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

doi:10.6023/A20080399

化学学报 ›› 2021, Vol. 79 ›› Issue (1): 87-92.DOI: 10.6023/A20080399 上一篇下一篇

研究论文

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

任江波^a, 王蕾^b, 郭锐^a, 唐永和^a, 周红梅^a, 林伟英^a^,^c^,^*()

a 广西大学光功能材料与化学生物学研究院化学化工学院南宁 530004
b 南宁市食品药品检验所南宁 530007
c 济南大学光功能材料与成像研究院化学化工学院材料科学与工程学院济南 250022

投稿日期:2020-08-31 发布日期:2020-10-17
通讯作者: 林伟英
作者简介:
* E-mail: weiyinglin@163.com; Tel.: +86-0771-3236493; Fax: +86-0771-3236493
基金资助:
国家自然科学基金(21672083); 国家自然科学基金(21877048); 国家自然科学基金(22077048); 广西大学启动基金(A3040051003)

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

Jiangbo Ren^a, Lei Wang^b, Rui Guo^a, Yonghe Tang^a, Hongmei Zhou^a, Weiying Lin^a^,^c^,^*()

a Institute of Optical Materials and Chemical Biology, School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China
b Nanning Institute for Food and Drug Control, Nanning 530007, China
c Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, Jinan University, Jinan 250022, China

Received:2020-08-31 Published:2020-10-17
Contact: Weiying Lin
Supported by:
the National Natural Science Foundation of China(21672083); the National Natural Science Foundation of China(21877048); the National Natural Science Foundation of China(22077048); the Startup Fund of Guangxi University(A3040051003)

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摘要/Abstract

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.

关键词: 溶酶体, 细胞核, pH, 萘酰亚胺, 荧光成像

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-1exhibited 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.

Key words: Lysosome, Cell nucleus, pH, Fluorescent imaging, Naphthalimide

引用此文

任江波, 王蕾, 郭锐, 唐永和, 周红梅, 林伟英. 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[J]. 化学学报, 2021, 79(1): 87-92.

Jiangbo Ren, Lei Wang, Rui Guo, Yonghe Tang, Hongmei Zhou, Weiying Lin. A Naphthalimide-Based Fluorescent Probe for Detecting Intracellular pH and Its Biological Imaging Application[J]. Acta Chimica Sinica, 2021, 79(1): 87-92.

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图/表 9

图式1. 探针NpH-1对pH值变化的响应机理

Scheme 1. The mechanism of the probe NpH-1 response to pH

图式2. 探针NpH-1的合成路线

Scheme 2. The synthesis of the probe NpH-1

图1. 探针NpH-1 (10 μmol/L)在B-R缓冲液中的紫外-可见吸收光谱 (pH值范围3.0~10.0)

Figure 1. The UV-Vis absorption spectra of the probeNpH-1 (10 μmol/L) in PBS buffers (pH range of 3.0~10.0).

图2. (a) 探针NpH-1(10 μmol/L)在3.0~10.0的pH值范围内的荧光发射光谱; (b)探针NpH-1(10 μmol/L) 在3.0~10.0的pH值范围内540 nm处荧光强度(λ ex=440 nm)

Figure 2. (a) The emission spectra of the probeNpH-1 at 540 nm in pH range of 3.0~10.0; (b) the pH titration of the probeNpH-1. (λ ex=440 nm)

图3. 探针NpH-1(10 μmol/L)在pH值为4.0, 7.4和9.0时有无光照条件下的荧光强度随时间的变化曲线(λ ex=440 nm, λ em=540 nm)

Figure 3. The curve of fluorescent emission intensity variation with time of the probeNpH-1(10 μmol/L)at pH 4.0, 7.4 and 9.0. (λ ex=440 nm, λ em=540 nm)

图4. 探针NpH-1(10 μmol/L)在pH为4.0和8.0之间的可逆循环(λ ex=440 nm,λ em=540 nm)

Figure 4. The reversible cycle of the fluorescent emission of the probe the probeNpH-1(10 μmol/L)between pH 4.0 and 8.0.

