[体外细菌荧光素酶生物发光法定量检测烟酰胺腺嘌呤二核苷酸（NADH）]

[Quantitative detection of NADH by in vitro bacterial luciferase bioluminescent].

作者信息

Mei Cexia, Wang Jingxue, Lin Hong, Wang Jing

机构信息

Food Safety Laboratory, Food Science and Engineering Department, Ocean University of China, Qingdao 266003.

出版信息

Wei Sheng Wu Xue Bao. 2009 Sep;49(9):1223-8.

PMID:20030062

Abstract

OBJECTIVE

The study aimed at establishing the bacterial luciferase: FMN-NADH oxidoreductase bioluminescent system in vitro and evaluating its potential for quantitative detection of NADH.

METHODS

By optimizing the amount of substrates, we set up the coupled luciferase: FMN-NADH oxidoreductase bioluminescent system in vitro, based on the crude extract from Photobacterium leiognathi YL.

RESULTS

The in vitro coupled bacterial luciferase: FMN-NADH oxidoreductase bioluminescent system was: 1 mL crude extract, 27 mmol/L Dodecane 100 microL, 10 mmol/L FMN-Na 0.5 microL and 0.14 mmol/L NADH 300 microL. Furthermore, we developed a method for quantitative detection of NADH according to the bioluminescence of NADH catalyzed of bacterial luciferase: FMN-NADH oxidoreductase system in vitro. A good linear relationship of NADH concentration was in a range of 1.0 x 10(-10) to 1.0 x 10(-8) mol/L.

CONCLUSION

The bacterial luciferase: FMN-NADH oxidoreductase system used to measure NADH concentration was a good attempt to detect living bacterial cells in the fields of environment, food sanitation and other related.

摘要

目的

本研究旨在建立细菌荧光素酶：FMN - NADH氧化还原酶体外生物发光体系，并评估其对NADH进行定量检测的潜力。

方法

通过优化底物用量，基于鱼发光杆菌YL的粗提物建立了体外偶联荧光素酶：FMN - NADH氧化还原酶生物发光体系。

结果

体外偶联细菌荧光素酶：FMN - NADH氧化还原酶生物发光体系为：1 mL粗提物、27 mmol/L正十二烷100 μL、10 mmol/L FMN - Na 0.5 μL和0.14 mmol/L NADH 300 μL。此外，根据细菌荧光素酶：FMN - NADH氧化还原酶体系体外催化NADH产生的生物发光，我们开发了一种定量检测NADH的方法。NADH浓度在1.0×10⁻¹⁰至1.0×10⁻⁸ mol/L范围内具有良好的线性关系。