Yang Mei,Chenxi Li,Si Yang,Wenliang Chen,Rong Liu,Kexin Xu
Since mercury is one of the most toxic heavy metals in water, its detection is critical for environmental protection. Various biosensors based on T-Hg 2+ -T sensing strategies have been developed; however, their mechanisms are highly dependent on the buffer conditions and are not suitable for the rapid high-throughput analysis of many complex water samples. In this work, a self-assembled biosensor for the high-throughput detection of Hg 2+ based on a cleavable PS-RNA-modified probe and homogeneous photochemical chemiluminescence assay was developed. In the presence of Hg 2+ , the probe was cleaved, and the singlet oxygen channel was destroyed, which decreased the fluorescence signal. This biosensor is highly selective for Hg 2+ , with a limit of detection of 1.4 nM (S/N = 3) in a linear range of 5 nM - 5 μM (R 2 = 0.991). The recoveries of standard addition methods in different water samples indicate that this feasible, simple method is a promising tool to monitor mercury ions in environmental applications.
Yang Mei,Si Yang,Chenxi Li,Xinyu Li,Yuewen Yu,Rong Liu,Wenliang Chen,Xiaoli Wang,Kexin Xu
Mercury ion (Hg 2+ ) detection plays a crucial role in safeguarding the environment and protecting human health. In this study, we developed a microfluidic biosensing platform that utilizes a phosphorothioate-RNA (PS-RNA) probe and gold-coated magnetic nanoparticles to quantify Hg 2+ visually. The PS-RNA probe was linked to gold-coated magnetic nanoparticles via Au–S interaction and biotin–avidin interaction and then combined with polystyrene microspheres to form a self-assembled structure called a magnetic bead-AuNP-probe-polystyrene (MAPP) bead. We designed and fabricated a microfluidic chip that contained only a magnetic separator and a particle trap. The presence of Hg 2+ cut the PS-RNA probe, leading to the separation of magnetic beads and polystyrene microspheres. The MAPP was removed by the magnetic separator, while the cut polystyrene microspheres flowed along the channel and accumulated in the particle trap, forming a long visible strip. The length of the polystyrene microsphere strip was proportional to the Hg 2+ concentration. The microfluidic biosensing platform exhibited high sensitivity and specificity for Hg 2+ detection, with a detection limit of 21.9 nM. By integrating the microfluidic magnetic manipulation system with a sensitive nucleic acid probe, we developed an automated and visual biosensor that is a valuable reference for the on-site visual detection of heavy metal ions.
Summary Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5′- and 3′-cleavage products simultaneously. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022) .
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