用于检测细胞内谷胱甘肽的比率型荧光探针

doi:10.7503/cjcu20150725

摘要/Abstract

摘要：

设计合成了1种用于检测生物巯基的比率型荧光探针(4), 并考察了其对谷胱甘肽的识别作用. 在4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中, 探针4可与谷胱甘肽快速反应, 溶液颜色由淡黄色变为粉红色, 从而实现“裸眼”检测, 且在608 nm处的荧光信号增强. 在1.6×10^-5~2×10^-4 mol/L范围内, 探针4能够定量检测谷胱甘肽, 检出限为8.9×10^-7 mol/L. 此外, 探针4还可用于MCF-7细胞中谷胱甘肽的成像.

关键词: 荧光探针, 谷胱甘肽, 细胞成像

Abstract:

A fluorescent probe(4) for the detection of biothiols was designed and synthesized and its properties for labeling glutathione was investigated. After reaction with glutathione in 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid(HEPES) buffer, the color of solution changed from light yellow to pink, which could be detected by naked eyes. Meanwhile, the fluorescence was enhanced at 608 nm. Probe 4 was useful for measuring glutathione at concentrations ranging from 1.6×10^-5 to 2×10^-4 mol/L with a detection limit of 8.9×10^-7 mol/L. Besides, probe 4 was sensitive to glutathione in MCF-7 cells, which emitted red fluorescence when it was incubated with MCF-7 cells.

Key words: Fluorescent probe, Glutathione, Intracellular imaging

中图分类号:

O657
O625.75

TrendMD:

杨润洁, 唐尧, 朱维平. 用于检测细胞内谷胱甘肽的比率型荧光探针. 高等学校化学学报, 2016, 37(4): 643.

YANG Runjie, TANG Yao, ZHU Weiping. Ratiometric Fluorescent Probe for the Detection of Glutathione in Living Cells^†. Chem. J. Chinese Universities, 2016, 37(4): 643.

图/表 8

Scheme 1 Synthetic routes of probe 4

Table 1 Appearance, melting points, yields, HRMS and elemental analysis data of compounds 3 and 4

Compd.	Appearance	m. p./℃	Yield(%)	HRMS(ESI) [M-H]^-(calcd.), m/z	Elemental analysis(%, calcd.)
Compd.	Appearance	m. p./℃	Yield(%)	HRMS(ESI) [M-H]^-(calcd.), m/z	C	H	N
3	Red solid	293.6—294.2	53	302.0930(302.0934)	70.87(71.29)	4.18(4.29)	13.72(13.86)
4	Yellow solid	﹥300	40	532.0564(532.0563)	53.03(54.03)	2.94(2.81)	13.06(13.13)

Table 2 1H NMR and 13C NMR data of compounds 3 and 4

Compd.	¹H NMR(DMSO-d₆, 400MHz), δ	¹³C NMR(DMSO-d₆, 100 MHz), δ
3	10.63(s, 1H, OH), 7.91(d, 1H, CH═C), 7.81(d, 2H, ArH), 7.02(d, 1H, CH═C), 6.9(d, 2H, ArH), 1.77(s, 6H, CH₃)	177.3, 175.8, 162.3, 148.3, 132.3, 125.7, 116.4, 112.9, 112.1, 111.7, 111.2, 99.0, 96.6, 53.2, 25.3
4	9.14(s, 1H, ArH),8.61(dd, 1H, ArH), 8.27(d, 1H, ArH), 8.00(d, 2H, ArH), 7.89(d, 1H, CH═C), 7.34(d, 2H, ArH), 7.23(d, 1H, CH═C), 1.79(s, 6H, CH₃)	176.9, 174.6, 151.6, 150.3, 148.1, 144.9, 134.4, 133.6, 131.4, 130.5, 127.5, 122.8, 121.1, 116.9, 112.5, 111.7, 110.6, 100.8, 99.5, 54.9, 54.9, 25.0

Fig.1 UV-Vis absorption(A) and fluorescence spectra(B) of probe 4(10 μmol/L) before(a) and after(b) reaction with glutathione(200 μmol/L) in HEPES buffer(0.1 mol/L, pH=7.4, 1% DMSO)Inset of (A): color changes of solution before(a) and after(b) reaction.

