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 (Hg²⁺) based on the coordination chemistry of thymine-Hg²⁺-thymine (T-Hg²⁺-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 Hg²⁺ via the T-Hg²⁺-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 Hg²⁺ 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 Hg²⁺ in real samples of Zhujiang river water and tangerine peel, demonstrating its potential in environmental monitoring and food safety applications.