Bi-functionality of glyoxal caged nucleic acid coupled with CRISPR/Cas12a system for Hg²⁺ determination 

A highly sensitive and selective fluorescence method has been conducted for the detection of Hg²⁺ based on aminophenylboronic acid–modified carboxyl magnetic beads (CMB@APBA) and CRISPR/Cas12a system mediated by glyoxal caged nucleic acid (gcDNA). As a bi-functional DNA linker, gcDNA offers advantages of simultaneous recognition by boronic acid and complementary DNA/RNA. Under acidic condition, gcDNA can be immobilized on CMB@APBA through the formation of borate ester bond. The formed boric acid–esterified gcDNA can further bind with complementary CRISPR RNA through A-T base pairing to activate Cas12a with k_cat/K_m ratio of 3.4 × 10⁷ s⁻¹ M⁻¹, allowing for amplified signal. Hg²⁺ can specifically combine with CMB@APBA, resulting in the release of gcDNA from CMB@APBA and the following inhibition on the activation of CRISPR/Cas12a system around magnetic bead. Under optimal conditions, the method exhibits a linear range from 20 to 250 nM, with a detection limit of 2.72 nM. The proposed method can detect Hg²⁺ in milk and tea beverages, providing a great significance for on-site monitoring of Hg²⁺ contamination in food.