一种罗丹明内酰胺类高分子pH荧光探针的合成及性能

doi:10.16085/j.issn.1000-6613.2023-0262

摘要/Abstract

摘要：

用罗丹明B、丙烯酰胺等为主要原料，设计制备出一种高分子pH荧光探针PRAM，在结构表征的基础上，探究此探针的光物理性质和活细胞的荧光成像。结果表明，探针PRAM的pK_a为4.30，在pH为3.0~7.0区间内，对H⁺表现出高度灵敏性和选择性，可实时监测体系pH变化。在pH为5.0~6.0区间内，其585nm下的荧光强度与pH呈现出良好的线性关系，线性相关系数为0.9955，可用来精确测定体系的pH。在常见的金属离子存在下，PRAM表现良好的抗干扰性和选择性。同时还具备很好的可逆性，可重复利用。PRAM具有良好的水溶性和膜透性，弱酸性条件下可快速穿透细胞膜，并能实现活细胞pH变化的荧光成像。因此，可用于弱酸环境下细胞器（如溶酶体、癌细胞等）内生理pH变化的检测。

关键词: 高分子荧光探针, 罗丹明B酰肼, pH探针, 细胞成像, 合成

Abstract:

A class of polymeric pH fluorescent probe PRAM was designed and prepared using Rhodamine B and acrylamide as the main raw materials. The structure of PRAM was characterized. The photophysical properties of this probe and the fluorescence imaging of living cells were investigated. The experimental results showed that the pK_a value of the probe was 4.30, and it had high selectivity and sensitivity to H⁺ in the pH range of 3.0—7.0, allowing real-time monitoring of system pH changes. Furthermore, its fluorescence intensity at 585 nm showed a good linear relationship with pH in the pH range of 5.0—6.0, with a linear correlation coefficient of 0.9955, which could be used to accurately determine the pH of the system. PRAM still performed well in terms of interference resistance and selectivity in the presence of common metal ions. PRAM had good water solubility and membrane permeability, allowing rapid penetration of cell membranes under weakly acidic conditions, and enables fluorescence imaging of pH changes in living cells. Therefore, it could be used to detect physiological pH changes within organelles (e.g., lysosomes, cancer cells, etc.) in a weakly acidic environment.

Key words: polymer fluorescent probe, Rhodamine B hydrazide, pH probes, cell imaging, synthesis

中图分类号:

O657.31

李桂贞, 胡英元, 章博, 杨耀祖, 翟荣佳, 赵鑫. 一种罗丹明内酰胺类高分子pH荧光探针的合成及性能[J]. 化工进展, 2024, 43(1): 473-479.

LI Guizhen, HU Yingyuan, ZHANG Bo, YANG Yaozu, ZHAI Rongjia, ZHAO Xin. Synthesis and properties of a Rhodamine lactam-based polymeric pH fluorescent probe[J]. Chemical Industry and Engineering Progress, 2024, 43(1): 473-479.

图/表 7

图1 探针PRAM对pH变化的响应机理

图2 PRAM的合成路线

图3 PRAM在不同pH水溶液下的紫外-可见吸收光谱

图4 PRAM的荧光强度与pH的关系

图5 探针PRAM的选择性和抗干扰性

图6 探针PRAM在585nm处的荧光强度在pH为3.0和7.0下的变化

图7 TM3细胞的荧光成像[细胞可见光(a)、荧光成像(g)和细胞加入PRAM后的可见光(b)、荧光成像(h)；加入PRAM后，pH分别为4.0 (c)、(i)、5.0 (d)、(j)、6.0 (e)、(k)、7.0 (f)、(l)的可见光和荧光成像]