Fig.2 UV-Vis absorption spectra of probe 4(10 μmol/L) with different concentrations of GSH(A) and the linear relationship of A568/A394 vs. the concentrations of GSH over 0—200 μmol/L(B)c(GSH)/(μmol·L-1): 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200.

Fig.3 UV-Vis absorption(A) and fluorescence spectra(B) changes of probe 4(10 μmol/L) to glutathione(200 μmol/L) in 15 min

Fig.4 Fluorescence emission changes of probe 4(10 μmol/L) in HEPES upon addition of different amino acids(200 μmol/L)

Fig.5 Bright-field(A, C) and fluorescence(B, D) images of MCF-7 cells(A, B) and MCF-7 cells incubated with 50 μmol/L probe 4 for 30 min(C, D)

参考文献 22

[1]	Wood Z. A., Schröder E., Robin H. J., Poole L. B., Trends Biochem. Sci., 2003, 28(1), 32—40
[2]	Pastore A., Piemonte F., Locatelli M., Lo R. A., Gaeta L. M., Tozzi G., Federici G., Clin. Chem., 2001, 47(8), 1467—1469
[3]	Wu G., Fang Y. Z., Yang S., Lupton J. R., Turner N. D., J. Nutr., 2004, 134(3), 489—492
[4]	Dalton T. P., Shertzer H. G., Puga A., Annu. Rev. Pharmacol., 1999, 39(1), 67—101
[5]	Stipanuk H. M., Dominy E. J., Lee J. I., Coloso M. R., J. Nutr., 2006, 136(6), 1652—1659
[6]	Wu G. Y., Fang Y. Z., Yang S., Lupton R. J., Turner D. N., Recent Adv. Nutr. Sci., 2003, 134(3), 489—492
[7]	Carmel R.,Jocobsen D. W., Homocysteine in Health and Disease, Cambridge University Press, Cambridge, 2001, 12—18
[8]	Townsend D. M., Tew K. D., Tapiero H., Biomed. Pharmacother., 2003, 57(34), 145—155
[9]	Estrela J. M., Ortega A., Obrador E., Crit. Rev. Clin. Lab. Sci., 2006, 43(2), 143—181
[10]	Li H. L., Fan J. L., Liu X. J., Sun S. G., Peng X. J., Chem. J. Chinese Universities, 2010, 31(9), 1725—1728
	(李宏林, 樊江莉, 刘晓键, 孙世国, 彭孝军. 高等学校化学学报, 2010, 31(9), 1725—1728)
[11]	Wang P., Liu J., Lv X., Liu Y., Zhao Y., Guo W., Org. Lett., 2012, 14(2), 520—523
[12]	Guo Z., Nam S., Park S., Yoon J., Chem. Sci., 2012, 3(9), 2760—2765
[13]	Yang X. F., Huang Q., Zhong Y., Li Z., Li H., Lowry M., Escobedo J. O., Strongin R. M., Chem. Sci., 2014, 5(6), 2177—2183
[14]	Hou J. T., Yang J., Li K., Yu K. K., Yu X. Q., Sensors Actuators B, 2015, 214, 92—100
[15]	Samuel J. L., Nicholas R. C., Hsiao-lu D. L., Reichel S., Na L., Robert J. T., Moerner W. E., J. Am. Chem. Soc., 2008, 130, 9204—9205
[16]	Marissa K. L., Jarrod W., Robert J. T., Jiang H. R., Moerner W. E., Chem. Sci., 2013, 4, 220—225
[17]	Li M., Wu X., Wang Y., Li Y., Zhu W. H., James T. D., Chem. Commun., 2014, 50(14) 1751—1753
[18]	Wang S. P., Deng W. J., Sun D., Yan M., Zheng H., Xu J. G., Org. Biomol. Chem., 2009, 7, 4017—4020
[19]	Ji S. M., Guo H. M., Yuan X. L., Li X. H., Ding H. D., Gao P., Zhao C. X., Wu W. T., Wu W. H., Zhao J. Z., Org. Lett., 2010, 12(12), 2876—2879
[20]	Tao Z., Kun P. G., Ling Q., Yu Q. S., Synth. Commun., 2006, 36(10), 1367—1372
[21]	Avetisyan A. A., Alvandzhyan A. G., Avetisyan K. S., Russ. J. Org. Chem., 2009, 45(12), 1871—1872
[22]	Huang C. S., Jia T., Yu C. J., Zhang A. M., Jia N. Q., Biosens. Bioelectron., 2015, 63, 513—518

