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

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 (ΔEP) 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 ΔEP. 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.