Computational Design of Protein-Based Biosensors for Nicotine

Author: Friesenhahn, Nicholas James

Year: 2026

Degree: Dissertation (Ph.D.)

Advisor: Mayo, Stephen L.

Committee Members: Shapiro, Mikhail G.; Chou, Tsui-Fen; Mayo, Stephen L.; Tirrell, David A.

Option: Chemical Engineering

DOI: 10.7907/mw91-pe18

Abstract

It has been observed that among individual users of nicotine-containing products and between nicotine delivery systems (smoking, vaping, patches, etc.), there is a large degree of variability in the pharmacokinetic profile of nicotine. The time course of [nicotine], [nicotine]t, is crucial to understanding why some nicotine-containing products are addictive, while others aid in smoking cessation via nicotine-replacement therapy. The current methods for measuring [nicotine]t via blood draws and LC-MS analysis are cost-prohibitive for the number of nicotine users, estimated at 1.2 billion globally. To enable [nicotine]t measurements at the necessary scale, we have developed a series of protein-based intensity-based nicotine sensing fluorescent reporters (iNicSnFRs). These sensors are comprised of a nicotine-binding moiety engineered from the OpuBC periplasmic binding protein of Thermoanaerobacter sp. X513 and circularly permuted superfolderGFP. Through a variety of computational protein design and experimental techniques, we discovered mutations that improved the biosensor’s sensitivity, as measured by EC50, 61-fold, from iNicSnFR3a with a 27 μM EC50 to iNicSnFR12-D72A with a 440 nM EC50. Notably, iNicSnFR12-D72A, with its series-best 11.6 μM-1 fluorescence response in the linear regime of the dose-response relation, is able to resolve 10 nM nicotine, the lowest [nicotine] measured between smoking and vaping sessions in plasma via the blood draw method. With unparalleled sensitivity and robust thermostability profile, iNicSnFR12-D72A meets many requirements for a sensor to be used in biofluids as a continuous nicotine monitor, an important tool in reducing nicotine addiction and associated morbidity and mortality from tobacco-related diseases.