Tailoring the Interfacial Ionic Microenvironment of the ssDNA Brush to Quantify 250 Zeptomoles of Targets

A promising approach for the electrochemical detection of nucleic acids is to measure changes in the redox behavior of methylene blue (MB) tethered to the free end of an ssDNA probe immobilized on an Au electrode via an Au-S bond. The change in redox current results from the modulation of MB proximity to the electrode due to conformational changes upon probe-target binding. A critical limitation of these electrochemical beacon methods is the intrinsic conformational heterogeneity of probes that are partly adsorbed (DN) and standing-up (UP), which limits sensitivity and reliability. An opto-electrochemical instrument called SEED has been developed, in which a fast scan at 400 V/s inhibits (diffusion-controlled) MB redox from the UP chains to deconvolute the heterogeneity effect. The optical contrast is due to color flip-flops between blue MB and colorless leucomethylene-blue; thus, the background, due to high capacitance from ion vibration at a fast scan rate, is eliminated. To evaluate sensing performance, irrespective of sample volume and binding conditions, the sensitivity to the percentage of probe binding is measured and compared with cyclic voltammetry (CV). A "reactivity amplification" was discovered where ∼2% binding produces a 27% change in the SEED signal, enabling detection of 250 zeptomoles of binding on a ∼20 µm spot. The study underscores and leverages the subtle effects of the interfacial ionic microenvironment, especially the local pH, to rationally identify the appropriate buffer chemistry. By measuring local redox, SEED allows combinatorial electrochemical sensing of binding across an array of different probe sequences on a monolithic electrode.