Metal ion-complexed DNA probe coupled with CRISPR/Cas12a amplification and AuNPs for sensitive colorimetric assay of metallothionein in fish

The accurate and sensitive detection of metallothionein (MT) is of great significance in the fields of biomedical, toxicological and environmental sciences. In this work, based on the high affinity interaction between MT and the heavy metal ions of Hg 2 + and the significant signal amplification capability of Cas12a/crRNA enzyme as well, we report a simple and highly sensitive method for visual detection of MT, a biomarker in fish for heavy metal ion -induced water bio-pollution. The target MT molecules bind Hg 2 + in the Hg 2 + - complexed hairpin DNA probes to unfold the hairpin structure into ssDNAs, which hybridize with the partial dsDNA duplexes via strand displacement to yield specific sequence -containing dsDNAs. Cas12a/crRNA recognizes these specific sequences to activate its enzyme activity to cyclically cleave the ssDNA linkers in the blue colored gold nanoparticle aggregates to transit their color into red to realize visual detection of MT. Owing to the signal amplification by Cas12a/crRNA, as low as 25 nM of MT can be visually detected with naked eye. In addition, our colorimetric detection method has high selectivity for MT against other interference proteins and can detect MT in the livers and kidneys of crucian carps bought from a local supermarket. Moreover, the developed assay overcomes the limitations of conventional MT detection methods in terms of complexity, high cost and low sensitivity and can therefore offer new methods for monitoring water bio-pollutions.