Chemical Equilibrium-Based Mechanism for the Electrochemical Reduction of DNA-Bound Methylene Blue Explains Double Redox Waves in Voltammetry

Abstract

Methylene blue is widely used as a redox reporter in DNA-based electrochemical sensors and, in particular, it is the benchmark DNA-bound reporter used in electrochemical, aptamer-based sensors (E-ABs). Our group recently published an approach to interrogate E-ABs via cyclic voltammetry, which uses the cathodic to anodic peak-to-peak voltage separation (ΔE P) from methylene blue to report on the electron-transfer kinetics and binding state of these sensors. Although effective at scanning rates ≤10 V·s–1, the method is limited at faster scanning rates because cyclic voltammograms of methylene blue-modified, electrode-bound DNA present double faradaic waves that prevent the accurate estimation of ΔE P. These double waves have been observed in previous works, but their origin was unknown. In response, here we investigated the origin of these redox waves by developing a numerical model that incorporates methylene blue’s chemical equilibria in phosphate buffer to predict the shape and magnitude of cyclic voltammograms with 85% or better accuracy from single- and double-stranded DNA. Our model confirms that the peak splitting observed at scanning rates >10 V·s–1 originates from the protonation equilibrium of the radical intermediate species formed after methylene blue receives the first electron. Moreover, the model reveals a strong interaction between the proton transferred during the reduction of methylene blue and the chemical make of blocking self-assembled monolayers typically used in the fabrication of E-ABs. This interaction affects the apparent rate of the first electron-transfer step, accelerating or decelerating it depending on the hydrophobicity and polarity of the blocking monolayer. By expanding our understanding of the effect that monolayer chemistries have on methylene blue’s protonation rates and E-AB signaling, this work may serve the rational design of future sensors with tunable electron-transfer kinetics.

Study of surface modification strategies to create glassy carbon-supported, aptamer-based sensors for continuous molecular monitoring

“…Likewise, strong dampening of the faradaic current from ferricyanide also occurred with mercaptohexanoic acid monolayers, which contain surface carboxylic groups that are negatively charged at the pH of our electrolyte (pH = 7.4). Electrostatic repulsion prevents ferricyanide molecules (which are negatively charged) from approaching the electrode surface to transfer electrons, dampening the net faradaic current regardless of packing density (which is approximately the same as that achieved by mercaptohexanol monolayers) [ 41 ]. Thus, the approach of measuring electron transfer from ferricyanide to determine the extent of surface coverage by a given monolayer can only be used qualitatively.…”

Redox Reporter ‐ Ligand Competition to Support Signaling in the Cocaine‐Binding Electrochemical Aptamer‐Based Biosensor

“…The widespread use of methylene blue stems from a redox potential that is conveniently positioned in a potential window devoid of other parasitic electrochemical processes (i. e., oxygen reduction and gold oxidation) on gold electrodes coated with an alkanethiol self‐assembled monolayer. Though undergoing a complex proton‐coupled electron transfer mechanism, [16] methylene blue strikingly shows, in contrast to other well‐known outer‐sphere redox reporters like ferrocene, reversible and stable electrochemical behavior when attached to DNA [13] . E‐AB sensors relying on methylene blue, however, show dissociation constants several order of magnitudes greater than values determined using solution‐based techniques (i. e., isothermal titration calorimetry (ITC), fluorescence, etc.…”

The challenge of long-term stability for nucleic acid-based electrochemical sensors

“…However, its negative reduction potential overlaps with the reduction of molecular oxygen (which includes the formation of the highly reactive radical superoxide, Figure 1c ), a reaction that likely accelerates the degradation of the underlying monolayer [ 12 ]. Additionally, because methylene blue undergoes a proton transfer during reduction, signaling with methylene blue is pH dependent [ 27 ]. Therefore, finding alternative redox reporters with a more positive reduction potential and insensitivity to sample pH remains of interest in the field.…”