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

Mengfang Chang,Jinquan Chen,Sanjun Zhang,Xiantong Yu,Qingxun Hu,Xiaodan Cao,Yuzheng Zhao,Aoxue Wang,Jianhua Xu,Lei Li,Yi Yang,Hanyang Hu

doi:10.1038/s41598-017-04051-7

Abstract

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 (αiτi) 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.

Highlights

The ratio of reduced nicotinamide adenine dinucleotide (NADH) to its oxidized form NAD+ is a key indicator that reflects the overall redox state of many physiological or pathological processes, including energy metabolism, mitochondrial function, calcium homeostasis, gene expression, cell death, embryonic development, blood flow, aging, cancer, and diabets[1,2,3,4,5,6,7,8,9,10]
Computational and mathematical modeling of fluorescence lifetime imaging can discriminate between NADH and NADPH25, and the fluorescent co-enzymes NADH and flavin adenine dinucleotide (FAD)[26]
Compared to the NADH/NAD+ biosensor used previously, we removed the mCherry required for emission-ratiometric measurements[14] and kept only Peredox

Summary

Introduction

The ratio of reduced nicotinamide adenine dinucleotide (NADH) to its oxidized form NAD+ is a key indicator that reflects the overall redox state of many physiological or pathological processes, including energy metabolism, mitochondrial function, calcium homeostasis, gene expression, cell death, embryonic development, blood flow, aging, cancer, and diabets[1,2,3,4,5,6,7,8,9,10]. Genetically encoded NADH/NAD+ biosensors have been developed based on circular permutation of fluorescent proteins (cpFPs)[14,15,16]. Only one emission peak of cpFP-based biosensors makes it difficult to achieve ratiometric detection To overcome this problem, Hung et al constructed an emission-ratiometric biosensor by fusing mCherry, a red fluorescent protein, to C-terminal of cpGFP-based NADH/NAD+ probe Peredox[14]. Peredox and mCherry were excited at 405 and 578 nm, respectively, and the ratio of fluorescence intensities detected at 525 and 629 nm could be related to NADH/NAD+ levels[14] This large protein tends to aggregate, and the emission ratios of dual FP sensors were subject to prep-to-prep variations and variations due to different FP maturation processes and photobleaching rates[14, 23]. This fluorescence lifetime (average arrival time of the detected photons ) gave a quantitative measurement of the NADH/NAD+ ratio, the dynamic range was even smaller than that of the steady-state fluorescence[14, 27]

Methods

Results

Conclusion