Dicyanostilbene-derived Two-photon Fluorescence Probe for Lead Ions†

doi:10.7503/cjcu20170672

Abstract

Abstract:

One-photon microscopy(OPM) uses a single photon of higher energy to excite the fluorophore, while two-photon microscopy(TPM) uses two photons of lower energy as the excitation source. Therefore, TPM has such distinct advantages as improvement of penetration depth, localized two-photon excitation, reduction of photo-damage and photobleaching, small absorption coefficient of light in tissue, and lower tissue autofluorescence. These advantages make TPM more popular among biologists. Lead is a common contaminant and has inherent toxic effects on human health and environment. Pb²⁺ ion contamination can affect a wide variety of diseases such as anemia, irritability, memory loss, and muscle paralysis. For these reasons, there is an urgent need to develop some effective two-photon fluorescence probes to detect trace amounts of Pb²⁺ ions in live cells and tissues. A novel two-photon fluorescence probe for Pb²⁺ derived from 4-methyl-2,5-dicyano-4'-amino stilbene as a two-photon fluorophore and bis[2-(2-aminophenylsulfanyls)ethyl]amine as a novel Pb²⁺ ligand was developed. The probe possessed small molecule size, large two-photon absorption cross-section(1020 GM), noncytotoxic effect, long-wavelength emission at 609 nm, large Stokes shift(209 nm), excellent photostability, moderate water-solubility, good cell-permeability and pH-insensitivity in the biologically relevant range. The probe was able to selectively detect Pb²⁺ ions in live cells and living tissues without interference from other metal ions and the membrane-bound probes, and its quenching constant( $K sv TP$ ) was 7.58×10⁵ L/mol.

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

CLC Number:

O625

TrendMD:

HUANG Chibao,PAN Qi,CHEN Huashi,LIANG Xing,LÜ Guoling. Dicyanostilbene-derived Two-photon Fluorescence Probe for Lead Ions^†[J]. Chem. J. Chinese Universities, 2018, 39(5): 897.

Figures/Tables 7

Fig.1 Structure of DPb chelating lead ion

Fig.2 Relative fluorescence intensity of 1 μmol/L DPb in the presence of 20 mmol/L Pb2+ and other ions(empty bars) followed by addition of 40 μmol/L Pb2+(filled bars) Note：a—q: Pb2+, K+, Ca2+, Mg2+, Ba2+, Fe2+, Fe3+, Zn2+, Co2+, Ni2+, Cr3+, Cd2+, Mn2+, Cu2+, Ag+, Na+, Hg2+. Data were measured in 30 mmol/L MOPS buffer(100 mmol/L KCl+10 mmol/L EGTA, pH=7.2). λex1=400 nm, λex2=790 nm.

Fig.3 Absorption(A) and emission spectra(B) of DPb(1 μmol/L) in 30 mmol/L MOPS buffer(100 mmol/L KCl+10 mmol/L EGTA, pH=7.2) upon the addition of Pb2+(0—40 μmol/L), respectively Note：λex1=400 nm, λex2=790 nm. (A) Left: absorption spectra; right: one-photon emission spectra; (B) two-photon emission spectra.

Fig.4 Best fitting curve between changes in one-photon absorption intensities of DPb(10 μmol/L) at 400 nm and Pb2+ concentrations Note：Data were measured in 30 mmol/L MOPS buffer (100 mmol/L KCl+10 mmol/L EGTA, pH=7.2).

Fig.5 TPM images of 1 μmol/L DPb-labeled mouse fibroblast collected at 550—650 nm before(A) and after(B) the addition of 20 μmol/L Pb2+ to the imaging solution Note： The two-photon excitation fluorescence(TPEF) images were collected upon excitation at 790 nm with a femtosecond pulse. Cells shown are representative images from replicate experiments(n=5).

Fig.6 TPM images of a mouse brain tissue slice stained with 10 μmol/L DPb at a depth of ca. 120 μm with magnification 100× before(A) and after(B) the addition of 20 μmol/L Pb2+ to the imaging solution Note：The two-photon excitation fluorescence(TPEF) images were collected at 550—650 nm upon excitation at 790 nm with a femtosecond pulse. Cells shown are representative images from replicate experiments(n=5).

Fig.7 Normalized fluorescence decay curves of DPb(10 μmol/L) solutions at 609 nm before and after the addition of Pb2+ excited by one-photon absorption(403 nm) and two-photon absorption(790 nm) Note：(A) OPFL(DPb-Pb2+); (B) TPFL(DPb-Pb2+); (C) OPFL(DPb); (D) TPFL(DPb). OPFL and TPFL abbreviated from one- and two-photon fluorescence lifetime, respectively.

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