基于尼罗红类ONOO–近红外荧光探针的开发及其成像应用

doi:10.6023/A23050225

摘要/Abstract

过氧亚硝酸根(ONOO^–)在氧化应激和炎症过程中的生成与细胞损伤、炎症反应和免疫调节等密切相关, 因此, 开发高灵敏且高度选择性的ONOO^–检测方法对于深入理解疾病发生和发展机制、提高疾病的早期诊断和治疗水平具有极其重要意义. 设计合成了一种选择性检测ONOO^–的新型荧光探针NR-Pro. 该探针通过使用烷基链将2-羟基尼罗红衍生物(NR-OH)与4,4'-氮杂二基二苯酚基团有机结合, 在ONOO^–作用下, 释放出近红外染料NR-OH. 实验研究表明, 该探针在658 nm处对ONOO^–表现出显著的“OFF-ON”型荧光信号响应, 且对ONOO^–浓度在0~10 μmol/L范围内呈现良好的线性关系, 检出限低至17.7 nmol/L. 这项研究进一步证实了NR-Pro探针在检测RAW264.7细胞中外源和内源ONOO^–方面具有优良的成像能力, 这为早期诊断和治疗疾病提供了新的思路.

关键词: 近红外, 荧光探针, 过氧亚硝酸根, 尼罗红, 细胞成像

The production of peroxynitrite (ONOO^–) in situations of oxidative stress and inflammation has a profound impact on cellular damage, inflammatory responses, and immune regulation. Thus, the advancement of highly sensitive and selective techniques to detect ONOO^– is of great significance. These methods are crucial for gaining insights into disease mechanisms, improving early disease diagnosis, and enhancing treatment effectiveness. In this study, we have designed and synthesized a novel fluorescent probe, NR-Pro, specifically for the selective detection of ONOO^–. The probe incorporates a 2-hydroxy Nile red derivative (NR-OH) that is organically bound to a 4,4'-azabediyldiphenol group via an alkyl chain. Upon interaction with ONOO^–, the probe releases the near-infrared dye NR-OH, leading to a significant “OFF-ON” fluorescence signal response at 658 nm. The mechanism involves the initial attack of ONOO^– on the 4,4'-aza-diyl diphenol group, resulting in the formation of an alkylamine-modified Nile red derivative and the release of two 1,4-benzoquinone molecules. Subsequently, the alkylamine group undergoes further oxidation by ONOO^–, leading to the liberation of the near-infrared fluorophore NR-OH and acrolein through a 1,3-elimination reaction with the involvement of water. Experimental investigations have demonstrated that the probe exhibits a favorable linear relationship with ONOO^– concentration in the range of 0 to 10 μmol/L, with an exceptionally low detection limit of 17.7 nmol/L. Furthermore, the fluorescence emission of NR-Pro remained unaffected by various biological species, including reactive oxygen species (ROSs), metal ions, enzymes, anions, and amino acids. NR-Pro also exhibited minimal cytotoxicity and very high photostability, suggesting its potential suitability for cell imaging applications. Moreover, our study confirms the remarkable imaging capability of the NR-Pro probe in detecting both exogenous and endogenous ONOO^– in RAW264.7 cells. These findings present novel insights for the early diagnosis and treatment of diseases, paving the way for potential advancements in the field.

Key words: near-infrared, fluorescent probe, peroxynitrite, Nile red, cell imaging

引用此文

贺晓梦, 袁方, 张素雅, 张健健. 基于尼罗红类ONOO^–近红外荧光探针的开发及其成像应用[J]. 化学学报, 2023, 81(11): 1515-1521.

Xiaomeng He, Fang Yuan, Suya Zhang, Jianjian Zhang. Development of a Near-Infrared Fluorescent Probe Based on Nile Red for ONOO^– and Its Imaging Applications[J]. Acta Chimica Sinica, 2023, 81(11): 1515-1521.

