Label-free fluorescence detection of mercury ions based on thymine-mercury-thymine structure and CRISPR-Cas12a.

In this work, we developed a novel label-free fluorescent sensor for the highly sensitive detection of mercury ions (Hg) based on the coordination chemistry of thymine-Hg-thymine (T-Hg-T) structures and the properties of CRISPR-Cas12a systems. Most notably, two T-rich sequences (a blocker and an activator) were designed to form stable double-stranded structures in the presence of Hg via the T-Hg-T base pairing. The formation of T-T mismatched double-stranded DNA between the blocker and the activator prevented the cleavage of G-rich sequences by Cas12a, allowing them to fold into G-quadruplex-thioflavin T complexes, resulting in significantly enhanced fluorescence. Under the optimized conditions, the developed sensor showed an excellent response for Hg detection in the linear range of 0.05 to 200 nM with a detection limit of 23 pM. Moreover, this fluorescent sensor exhibited excellent selectivity and was successfully used for the detection of Hg in real samples of Zhujiang river water and tangerine peel, demonstrating its potential in environmental monitoring and food safety applications.