相关文章 15

[1]	赵永梅, 穆叶舒, 洪琛, 罗稳, 田智勇. 双萘酰亚胺衍生物用于检测水溶液中的苦味酸[J]. 高等学校化学学报, 2022, 43(3): 20210765.
[2]	唐倩, 但飞君, 郭涛, 兰海闯. 喹啉酮-香豆素类Hg²⁺比色荧光探针的合成及应用[J]. 高等学校化学学报, 2022, 43(2): 20210660.
[3]	王迪, 钟克利, 汤立军, 侯淑华, 吕春欣. 席夫碱共价有机框架的合成及对I ^‒ 的识别[J]. 高等学校化学学报, 2022, 43(10): 20220115.
[4]	黄珊, 姚建东, 宁淦, 肖琦, 刘义. 石墨烯量子点荧光探针对碱性磷酸酶活性的高效检测[J]. 高等学校化学学报, 2021, 42(8): 2412.
[5]	李安然, 赵冰, 阚伟, 宋天舒, 孔祥东, 卜凡强, 孙立, 殷广明, 王丽艳. 基于菲并咪唑的ON⁃OFF⁃ON双比色荧光探针及细胞成像[J]. 高等学校化学学报, 2021, 42(8): 2403.
[6]	杨新杰, 赖艳琼, 李秋旸, 张艳丽, 王红斌, 庞鹏飞, 杨文荣. 基于环状DNA-银纳米簇荧光探针对微囊藻毒素-LR的传感检测[J]. 高等学校化学学报, 2021, 42(12): 3600.
[7]	谌委菊, 陈诗雅, 薛曹叶, 刘波, 郑晶. 缺氧响应荧光探针的成像及治疗应用[J]. 高等学校化学学报, 2021, 42(11): 3433.
[8]	黄加玲,刘凤娇,王婷婷,刘翠娥,郑凤英,王振红,李顺兴. 氮硫共掺杂碳量子点对胃液pH值的精确检测[J]. 高等学校化学学报, 2020, 41(7): 1513.
[9]	凌云云,李莉,梁修蓉,夏云生. 基于金@二氧化锰纳米片超级纳米粒子从比色到单颗粒光谱检测谷胱甘肽[J]. 高等学校化学学报, 2020, 41(4): 623.
[10]	吴倩, 程丹, 吕芸, 袁林, 张晓兵. 大斯托克斯位移远红光至近红外荧光探针用于检测肝损伤过程中过氧化亚硝酸盐的动态变化[J]. 高等学校化学学报, 2020, 41(11): 2426.
[11]	郑姗, 刘洋, 陈飘飘, 邢怡晨, 黄朝表. 基于PbS QDs/TiO₂ NPs构建新型谷胱甘肽光电化学传感器[J]. 高等学校化学学报, 2019, 40(9): 1866.
[12]	王金金, 戚少龙, 杜建时, 杨清彪, 宋岩, 李耀先. 苯并噻唑类荧光探针的合成及对N₂H₄·H₂O和HS $O 3 -$ 的检测性能[J]. 高等学校化学学报, 2019, 40(7): 1397.
[13]	黄池宝, 潘淇, 陈华仕, 梁兴, 吕国岭. 双氰基二苯代乙烯型双光子荧光环境敏感探针[J]. 高等学校化学学报, 2019, 40(6): 1128.
[14]	沐亚新, 阿丽, 庄欠粉, 王勇, 倪永年. 超小荧光二硫化钨量子点的水热合成及细胞成像应用[J]. 高等学校化学学报, 2019, 40(4): 693.
[15]	张勇,申城,幸志荣,陈归柒,卢资,侯志兵,陈雪梅. 可视化检测次氯酸的苯并咪唑类荧光增强型探针[J]. 高等学校化学学报, 2019, 40(12): 2480.