Smartphone-integrated paper-based biosensor for sensitive fluorometric ethanol quantification

The development of fluorometric paper-based analytical devices (fPADs) integrated with smartphone for fluorometric quantification of ethanol in an instrument-free and portable setup is described. The NAD⁺-dependent alcohol dehydrogenase immobilized within chitosan modified paper substate was utilized as a bio-recognition element and enzymatically generated NADH was used as a fluorescent probe. 3D-printed imaging setup which houses a paper chip holder and UV-light emitting device (LED) was developed for rapid, accurate capture of the fluorescent images. The biocompatible chitosan layer covering the paper provides a feasible environment for enzyme immobilization and enhances the fluorescence signal. The developed fPADs exhibited high sensitivity for ethanol detection and has a linear range for ethanol detection from 17 µM to 8.75 mM (R² =0.99). Additionally, the fPADs were applied to quantify ethanol in four different wine samples including red, white, rose, and sparkling wines successfully. Moreover, the fPADs produce reproducible signals without loss of enzyme activity for at least 14 days and ~80% activity remained till 28 days. Thus, the proposed approach can provide a facile, affordable, portable, and instrument-free tool for the onsite quantification of ethanol in real samples and is applicable for food quality controls.