Université Grenoble Alpes, CNRS, UMR 5525, VetAgro Sup, Grenoble INP, TIMC, 38000 Grenoble, France.
Biomolecules. 2023 Feb 26;13(3):442. doi: 10.3390/biom13030442.
There is a variety of fluorescent probes for pH measurements and which are mainly used for biological systems. In general, they can be classified into two groups. The first group includes fluorescent pH probes which exhibit a single fluorescence emission peak. For these probes, the fluorescence excitation profile is pH-dependent and the shape of the emission spectra remains almost constant. Hence, the ratiometric pH measurement-which makes pH determination independent of the probe concentration-is implemented when the excitation is performed at two excitation wavelengths and the fluorescence emission is measured at one wavelength. The second group exhibits a dual fluorescence emission peak. Here, each protonated or deprotonated form exhibits characteristics emission and/or absorption spectra. Shifts between spectra obtained for protonated and deprotonated species can be exploited in order to perform a ratiometric measurement. In this study we present a methodology that evaluates the precision of the ratiometric measurements based on multiple wavelengths excitation to determine the optimum wavelengths combination for pH determination in biological samples. This methodology using the BCECF probe is applied to measure the pH drift in cell culture medium. It exhibits a high precision and significantly extends the range of validity for pH measurements spanning from very acidic to basic.
有各种各样的用于 pH 值测量的荧光探针，它们主要用于生物系统。一般来说，它们可以分为两类。第一类包括荧光 pH 探针，它们只显示一个荧光发射峰。对于这些探针，荧光激发轮廓随 pH 值变化，而发射光谱的形状几乎保持不变。因此，当在两个激发波长处进行激发并在一个波长处测量荧光发射时，实现了比率 pH 测量，从而使 pH 值测定与探针浓度无关。第二类探针则显示出双荧光发射峰。在这里，每个质子化或去质子化形式都具有特征的发射和/或吸收光谱。可以利用质子化和去质子化物种之间的光谱位移来进行比率测量。在这项研究中，我们提出了一种基于多波长激发评估比率测量精度的方法，以确定用于生物样品 pH 值测定的最佳波长组合。该方法使用 BCECF 探针来测量细胞培养基中的 pH 值漂移。它具有很高的精度，并显著扩展了 pH 值测量的有效范围，从非常酸性到碱性。
