衍生于双氰基二苯代乙烯的用于活体成像的双光子荧光锌离子探针

doi:10.7503/cjcu20140891

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (4): 646.doi: 10.7503/cjcu20140891

衍生于双氰基二苯代乙烯的用于活体成像的双光子荧光锌离子探针

黄池宝^1,²(), 梁兴¹, 曾启华¹, 陈华仕¹, 曾伯平¹, 易道生², 陈晓远²

1. 遵义师范学院化学化工学院, 遵义 563002
2. 韶关学院英东农业科学与工程学院, 韶关 512005

收稿日期:2014-10-08 出版日期:2015-04-10 发布日期:2015-03-27
作者简介:联系人简介: 黄池宝, 男, 博士, 教授, 主要从事双光子荧光探针研究. E-mail: huangchibao@163.com
基金资助:
中国博士后科学基金(批准号: 20100471224)、广东省自然科学基金(批准号: s2011040000536)、湖南省科技计划项目(批准号: 2011RS4020)、深圳大学省部重点实验室开放基金项目(批准号: 201202)、韶关市科技计划项目(批准号: 2011CX/K19)和贵州省教育厅自然科学重点研究项目(批准号: 黔教合KY字[2014]296)

Dicyanostilbene-derived Two-photon Fluorescence Probe for Free Zinc Ions in Live Cells and Living Tissues^†

HUANG Chibao^1,^2,^*(), LIANG Xing¹, ZENG Qihua¹, CHEN Huashi¹, ZENG Boping¹, YI Daosheng², CHEN Xiaoyuan²

1. Chemistry and Chemical Engineering College, Zunyi Normal University, Zunyi 563002, China
2. School of Yingdong Agricultural Science and Engineering,Shaoguang University, Shaoguan 512005, China

Received:2014-10-08 Online:2015-04-10 Published:2015-03-27
Contact: HUANG Chibao E-mail:huangchibao@163.com
Supported by:
† Supported by the China Postdoctoral Science Foundation Funded Project(No.20100471224), the Guangdong Provincial Natural Science Foundation, China(No.S2011040000536), the Planned Science and Technology Project of Hunan Province, China(No.2011RS4020), the Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, China(No.201202), the Planned Science and Technology Project of Shaoguan, China(No.2011CX/K19) and the Natural Science Key Research Project of Department of Education of Guizhou Province, China(No.Guizhou Province Office of Education Contract KY [2014]296)

摘要/Abstract

摘要：

制备了衍生于双氰基二苯代乙烯的双光子荧光锌离子探针, 该探针以4-(2-吡啶甲基)哌嗪为锌离子受体, 当络合锌离子时, 探针的荧光强度增强了72.5倍和580 GM的双光子吸收截面. 该探针无细胞毒性且在体内环境中无pH敏感性, 其解离常数 $K d TP$ =(0.52±0.01) μmol/L. 性能测试结果表明, 该探针能选择性地检测活细胞中的游离锌离子, 耐时长达约1500 s, 且能探测活体组织80~150 μm深处的锌离子, 不受其它金属离子与生物膜的干扰.

关键词: 双光子荧光探针, 锌离子, 活体成像, 双光子发射截面

Abstract:

A novel two-photon fluorescence probe for Zn²⁺ derived from dicyanostilbene as a two-photon fluorophore and 4-(pyridine-2-ylmethyl)piperazine as a novel Zn²⁺ ligand was developed, and its structure was characterized by ¹H NMR, IR and elemental analysis. By UV-Vis absorption and absorption-titration experiments, one- and two-photon excited fluorescence experiments in femtosecond pulses, as well as cell-imaging and tissue-imaging experiments, the probe showed a 72.5-fold fluorescence enhancement in response to Zn²⁺, a large two-photon action cross-section(580 GM), a noncytotoxic effect, and pH insensitivity in the biolo-gically relevant range, with a dissociation constant( $K d TP$ ) of (0.52±0.01) μmol/L. The probe could detect free Zn²⁺ ions selectively in live cells for about 1500 s and in living tissues at a depth of 80—150 μm without interference from other metal ions or membrane-bound probes.

Key words: Two-photon fluorescence probe, Zinc ion, Live-imaging, Two-photon absorption across-section

中图分类号:

O655.25

TrendMD:

黄池宝, 梁兴, 曾启华, 陈华仕, 曾伯平, 易道生, 陈晓远. 衍生于双氰基二苯代乙烯的用于活体成像的双光子荧光锌离子探针. 高等学校化学学报, 2015, 36(4): 646.

HUANG Chibao, LIANG Xing, ZENG Qihua, CHEN Huashi, ZENG Boping, YI Daosheng, CHEN Xiaoyuan. Dicyanostilbene-derived Two-photon Fluorescence Probe for Free Zinc Ions in Live Cells and Living Tissues^†. Chem. J. Chinese Universities, 2015, 36(4): 646.

