Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
A rhodamine chromene-based turn-on fluorescence probe for selectively imaging Cu2+ in living cell
Graphical abstract
Rhodamine chromene-based “turn-on” fluorescence probe can monitor the intracellular Cu2+ level in living HeLa cells with high sensitivity and selectivity in shorter time.
Highlights
► A new fluorescence probe was synthesized to improve membrane-permeable property. ► The probe can monitor Cu2+ in living cells with high sensitivity and selectivity. ► The probe can monitor intracellular Cu2+ in HeLa cells in shorter time.
Introduction
Copper, after zinc and iron, ranks third in abundance in human bodies among the essential heavy metals [1]. Copper plays a critical role in the physiology of living organisms and has multiple functions, as iron absorption, hemopoiesis, and diverse enzyme activities and in the redox processes. However, alteration in the cellular homeostasis of copper ions is connected to various severe neurodegenerative diseases such as Menkes syndrome, Wilson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease [2]. Furthermore, a higher level of Cu2+ is detected in tumors with a possible role in promoting angiogenesis (new blood vessel growth) [3]. Moreover, with excessive loading, Cu2+ is highly toxic to organisms [4]. Therefore, the quantitative detection of intracellular Cu2+ is of great importance for elucidating its complex physiological and pathological roles.
The design and synthesis of chemosensors for copper ions has become a very highly focused area of research, as a result of the demand for more sensitive and selective chemosensors for imaging in living cells and in vivo purposes [5]. A number of fluorescent probes based on coumarin [6], 1,3,5-triphenylbenzene fluorophore [7], N-(quinolin-8-yl)acetamide [8], dansyl-anthracene dyads [9], nanoparticle [10], polymer-based [11] with high sensitivity and specificity toward Cu2+ have been developed.
It is known that the rhodamine framework is an ideal mode to construct fluorescent chemosensors, because the spirolactam ring-opening process leads to a turn-on fluorescence change with excellent photophysical properties, such as long absorption and emission wavelengths, large absorption coefficient, and high fluorescence quantum yield [12]. An additional advantage of such a rhodamine-based sensing system is that the ring-opening process is also accompanied by a vivid color change from colorless to pink, thus enabling the metal detection with the naked eye [13].
Although a number of rhodamine-based fluorescent probes for transition metals including zinc, iron, chromium and mercury have been developed over the past decades [14], [15], [16], [17], relatively few copper-selective probes have been reported [5](a), [18]. Among these probes, membrane-permeable chemosensors that allow monitoring of intracellular copper are rare [5](a), [5](b), [18](b), [18](c), [18](d). Thus, the development of turn-on fluorescent probes with high sensitivity and selectivity for monitoring Cu2+ in living cells remains a significant challenge. Due to the good stability of Schiff base ligands with Cu2+ to form complexes [19], rhodamine Schiff base ligands based fluorescence probe have attracted a great deal of attention in recent years [14](a), [17](a), [18](a), [18](e), [20]. In our previous paper, we described a rhodamine chromene-based fluorescence probe to monitor the intracellular Cu2+ level in living cells [21]. Due to the less membrane permeation ability of the probe, the incubation of the probe needs 5 h to take fluorescence microscope images of living HeLa cells. Hence, in order to increase the membrane permeation ability of the probe, we modified the structure of the probe by introducing a chlorine atom to chromene moiety because the predicted lipophilicity of the probe with chlorine instead of methoxyl group is stronger. In addition, a smaller size of chlorine atom compared to methoxyl group was expected to improve the cell permeability of the probe. The results showed that the modified probe not only possess higher fluorescence intensity but also have favorable membrane permeation ability for the detection of the intracellular Cu2+ level in living cells.
Section snippets
Materials and characterization
Deionized water was used throughout the experiment. All the reagents were purchased from commercial suppliers and used without further purification. The solutions of metal ions were prepared from NaNO3, Mg(NO3)2·6H2O, Al(NO3)3·9H2O, KNO3, Ca(NO3)2·4H2O, Cr(NO3)3·9H2O, 50% (wt.) Mn(NO3)2 (aq.), Fe(NO3)3·9H2O, Co(NO3)2·6H2O, Ni(NO3)2·6H2O, Cu(NO3)2·3H2O, Zn(NO3)2·6H2O, AgNO3, Cd(NO3)2·4H2O, Ba(NO3)2, Hg(NO3)2·H2O, Pb(NO3)2, CuCl2·2H2O, CuSO4·5H2O, Cu(Ac)2·H2O, NaCl, NaAc and Na2SO4, respectively,
Synthesis and characterization of 4
The synthetic strategy adopted for the synthesis of novel probe 4 is outlined in Scheme 1. Probe 4 was synthesized in a facile manner from rhodamine B (1) and 6-chloro-4-oxo-4H-chromene-3-carbaldehyde (3) by a two-step reaction. Briefly, compound 2 was first synthesized from rhodamine B and hydrazine hydrate following the literature procedure [20a]. Probe 4 was then easily synthesized by the reaction of 2 with 3 in 79% yield according to our previous report [21].
Structure of probe 4 was
Conclusion
In summary, we have developed a novel rhodamine chromene-based turn-on fluorescence probe to monitor intracellular Cu2+ level in living cells. The probe switches to a highly fluorescent complex upon Cu2+ chelation under physiological conditions. The high sensitivity and selectivity of the probe are demonstrated by a remarkable fluorescence enhancement and the lack of interference from up to 16 other metal ions. Furthermore, this probe that possesses chlorine group instead of methoxyl group has
Acknowledgments
This study was supported by 973 Program (2010CB933504) and the National Natural Science Foundation of China (20972088 and 90813022).
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These two authors are equally contributed to this work.