Fluorescent Probe for Bisulfite/Sulfite Imaging in Mitochondria†

doi:10.7503/cjcu20140542

Abstract

Abstract:

A new type of pentamethine cyanine dye with bromo group at the central position Cy5-Br was designed and synthesized, and the structure is not reported as far as we know. Cy5-Br can identify SO₂ derivatives bisulfite and sulfite with a good linear response. Furthermore, Cy5-Br has low cytotoxic to living cells, can localize in mitochondria, and image its bisulfite and sulfite ions concentration real-time change in living cells by the laser confocal fluorescence microscope.

Key words: Sulfite, Bisulfate ion, Mitochondria, Fluorescent imaging

CLC Number:

O657

TrendMD:

JIANG Na, FAN Jiangli, ZHANG Shuai, PENG Xiaojun. Fluorescent Probe for Bisulfite/Sulfite Imaging in Mitochondria^†[J]. Chem. J. Chinese Universities, 2014, 35(12): 2534.

Figures/Tables 12

Scheme 1 Synthetic routes of Cy5-Br

Fig.1 Normalized absorbance(A) and emission spectra(B) of Cy5-Br in different solvents

Table 1 Spectral data of Cy5-Br in different solvents

Solvent	λ_abs/nm	$λ em a$ /nm	Δλ^b/nm	ε^c	$Φ f d$ (%)
THF	655	668	13	2.23	12.0
DCM	664	682	18	2.72	9.7
DMSO	657	675	18	2.47	1.4
Acetonitrile	648	668	20	2.31	7.4
Ethanol	654	668	14	2.55	10.1
Methanol	651	668	17	2.41	6.7
Water	645	663	18	0.53	3.7

Table 1 Spectral data of Cy5-Br in different solvents

Solvent	λ_abs/nm	$λ em a$ /nm	Δλ^b/nm	ε^c	$Φ f d$ (%)
THF	655	668	13	2.23	12.0
DCM	664	682	18	2.72	9.7
DMSO	657	675	18	2.47	1.4
Acetonitrile	648	668	20	2.31	7.4
Ethanol	654	668	14	2.55	10.1
Methanol	651	668	17	2.41	6.7
Water	645	663	18	0.53	3.7

Fig.2 Normalized absorbance(a) and emission spectra(b) of Cy5-Br in PBS-CH3CN

Fig.3 Absorption(A, C) and fluorescence spectra(B, D) of Cy5-Br(5 μmol/L) after incubation with different concentrations of sulfite(A, B) and bisulfite(C, D) PBS-CH3CN system at 37 ℃, λex = 610 nm. c/(μmol·L-1) of (A, B, D): a. 16.7; b. 33.3; c. 50.0; d. 66.7; e. 83.3; f. 100.0; g. 116.7; h. 133.3; i. 150.0; j. 166.7; k. 183.3; l. 200.0; m. 216.7; n. 233.3; o. 250.0; p. 266.7; q. 283.3; r. 300.0; s. 316.7; t. 333.3; u. 350.0.c/(μmol·L-1) of (C): a. 0; b. 3.3; c. 6.7; d. 10.0; e. 13.3; f. 16.7; g. 33.3; h. 50.0; i. 66.7; j. 83.3; k. 100.0; l. 116.7; m. 133.3; n. 150.0; o. 166.7; p. 183.3; q. 200.0; r. 216.7.

Fig.4 Fluorescence intensity at 665 nm of probe Cy5-Br(5 μmol/L) after incubation with different concentrations of sulfite(A) and bisulfite(B)

Fig.5 Time-dependent fluorescence changes at 665 nm of Cy5-Br(5 μmol/L) with 500 μmol/L SO32-(A) and HSO3-(B) in PBS-CH3CN system

Fig.6 Fuorescence intensity at 665 nm of Cy5-Br (5 μmol/L) under different pH values λex=610 nm.

Fig.7 Fluorescence intensity at 665 nm of Cy5-Br in PBS-CH3CN system in the presence of various ions a. Blank; b. S O 3 2 - ; c. HS O 3 - ; d. Al3+; e. Cd2+; f. Cu2+; g. Fe3+; h. K+; i. Mn2+; j. Na+; k. N H 4 + ; l. Ni2+; m. Zn2+; n. C O 3 2 - ; o. HP O 4 2 - ; p. I-; q. H2P O 4 - ; r. S2-; s. Cl O 4 - ; t. Cl-; u. N O 3 - ; v. S O 4 2 - ; w. N O 2 - ; x. GSH; y. Cys; z. ATP.

Fig.8 Confocal fluorescence images of Cy5-Br in HeLa cells The cells were incubated with Cy5-Br(1.0 μmol/L) for 15 min, then washed by PBS for three times. (A) The red channel; (B) the bright channel.