图5. 探针NpH-1(10 μmol/L)在各种离子和分子(200 μmol/L)在pH为4.0和7.4的溶液中在540 nm处的荧光发射强度. 1. α-D-葡萄糖, 2. L-苯丙氨酸, 3. L-丙氨酸, 4. L-蛋氨酸, 5. L-精氨酸, 6. L-抗坏血酸, 7. L-亮氨酸, 8. L-脯氨酸, 9. L-色氨酸, 10. L-丝氨酸, 11. L-天冬氨酸, 12. L-缬氨酸, 13. L-异亮氨酸, 14.L-组氨酸, 15. 丙酮醛, 16. 醋酸钠, 17. 二氯化钴, 18. 氟化钠, 19. 甘氨酸, 20. 硫氰化钠, 21. 硫酸钠, 22. 氯化钾, 23. 二氯化锰, 24. 氯化钠, 25. 碳酸钠, 26. 碳酸氢钠, 27. 无水氯化镁, 28. 五水合硫代硫酸钠, 29. 五水合硫酸铜, 30. 亚硫酸钠, 31. 亚硝酸钠.

Figure 5. Fluorescence intensity ofNpH-1(10 μmol/L) atλ em=540 nm in solution at pH 4.0 and 7.4 with various species (200 mmol/L). 1.α-D-glucose; 2.L-phenylalanine; 3.L-alanine; 4.L-Methionine; 5. L-arginine; 6. L-ascorbic acid; 7.L-leucine; 8. L-proline; 9. L-tryptophan; 10.L-serine; 11.L-aspartic acid; 12.L-valine; 13.L-isoleucine; 14.L-histidine; 15. Methylglyoxal; 16. NaOAc; 17. CoCl2; 18. NaF; 19. glycine; 20. NaCNS; 21. Na2SO4; 22. KCl; 23. MnCl2; 24. NaCl; 25. Na2CO3; 26. NaHCO3; 27. MgCl2; 28. Na2S2O3•5H2O; 29. Cu2SO4•5H2O; 30. Na2SO3; 31. NaNO2.

图6. (a~c) HeLa细胞用探针NpH-1 (10 μmol/L)处理后的细胞成像图; (d~f) HeLa细胞用氯喹(10 μmol/L)处理30 min后加入探针NpH-1 (10 μmol/L)的细胞成像图

Figure 6. (a~c) The imaging photograph of HeLa cell treated with the probeNpH-1 (10 μmol/L); (d~f) the imaging photograph of HeLa cell treated with chloroquine (10 μmol/L) for 30 min then addition of the probeNpH-1 (10 μmol/L).

图7. (a1)~(a4) 探针NpH-1(10 μmol/L) 与Hoechst 33342(10 μmol/L)的共定位成像图; (b1)~(b4) 探针NpH-1(10 μmol/L) 与MitoTrackerTM Deep Red(10 μmol/L)的共定位成像图; (c1)~(c4) 探针NpH-1(10 μmol/L) 与LysoTrackerTM Deep Red (10 μmol/L)的共定位成像图; (d1)~(d4) 探针NpH-1 (10 μmol/L) 与Golgi-Tracker Red (10 μmol/L)的共定位成像图; (e1)~(e4) 探针NpH-1 (10 μmol/L) 与ER-Tracker Blue (10 μmol/L)的共定位成像图.

Figure 7. (a1)~(a4) The colocalization photograph of the probe NpH-1 (10 μmol/L) with Hoechst 33342(10 μmol/L); (b1)~(b4) The colocalization photograph of the probeNpH-1(10 μmol/L) with MitoTrackerTM Deep Red (10 μmol/L); (c1)~(c4) The colocalization photograph of the probeNpH-1 (10 μmol/L) with LysoTrackerTM Deep Red; (d1)~(d4) The colocalization photograph of the probeNpH-1(10 μmol/L) with Golgi-Tracker Red; (e1)~(e4) The colocalization photograph of the probeNpH-1 (10 μmol/L) with ER-Tracker Blue.

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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

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

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