图/表 9

Scheme 1 Synthetic procedure of compound DZn

Table 1 High resolution mass spectrum[HRMS(EI)] data of compounds 2—7

Compd.	Ref.	HRMS(calcd.), m/z	Compd.	Ref.	HRMS(calcd.), m/z
2	[17]	263.9207(263.9200)	5	[18]	292.0977(292.0977)
3	[17]	313.8803(313.8801)	6	[19]	245.0374(245.0352)
4	[18]	233.9747(233.9742)	7	[18]	383.0952(383.0952)

Scheme 2 Two-photon PET mechanism of DZn

Fig.1 Relative fluorescence intensities of 1 μmol/L DZn in the presence of 20 mmol/L of some metal ions(empty bars) followed by addition of 1 μmol/L of Zn2+(filled bars) a. Zn2+; b. K+; c. Ca2+; d. Mg2+; e. Ba2+; f. Fe2+; g. Fe3+; h. Pb2+; i. Co2+; j. Ni2+; k. Cr3+; l. Cd2+; m. Mn2+; n. Cu2+; o. Ag+; p. Na+; q. Hg2+.

Table 2 Photophysical data for DZn and AZn2

Compd.	$λ Abs a$ /nm	$λ em b$ /nm	η^c	$λ ex d$ /nm	$δ max e$	ηδ_max
DZn	403	610	0.02	810	400	8
DZn-Zn²⁺	403	610	0.62	810	935	580
AZn2^[12]	365	494	0.012	780	ND	ND
AZn2-Zn^2+[12]	365	499	0.65	780	140	95

Table 2 Photophysical data for DZn and AZn2

Compd.	$λ Abs a$ /nm	$λ em b$ /nm	η^c	$λ ex d$ /nm	$δ max e$	ηδ_max
DZn	403	610	0.02	810	400	8
DZn-Zn²⁺	403	610	0.62	810	935	580
AZn2^[12]	365	494	0.012	780	ND	ND
AZn2-Zn^2+[12]	365	499	0.65	780	140	95

Table 3 1H NMR data for DZn with various zinc ion concentrations

c(Zn²⁺)/ (mmol·L^-1)	¹H NMR(DMSO-d₆, 400 MHz), δ
c(Zn²⁺)/ (mmol·L^-1)	H_a	H_b	H_c	H_d	H_e	H_f	H_g	H_h	H_i	H_j	H_k	H_l	H_m
0	8.508	8.419	7.920	7.785	7.567	7.478	7.281	7.066	6.982	3.669	3.262	2.584	2.502
9.5	8.563	8.421	7.923	7.908	7.572	7.486	7.411	7.074	6.993	3.789	3.338	2.676	2.502
95	8.597	8.418	7.920	7.989	7.570	7.490	7.586	7.075	6.997	3.869	3.381	2.741	2.500

Fig.2 Bright-field image(A) and two-photon microscopy(TPM) images(B—D) of 1 μmol/L DZn-labeled mouse fibroblast collected at 550—650 nm (B) Before addition of 10 mmol/L SNOC; (C) after addition of 10 mmol/L SNOC; (D) after addition of 0.1 mmol/L TPEN to(C). The two-photon excitation fluorescence(TPEF) images were collected upon excitation at 810 nm with a femtosecond pulse. Cells were showed representative images from replicate experiments(n=5). SNOC is an endogenous NO donor that triggers the release of Zn2+. TPEN is a membrane-permeable Zn2+ chelator that can remove Zn2+ effectively.

Fig.3 TPM images of a mouse brain tissue slice (A) Before addition of 30 mmol/L SNOC; (B) after addition of 30 mmol/L SNOC; (C) after addition of 0.3 mmol/L TPEN to(B); (D) relative TPEF intensity of a mouse brain tissue slice stained with 10 μmol/L DZn collected at 550—650 nm as a function of time. The TPEF images were collected at 550—650 nm at a depth of ca. 120 μm with magnification 100× upon excitation at 810 nm with a femtosecond pulse.

Fig.4 TPM images of a mouse brain tissue slice stained with 10 μmol/L DZn by magnification 100× at different penetration depth of 80(A), 100(B), 120(C) and 150 μm(D) The TPEF images were collected at 550—650 nm upon excitation at 810 nm with a femtosecond pulse.

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衍生于双氰基二苯代乙烯的用于活体成像的双光子荧光锌离子探针

Dicyanostilbene-derived Two-photon Fluorescence Probe for Free Zinc Ions in Live Cells and Living Tissues^†

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衍生于双氰基二苯代乙烯的用于活体成像的双光子荧光锌离子探针

Dicyanostilbene-derived Two-photon Fluorescence Probe for Free Zinc Ions in Live Cells and Living Tissues†

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Dicyanostilbene-derived Two-photon Fluorescence Probe for Free Zinc Ions in Live Cells and Living Tissues^†