Described herein is an amperometric biosensor, e.g., chronoamperometric biosensor for the measurement of the concentration of nicotine. Also disclosed herein is a wearable nicotine biosensor device and a biosensor that detects nicotine in smoke. The biosensor disclosed herein comprises a nicotine-catalyzing enzyme, such as NicA2 or mutant NicA2 enzymes. Also described herein are systems comprising said amperometric biosensor, e.g., chronoamperometric biosensor and methods of using said chronoamperometric biosensor.
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