Radial Flow Assay Using Gold Nanoparticles and Rolling Circle Amplification to Detect Mercuric Ions

A novel colorimetric assay employing oligonucleotide-conjugated gold nanoparticle (AuNP probes) and rolling circle amplification (RCA) was developed for simple detection of mercuric ions (Hg²⁺). The thymine-Hg²⁺-thymine (T-Hg²⁺-T) coordination chemistry makes our detection system selective for Hg²⁺. In the presence of Hg²⁺, the thymine 12-mer oligonucleotide is unable to act as a primer for RCA due to the formation of T-Hg²⁺-T before the RCA reaction. However, in the absence of Hg²⁺, DNA coils as RCA products are generated during the RCA reaction, and is further labeled with AuNP probes. Colorimetric signals that depend on the amount of DNA coil-AuNP probe complexes were generated by drop-drying the reaction solution on nitrocellulose-based paper. As the reaction solution spread radially because of capillary action, the complexes formed a concentric red spot on the paper. The colorimetric signals of the red spots were rapidly measured with a portable spectrophotometer and determined as the ΔE value, which indicates the calculated color intensity. Our assay displays great linearity (detection limit: 22.4 nM), precision, and reproducibility, thus demonstrating its utility for Hg²⁺ quantification in real samples. We suggest that our simple, portable, and cost-effective method could be used for on-site Hg²⁺ detections.