In this work, we introduce the use of intermittent pulse amperometry (IPA) as a technique for the millisecond (ms) interrogation of electrochemical, aptamer-based (E-AB) sensors. This type of regenerative and robust sensor uses thiolated oligonucleotides (DNA or RNA) with a distal-appended redox reporter. Target-induced conformation changes alter the charge transfer rate between the redox reporter attached to the aptamer and the underlying electrode surface. We show that IPA allowed quantification of the observed binding rates (Kobs on) for two representative E-AB sensors for small molecule target (the aminoglycoside antibiotic, tobramycin). The introduction of IPA to monitor this type of sensor extensively improves the temporal resolution offered by traditional voltammetric methods, that provide at best responses on the second (s) timescale. To take advantage of the rapid interrogation (ms timescale) made possible by IPA, we combined E-AB sensors with a flow injection analysis platform to determine Kobs, the bimolecular rate constant of aptamer-target binding (kon), and the monomolecular rate constant of dissociation (koff) for small molecule targets. Traditional techniques to study surface binding kinetics often fail to determine apparent binding affinities, and therefore this limits a complete understanding of molecular recognition parameters such as the kinetics of target-induced conformation changes of aptamers, binding stoichiometries, and equilibrium constants. The use of IPA allowed a label-free approach to determine fast-binding kinetics in the ms timescale for small molecule targets, on a real aptamer-modified electrode surface.

Fast interrogation of electrochemical, aptamer-based (E-AB) sensors for the characterization of small molecule-aptamer binding kinetics

2019 Fall National Meeting

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