Li Yinhui, Wang Yijun, Yang Sheng, Zhao Yirong, Yuan Lin, Zheng Jing, Yang Ronghua
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, China.
Anal Chem. 2015 Feb 17;87(4):2495-503. doi: 10.1021/ac5045498. Epub 2015 Jan 30.
Intracellular pH is an important parameter associated with cellular behaviors and pathological conditions. Quantitative sensing pH and monitoring its changes by near-infrared (NIR) fluorescence imaging with high resolution in living systems are essential but challenging due to the lack of effective probes. To achieve good adaptability, in this study, a class of resolution-tunable ratiometric NIR fluorescent probes, which possess a stable NIR hemicyanine skeleton bearing different substituents, are rationally designed and synthesized, enabling detection through noninvasive optical imaging of organisms. Based on the protonation/deprotonation of the hydroxy group, a marked NIR emission shift provides a ratio signal in response to pH. Meanwhile, two states exhibit good photostability, sensitivity and reversibility, conducive to longtime monitoring of persistent pH changes without disturbance of other biological active species. Among the series, NIR-Ratio-BTZ modified with an electron-withdrawing substituent of benzothiazole exhibited the largest emission shift of about 76 nm from 672 to 748 nm with the pH environment changing from acidic to basic, which could be considered as a good candidate for high resolution pH imaging in live cells, tissues and organisms. Moreover, NIR-Ratio-BTZ has an ideal pK(a) value (pK(a) ≈ 7.2) for monitoring the minor fluctuations of physiological pH near neutrality. The ratiometric fluorescence measurement is beneficial to ensure the accuracy of quantitative measuring pH changes, as well as the real-time monitoring pH-related physiological effects both in living cells and living mice. The results demonstrate that NIR-Ratio-BTZ is a highly sensitive ratiometric pH probe in vivo, giving it potential for biological applications.
细胞内pH值是与细胞行为和病理状况相关的重要参数。在生物系统中，通过近红外（NIR）荧光成像进行高分辨率的定量pH值传感和监测其变化至关重要，但由于缺乏有效的探针而具有挑战性。为了实现良好的适应性，在本研究中，合理设计并合成了一类分辨率可调的比率型近红外荧光探针，该探针具有带有不同取代基的稳定近红外半菁骨架，能够通过对生物体的无创光学成像进行检测。基于羟基的质子化/去质子化，显著的近红外发射位移提供了响应pH值的比率信号。同时，两种状态表现出良好的光稳定性、灵敏度和可逆性，有利于长期监测持续的pH值变化而不受其他生物活性物质的干扰。在该系列中，用苯并噻唑吸电子取代基修饰的NIR-Ratio-BTZ在pH环境从酸性变为碱性时，表现出约76 nm的最大发射位移，从672 nm变为748 nm，这可被视为活细胞、组织和生物体中高分辨率pH成像的良好候选者。此外，NIR-Ratio-BTZ具有理想的pK(a)值（pK(a)≈7.2），用于监测接近中性的生理pH值的微小波动。比率荧光测量有利于确保定量测量pH值变化的准确性，以及实时监测活细胞和活小鼠中与pH相关的生理效应。结果表明，NIR-Ratio-BTZ是一种体内高灵敏度的比率型pH探针，具有生物应用潜力。
