PROJECT SUMMARY The use of 10-14 day continuous glucose monitors for diabetes management is a historical achievement in modern diagnostics, but unfortunately it remains an isolated success despite acute needs for the real-time monitoring of many other molecules across the broader field of human disease management (cardiac, drug dosing, fertility, etc.). The limitation is that glucose sensors are enzymatic, limiting their generalizability to other analytes (i.e., enzymes oxidize/reduce the target molecule). Unlike enzymatic sensors, electrochemical aptamer-based (EAB) sensors are broadly generalizable, demonstrated by several examples of real-time, in- vivo molecular monitoring at nanomolar to micromolar concentrations. Unfortunately, in-vivo device longevity remains a significant challenge for the clinical adoption of EAB sensors. EAB sensors conventionally use a monolayer of redox-tagged aptamers and alkylthiol blocking molecules on a gold working electrode. These self-assembled monolayers (SAMs) rapidly degrade on gold electrodes in real biofluids at body temperature of 37 °C. The mechanisms of degradation of EAB SAMs have not been well- understood in complex biofluids, which then limits the ability to pursue techniques to improve longevity. Our preliminary data now provides major insights into the true mechanisms of SAM degradation. With this improved understanding of degradation, its is now feasible to pursue stable operation for at least 5 days. Multi-day operation would then allow EABs to be credibly pursued for applications beyond glucose, and for the first time, proper research could begin on resolving the next expected longevity bottlenecks that would likely prevent 1-2 week operation (e.g. fouling, nuclease attack, etc.). The central hypothesis is that at minimum 5 day EAB sensor operation can be achieved through a blocking layer with superior stability achieved by either (1) electrochemically stabilizing an alkylthiol blocking layer during sensor fabrication, or (2) replacing an alkylthiol blocking layer with a inorganic dielectric film that has a similar density of defects supporting efficient electron transfer. 5 day operation would provide a leap forward in clinical relevance, and is 10-20X greater the typical limit of 6-12 hours. 5 day operation would then position the PI Heikenfeld to pursue clinical research, and would ignite critically-needed partnerships with industry leaders in glucose sensors. The PI Heikenfeld is a new NIH investigator, but is well-prepared to pursue this longevity breakthrough given his deep expertise in biosensors, and his co-PI’s White and Porter’s expertise in EAB sensors and electrochemical blocking layers.

PROJECT NARRATIVE This proposal will create breakthrough stability for the blocking-layers used in aptamer-based biosensors, and use that improved stability to: (1) achieve at least 5-day operating longevity for the biosensors; (2) create new fundamental knowledge of biofouling kinetics and other sensor degradation mechanisms. With improved operating longevity, aptamer biosensors are then well-positioned to mirror the historical achievement for 10-14 day continuous glucose meters, but instead applied to new applications such as cardiac health and drug metabolism monitoring.