活细胞线粒体中SO32-/HSO3-的荧光成像

doi:10.7503/cjcu20140542

摘要/Abstract

摘要：

在五甲川菁染料的基础上, 合成了一种新型的中位溴取代的五甲川菁染料Cy5-Br, 其结构通过质谱和核磁共振波谱表征. 性能测试结果表明, 该染料作为荧光探针的发射波长可达665 nm, 具有较低的细胞毒性, 而且对二氧化硫衍生物S $O 3 2 -$ 和HS $O 3 -$ 具有很好的选择性. 该探针在665 nm处的荧光发射强度随着S $O 3 2 -$ /HS $O 3 -$ 浓度的增加不断降低, 具有良好的线性荧光响应. Cy5-Br可以进入活细胞线粒体, 对S $O 3 2 -$ 和HS $O 3 -$ 的浓度进行实时共聚焦荧光成像.

关键词: 亚硫酸根, 亚硫酸氢根, 线粒体, 荧光成像

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

中图分类号:

O657

TrendMD:

姜娜, 樊江莉, 张帅, 彭孝军. 活细胞线粒体中S $O 3 2 -$ /HS $O 3 -$ 的荧光成像. 高等学校化学学报, 2014, 35(12): 2534.

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

图/表 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.

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