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.

背景：一种简单，快速且经济的测定乙醇的分析方法，对于临床，生物学，法医和法律合法的目的非常重要。

方法：将Ni2 + -NTA树脂作为固定化基质，用于his6-tagged ADH的简单一步纯化/固定化。使用制备的ADH酶热敏电阻测量了浓度范围为0.5-50％V / V的不同醇，即甲醇，乙醇和丙醇。青岛啤酒的乙醇含量作为含酒精的真实样品进行了测试。还通过重复测量研究了所制备的ADH酶热敏电阻的再现性和稳定性。

结果：与热生物传感器中使用的可控孔玻璃（用于固定酶的常用支持物）相比，Ni2 + -NTA树脂的使用不仅可通过组氨酸标记的ADH与ADH结合而导致简单的一步纯化/固定。 Ni2 + -NTA树脂，但也使固定化支持物可重复使用。所制备的生物传感器可用于通过量热法测定乙醇和甲醇。乙醇和甲醇的线性范围分别为1 -32％（V / V）和2-20％（V / V）。乙醇和甲醇的检出限分别为0.3％（V / V）和1％（V / V）。青岛啤酒的乙醇测试浓度为4.5％V / V，与标记酒精体积（ABV）4.80％相当。

结论：Ni2 + -NTA树脂作为ET传感器中的固定基质，可为His6标签重组酶和可重复使用的固定基质提供简单的一步纯化/固定。制备的生物传感器具有良好的重复性和稳定性。这种新的生物传感器在定性和定量测试中都显示出对乙醇和甲醇进行快速，简单且经济高效的分析的巨大希望。