Fig.9 Confocal fluorescence images of HeLa cells stained with Mito Tracker Green FM(2.0 μmol/L)(A) and Cy5-Br(1.0 μmol/L)(B) for 15 min, merged image of(A) and(B, C), bright-field image(D), merged image of (C) and bright-field image(E) and correlation plot of Mito Tracker Green FM and Cy5-Br intensities(F)

Fig.10 Confocal fluorescence intensity images of Cy5-Br in HeLa cells (A) The cells were incubated with Cy5-Br(1.0 μmol/L) for 15 min, then washed by PBS for three times;(B—D) HeLa cells were incubated with 20.0 μmol/L HS O 3 - for 5, 10 and 15 min, respectively.

References 19

[1]	Eto K., Asada T., Arima K., Makifuchi T., Kimura H., Biochem. Bioph Res. Co., 2002, 293(5), 1485—1488
[2]	Reiffenstein R. J., Hulbert W. C., Roth S. H., Annu. Rev. Pharnacol., 1992, 32, 109—134
[3]	Sun Y. Q., Liu J., Zhang J. Y., Yang T., Guo W., Chem. Commun., 2013, 49, 2637—2639
[4]	Gu X. F., Liu C. H., Zhu Y. C., Zhu Y. Z., J. Agric. Food Chem., 2011, 59(22), 11935—11939
[5]	Koch M., Köppen R., Siegel D., Witt A., Nehls I., J. Agric. Food Chem., 2010, 58(17), 9463—9467
[6]	Lin L., Cao X. N.,, Zhang W., Zhou Y. Y., Li J. H., Jin L. T., Chin. J. Anal. Chem., 2006, 34(1), 31—34
	(林丽, 曹旭妮, 张文, 周宇艳, 李金花, 金利通. 分析化学, 2006, 34(1), 31—34)
[7]	Yao C. F., Lü M. X., Hu W. Y., Zeng H. P., Chin. J. Org. Chem., 2011, 31(12), 2088—2094
	(姚春凤, 吕梅香, 胡雯艳, 曾和平. 有机化学, 2011, 31(12), 2088—2094)
[8]	Xu H., Dai Y. N., Shan H. Y., Fei Q., Xun Y. F., Li G. H., Feng G. D., Chem. J. Chinese Universities, 2014, 35(4), 736—740
	(徐惠, 代艳娜, 单洪岩, 费强, 郇延富, 李光华, 冯国栋. 高等学校化学学报, 2014, 35(4), 736—740)
[9]	Sun Y. Q., Wang P., Liu J., Zhang J. Y., Guo W., Analyst,2012, 137, 3430—3433
[10]	Chen S., Hou P., Wang J. X., Song X. Z., RSC Advances, 2012, 2, 10869—10873
[11]	Yang Y. T., Huo F. J., Zhang J. J., Xie Z. H., Chao J. B., Yin C. X., Tong H. B., Liu D. S., Jin S., Cheng F. Q., Yan X. X., Sensors and Actuators B, 2012, 166, 665—670
[12]	Yang X. F., Zhao M. L., Wan G., Sensors and Actuators B, 2011, 152, 8—13
[13]	Xu J., Liu K., Di D. L., S. J., Guo Y., Inorg. Chem. Commun., 2007, 10, 681—684
[14]	Wang G., Development of Fluorescent Probes for Bisulfite and Sulfide Anion, Northwest University, Xi’an, 2012
	(王刚. 检测亚硫酸氢根和硫离子的荧光探针的合成及性质研究, 西安: 西北大学, 2012)
[15]	Morton M., Landfield H., J. Am. Chem. Soc., 1952, 74(14), 3523—3526
[16]	Mishra A., Behera R. K., Behera P. K., Mishra B. K., Behera G. B., Chem. Rev., 2000, 100, 1973—2011
[17]	Wang L., Peng X. J., Dai B., Wang B. S., Liu F., Qiang X. X., Chem. J. Chinese Universities, 2010, 31(7), 1381—1385
	(王丽, 彭孝军, 戴彬, 王炳帅, 刘飞, 强新新. 高等学校化学学报, 2010, 31(7), 1381—1385)
[18]	Leung C. W. T., Hong Y., Chen S. J., Zhao E. G., Lam J. W. Y., Tang B. Z., J. Am. Chem. Soc., 2013, 135, 62—65
[19]	Jiang N., Fan J. L., Zhang S., Wu T., Wang J. Y., Gao P., Qu J. L., Zhou F., Peng X. J., Sensors and Actuators B, 2014, 190, 685—693

