文献类型：期刊文献

英文题名：Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD+ with Genetically Encoded Fluorescent Biosensors

作者：Chang, Mengfang[1];Li, Lei[1];Hu, Hanyang[2,3,4];Hu, Qingxun[2,3,4];Wang, Aoxue[2,3,4];Cao, Xiaodan[1];Yu, Xiantong[1];Zhang, Sanjun[1];Zhao, Yuzheng[2,3,4];Chen, Jinquan[1];Yang, Yi[2,3,4];Xu, Jianhua[1]

机构：[1]East China Normal Univ, State Key Lab Precis Spect, 3663 North Zhongshan Rd, Shanghai 200062, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg Technol, State Key Lab Bioreactor Engn, Synthet Biol & Biotechnol Lab, 130 Mei Long Rd, Shanghai 200237, Peoples R China;[3]CAS Ctr Excellence Brain Sci, Optogenet & Synthet Biol Interdisciplinary Res Ct, 130 Mei Long Rd, Shanghai 200237, Peoples R China;[4]East China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, 130 Mei Long Rd, Shanghai 200237, Peoples R China

年份：2017

卷号：7

外文期刊名：SCIENTIFIC REPORTS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000404118700099)】；

基金：We thank Prof. Yang Tian for the help of FLIM measurements of cells. This work was partly supported by the Science and Technology Commission of Shanghai Municipality (15520711500, 15ZR1411700, 15ZR1410100, 14XD1401400, 15YF1402600), NSFC (91313301, 31225008, 31470833, 91649123, 31671484), the Lift Engineering for Young Talent of China Association for Science and Technology, the Program of Introducing Talents of Discipline to Universities (B12024), and Jorris project.

语种：英文

摘要：In this paper, we propose a novel and sensitive ratiometric analysis method that uses the fractional intensities of time-resolved fluorescence of genetically encoded fluorescent NADH/NAD(+) biosensors, Peredox, SoNar, and Frex. When the conformations of the biosensors change upon NADH/NAD(+) binding, the fractional intensities (alpha(i)tau(i)) have opposite changing trends. Their ratios could be exploited to quantify NADH/NAD(+) levels with a larger dynamic range and higher resolution versus commonly used fluorescence intensity and lifetime methods. Moreover, only one excitation and one emission wavelength are required for this ratiometric measurement. This eliminates problems of traditional excitation-ratiometric and emission-ratiometric methods. This method could be used to simplify the design and achieve highly sensitive analyte quantification of genetically encoded fluorescent biosensors. Wide potential applications could be developed for imaging live cell metabolism based on this new method.