Surface Charge Effects to Differentiate Structurally Similar Chemicals with Electrochemical Aptamer Biosensors

Electrochemical aptamer-based sensors (EABs) can directly detect a wide variety of targets in both in vitro and in vivo settings, ranging from small molecules to whole cells. However, these biosensors often face cross-reactivity issues when measuring targets in samples containing chemically similar molecules, such as precursors or metabolites. While progress has been made in selecting highly specific aptamers, discovering these reagents remains slow and costly. In this talk, we demonstrate a novel strategy to distinguish molecules with minuscule differences in chemical composition by tuning the charge state of the surface on which aptamer probes are immobilized. First, we show that this strategy successfully differentiates doxorubicin (DOX) from many structurally similar analytes, including its primary metabolite, doxorubicinol (DOXol). We further investigate the effects of monovalent and short-chained zwitterionic molecules on EAB sensing to distinguish structurally similar aminoglycosides, demonstrating enhanced robustness in biological media. To improve specificity further, we integrated various blocking layers into nanoporous gold electrodes. These modifications allowed us to interrogate unique signal traces arising from chemical interactions between the aptamers and different monolayers. We found that incorporating negatively charged monolayers can cause the aptamer to switch its binding preference from the primary target to a secondary target. Conversely, partially positive monolayers can restrict aptamer binding altogether. The findings presented in this work will aid in the development of next-generation EABs capable of multiplex sensing of therapeutics for wearable or in situ sensing applications. By modifying the local environment with tailored blocking layers, we can achieve enhanced specificity and performance in complex biological systems.