Nichols, A. L.; Marotta, C. B.; Wagenaar, D. A.; Mayo, S. L.; Dougherty, D. A.; Lester, H. A.

The reinforcing and addictive properties of nicotine result from concentration- and time-dependent activation, desensitization, and upregulation of nicotinic acetylcholine receptors. However, time-resolved [nicotine] measurement in people who consume nicotine is challenging, as current approaches are expensive, invasive, tedious, and discontinuous. To address the challenge of continuous nicotine monitoring in human biofluids, we report the encapsulation of a purified, previously developed fluorescent biosensor protein, iNicSnFR12, into acrylamide hydrogels and PEGDA hydrogels. We optimized the hydrogels for optical clarity and straightforward slicing. With fluorescence photometry of the hydrogels in microscopes and a miniscope, [nicotine] is detected at the smoking- and vaping- relevant level of 10 - 100 nM (1.62 - 16.2 ng/ml). Concentration-response relations are consistent with previous measurements on isolated iNicSnFR12. Leaching of iNicSnFR12 from the hydrogel and inactivation of iNicSnFR are minimal for several days, and nicotine can be detected for at least 10 months after casting. This work provides the molecular, photophysical, and instrumental bases for personal, wearable continuous [nicotine] monitoring, with straightforward extensions to existing, homologous \'iDrugSnFR\' proteins for other abused and prescribed drugs.