Terminal Deoxynucleotidyl Transferase-Mediated CRISPR Sensing Platform for Simple and Point-of-care Detection of Cobalt Pollution

The excessive use of cobalt in various chemical industries and arbitrary discharge of industrial wastewater have led to increased cobalt pollution in soil and water resources, increasing the risk of human exposure to high concentrations of cobalt and necessitating an urgent need for on-site monitoring platform for cobalt pollution. In this study, the terminal deoxynucleotidyl transferase (TdT)-CRISPR platform has been developed. In this platform, cobalt as a cofactor of TdT, can significantly improve the tailing efficiency of TdT-mediated extension. Therefore, when cobalt is present, the detection probe can be extended with poly(T) tails through the TdTmediated extension, which can be subsequently served as the DNA activator for Cas12a, leading to the cleavage of fluorescence reporter molecules and triggering turn-on fluorescence signals. Consequently, this dual amplification sensing strategy of TdT-CRISPR platform demonstrated exceptional sensitivity (0.83 nM) and high specificity for cobalt over other ions. Furthermore, the method was successfully employed for the detection of cobalt in tap water and river samples. CRISPR-lateral flow assays (CRISPR-LFAs) were evaluated in this study for the simple and point-of-care detection of cobalt pollution. The assays are capable of detecting cobalt concentrations as low as 50 nM, which is significantly lower than the environmental standards of 16.9 mu M, through strip analysis with the naked eye. These results commonly suggest that the TdT-CRISPR platform holds significant promise for monitoring cobalt pollution, providing a robust and sensitive solution for on-site detection and contributing to the mitigation of cobalt contamination risks in environmental matrices.