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

doi:10.7503/cjcu20140891

Abstract

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

CLC Number:

O655.25

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2015, 36(4): 646.

Figures/Tables 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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