Enhanced sensitivity of a fluorometric biosensor for alcohol metabolites with an enzymatic cycling reaction

Abstract

Assessing ethanol (EtOH) and acetaldehyde (AcH), metabolites of alcohol, enables more precise evaluation of alcohol metabolism. In this study, we developed a biosensor capable of concurrently measuring EtOH and AcH solutions with high sensitivity, utilizing a cycling reaction mediated by two enzymes: alcohol dehydrogenase (ADH) and alcohol oxidase (AOD). Within the cycling reaction, β-nicotinamide adenine dinucleotide (NADH, λex = 340 nm, λem = 490 nm) is oxidized when AcH is reduced to EtOH by ADH, while the EtOH produced in this reaction is oxidized by AOD, regenerating AcH. By detecting the consumption of oxygen or NADH in this reaction cycle, highly sensitive measurements of AcH and EtOH can be achieved. Initially, an AOD-ADH electrosensor was constructed to validate the feasibility of the cycling reaction by applying EtOH solution to the sensor. This electrosensor quantifies alcohol metabolites via the oxygen consumed in the cycling reaction, demonstrating a dynamic range for EtOH solution from 700 nM to 500 µM—a sensitivity two-fold greater than that achieved with the AOD membrane (1.4 µM–1 mM) alone. Subsequently, an ADH-AOD fluorosensor was developed, measuring alcohol metabolites via NADH consumption within the cycling reaction. The ADH-AOD fluorosensor demonstrated a dynamic range of 59 pM–100 µM for AcH solution, representing approximately a 1000-fold increase in sensitivity compared to the ADH membrane (70 nM–100 µM) alone. In future work, it is anticipated that utilizing this cycling reaction for the highly sensitive measurement of skin gases (EtOH and AcH) and facilitating non-invasive metabolic evaluations.