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Change in the dynamics of single-stranded DNA or RNA probes tethered to an Au electrode on immunospecific binding to the analyte is a versatile approach to quantify a variety of molecules, such as heavy metal ions, pesticides, proteins, and nucleic acids (NAs). A widely studied approach is the electrochemical beacon method where the redox of a dye attached to the probe decreases as its proximity to the underlying electrode changes on binding. The limit of quantification (LOQ) defined by the semilog dependence of the signal on target concentration is in the picomolar range. Here, a method was studied where, by differential reflectivity, multiple reactions were measured on a monolith electrode. An alternative contrast mechanism was discovered, which led to an approach to enhance the LOQ to 10 aM and increase the dynamic range to 7 orders of magnitude using similar probes and binding conditions. Quantitative analysis on sequences with the G-C fraction ranging from 37 to 72% was performed. The approach will allow for the development of a label-free, enzyme-free microarray to detect biomolecules including NAs and proteins on a single electrode at quantification from 10 aM to 0.1 nM with high specificity.

Electrochemical characteristics of immobilized double-stranded DNA (dsDNA) on a Au electrode were studied as a function of coverage using a home-built optoelectrochemical method. The method allows probing of local redox processes on a 6 μm spot by measuring both differential reflectivity (SEED-R) and interferometry (SEED-I). The former is sensitive to redox ions that tend to adsorb to the electrode, while SEED-I is sensitive to nonadsorbing ions. The redox reaction maxima, R(max) and Δ(max) from SEED-R and SEED-I, respectively, are linearly proportional to amperometric peak current, I(max). The DNA binding is measured by a redox active dye, methylene blue, that intercalates in dsDNA, leading to an R(max). Concomitantly, the absence of Δ(max) for [Fe(CN)(6)](4−/3−) by SEED-I ensures that there is no leakage current from voids/defects in the alkanethiol passivation layer at the same spot of measurement. The binding was regulated electrochemically to obtain the binding fraction, f, ranging about three orders of magnitude. A remarkably sharp transition, f = f(T) = 1.25 × 10(−3), was observed. Below f(T), dsDNA molecules behaved as individual single-molecule nanoelectrodes. Above the crossover transition, R(max), per dsDNA molecule dropped rapidly as f(−1/2) toward a planar-like monolayer. The SEED-R peak at f ~ 3.3 × 10(−4) (~270 dsDNA molecules) was (statistically) robust, corresponding to a responsivity of ~0.45 zeptomoles of dsDNA/spot. Differential pulse voltammetry in the single-molecule regime estimated that the current per dsDNA molecule was ~4.1 fA. Compared with published amperometric results, the reported semilogarithmic dependence on target concentration is in the f > f(T) regime.