A 3D-Printed Breath Analyzer Incorporating CeO₂ Nanoparticles for Colorimetric Enzyme-Based Ethanol Sensing

Breath biosensors are a viable cost-effective platform for ethanol detection providing features such as portability, simplicity, and real-time in situ analysis. Herein, we describe a custom-made 3D-printed device containing a nanoparticle-based enzyme biosensor, designed to allow the user to blow breath on the sensor surface and obtain an immediate measure of the alcohol level in breath. The entire assembly enables sample collection, analysis, and signal generation, which could be visualized by the naked eye or by image capturing with a smart phone for color measurement. The biosensor unit was fabricated onto paper that was modified with alcohol oxidase (ALOx) enzyme and cerium oxide nanoparticles (CeNPs), used as a combined peroxidase-like mimetic and chromogenic reagent. The enzymelike chromogenic behavior of the nanoparticles facilitates production of a robust biosensor with all the reagents integrated within the sensing body. Because the biosensor uses biomimetic nanoparticles in lieu of peroxidase enzymes and soluble redox reagents, the detection system has increased stability and low cost. The biosensor showed a linear range of 0.02–1.2% (0.2–1.2 g/L) and a limit of detection of 0.001% and was able to measure breath ethanol concentrations at levels comparable to electronic breathalyzers, at a low cost and with high sensitivity. The device is sensitive, selective, portable, and simple to operate and can be used broadly for ethanol detection in multiple applications, in law enforcement, forensics, and food and beverage authentication. The method is inexpensive, and the 3D device and the biosensor can be mass-produced, which provides a route to extend this technology for noninvasive monitoring of other volatile compounds in breath for disease diagnostics and other applications.