Development of a DNAzyme-based colorimetric biosensor assay for dual detection of Cd²⁺ and Hg²

A colorimetric biosensor assay has been developed for Cd²⁺ and Hg²⁺ detection based on Cd²⁺-dependent DNAzyme cleavage and Hg²⁺-binding-induced conformational switching of the G-quadruplex fragment. Two types of multifunctional magnetic beads (Cd-MBs and Hg-MBs) were synthesized by immobilizing two functionalized DNA sequences on magnetic beads via avidin-biotin chemistry. For Cd²⁺ detection, Cd-MBs are used as recognition probes, which are modified with a single phosphorothioate ribonucleobase (rA) substrate (PS substrate) and a Cd²⁺-specific DNAzyme (Cdzyme). In the presence of Cd²⁺, the PS substrate is cleaved by Cdzyme, and single-stranded DNA is released as the signal transduction sequence. After molecular assembly with the other two oligonucleotides, duplex DNA is produced, and it can be recognized and cleaved by FokI endonuclease. Thus, a signal output component consisting of a G-quadruplex fragment is released, which catalyzes the oxidation of ABTS with the addition of hemin and H₂O₂, inducing a remarkably amplified colorimetric signal. To rule out false-positive results and reduce interference signals, Hg-MBs modified with poly-T fragments were used as Hg²⁺ accumulation probes during the course of Cd²⁺ detection. On the other hand, Hg-MBs can perform their second function in Hg²⁺ detection by changing the catalytic activity of the G-quadruplex/hemin DNAzyme. In the presence of Hg²⁺, the G-quadruplex structure in Hg-MBs is disrupted upon Hg²⁺ binding. In the absence of Hg²⁺, an intensified color change can be observed by the naked eye for the formation of intact G-quadruplex/hemin DNAzymes. The biosensor assay exhibits excellent selectivity and high sensitivity. The detection limits for Cd²⁺ and Hg²⁺ are 1.9 nM and 19.5 nM, respectively. Moreover, the constructed sensors were used to detect environmental water samples, and the results indicate that the detection system is reliable and could be further used in environmental monitoring. The design strategy reported in this study could broadly extend the application of metal ion-specific DNAzyme-based biosensors.