State Key Laboratory of Precision Spectroscopy, East China Normal University, 3663 North Zhongshan Road, Shanghai, 200062, China.
Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Mei Long Road, Shanghai, 200237, China.
Sci Rep. 2017 Jun 23;7(1):4209. doi: 10.1038/s41598-017-04051-7.
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 τ ) 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.
在本文中，我们提出了一种新颖而灵敏的比率分析方法，该方法使用基因编码的荧光 NADH/NAD 生物传感器（Peredox、SoNar 和 Frex）的时间分辨荧光的分数强度。当生物传感器的构象在 NADH/NAD 结合时发生变化时，分数强度（ατ）呈现相反的变化趋势。它们的比值可以用于定量 NADH/NAD 水平，与常用的荧光强度和寿命方法相比，具有更大的动态范围和更高的分辨率。此外，这种比率测量仅需要一个激发和一个发射波长。这消除了传统的激发比率和发射比率方法的问题。这种方法可用于简化设计并实现基因编码荧光生物传感器的高灵敏度分析物定量。基于这种新方法，可以开发出广泛的潜在应用，用于对活细胞代谢进行成像。
