Hydrogel Encapsulation of a Designed Fluorescent Protein Biosensor for Continuous Measurements of Sub-100 nM Nicotine

The reinforcing, addictive, and therapeutic properties of nicotine depend strongly on the concentration and time dependence of nicotine in human biofluids. This pattern, termed [nicotine]_t, varies markedly among methods of nicotine consumption and, for a given method, among individual users. Measuring [nicotine]_t presents challenges: current approaches are expensive, invasive, tedious, and discontinuous. We report the entrapment of a purified, previously developed fluorescent biosensor protein, iNicSnFR12, in hydrogels. We optimized poly(ethylene glycol) diacrylate hydrogels for optical clarity and straightforward slicing. With fluorescence photometry of the hydrogels in a microscope or an integrated miniscope, [nicotine] is detected within a few min at the smoking- and vaping-relevant level of 10-100 nM (1.62-16.2 ng/mL), in a 250 μm thick hydrogel at the end of a 400 μm diameter multimode fiber optic. Concentration-response relationships are consistent with previous measurements of isolated iNicSnFR12. Leaching of iNicSnFR12 from the hydrogel and inactivation of iNicSnFR12 are minimal for several days, and reversible nicotine-induced fluorescence occurs at least 10 months after casting. This work provides the molecular, photophysical, and mechanical bases for minimally invasive, personal, wearable, continuous [nicotine] monitoring with straightforward extensions to existing, homologous "iDrugSnFR" proteins for other abused and prescribed drugs.