Related Articles 15

[1]	CHANG Liying, LING Xinyu, CHEN Heqi, WANG Xue, LIU Tao. Application of Gene Editing in Mitochondrial Diseases [J]. Chem. J. Chinese Universities, 2022, 43(Album-4): 20220363.
[2]	WANG Jinjin,QI Shaolong,DU Jianshi,YANG Qingbiao,SONG Yan,LI Yaoxian. Synthesis of Benzothiazole Fluorescent Probe for Detection of N₂H₄·H₂O and HS $O 3 - †$ [J]. Chem. J. Chinese Universities, 2019, 40(7): 1397.
[3]	AI Shujuan, ZONG Chengxing, WU Wei, FENG Jingjing, JIN Can, FU Fengzhi, LIU Jing, SUN Donglan, ZHENG Qin, GUO Yeping. Effect of Derivatives of Glycerol Sulfite as Electrolyte Additive on Electrochemical Performance of Lithium Ion Battery^† [J]. Chem. J. Chinese Universities, 2018, 39(11): 2520.
[4]	CHEN Jingxin, LIAN Wenhua, CHENG Yuanchu, XU Jianhua, ZHANG Lianru. Effect of Curcumin on Yeast Cells by Fluorescence Analysis^† [J]. Chem. J. Chinese Universities, 2018, 39(1): 71.
[5]	HU Shuang, TENG Lirong, ZHANG Junrong, MENG Jiatong, ZHUANG Zhe, DU Mengyan, WANG Di, LIAO Weiwei. Synthesis of a Novel Compound and Mediated Neuro-protection in Differentiated PC12 Cells via Mitochondria Related Pathway [J]. Chem. J. Chinese Universities, 2015, 36(11): 2322.
[6]	XIE Jiang, ZHU Jian-Hua. Synthesis and Imaging Study on the FITC Labeled Folic Derivative [J]. Chem. J. Chinese Universities, 2011, 32(7): 1532.
[7]	YE She-Fang^1, ZHONG Li-Ming¹, WU Yi-Hui¹, ZHANG Qi-Qing^1,2 . Multi-walled Carbon Nanotubes Exposure Induces Oxidative Stress and Depolarizes Mitochondrial Membrane Potential in Cultured A549 Cells [J]. Chem. J. Chinese Universities, 2009, 30(3): 497.
[8]	ZHAO Wen-Jing, NIU Feng-Lan^*. Trihydroxybenzoic Acid on Induction of the Apoptosis of Human Hepatocarcinoma SMMC-7721 Cells by Mitochondrial-dependent Pathway [J]. Chem. J. Chinese Universities, 2009, 30(2): 320.
[9]	HE Xiao-Xiao, YUAN Yuan, SHI Hui, WANG Ke-Min*, HE Ding-Gen, QIN Di-Lan. Study on the Target Interaction Between Mitochondria and Signal Peptide Functionalized Silica Nanoparticles [J]. Chem. J. Chinese Universities, 2009, 30(12): 2376.
[10]	LI Li^1,3, WU Feng^1,3, CHEN Ren-Jie^2,3, WU Sheng-Xian¹. Electrochemical Behavior of Butylene Sulfite as Novel Film-forming Electrolyte Additive for Lithium Ion Batteries [J]. Chem. J. Chinese Universities, 2007, 28(2): 293.
[11]	LIU Hui-Xue, YANG Xiao-Da*, WANG Kui. Effects of Lanthanide Ions(La³⁺, Gd³⁺, Yb³⁺) on Production of Reactive Oxygen Species in Mitochondria [J]. Chem. J. Chinese Universities, 2006, 27(6): 999.
[12]	XU Li, LI Xiang-Lu, HUANG Yi-Bing, WU Xiao-Xia, HOU Rui-Zhen, WANG Hua, WANG Na, ZHANG Xue-Zhong. Protection of Small Molecule Corn Peptide Leu-Asp-Tyr-Glu from Mitochondria Against Oxidative Damage [J]. Chem. J. Chinese Universities, 2004, 25(6): 1073.
[13]	XIONG Ya, TAN Zhi-Qun, LIU Yi, PAN Zheng-Jun, WU Ding-Quan, KANG Li-Shan, QU Song-Sheng. Studies on the Metabolism of Mitochondria and Sub-mitochondria and the Effect of Laccase on It by Microcalorimetry [J]. Chem. J. Chinese Universities, 1997, 18(5): 753.
[14]	XING Hui, FENG Yan, GAO Gui, CHEN Si-Geng, YANG Tong-Shu. Protection of Myocardial Mitochondria Against the Active Oxygen Injury by Horseradish Peroxidase [J]. Chem. J. Chinese Universities, 1997, 18(10): 1628.
[15]	ZHANG Jin-Sheng, ZHANG Han-Qi, DUAN Yi-Xiang, XU Fang, JIN Qin-Han, WANG Da-Ning, ZOU Ming-Qiang, LIU Li. A Study on Determination of Sulfide and Sulfite By Microwave Induced Plasma Atomic Emission Spectrometry with Gas Generation for Sample Introduction [J]. Chem. J. Chinese Universities, 1992, 13(11): 1364.