The desire to improve and decentralize diagnostic platforms to facilitate highly precise and personalized medicine has motivated the development of a large number of electrochemical sensing technologies. Such a development has been facilitated by electrochemistry's unparalleled ability to achieve highly specific molecular measurements in complex biological fluids, without the need for expensive instrumentation. However, for decades, progress in the field had been constrained to systems that depended on the chemical reactivity of the analyte, obstructing the generalizability of such platforms beyond redox- or enzymatically active clinical targets. Thus, the pursuit of alternative, more general strategies, coupled to the timely technological advances in DNA sequencing, led to the development of DNA-based electrochemical sensors. The analytical value of these arises from the structural customizability of DNA and its ability to bind analytes ranging from ions and small molecules to whole proteins and cells. This versatility extends to interrogation methods, as DNA-based sensors work through a variety of detection schemes that can be probed via many electroanalytical techniques. As a reference for those experienced in the field, and to guide the unexperienced scientist, here we review the specific advantages of the electroanalytical methods most commonly used for the interrogation of DNA-based sensors.

Electrochemical, aptamer-based (E-AB) sensors support continuous, real-time measurements of specific molecular targets in complex fluids such as undiluted serum. They achieve these measurements by using redox-reporter-modified, electrode-attached aptamers that undergo target binding-induced conformational changes which, in turn, change electron transfer between the reporter and the sensor surface. Traditionally, E-AB sensors are interrogated via pulse voltammetry to monitor binding-induced changes in transfer kinetics. While these pulse techniques are sensitive to changes in electron transfer, they also respond to progressive changes in the sensor surface driven by biofouling or monolayer desorption and, consequently, present a significant drift. Moreover, we have empirically observed that differential voltage pulsing can accelerate monolayer desorption from the sensor surface, presumably via field-induced actuation of aptamers. Here, in contrast, we demonstrate the potential advantages of employing cyclic voltammetry to measure electron-transfer changes directly. In our approach, the target concentration is reported via changes in the peak-to-peak separation, ΔE_P, of cyclic voltammograms. Because the magnitude of ΔE_P is insensitive to variations in the number of aptamer probes on the electrode, ΔE_P-interrogated E-AB sensors are resistant to drift and show decreased batch-to-batch and day-to-day variability in sensor performance. Moreover, ΔE_P-based measurements can also be performed in a few hundred milliseconds and are, thus, competitive with other subsecond interrogation strategies such as chronoamperometry but with the added benefit of retaining sensor capacitance information that can report on monolayer stability over time.

The measurement of serum vancomycin levels at the clinic is critical to optimizing dosing given the narrow therapeutic window of this antibiotic. Current approaches to quantitate serum vancomycin levels are based on immunoassays, which are multistep methods requiring extensive processing of patient samples. As an alternative, vancomycin-binding electrochemical, aptamer-based sensors (E-ABs) were developed to simplify the workflow of vancomycin monitoring. E-ABs enable the instantaneous measurement of serum vancomycin concentrations without the need for sample dilution or other processing steps. However, the originally reported vancomycin-binding E-ABs had a dissociation constant of 45 μM, which is approximately 1 order of magnitude higher than the recommended trough concentrations of vancomycin measured in patients. This limited sensitivity hinders the ability of E-ABs to accurately support vancomycin monitoring. To overcome this problem, here we sought to optimize the length of the vancomycin-binding aptamer sequence to enable a broader dynamic range in the E-AB platform. Our results demonstrate, via isothermal calorimetry and E-AB calibrations in undiluted serum, that superior affinity and near-equal sensor gain in vitro can be achieved using a one-base-pair-longer aptamer than the truncated sequence originally reported. We validate the impact of the improved binding affinity in vivo by monitoring vancomycin levels in the brain cortex of live mice following intravenous administration. While the original sequence fails to resolve vancomycin concentrations from baseline noise (SNR = 1.03), our newly reported sequence provides an SNR of 1.62 at the same dose.

Electrochromic materials can be used in self-powered electrochemical sensors to display quantitative information without the need for silicon-based electronics or external instrumentation.