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图/表 8

图1. (a) NR-Pro (5 μmol/L)与不同浓度ONOO– (0~50 μmol/L)在PBS (pH 7.4, 20 mmol/L, 20% MeCN)中反应30 min后的荧光光谱; (b)反应体系在658 nm处的荧光强度与ONOO–浓度的关系

Figure 1. Fluorescence spectra of NR-Pro (5 μmol/L) and ONOO– (0~50 μmol/L) in PBS (pH 7.4, 20 mmol/L, 20% MeCN) for 30 min (a); the embedded graph (b) shows the relationship between fluorescence intensity and ONOO– concentration of the reaction system at 658 nm

图2. NR-Pro (5 μmol/L)与ONOO– (20 μmol/L)在PBS (pH 7.4, 20 mmol/L, 20% MeCN)中反应的荧光动力学曲线(a)和光稳定性曲线(b)

Figure 2. Fluorescence kinetic curves (a) and photostability curves (b) of NR-Pro (5 μmol/L) and ONOO– (20 μmol/L) in PBS (pH 7.4, 20 mmol/L, 20% MeCN)

图3. pH对NR-Pro (5 μmol/L)与ONOO– (20 μmol/L)反应体系荧光强度的影响.

Figure 3. Effect of pH on fluorescence intensity of NR-Pro (5 μmol/L) and ONOO– (20 μmol/L) reaction systems

图4. NR-Pro (5 μmol/L)与不同分析物(50 μmol/L)在PBS (pH 7.4, 20 mmol/L, 20% MeCN)中反应30 min后的荧光强度. 其中分析物1~25: Blank, ONOO–, H2O2, $\mathop{{O}}_{2}^{{\mathop{}^{-·}}}$, ClO–, TBO?, TBHP, NO, $NO_{2}^{-}$, Na2S, GSH, Lys, Cys, Na+, K+, Mg2+, Fe3+, Ca2+, Fe2+, Zn2+, ?OH, 1O2, β-半乳糖苷酶, 胰蛋白酶, L-抗坏血酸

Figure 4. Fluorescence intensity of NR-Pro (5 μmol/L) and different analytes (50 μmol/L) after 30 min reaction in PBS (pH 7.4, 20 mmol/L, 20% MeCN). Among them, analytes 1~25: Blank, ONOO–, H2O2, $\mathop{{O}}_{2}^{{\mathop{}^{-·}}}$, ClO–, TBO?, TBHP, NO, $NO_{2}^{-}$, Na2S, GSH, Lys, Cys, Na+, K+, Mg2+, Fe3+, Ca2+, Fe2+, Zn2+, ?OH, 1O2, β-galactosidase, trypsin, L-ascorbic acid Fluorescence intensity of NR-Pro (5 μmol/L) and different analytes (50 μmol/L) after 30 min reaction in PBS (pH 7.4, 20 mmol/L, 20% MeCN). Among them, analytes 1~25: Blank, ONOO–, H2O2, $\mathop{{O}}_{2}^{{\mathop{}^{-·}}}$, ClO–, TBO?, TBHP, NO, $NO_{2}^{-}$, Na2S, GSH, Lys, Cys, Na+, K+, Mg2+, Fe3+, Ca2+, Fe2+, Zn2+, ?OH, 1O2, β-galactosidase, trypsin, L-ascorbic acid

图5. 探针NR-Pro检测ONOO–可能的作用机制

Figure 5. Possible mechanism of ONOO– detection by probe NR-Pro

图6. 探针NR-Pro与ONOO–反应的高效液相色谱图. (I) NR-Pro; (II, III) NR-Pro (5 μmol/L)与ONOO– (25和50 μmol/L)在PBS (pH 7.4, 20 mmol/L, 20% MeCN)中反应的溶液; (IV) NR-OH. 流动相: MeCN/H2O (70/30, V/V), 流速(1 mL/min)