参考文献 25

1	赵秋丽, 卞洁鹏, 杨庆浩, 等. 聚集诱导发红光材料在生物成像领域的应用[J]. 材料导报, 2019, 33(3): 522-535.
	ZHAO Qiuli, BIAN Jiepeng, YANG Qinghao, et al. Application of aggregation-induced red emission materials in bioimaging[J]. Materials Reports, 2019, 33(3): 522-535.
2	池雨, 高云玲, 潘勇，等. 氮杂氟硼荧（Aza-BODIPY）类荧光探针的研究进展[J]. 化工进展, 2018, 37(3): 1137-1144.
	CHI Yu, GAO Yunling, PAN Yong, et al. New progress in Aza-BODIPY-based fluorescence probes[J]. Chemical Industry and Engineering Progress, 2018, 37(3): 1137-1144.
3	彭丽, 光善仪, 徐洪耀. 一种新型罗丹明席夫碱(RHBS)制备及多功能性研究[J]. 功能材料, 2020, 51(4): 4013-4017.
	PENG Li, GUANG Shanyi, XU Hongyao. Preparation and multifunctional properties of a new rhodamine Schiff base (RHBS) [J]. Journal of Functional Materials, 2020, 51(4): 4013-4017.
4	于雪, 包青青, 姜超, 等. 花菁类近红外荧光探针在生物检测中的应用研究进展[J]. 化工进展, 2019, 38(12): 5492-5503.
	YU Xue, BAO Qingqing, JIANG Chao, et al. Applied research progress on cyanine-based near-infrared fluorescent probe in biological detection[J]. Chemical Industry and Engineering Progress, 2019, 38(12): 5492-5503.
5	VINGTDEUX Valérie, HAMDANE Malika, BEGARD Séverine, et al. Intracellular pH regulates amyloid precursor protein intracellular domain accumulation[J]. Neurobiology of Disease, 2007, 25(3): 686-696.
6	任江波, 王蕾, 郭锐, 等. 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.
	REN Jiangbo, WANG Lei, GUO Rui, et al. A naphthalimide-based fluorescent probe for detecting intracellular pH and its biological imaging application[J]. Acta Chimica Sinica, 2021, 79(1): 87-92.
7	刘涛, 杨夕强, 高洪成, 等. 罗丹明-乙二胺荧光探针用于pH的检测[J]. 承德石油高等专科学校学报, 2020, 22(3): 32-34.
	LIU Tao, YANG Xiqiang, GAO Hongcheng, et al. Rhodamine-ethylenediamine fluorescent sensor for pH detection[J]. Journal of Chengde Petroleum College, 2020, 22(3): 32-34.
8	公少华, 张霞, 李娜, 等. 荧光纳米探针用于细胞器pH检测的研究进展[J]. 高等学校化学学报, 2020, 41(9): 1933.
	GONG Shaohua, ZHANG Xia, LI Na, et al. Recent progress of fluorescent nanoprobes for organelle pH detection[J]. Chemical Journal of Chinese Universities, 2020, 41(9): 1933-1944.
9	Misbah NAZ, DAI Zhicong, HUSSAIN Sajid, et al. The soil pH and heavy metals revealed their impact on soil microbial community[J]. Journal of Environmental Management, 2022, 321:115770.
10	PALPURINA Salza WAGNER Viktoria, VON WEHRDEN Henrik, et al. The relationship between plant species richness and soil pH vanishes with increasing aridity across Eurasian dry grasslands[J]. Global Ecology and Biogeography, 2017, 26(4): 425-434.
11	MENG Zuchao, ZHANG Hongfang, ZHENG Jianbin. An electrochemical sensor based on titanium oxide-carbon nanotubes nanocomposite for simultaneous determination of hydroquinone and catechol[J]. Research on Chemical Intermediates, 2015, 41(5): 3135-3146.
12	NAIME J, MAMUN M S, SAAD A M ALY, et al. Synthesis, characterization and application of a novel polyazo dye as a universal acid-base indicator[J]. RSC Advances, 2022, 12(43):28034-28042.
