of nucleic acids.