High-sensitivity isopropanol Biomeasurement using enzyme cycling reactions.

Elevated isopropanol (IPA) levels in biological fluids are potential biomarkers for ketoacidosis. This study developed a high-sensitivity IPA detection system using an enzyme cycling reaction with secondary alcohol dehydrogenase (S-ADH). The system employs a dual-coenzyme mechanism where IPA is repeatedly oxidized and regenerated through sequential reactions with thio-NAD+ and NADH, producing accumulating thio-NADH that is measured spectrophotometrically at 405 nm. After optimization of reaction conditions (pH 8.5, 500 μM thio-NAD+, 50 μM NADH), the cycling reaction achieved a 6-fold improvement in sensitivity compared to single-step detection. The detection range expanded from 22.5 μM-1 mM to 3.9 μM-1 mM, encompassing physiological IPA concentrations in healthy human saliva (5.51-24.6 μM). This enhanced sensitivity enables non-invasive ketoacidosis monitoring through saliva analysis, demonstrating the potential of enzyme cycling-based biosensors for clinical applications.