Calorimetric Sensor for Ethanol Using Ni²⁺-nitrilotriacetic Acid (NTA) Resin Immobilized Alcohol Dehydrogenase (ADH)

Background: A simple, fast and economic analytical method for the determination of ethanol is important for clinical, biological, forensic and physico-legal purposes. Methods: Ni2+-NTA resin was used as an immobilization matrix for the simple one-step purification/ immobilization of his6-tagged ADH. Different alcohols with a concentration range of 0.5-50% V/V, namely methanol, ethanol and propanol were measured using prepared ADH enzyme thermistor. The ethanol content of Tsingtao beer was tested as a real sample containing alcohol. Reproducibility and stability of prepared ADH enzyme thermistor were also investigated by repeated measurements. Results: In comparison to the controlled pore glass (a common used support for the immobilization of enzyme) used in thermal biosensor, the use of Ni2+-NTA resin not only led to simple one-step purification/ immobilization by his6-tagged ADH binding to Ni2+-NTA resin, but also made the immobilizing supports reusable. The prepared biosensor can be used to determine ethanol and methanol by the calorimetric measurement. A linear range of 1 -32% (V/V) and 2-20% (V/V) was observed for ethanol and methanol, respectively. The detection limits were 0.3% (V/V) and 1% (V/V) for ethanol and methanol, respectively. The tested ethanol concentration of Tsingtao beer was 4.5% V/V, which is comparable with the labeled alcohol by volume (ABV) 4.80%. Conclusion: Ni2+-NTA resin, as an immobilization matrix in ET sensor, provides a simple one-step purification/immobilization for His6-tagged recombinase and a reusable immobilization matrix. The prepared biosensor exhibits good repeatability and stability. Such a new biosensor shows great promise for rapid, simple, and cost-effective analysis of ethanol and methanol, both in qualitative and in quantitative tests.