Anal Chem. 2015;87(10):5117-24. doi: 10.1021/ac504386x.
Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitors—cyanide and ethanol—are similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.
细胞内 NADH 构象越来越被认为是一种内源性的光学生物标志物和代谢指标。最近，我们报道了一种基于紫外激发自发荧光光谱形状定量的实时跟踪代谢的方法。在这里，我们使用纳秒门控光谱采集，结合光谱形状定量，将与蛋白结合的 NADH 构象相关的长激发态寿命自发荧光（通常与蛋白结合的 NADH 构象相关）与自发荧光信号整体分开进行监测。我们观察到，当使用时间积分检测时，两种 NADH 氧化抑制剂（氰化物和乙醇）对酿酒酵母的自发荧光响应相似，但使用时间门控检测很容易区分。结果与使用光谱相量分析评估的多种 NADH 构象的观察结果一致。此外，由于使用了众所周知的氧化抑制剂，光谱形状的变化可以与参与不同代谢途径的 NADH 构象相关联，从而使光谱响应具有生物分析的实用性。
