Explaining the Decay of Nucleic Acid-Based Sensors under Continuous Voltammetric Interrogation

Abstract

Nucleic acid-based electrochemical sensors (NBEs) can support continuous and highly selective molecular monitoring in biological fluids, both in vitro and in vivo, via affinity-based interactions. Such interactions afford a sensing versatility that is not supported by strategies that depend on target-specific reactivity. Thus, NBEs have significantly expanded the scope of molecules that can be monitored continuously in biological systems. However, the technology is limited by the lability of the thiol-based monolayers employed for sensor fabrication. Seeking to understand the main drivers of monolayer degradation, we studied four possible mechanisms of NBE decay: (i) passive desorption of monolayer elements in undisturbed sensors, (ii) voltage-induced desorption under continuous voltammetric interrogation, (iii) competitive displacement by thiolated molecules naturally present in biofluids like serum, and (iv) protein binding. Our results indicate that voltage-induced desorption of monolayer elements is the main mechanism by which NBEs decay in phosphate-buffered saline. This degradation can be overcome by using a voltage window contained between −0.2 and 0.2 V vs Ag|AgCl, reported for the first time in this work, where electrochemical oxygen reduction and surface gold oxidation cannot occur. This result underscores the need for chemically stable redox reporters with more positive reduction potentials than the benchmark methylene blue and the ability to cycle thousands of times between redox states to support continuous sensing for long periods. Additionally, in biofluids, the rate of sensor decay is further accelerated by the presence of thiolated small molecules like cysteine and glutathione, which can competitively displace monolayer elements even in the absence of voltage-induced damage. We hope that this work will serve as a framework to inspire future development of novel sensor interfaces aiming to eliminate the mechanisms of signal decay in NBEs.

Building a Nucleic Acid Nanostructure with DNA–Epitope Conjugates for a Versatile Approach to Electrochemical Protein Detection

“…Interestingly, this sensor’s response to antibody addition was smaller than other antibody sensing responses in our previous reports, , such that the 10–15% signal drifting effect in buffer became more prominent (Figure , marked region). The signal drift in these and similar sensors is well-known and is the subject of ongoing research in multiple groups. − Drift can be accounted for in calibrations, and recent work has proven that electrochemical desorption of the monolayers on the electrode surface can be a major driving force for the signal drift . Since we carried out SWV at multiple frequencies and scanned from −0.450 to 0 V in all measurements, it is likely that EC desorption of MCH or thiol-DNA was a significant factor in our work as well.…”

Perspective—The Feasibility of Continuous Protein Monitoring in Interstitial Fluid

“…28 Researchers recently reported no loss in DNA signal following continuous interrogation in the voltage window of −0.2 V to 0.2 V vs Ag|AgCl. 26 While promising, this voltage range is outside the reduction potential of methylene blue, the benchmark redox tag. Recent work in the field led to the development of novel redox tags ECS Sensors Plus, 2023 2 027001 including, a tetrathiafulvalene based reporter (E°' = 0.05 V vs Ag|AgCl), 29 and an osmium-based redox reporter(E°' = 0.18 V vs Ag|AgCl).…”

“…L-cysteine) commonly found in biofluids. 26 Scanning sensors at negative potentials is one major source of sensor degradation caused by: the reduction of oxygen into reactive oxygen species, thereby catalyzing the loss of monolayer elements from the sensor surface, 27 the phosphate backbone of DNA being strained away from the electrode surface causing electrochemical desorption of aptamer, 25 and increased fouling from attracted proteins. 28 Researchers recently reported no loss in DNA signal following continuous interrogation in the voltage window of −0.2 V to 0.2 V vs Ag|AgCl.…”

Au‐Modified Carbon Electrodes Produced by Laser Scribing for Electrochemical Analysis of Probiotic Activity

“…[20,28,42,43] Thus, gold was selected as the best material because it produced the lowest electrode resistance while opening the door for other analytical applications. [44,45] Thus, all electrochemical experiments described below were performed using Au-modified carbon electrodes and were all produced by the laser scribing method.…”