Application Of Coupled Enzymes Bioluminescent System From Luminous Bacteria In Rapid Detection

Coupled enzymes bioluminescent system (CEBS) from luminous bacteria hasbeen established and applied to rapid determination of NADH. Based on the rapiddetermination of NADH by CEBS and enzymatic reactions related to NADH, rapiddetection methods of ethanol and pyruvic acid by CEBS from Photobacteriumleiognathi YL were established. In addition, the relation between glucose orglucose-6-phosphate and their luminescence intensity in CEBS was also studied.These novel methods not only have the advantages of traditional enzymatic detectionmethods such as mild reaction conditions, rapid reaction velocity, and high specificity,but also meet the requirements of ready-to-use detection. This work providedtheoretical basis for the development of a novel portable instrument for rapiddetection in the future.1. A new method of ethanol rapid determination by CEBS is established based onthe reaction catalyzed by ethanol dehydrogenase, and NAD+is converted to NADHthrough this reaction. The determination of ethanol in samples is performed by theluminescence intensity of NADH in the CEBS. The linear range of this new method isfrom0.002mol/L to0.1mol/L, and the detection limit is1.36×10-4mol/L. Otheralcohols such as methanol were also detected using this method, and they had noeffect on the determination of ethanol. This method was applied to the ethanoldetermination in beer samples, with recovery experiment conducted. The resultshowed that the new method is with good repeatability and accuracy.2. A new method of pyruvic acid rapid determination by CEBS is establishedbased on the reaction catalyzed by lactate dehydrogenase, and NADH is converted to NAD+through this reaction. The determination of pyruvic acid in samples is throughthe variation of luminescence intensity of NADH after the reaction in CEBS. Thelinear range of this new method is from0.00014mol/L to0.001mol/L, and thedetection limit is8.537×10-5mol/L. This method was applied to the pyruvic aciddetermination in serum samples, and the recovery experiment was also conducted.The result showed that the new method is with good sensitivity, high precision andspecificity.3. The relation between glucose or glucose-6-phosphate and their luminescenceintensity in CEBS was studied based on the reaction catalyzed by hexokinase andglucose-6-phosphate dehydrogenase. Glucose is converted to glucose-6-phosphateand then deoxidized NADP+to NADPH. The determination of glucose andglucose-6-phosphate are by means of the luminescence intensity of NADPH in theCEBS. Compared with ultraviolet spectrophotometer method, the linear range ofcoupled enzymes bioluminescent method decreases by100times and the linearrelation is better, and the detection limit of coupled enzymes bioluminescent methodfor glucose-6-phosphate detecting is6.468×10-7mol/L. This work lays a foundation ofthe establishment of coupled enzymes bioluminescent method for rapid detection ofglucose and glucose-6-phosphate.