A mitochondria-targeted ratiometric two-photon fluorescent probe for biological zinc ions detection
Introduction
Zinc ion has been known as the second most abundant transition metal ion in the human body, which plays critical role in neurotransmission, enzymatic regulation and cell apoptosis (Xie and Smart, 1991, Berg and Shi, 1996, Vallee and Falchuk, 1993, Bush, 2000, Li et al., 2009). Recent studies revealed that the over-taken of Zn2+ in mitochondria will lead to the accumulation of reactive oxygen species (especially H2O2) and the dysfunction of mitochondria (Sensi et al., 2000, Sensi et al., 2003a, Sensi et al., 2003b, Sensi et al., 2009, Chyan et al., 2014). Fluorescent probe has been evaluated as the most powerful tool to monitor biologically relevant species for their high sensitivity and spatial resolution (Egawa et al., 2013, Chen et al., 2013, Radford et al., 2013, Huang et al., 2013, Bae et al., 2013, Jung et al., 2014, Zhang et al., 2014a, Zhang et al., 2014b, Hettiarachchi et al., 2014, Zhou et al., 2014a, Zhou et al., 2014b). During last years, lots of fluorescent probes for Zn2+ have been developed to detect the cytosolic Zn2+ to understand its role in living system (Zhang et al., 2014a, Zhang et al., 2014b, Hettiarachchi et al., 2014, Zhou et al., 2014a, Zhou et al., 2014b, Qian et al., 2009, Du et al., 2010, Meng et al., 2012, Guo et al., 2012, Lin et al., 2013, Divya et al., 2014, Hagimori et al., 2015, Lee et al., 2015). Unfortunately, most of the reported Zn2+ fluorescent probes failed to target the mitochondria.
Most of reported fluorescent probes are designed based on single-photon fluorescence technology, which requires excitation with short-wavelength light (ca. 350–550 nm) that limits their application in subcellular organelles and deep-tissue, owing to the shallow penetration depth (less than 80 μm) as well as to photo-bleaching, photo-damage, and cellular auto fluorescence (Sensi et al., 2003a, Sensi et al., 2003b, Que et al., 2008, Tomat et al., 2010, McRae et al., 2009, Meng et al., 2006, Zhou et al., 2010). Recently, Two-photon fluorescence (TPF) probes, which can be excited by two-photon absorption in the NIR wavelength, provided an opportunity to overcome the problems originated from the single-photon fluorescence technology (Denk et al., 1990, Jing et al., 2012, Kim et al., 2014, Kim and Cho, 2015, Masanta et al., 2011, Meng et al., 2012, Park et al., 2012, Sarkar et al., 2014, Sarkar et al., 2013, Wang et al., 2014, Yao and Belfield, 2012, Yin et al., 2015, Zhang et al., 2014a, Zhang et al., 2014b, Zhang et al., 2013, Zhou et al., 2014a, Zhou et al., 2014b). However, most of the reported two-photon fluorescent probes Zn2+ are “turn-on” ones, using enhancement of the fluorescence intensity at only one wavelength as the detection signal. This design may cause difficulty for quantitative determination and quantitative bio-imaging due to the background interference (Baek et al., 2012, Rathore et al., 2014). By comparison, ratiometric probes are better choices that can overcome this particular limitation, because they allow quantitative detection of the analyte by measuring the ratio of emissions at two different wavelengths (Meng et al., 2012, Dunn et al., 1994, Dittmer et al., 2009, Xue et al., 2012, Qin et al., 2011). Therefore, mitochondria-targetable ratiometric fluorescent probes for Zn2+ are still highly needed.
Herein, we design a new two-photon ratiometric probe (Mito-MPVQ) for mitochondrial Zn2+ based on 6-substituted quinoline group, an efficient two photon fluorophore we reported before (Meng et al., 2012) (Scheme 1). Triphenylphosphonium(TPP) group, which was widely used as the mitochondria targeting group (Masanta et al., 2011, Xue et al., 2012, Komatsu et al., 2005, Murphy and Smith, 2007, Dickinson and Chang, 2008, Ross et al., 2008, Dickinson et al., 2010, Dodani et al., 2011), was linked to the fluorescent group to deliver the probe selectively to mitochondria. In order to eliminate the influence of the TPP group to the detection of the Zn2+, a long aliphatic linker was used to separate TPP group from the fluorescent group. We speculate that the new probe will selectively located into mitochondria and give good ratiometric two-photon detection signal to mitochodrial Zn2+. Mito-MPVQ was synthesized from simple starting material (4-Iodoaniline) via 6-steps procedure with overall yield of 15.0% (Scheme S1).
Section snippets
General procedures
All reagents and solvents were commercially purchased. All reactions were magnetically stirred and monitored by thin layer chromatography (TLC). Flash chromatography (FC) was performed using silica gel 60 (200–300 mesh). 1H NMR spectra were recorded on Bruker-400 MHz spectrometers and 13C NMR spectra were recorded on 100 MHz spectrometers. Fluorescence spectra were obtained using a HITACHIF-2500 spectrometer. UV–vis absorption spectra were recorded on a Tech-comp UV 1000 spectrophotometer. MS
UV/vis and fluorescence spectra responses
All spectroscopic measurements are carried out in the methanol–water solutions (1:1, v/v, 50 mM PBS buffer, pH=7.4). As shown in Fig. 1, the emission spectra of Mito-MPVQ exhibited a large red-shift of 68 nm (from 420 nm to 488 nm) with an iso-emissive point at 434 nm. The ratio of emission intensity (I488 nm/I420 nm) increases dramatically from 0.45 to 3.79 (ca. 8-fold ). There is a linear relationship between the ratio of emission intensity (I488 nm/I420 nm) of Mito-MPVQ and the concentration of the Zn
Conclusion
In summary, we have developed a mitochondria-targeted ratiometric two-photon fluorescent probe (Mito-MPVQ) for biological Zn2+ detection. Mito-MPVQ shows large red shifts from 420 nm to 488 nm and excellent ratiometric detection signal to Zn2+. The interaction of Mito-MPVQ with Zn2+ was verified by NMR titration and theoretical calculation. Mito-MPVQ exhibits large two-photon absorption cross sections at nearly 720 nm and steady fluorescence within the biologically relevant pH range. Morever, cell
Acknowledgments
This work was supported by NSFC (Grant no. 21102002, 21272223, 21372005), NIHR01 GM101279, the Education department of Anhui Province (KJ2011A018) and 211 Project of Anhui University.
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Contributed equally to the work.