一种基于萘酰亚胺的检测细胞内pH值的荧光探针及其生物成像应用

细胞内溶酶体的pH值对细胞自噬、吞噬、酶加工等各项生命活动有着重要影响. 细胞核是真核细胞中最大的细胞器, 控制着生物体内的遗传和代谢过程, 参与代谢过程的酶对pH值的变化很敏感. 因此, 研究细胞体内的pH值变化至关重要. 我们设计并以简单的两步反应合成了一种新型荧光探针 NpH-1. 该探针以萘酰亚胺作为荧光团, 以吗啉基团作为对pH值响应的位点, 通过光诱导电子转移(PET)机制调控荧光, 能够对pH值变化响应. 我们在缓冲范围为1.81到11.92的Britton-Robison缓冲液中测定了 NpH-1对pH值变化响应的光谱性质. 在pH值3.0道10.0的范围内, NpH-1能够对pH值的变化产生快速可逆的响应, 其p Ka值为5.41. 探针具有很高的光稳定性. NpH-1具有很低的细胞毒性, 能够用于活细胞成像. 我们用氯喹刺激HeLa细胞, 使细胞的pH值发生变化, 并用探针 NpH-1监测了这一过程中的pH值变化. 另外, 还对 NpH-1进行了溶酶体、线粒体、高尔基体、内质网和细胞核的共定位实验, 结果表明, 探针主要分布在溶酶体和细胞核中, 这意味着 NpH-1可以用于检测复杂细胞环境中的pH值变化.

A Naphthalimide-Based Fluorescent Probe for Detecting Intracellular pH and Its Biological Imaging Application

The pH of lysosome in cells has significant affect to various biological activities,including autophagy,phagocytosis,enzyme processing and so on.The metastasis and apoptosis of cancer cells are closely related to change of the lysosomal pH.The nucleus is the largest organelle in eukaryotic cells which control the most important genetic and metabolic processes,and the enzymes involved in metabolic processes are sensitive to changes of pH.Therefore,it is very important to investigate the pH change in cells.Herein,we designed and synthesized a novel fluorescent probe NpH-1 via a simple two-step reaction.The probe uses naphthalimide as the fluorescent group and morpholine group as the pH responsive site,and regulates the fluorescence through the photoinduced electron transfer(PET)mechanism.The spectroscopic properties of NpH-1 response to pH changes were measured in the Britton-Robison buffers,whose buffering range was from 1.81 to 11.92.In the pH range from 3.0 to 10.0,NpH-1 exhibited a very rapid and reversible response to pH change.The pKa of NpH-1 was 5.41.It showed a very high photostability.Moreover,the fluorescence emission of NpH-1 was not influenced by biological species including metal ions,anions and amino acids.NpH-1 showed very low cytotoxicity,which indicated that it could be applied for cell imaging.Thus,we monitored the pH changes in HeLa cells via stimulating them by Chloroquine.The cell imaging results showed that NpH-1 could be utilized for detection of pH changes in living cells.Furthermore,the colocalization experiments of the probe NpH-1 for various organelles including lysosome,mitochondria,Golgi,endoplasmic reticulum and cellular nucleus were carried out,and the results exhibited the probe NpH-1 mainly distributed in lysosome and nucleus,which implied that the probe could be applied for detection of pH change in complex intracellular environment.