13	LUNKAD Raju, BIEHL Philip, MURMILIUK Anastasiia, et al. Simulations and potentiometric titrations enable reliable determination of effective pK_a values of various polyzwitterions[J]. Macromolecules, 2022, 55(17): 7775-7784.
14	陈俊, 冯燕, 孟祥明. 一种线粒体靶向香豆素基pH荧光探针合成及性能[J]. 发光学报, 2021, 42(4): 462-469.
	CHEN Jun, FENG Yan, MENG Xiangming. Synthesis and properties of a mitochondria-targeted coumarin-based pH fluorescent probe[J]. Chinese Journal of Luminescence, 2021, 42(4): 462-469.
15	曹西颖, 罗时荷, 杨崇玲, 等. 有机聚合物荧光传感器的研究进展[J]. 有机化学, 2020, 40(12): 4046-4059.
	CAO Xiying, LUO Shihe, YANG Chongling, et al. Research progress of organic polymer fluorescence sensor[J]. Chinese Journal of Organic Chemistry, 2020, 40(12): 4046-4059.
16	王宇. 聚合物基荧光探针的制备及其性能研究[D]. 无锡: 江南大学, 2012.
	WANG Yu. Preparation and property study of polymer-based fluorescent probes[D]. Wuxi: Jiangnan University, 2012.
17	杨栋梁. 共轭聚合物荧光探针及其离子检测与生物成像研究[D]. 南京: 南京邮电大学, 2018.
	YANG Dongliang. Fluorescent probes based on conjugated polymers and their applications for ions detection and bioimaging[D]. Nanjing: Nanjing University of Posts and Telecommunications, 2018.
18	郭文波. 含有罗丹明的有机聚合物金属离子光谱探针研究[D]. 苏州: 苏州大学, 2020.
	GUO Wenbo. Research on rhodamine-based organic polymeric spectral probes for metal ions[D]. Suzhou: Suzhou University, 2020.
19	张哲. 近红外荧光聚合物纳米探针的设计制备及生物成像应用研究[D]. 长春: 吉林大学, 2019.
	ZHANG Zhe. Design and bio-imaging applications of near-infrared fluorescent polymer nanoparticles[D]. Changchun: Jililn University, 2019.
20	王钊. 聚合物基罗丹明6G衍生物型Hg²⁺荧光探针的制备及性能研究[D]. 长沙: 湖南大学, 2015.
	WANG Zhao. The research on the synthesis and properties of polymer based derivatives of rhodamine 6G fluorescent probe for Hg²⁺ [D]. Changsha: Hunan University, 2015.
21	XUE Xiaolei, WANG Yang, CHEN Shaojin, et al. Monitoring intracellular pH using a hemicyanine-based ratiometric fluorescent probe[J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2023, 284: 121778.
22	张钰绮, 陈永虎, 刘华丽, 等. 葡萄糖修饰罗丹明B的荧光探针的合成及其性质研究[J]. 化学试剂, 2021, 43(2): 243-246.
	ZHANG Yuqi, CHEN Yonghu, LIU Huali, et al. Synthesis and application of fluorescent probes of rhodamine B modified with glucose[J]. Chemical Reagents, 2021, 43(2): 243-246.
23	MINAMI Kimitaka, SUZUKI Takashi, AIZAWA Takafumi, et al. Proton concentration of supercritical water and high-concentrated carbon dioxide mixture using UV-vis spectroscopy[J]. Fluid Phase Equilibria, 2007, 257(2): 177-182.
24	翟荣佳, 赵鑫, 刘娅静, 等. 一种罗丹明类Hg²⁺和pH双功能荧光探针及其选择识别性能[J]. 化学世界, 2015, 56(11): 680-685.
	ZHAI Rongjia, ZHAO Xin, LIU Yajing, et al. Synthesis and performance of a new fluorescent probe based on rhodamine for selective recognition of Hg²⁺ and pH[J]. Chemical World, 2015, 56(11): 680-685.
25	YU Kangkang, LI Kun, HOU Jiting, et al. Rhodamine-based lysosome-targeted fluorescence probes: High pH sensitivity and their imaging application in living cells[J]. RSC Advances, 2014, 4(64): 33975-33980.

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