Hou Shuqi, Xu Junhong, Ma Qiujuan, Mao Guojiang, Ma Yijie, Lian Yujie, Ning Cui, Xia Ruxue
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, PR China.
Department of Electrical Engineering, North China University of Water Resources and Electric Power, Zhengzhou 450011, PR China.
J Pharm Biomed Anal. 2025 Nov 15;265:116987. doi: 10.1016/j.jpba.2025.116987. Epub 2025 May 26.
Cysteine (Cys) is a small molecule thiol and an important component of proteases in lysosomes. Developing fluorescent probes specifically for detecting lysosomal Cys holds significant importance for elucidating its functions in lysosomal redox homeostasis and protease activity. In this study, a near-infrared fluorescent probe was developed to detect Cys within lysosomes. A near-infrared fluorophore was constructed by conjugating dicyanoisophorone (DCI) with 1,8-naphthalimide. The acrylate unit functioned as the recognition moiety for cysteine, while the fluorophore was modified with morpholine to target lysosomes. When Cys was absent, the developed probe only emitted faint fluorescence, attributed to the inhibition of the intramolecular charge transfer (ICT) process. Upon Cys addition, the ICT process was activated, resulting in fluorescence emission at 688 nm. The linear range of the probe for Cys ranged from 0.2 to 10 μM and the detection limit was 0.036 μM. The near-infrared probe for Cys exhibited strong specificity, high sensitivity, short response time and a wide working pH range. Additionally, the probe exhibited negligible cytotoxicity and had been effectively exploited to determine endogenous and exogenous Cys in lysosomes of A549 cells. It had also been effectively used to detect Cys in zebrafish and mice.
半胱氨酸（Cys）是一种小分子硫醇，是溶酶体中蛋白酶的重要组成部分。开发专门用于检测溶酶体中Cys的荧光探针对于阐明其在溶酶体氧化还原稳态和蛋白酶活性中的功能具有重要意义。在本研究中，开发了一种近红外荧光探针来检测溶酶体内的Cys。通过将二氰基异佛尔酮（DCI）与1,8-萘二甲酰亚胺共轭构建了一种近红外荧光团。丙烯酸酯单元作为半胱氨酸的识别部分，而荧光团用吗啉修饰以靶向溶酶体。当不存在Cys时，所开发的探针仅发出微弱的荧光，这归因于分子内电荷转移（ICT）过程的抑制。加入Cys后，ICT过程被激活，导致在688nm处发射荧光。该探针对Cys的线性范围为0.2至10μM，检测限为0.036μM。用于Cys的近红外探针表现出很强的特异性、高灵敏度、短响应时间和宽工作pH范围。此外，该探针的细胞毒性可忽略不计，并已有效地用于测定A549细胞溶酶体中的内源性和外源性Cys。它也已有效地用于检测斑马鱼和小鼠中的Cys。