Figure 6. High performance liquid chromatogram of the reaction of probe NR-Pro with ONOO–. (I) NR-Pro; (II, III) NR-Pro (5 μmol/L) and ONOO– (25 and 50 μmol/L) in PBS (pH 7.4, 20 mmol/L, 20% MeCN); (IV) NR-OH. Mobile phase: MeCN/H2O (70/30, V/V), flow rate (1 mL/min) High performance liquid chromatogram of the reaction of probe NR-Pro with ONOO–. (I) NR-Pro; (II, III) NR-Pro (5 μmol/L) and ONOO– (25 and 50 μmol/L) in PBS (pH 7.4, 20 mmol/L, 20% MeCN); (IV) NR-OH. Mobile phase: MeCN/H2O (70/30, V/V), flow rate (1 mL/min)

图7. RAW264.7细胞中外源性ONOO–的荧光成像图. NR-Pro (10 μmol/L)与细胞孵育30 min, 用PBS洗净后进行不同处理: (a)对照组; (b)加入20 μmol/L ONOO–孵育30 min; (c)加入50 μmol/L ONOO–孵育30 min; (d)图像a~c中细胞的平均荧光强度. 标尺: 20 μm

Figure 7. Fluorescence imaging of exogenous ONOO– in RAW264.7 cells. NR-Pro (10 μmol/L) was incubated with cells for 30 min, washed with PBS and treated differently: (a) control group; (b) add 20 μmol/L ONOO– and incubate for 30 min; (c) add 50 μmol/L ONOO– and incubate for 30 min; (d) average fluorescence intensity of cells in images a~c. Scale bar: 20 μm

图8. RAW264.7细胞中内源性ONOO–的荧光成像图. (a)细胞与NR-Pro (10 μmol/L)孵育30 min; (b)细胞先用1 μg/mL LPS刺激4 h, 接着用1 μg/mL PMA刺激30 min, 最后再与NR-Pro (10 μmol/L)孵育30 min; (c) 100 μmol/L UA预处理细胞1 h后, 与LPS (1 μg/mL)孵育4 h, PMA (1 μg/mL)孵育30 min, 再与NR-Pro (10 μmol/L)孵育30 min; (d)图像a~c中细胞的平均荧光强度. 标尺: 20 μm

Figure 8. Fluorescence imaging of endogenous ONOO– in RAW264.7 cells. (a) Incubate the cells with NR-Pro (10 μmol/L) for 30 min; (b) cells were first stimulated with 1 μg/mL LPS for 4 h, then 1 μg/mL PMA for 30 min, and finally incubated with NR-Pro (10 μmol/L) for 30 min; (c) after 100 μmol/L UA pretreated cells for 1 h, incubate with LPS (1 μg/mL) for 4 h, PMA (1 μg/mL) for 30 min, and NR-Pro (10 μmol/L) for 30 min; (d) average fluorescence intensity of cells in images a~c. Scale bar: 20 μm

