Haynes Emily P, Canzano Mary, Tantama Mathew
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, MA 02481, USA.
Sensors (Basel). 2024 Nov 26;24(23):7531. doi: 10.3390/s24237531.
Biological activity is strongly dependent on pH, which fluctuates within a variety of neutral, alkaline, and acidic local environments. The heterogeneity of tissue and subcellular pH has driven the development of sensors with different pKa values, and a huge assortment of fluorescent sensors have been created to measure and visualize pH in living cells and tissues. In particular, sensors that report based on fluorescence lifetime are advantageous for quantitation. Here, we apply a theoretical framework to derive how the apparent pKa of lifetime-based pH sensors depends on fluorescence excitation wavelength. We demonstrate that theory predicts the behavior of two different fluorescent protein-based pH sensors in solution as proofs-of-concept. Furthermore, we show that this behavior has great practical value in living cells because it extends the sensing range of a single sensor by simply choosing appropriate detection parameters to match the physiological pH range of interest. More broadly, our results show that the versatility of a single lifetime-based sensor has been significantly underappreciated, and our approach provides a means to use a single sensor across a range of pH environments.
生物活性强烈依赖于pH值，pH值在各种中性、碱性和酸性局部环境中波动。组织和亚细胞pH值的异质性推动了具有不同pKa值的传感器的发展，并且已经创建了大量荧光传感器来测量和可视化活细胞和组织中的pH值。特别是，基于荧光寿命报告的传感器在定量方面具有优势。在这里，我们应用一个理论框架来推导基于寿命的pH传感器的表观pKa如何依赖于荧光激发波长。我们证明，理论预测了两种不同的基于荧光蛋白的pH传感器在溶液中的行为，作为概念验证。此外，我们表明这种行为在活细胞中具有很大的实用价值，因为通过简单地选择合适的检测参数以匹配感兴趣的生理pH范围，它扩展了单个传感器的传感范围。更广泛地说，我们的结果表明，单个基于寿命的传感器的多功能性一直被大大低估，我们的方法提供了一种在一系列pH环境中使用单个传感器的手段。
