Voltammetric Analysis of Quasi-Reversible Kinetics in Aptamer Self-Assembled Monolayers: Toward Rational E-AB Sensor Design

Electrochemical aptamer-based (E-AB) sensors are surface-bound systems, in which electron-transfer kinetics are quasi-reversible on typical measurement timescales. This combination represents a critical process; therefore, a systematic understanding of these processes is essential for the rational design and performance optimization of E-AB sensors; however, thorough investigations in this area remain limited. Here, we present a comprehensive voltammetric characterization of an E-AB sensor to detect vancomycin as a model target. By employing cyclic voltammetry (CV) and square wave voltammetry (SWV), we delineate how the charge transfer kinetics and aptamer conformational dynamics influence the overall sensor response. A detailed analysis of the cyclic voltammetry (CV) results, using the Laviron method and peak-to-peak separation trends, enables quantification of the heterogeneous electron-transfer rate constants (ks) in the target-free ("unbound") state as ks, ox = 10.3 ± 3.3 s⁻¹ and ks, red = 6.8 ± 2.2 s⁻¹; in contrast, target binding increased these values, reaching ks, ox = 43.6 ± 14.8 s⁻¹ and ks, red = 45.6 ± 11 s⁻¹. Additionally, our analysis reveals that aptamer packing density cannot be measured independently from the sensor's peak current, highlighting the necessity for a calibration approach independent of aptamer surface coverage. We further demonstrate a ratiometric SWV approach using two distinct frequencies, enabling robust calibration and mitigating sensor-to-sensor variability. This method reduced the relative standard deviation (RSD) to 2.2% (n = 9 sensors) compared to 10.5% for absolute current values. Taken together, this work provides insights into E-AB sensor operation, facilitating rational optimization of sensor design and interrogation strategies for clinical and industrial applications.