Exonuclease III-assisted CRISPR/Cas12a electrochemiluminescence biosensor for sub-femtomolar mercury ions determination

A novel ECL biosensor was developed for ultrasensitive mercury ions (Hg2+) determination based on gold nanoparticles-coated nitrogen-doped carbon dots ([email protected]) as luminophores and exonuclease III (Exo III) as well as CRISPR/Cas12a as signal amplifiers. The biosensor was fabricated by pre-modifying [email protected] hybrids and ferrocene-labeled single-stranded DNA (Fc-ssDNA) layer in sequence on the interface of a gold working electrode. The initial [email protected] ECL of the biosensor is quenched by Fc. Then, a thymine-rich ssDNA probe was designed for the recognition of Hg2+. In the presence of Hg2+, this thymine-rich ssDNA probe was folded to a DNA duplex based on T-Hg2+-T mismatches. Then, Exo III catalyzes the digestion of duplex of hairpin DNA, liberating short single-stranded DNA (ssDNA) product and releasing Hg2+. The produced ssDNA can be recognized by CRISPR/Cas12a and further trigger the collateral cleavage ability of CRISPR system, which can indiscriminately cut short Fc-ssDNA on the electrode, producing an ECL signal of [email protected] Under the optimized conditions, a detection limit of 0.45 fM was realized for Hg2+ determination. The efficiency and robustness of this biosensor were further verified by using environmental water and human serum samples, which demonstrated its potential application in the environment and biological science.