Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD(+) with Genetically Encoded Fluorescent Biosensors.
Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD(+) with Genetically Encoded Fluorescent Biosensors.
复制标题
使用时间分辨荧光的分数强度通过基因编码荧光生物传感器灵敏地定量 NADH/NAD( )
DOI：
10.1038/s41598-017-04051-7
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发表时间：
2017-06-23
影响因子：
4.6
通讯作者：
Xu J
中科院分区：
文献类型：
--
作者：
Chang M;Li L;Hu H;Hu Q;Wang A;Cao X;Yu X;Zhang S;Zhao Y;Chen J;Yang Y;Xu J
在本文中，我们提出了一种新的和敏感的比率分析方法，使用遗传编码的荧光NADH/NAD+生物传感器，Peredox，SoNar和Frex的时间分辨荧光的分数强度。当NADH/NAD+结合时，生物传感器的构象发生变化，
我
τ
我
）有相反的变化趋势。与常用的荧光强度和寿命方法相比，可以利用它们的比率以更大的动态范围和更高的分辨率定量NADH/NAD+水平。此外，这种比率测量仅需要一个激发波长和一个发射波长。这消除了传统的激发比率和发射比率方法的问题。该方法可用于简化基因编码荧光生物传感器的设计并实现高灵敏度的分析物定量。基于这种新的方法，可以开发出广泛的潜在应用，为活细胞代谢成像。
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 (α
i
τ
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) 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.