pH Resistant Ratiometric Measurement of Nicotinamide Adenine Dinucleotide Levels by Time-resolved Fluorescence Spectroscopy

Publication date: January 2019Source: Chinese Journal of Analytical Chemistry, Volume 47, Issue 1Author(s): Lei LI, Jia-Sheng ZHOU, Peng WANG, Chao-Qun MA, Zhuo-Wei ZHU, Jiao GU, Chun ZHU, Guo-Qing CHEN, San-Jun ZHANG, Jian-Hua XUAbstractCircular permutation fluorescent protein is a novel method to construct biosensors. The ratio of two excitation channels is employed to quantitatively calibrate the level of analysts. SoNar is one of them, which can be used to monitor cellular NADH/NAD+ levels. However, the 490 nm excitation channel of these biosensors is sensitive to pH environments, which is negative in real applications. In this work, we demonstrated that the fractional intensity ratio extracted from time-resolved fluorescence spectroscopy could be used to quantify NADH levels with one excitation (420 nm) and one emission channels. The 420-nm excitation channel was pH resistant. Comparing to average lifetime, the fractional intensity ratio had a 3.2-fold dynamic range, which was much wider than average lifetimes.Graphical AbstractSoNar, a novel genetically encoded biosensor, was proved to be sensitive to NADH concentrations. When detecting NADH, the environments of pH had a great influence on the steady-state fluorescence of SoNar. However, the time-resolved fluorescence was pH-resisitant, which had greater potentials to measure NADH quantitatively in real applications.
Source: Chinese Journal of Analytical Chemistry - Category: Chemistry Source Type: research