基于罗丹明荧光信号报告基团的细胞通透性铜离子荧光探针

doi:10.7503/cjcu20160402

摘要/Abstract

摘要：

分别以罗丹明B和罗丹明6G为荧光信号报告基团, 以增强水溶性为目的的羟乙基肼为修饰基团, 合成了反应型的Cu²⁺离子选择性荧光探针分子L1和L2. 紫外光谱和荧光光谱等分析结果表明, 探针分子L1和L2对Cu²⁺离子具有高灵敏度、高选择性的光谱识别行为. 探针分子对Cu²⁺离子的识别过程是通过Cu²⁺离子催化水解控制氧杂蒽荧光信号的螺环酰肼基团实现荧光信号的开启, 从而达到识别检测Cu²⁺离子的目的, 对Cu²⁺离子的检出限均可达到10^-8 mol/L量级. 同时, 探针分子对常见金属离子和铵离子均具有较强的抗干扰能力. 由于羟乙基肼的引入增强了探针的水溶性, 使得探针L1和L2具有良好的细胞通透性和低毒性, 实现了其对β-胰岛细胞(INS-1细胞)中Cu²⁺离子的荧光成像检测.

关键词: 罗丹明, 酰肼, 二价铜离子, 荧光探针, 荧光成像

Abstract:

Two rhodamine-based fluorescent turn-on probes L1 and L2 were designed and synthesized for the detection of Cu²⁺ ion. L1 and L2 exhibit high selectivity toward Cu²⁺ ion over a wide range of metal ions in MeCN/H₂O(volume ratio 1∶5). The detection limits of the probes are down to 10^-8 mol/L range. Moreover, L1 and L2 can function as excellent fluorescence probes for imaging of cellular Cu²⁺ by means of fluorescence microscopy.

Key words: Rhodamine, Hydrazide, Cupric ion, Fluorescence probe, Fluorescence imaging

中图分类号:

O657
O626

TrendMD:

米小龙, 焦晓洁, 刘畅, 何松, 曾宪顺. 基于罗丹明荧光信号报告基团的细胞通透性铜离子荧光探针. 高等学校化学学报, 2016, 37(10): 1784.

MI Xiaolong, JIAO Xiaojie, LIU Chang, HE Song, ZENG Xianshun. Rhodamine-based Cell Permeable Fluoresecent Turn-on Probes for Cupric Ion^†. Chem. J. Chinese Universities, 2016, 37(10): 1784.

图/表 8

Scheme 1 Synthetic routes for probes L1 and L2

Fig.1 UV-Vis spectra of probes L1(A) and L2(B)(1×10-5 mol/L) in the presence of nitrate salts of metal ions(5×10-5 mol/L ) in MeCN/H2O(volume ratio 1∶5) solventInset: histogram representing the absorbance enhancement of probe L1 at 553 nm and probe L2 at 521 nm, respectively, in the presence of different metal ions. a. No other ions; b. Ag+; c. Al3+; d. Ca2+; e. Cd2+; f. Co2+; g. Cr3+; h. Cu2+; i. Fe2+; j. Fe3+; k. Hg2+; l. K+; m. Mg2+; n. Na+; o. NH4+; p. Ni2+; q. Pb2+; r. Zn2+.

Fig.2 Fluorescence emission spectra of probes L1(A) and L2(B)(1×10-6 mol/L) in the presence of nitrate salts of metal ions(5×10-6 mol/L) in MeCN/H2O(volume ratio 1∶5)Inset: histogram representing the fluorescence enhancement of probe L in the presence of different metal ions. a. No other ions; b. Ag+; c. Al3+; d. Ca2+; e. Cd2+; f. Co2+; g. Cr3+; h. Cu2+; i. Fe2+; j. Fe3+; k. Hg2+; l. K+; m. Mg2+; n. Na+; o. NH4+; p. Ni2+; q. Pb2+; r. Zn2+. Excitation was performed at 520 nm(A) and 500 nm(B). Excitation/emission slit width: 2.5 nm/5 nm.

Fig.3 Fluorescent titration spectra of probes L1(1×10-6 mol/L) in the presence of different concentrations of Cu2+ in MeCN/H2O(volume ratio 1∶5)[Cu2+]/(μmol·L-1): 0, 0.2, 0.6, 1.0, 1.8, 2.6, 3.4, 5.0, 6.6, 8.2, 9.8, 11.4, 13.0, 14.6, 16.2, 19.4, 22.6, 25.8, 30.6. Excitation was performed at 520 nm. Excitation/emission slit width: 2.5 nm/5 nm. Inset: plot of fluorescent intensity of L1 at 580 nm versus [Cu2+].

Fig.4 Relative fluorescent intensity change ratios[(Fi-FL)/(FCu2+-FL)] of probes L1(A) and L2(B)(1×10-6 mol/L) upon addition of Cu2+(5×10-6 mol/L) in the presence of metal ions(5×10-6 mol/L) in MeCN/H2O(volume ratio 1∶5)a. Cu2+; b. Cu2++Ag+; c. Cu2++Al3+; d. Cu2++Ca2+; e. Cu2++Cd2+; f. Cu2++Co2+; g. Cu2++Cr3+; h. Cu2++Fe2+; i. Cu2++Fe3+; j. Cu2++Hg2+; k. Cu2++K+; l. Cu2++Mg2+; m. Cu2++Na+; n. Cu2++NH4+; o. Cu2++Ni2+; p. Cu2++Pb2+; q. Cu2++Zn2+. Excitation was performed at 520 nm(A) and 500 nm(B). Excitation/emission slit width: 2.5 nm/5 nm.

Fig.5 Profile of pH dependence of the fluorescence intensity of probes L1(A)(1×10-6 mol/L) at 580 nm and L2(B)(1×10-6 mol/L) at 550 nm in the absence(a) and presence(b) of Cu2+(5×10-6 mol/L) in MeCN/H2O(volume ratio 1∶5)Excitation was performed at 520 nm(A) and 500 nm(B). Excitation/emission slit width: 2.5 nm/5 nm.

Fig.6 Bright field images(A, D), fluorescence images(B, E) and overlay images(C, F) of INS-1 cells incubated with L1(A—C) as well as L1 and Cu2+(D—F) Excitation wavelength: 559 nm.

Fig.7 Bright field images(A, D), fluorescence images(B, E) and overlay images(C, F) of INS-1 cells incubated with L2(A—C) as well as L2 and Cu2+(D—F) Excitation wavelength: 488 nm.

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