Reverse-engineered ratiometric sensor assisted by dual redox-active DNA intercalators for ultrasensitive and reliable...

Traditional electrochemical DNA sensors for Hg²⁺ rely on target-induced conformational changes of Hg²⁺-binding DNA probes, which often suffer from limited signal output and complex probe design. To address these issues, this study introduces a novel ratiometric electrochemical DNA sensor based on a "reverse-engineering" strategy, where the signal molecule is pre-immobilized on the electrode instead of the Hg²⁺-binding DNA.

The double-helix DNA intercalator Nile blue (NB) with excellent redox activity was covalently grafted onto a gold electrode to generate an intense inherent signal. In addition, in a solution containing target Hg²⁺, the polythymine DNA modified onto gold nanoparticles (AuNP@polyT) undergoes a conformational transition to a double-helix structure via specific T-Hg²⁺-T chemistry. Then the AuNP@polyT-Hg²⁺ is captured onto the electrode surface through intercalation between NB and the Hg²⁺-induced DNA duplex, leading to a decrease in the NB electrochemical signal. Subsequently, another intercalator, methylene blue (MB), is adsorbed into the remaining base pair space of polyT-Hg²⁺, generating a new indicating signal. A ratiometric response for Hg²⁺ is thus achieved through the decrease in the NB signal and the increase in the MB signal. Within the Hg²⁺ concentration range of 0.1 nM to 100 μM, log(I_NB/I_MB) exhibits a favorable double logarithmic linear relationship with the logarithm of Hg²⁺ concentration, achieving a detection limit as low as 40 pM. The sensor was successfully applied to Hg²⁺ analysis in actual water and tea samples.

By combining T-Hg²⁺-T coordination chemistry with a dual-intercalator ratiometric readout, the proposed reverse-engineered ratiometric electrochemical sensor eliminates complex DNA probe immobilization, improves accuracy and anti-interference capability, and shows great promise for on-site monitoring of Hg²⁺ in environmental water and tea samples.