Programming a Target-Initiated Bifunctional DNAzyme Nanodevice for Sensitive Ratiometric Electrochemical Biosensing

Here, a bifunctional DNAzyme nanodevice (BFDN) with two detection paths toward the same target was intelligently designed and applied to construct a ratiometric electrochemical biosensor for highly reliable and sensitive mercury ion (Hg²⁺) detection. In the presence of the target Hg²⁺, the T-Hg²⁺-T pair could actuate the preassembled DNA four-branched nanostructure (DNA-4B) without cleavage capability transform to the BFDN with strong cleavage capability for triggering two synchronous Hg²⁺ detection paths, including a "signal-off" path (1) that consisted of a cascade DNAzyme cleavage reaction to dramatically decrease the ferrocene (Fc) response and a "signal-on" path (2) that accomplished the capture of significant amounts of methylene blue (MB) on the electrode surface under the assistant of DNAzyme2 (D2) in BFDN. This strategy not only effectively avoided the false positive signal compared with traditional single paths, but also proposed a new ratiometric method to successfully circumvent the deficiency that existed in previous ratiometric electrochemical biosensors. As a result, the reliable and sensitive Hg²⁺ detection was achieved in the range from 0.1 pM to 200 nM with a detection limit of 23 fM. Above all, here, the assembly of the BFDN is ingeniously coupled with amplification strategy, paving a promising avenue to promote the performances of simple multifunctional DNA nanomachines and facilitate the corresponding development of DNA nanomachines in biosensor platform.