参考文献 41

[1]	Nathan C.; Cunningham-Bussel A. Nat. Rev. Immunol. 2013, 13, 349. doi: 10.1038/nri3423 pmid: 23618831
[2]	Dickinson B. C.; Chang C. J. Nat. Chem. Biol. 2011, 7, 504. doi: 10.1038/nchembio.607 pmid: 21769097
[3]	Liu Y.; Teng L.; Lyu Y.; Song G.; Zhang X. B.; Tan W. Nat. Commun. 2022, 13, 2216. doi: 10.1038/s41467-022-29894-1
[4]	Wang Y.; Shi L.; Ye Z.; Guan K.; Teng L.; Wu J.; Yin X.; Song G.; Zhang X. B. Nano. Lett. 2020, 20, 176. doi: 10.1021/acs.nanolett.9b03556
[5]	Ferrer-Sueta G.; Campolo N.; Trujillo M.; Bartesaghi S.; Carballal S.; Romero N.; Alvarez B.; Radi R. Chem. Rev. 2018, 118, 1338. doi: 10.1021/acs.chemrev.7b00568 pmid: 29400454
[6]	Radi R. J. Biol. Chem. 2013, 288, 26464. doi: 10.1074/jbc.R113.472936
[7]	De Armas M. I.; Esteves R.; Viera N.; Reyes A. M.; Mastrogiovanni M.; Alegria T. G. P.; Netto L. E. S.; Tortora V.; Radi R.; Trujillo M. Free Radic. Biol. Med. 2019, 130, 369. doi: 10.1016/j.freeradbiomed.2018.10.451
[8]	Graham P. M.; Li J. Z.; Dou X.; Zhu H.; Misra H. P.; Jia Z.; Li Y. Mol. Cell Biochem. 2013, 378, 291. doi: 10.1007/s11010-013-1620-z pmid: 23529546
[9]	Li X.; Tao R. R.; Hong L. J.; Cheng J.; Jiang Q.; Lu Y. M.; Liao M. H.; Ye W. F.; Lu N. N.; Han F.; Hu Y. Z.; Hu Y. H. J. Am. Chem. Soc. 2015, 137, 12296. doi: 10.1021/jacs.5b06865
[10]	Islam M. T. Neurol. Res. 2017, 39, 73. doi: 10.1080/01616412.2016.1251711
[11]	Munn L. L. WIREs Syst. Biol. Med. 2017, 9, e1382. doi: 10.1002/wsbm.2017.9.issue-4
[12]	Xie X.; Tang F.; Liu G.; Li Y.; Su X.; Jiao X.; Wang X.; Tang B. Anal. Chem. 2018, 90, 11629. doi: 10.1021/acs.analchem.8b03207
[13]	Bartesaghi S.; Radi R. Redox Biol. 2018, 14, 618. doi: S2213-2317(17)30621-3 pmid: 29154193
[14]	Hu J. S.; Shao C.; Wang X.; Di X.; Xue X.; Su Z.; Zhao J.; Zhu H. L.; Liu H. K.; Qian Y. Adv. Sci. 2019, 6, 1900341. doi: 10.1002/advs.v6.15
[15]	Gao L.; Wang W.; Wang X.; Yang F.; Xie L.; Shen J.; Brimble M. A.; Xiao Q.; Yao S. Q. Chem. Soc. Rev. 2021, 50, 1219. doi: 10.1039/D0CS00115E
[16]	Zhang S. Y.; Ning L. L.; Song Z. H.; Zhao X. Y.; Guan F.; Yang X. F.; Zhang J. J. Anal. Chem. 2022, 94, 5805. doi: 10.1021/acs.analchem.1c05184 pmid: 35380780
[17]	Zhao X. Y.; Ning L. L.; Zhou X. M.; Song Z. H.; Zhang J. J.; Guan F.; Yang X. F. Anal. Chem. 2021, 93, 4894. doi: 10.1021/acs.analchem.0c05081
[18]	Wu W.; Zhang C.; Rees T. W.; Liao X.; Yan X.; Chen Y.; Ji L.; Chao H. Anal. Chem. 2020, 92, 6003. doi: 10.1021/acs.analchem.0c00259
[19]	Ueno T.; Nagano T. Nat. Methods 2011, 8, 642. doi: 10.1038/nmeth.1663
[20]	Chan J.; Dodani S. C.; Chang C. J. Nat. Chem. 2012, 4, 973. doi: 10.1038/nchem.1500
[21]	Zhang S.-Y.; Zhang J.-J. Fine Chem. 2020, 37, 2229. (in Chinese)
	( 张素雅, 张健健, 精细化工, 2020, 37, 2229.)
