Electrochemical aptamer-based (E-AB) biosensors afford
real-time
measurements of the concentrations of molecules directly in complex
matrices and in the body, offering alternative strategies to develop
innovative personalized medicine tools. While different electroanalytical
techniques have been used to interrogate E-AB sensors (i.e., cyclic
voltammetry, electrochemical impedance spectroscopy, and chronoamperometry)
to resolve the change in electron transfer of the aptamer’s
covalently attached redox reporter, square-wave voltammetry remains
a widely used technique due to its ability to maximize the redox reporter’s
faradic contribution to the measured current. Several E-AB sensors
interrogated with this technique, however, show lower aptamer affinity
(i.e., μM–mM) even in the face of employing aptamers
that have high affinities (i.e., nM−μM) when characterized
using solution techniques such as isothermal titration calorimetry
(ITC) or fluorescence spectroscopy. Given past reports showing that
E-AB sensor’s response is dependent on square-wave interrogation
parameters (i.e., frequency and amplitude), we hypothesized that the
difference in dissociation constants measured with solution techniques
stemmed from the electrochemical interrogation technique itself. In
response, we decided to compare six dissociation constants of aptamers
when characterized in solution with ITC and when interrogated on electrodes
with electrochemical impedance spectroscopy, a technique able to,
in contrast to square-wave voltammetry, deconvolute and quantify E-AB
sensors’ contributions to the measured current. In doing so,
we found that we were able to measure dissociation constants that
were either separated by 2–3-fold or within experimental errors.
These results are in contrast with square-wave voltammetry-measured
dissociation constants that are at the most separated by 2–3
orders of magnitude from ones measured by ITC. We thus envision that
the versatility and time scales covered by electrochemical impedance
spectroscopy offer the highest sensitivity to measure target binding
in electrochemical biosensors relying on changes in electron-transfer
rates.
Electrochemical aptamer‐based (E‐AB) biosensors have demonstrated capabilities in monitoring molecules directly in undiluted complex matrices and in the body with the hopes of addressing personalized medicine challenges. This sensing platform relies on an electrode‐bound, redox‐reporter‐modified aptamer. The electrochemical signal is thought to originate from the aptamer undergoing a binding‐induced conformational change capable of moving the redox reporter closer to the electrode surface. While this is the generally accepted mechanism, it is notable that there is limited evidence demonstrating conformational change or distance‐dependent change in electron transfer rates in E‐AB sensors. In response, we investigate here the signal transduction of the well‐studied cocaine‐binding aptamer with different analytical methods and found that this sensor relies on a redox‐reporter ‐ ligand competition mechanism rather than a ligand‐induced structure formation mechanism. Our results show that the covalently bound redox reporter, methylene blue, binds at or near the ligand binding site on the aptamer resulting in a folded conformation of the cocaine‐binding aptamer. Addition of ligand then competes with the redox reporter for binding, altering its electron transfer rate. While we show this for the cocaine‐binding aptamer, given the prevalence of methylene blue in E‐AB sensors, a similar competition‐based may occur in other systems.
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