Ultrasensitive Fluorescent Assay Based on a Rolling-Circle-Amplification-Assisted Multisite-Strand-Displacement-Reaction Signal-Amplification Strategy

Heavy metal ions are persistent environmental contaminants and pose a great threat to human health, which has prompted demand for new methods to selectively identify and detect these metal ions. Herein, a novel fluorescent assay based on a rolling-circle-amplification (RCA)-assisted multi-site-strand-displacement-reaction (SDR) signal-amplification strategy was proposed for the ultrasensitive detection of heavy metal ions with lead ions (Pb2+) as a model. The proposed strategy not only achieved the target recycling but also introduced RCA induced by released DNAzyme. Most importantly, the RCA product was adapted as the initiator to provide multiple sites for SDR, which could displace signal duplexes from RCA products to effectively avoid the self-quenching of signal-probe assembly on the RCA product. Therefore, the amplification efficiency and the detection sensitivity could be improved significantly. As expected, the proposed strategy demonstrated good performance for the determination of Pb2+ with a linear range from 0.1 to 50 nM and a detection limit down to 0.03 nM. Using this strategy for intracellular-Pb2+ detection, a favorable property was obtained. Furthermore, the proposed strategy could be also expanded for the determination of microRNA, proteins, and other biomolecules, offering a novel avenue for environmental assays and clinical diagnostics.