[22]	Li Y.; Ning L. L.; Yuan F.; Zhang T.; Zhang J. J.; Xu Z. G.; Yang X. F. Anal. Chem. 2020, 92, 5733. doi: 10.1021/acs.analchem.9b04806 pmid: 32193934
[23]	Zhao X. Y.; Ding M. B.; Ning L. L.; Yuan F.; Li J. C.; Guo Y.; Mu Y. G.; Zhang J. J. Acta Mater. Med. 2022, 1, 476.
[24]	Yuan F.; He X. M.; Lu Y. R.; Ning L. L.; Zhao X. Y.; Zhang S. Y.; Guan F.; Guo Y.; Zhang J. J. Anal. Chem. 2023, 95, 6931. doi: 10.1021/acs.analchem.3c00230
[25]	Lv X.; Wu Y.; Zhang B.-R.; Guo W. Acta Chim. Sinica 2023, 81, 359. (in Chinese) doi: 10.6023/A22120487
	( 吕鑫, 吴仪, 张勃然, 郭炜, 化学学报, 2023, 81, 359.) doi: 10.6023/A22120487
[26]	Li X.; Liang X.; Yin J.; Lin W. Chem. Soc. Rev. 2021, 50, 102. doi: 10.1039/D0CS00896F
[27]	Lu J.; Li Z.; Gao Q.; Tan J.; Sun Z.; Chen L.; You J. Anal. Chem. 2021, 93, 3426. doi: 10.1021/acs.analchem.0c04512
[28]	Wang N.; Wang H.; Zhang J.; Ji X.; Su H.; Liu J.; Wang J.; Zhao W. Chin. Chem. Lett. 2022, 33, 1584. doi: 10.1016/j.cclet.2021.09.046
[29]	Cui J.; Zang S.; Nie H.; Shen T.; Su S.; Jing J.; Zhang X. Sensor. Actuat. B-Chem. 2021, 328, 129069. doi: 10.1016/j.snb.2020.129069
[30]	Chen S.; Vurusaner B.; Pena S.; Thu C. T.; Mahal L. K.; Fisher E. A.; Canary J. W. Anal. Chem. 2021, 93, 10090. doi: 10.1021/acs.analchem.1c00911
[31]	Huang J.; Wang C.; Lin M.-G.; Zeng F.; Wu S.-Z. Acta Chim. Sinica 2021, 79, 331. (in Chinese) doi: 10.6023/A20100459
	( 黄靖, 王超, 林敏刚, 曾钫, 吴水珠, 化学学报, 2021, 79, 331.) doi: 10.6023/A20100459
[32]	Chen F.; Teng L.; Lu C.; Zhang C.; Rong Q.; Zhao Y.; Yang Y.; Wang Y.; Song G.; Zhang X. Anal. Chem. 2020, 92, 13452. doi: 10.1021/acs.analchem.0c02859
[33]	Zhou D. Y.; Li Y.; Jiang W. L.; Tian Y.; Fei J.; Li C. Y. Chem. Commun. 2018, 54, 11590. doi: 10.1039/C8CC07389A
[34]	Cheng D.; Peng J.; Lv Y.; Su D.; Liu D.; Chen M.; Yuan L.; Zhang X. J. Am. Chem. Soc. 2019, 141, 6352. doi: 10.1021/jacs.9b01374 pmid: 30897899
[35]	Martinez V.; Henary M. Chemistry 2016, 22, 13764.
[36]	Jose J.; Burgess K. Tetrahedron 2006, 62, 11021. doi: 10.1016/j.tet.2006.08.056
[37]	Sebok-Nagy K.; Miskolczy Z.; Biczok L. Photochem. Photobiol. 2005, 81, 1212. pmid: 15901209
[38]	Wang P.; Yu L.; Gong J.; Xiong J.; Zi S.; Xie H.; Zhang F.; Mao Z.; Liu Z.; Kim J. S. Angew. Chem., Int. Ed. 2022, 61, e202206894. doi: 10.1002/anie.v61.36
[39]	Lin K. K.; Wu S. C.; Hsu K. M.; Hung C. H.; Liaw W. F.; Wang Y. M. Org. Lett. 2013, 15, 4242. doi: 10.1021/ol401932p
[40]	Hooper D. C.; Scott G. S.; Zborek A.; Mikheeva T.; Kean R. B.; Koprowski H.; Spitsin S. V. FASEB J. 2000, 14, 691. doi: 10.1096/fasebj.14.5.691 pmid: 10744626
[41]	Wang Z.; Cong T. D.; Zhong W.; Lau J. W.; Kwek G.; Chan-Park M. B.; Xing B. Angew. Chem., Int. Ed. 2021, 60, 16900. doi: 10.1002/anie.v60.31