Synthesis of a Cu2+-Selective Probe Derived from Rhodamine and Its Application in Cell Imaging

A new fluorescent probe P based on rhodamine for Cu2+ was synthesized and characterized. The new probe P showed high selectivity to Cu2+ over other tested metal ions. With optimal conditions, the proposed probe P worked in a wide linear range of 1.0 × 10−6−1.0 × 10−5 M with a detection limit of 3.3 × 10−7 M Cu2+ in ethanol-water solution (9:1, v:v, 20 mM HEPES, pH 7.0). Furthermore, it has been used for imaging of Cu2+ in living cells with satisfying results.


Introduction
The selective detection of chemical species upon molecular recognition is of great significance in the host environment, particularly when the guest is ionic. Since fluoroionophores can provide chemical information on the ion concentrations, they are important subjects in metal ion analysis [1][2][3][4][5].
Among the metal ions, Cu 2+ is one of the important targets, because it exhibits toxicity under overloading conditions, which can causes neurodegenerative diseases [6]. Thus, it is necessary to trace the concentration of Cu 2+ in vitro and in vivo. Even though considerable efforts have been devoted to developing fluorescent probes for Cu 2+ over the last few decades [7][8][9], it is still of great importance to develop new Cu 2+ -selective probes.
Owing to their excellent fluorescence properties, rhodamine dyes have been used extensively for conjugation with biomolecules [1,4,7]. Many rhodamine B derivatives have been used as fluorescent chemosensors for the detection of different metal ions [1,10,11]. Inspired by these research works, we report here a new fluorescent probe P based on a rhodamine B derivative for Cu 2+ (Scheme 1). It showed a reversible "turn-on" fluorescent response for Cu 2+ in aqueous solution with remarkably high sensitivity and selectivity. Moreover, it has been demonstrated that P can be used as a fluorescent probe for monitoring Cu 2+ in living cells. Scheme 1. The synthesis route of probe P.

Reagents and Instruments
All reagents and solvents are of analytical grade and used without further purification. Nuclear magnetic resonance (NMR) spectra were measured with a Brucker AV 400 instrument, and chemical shifts were given in ppm from tetramethylsilane (TMS). Mass spectra (MS) were recorded on a Thermo TSQ Quantum Access Agilent 1100. Fluorescence emission spectra were conducted on a Hitachi 4600 spectrofluorometer. UV-Vis spectra were obtained on a Hitachi U-2910 spectrophotometer. Fluorescence imaging was performed by confocal fluorescence microscopy on an Olympus FluoView Fv1000 laser scanning microscope. pH was conducted with a pH-meter PBS-3C.

General Spectroscopic Methods
Metal ions and probe P were dissolved in deionized water and DMSO to obtain 1.0 mM stock solutions, respectively. Before spectroscopic measurements, the working solution was freshly prepared by diluting the high concentration stock solution to the corresponding solution. For all of the measurements, excitation and emission slit widths were 10 nm, and the excitation wavelength was 520 nm.

Cell Incubation and Imaging
HepG2 cells placed on coverslips were washed with phosphate-buffered saline (PBS), followed by incubating with 1 μM of CuCl2 (in PBS) for 30 min at 37 °C, and then washed with PBS three times. After incubating with 10 μM of probe P for 30 min at 37 °C, the cells were washed with PBS three times again. Fluorescence imaging of intracellular Cu 2+ in HepG2 cells was conducted by using a confocal fluorescence microscopy on an Olympus FluoView Fv1000 laser scanning microscope.

Effect of pH on P and P with Cu 2+
In order to investigate a suitable pH working range of P for the sensing of Cu 2+ , a pH titration experiment was performed firstly ( Figure 1). The results showed that the absorption of the free probe P can be negligible under a pH range from 4 to 9. After the addition of Cu 2+ , the absorption of probe P at 560 nm rapidly increased to a maximum value. The results showed that the probe P can be worked within a wide pH range of 5.3-7.0. As the pH of a natural water body is near neutral, therefore, further UV-Vis and fluorescent studies were carried out in ethanol-water solution (9:1, v:v, 20 mM HEPES, pH 7.0).

UV-Vis Spectral Response of P
As expected, probe P alone was colorless and scarcely showed absorption in the 500-600 nm region in ethanol-water solution (9:1, v:v, 20 mM HEPES, pH 7.0). However, upon addition of Cu 2+ , an intense absorption band centered at 560 nm appeared, presumably because of the chelation of Cu 2+ with the nitrogen atom of the amide group of P, which resulted in the formation of the open-ring form of rhodamine B. At the same time, other related metal ions (K + , Na + , Ca 2+ , Mg 2+ , Zn 2+ , Pb 2+ Co 2+ , Cd 2+ , Cr 3+ , Ni 2+ , Hg 2+ , Ag + , Fe 3+ and Al 3+ ) did not show any obvious absorption under similar conditions ( Figure 2).

Fluorescence Spectral Response of P
To further evaluate the selectivity of probe P, the fluorescence spectra (ex = 520 nm) of P (10 µM) were investigated in ethanol-water solution (9:1, v:v, 20 mM HEPES, pH 7.0) with the addition of respective metal ions (100 μM) ( Figure 3). Compared with other tested ions, only Cu 2+ generated a significant "turn-on" fluorescence response of the monomeric peak at 577 nm with a fluorescence enhancement up to 200-fold, and Hg 2+ had negligible interference. These results suggested that P had a higher selectivity toward Cu 2+ than the other metal ions. In order to investigate further the interaction of Cu 2+ with P, a fluorescent titration experiment was carried out. The results showed that the fluorescence intensity of P was enhanced at 577 nm upon the addition of various amounts of Cu 2+ in ethanol-water solution (9:1, v:v, 20 mM HEPES, pH 7.0), as depicted in Figure 4. Under the present conditions, when P was employed at the 10-μM level, the fluorescent intensity of P was proportional to the concentration of Cu 2+ in the range from 1.0 × 10 −6 to 1.0 × 10 −5 M with a detection limit of 3.3 × 10 −7 M Cu 2+ . This clearly demonstrated that chemosensor P could sensitively detect environmentally relevant levels of Cu 2+ .

Proposed Reaction Mechanism of P with Cu 2+
According to the obtained results above, the reaction mechanism is very likely due to the metal ion-induced ring opening of rhodamine spirolactam, rather than other possible reactions [12]. The Job's plot experiment was also explored to study the coordination mode of P with Cu 2+ , which confirmed a 1:1 stoichiometry for the P-Cu 2+ complex and strongly supported the above conclusion ( Figure 5). Thus, according to the obtained results, the coordination mode between P and Cu 2+ was proposed as shown in Scheme 2. On the other hand, the response of P to Cu 2+ was confirmed to be reversible by the EDTA titration. Upon addition of 50 µM EDTA to the mixture of P (10 µM) and Cu 2+ (10 µM) in ethanol-water solution (9:1, v:v. 20 mM HEPES, pH 7.0), the color changed from pink to almost colorless, and a ~93% fluorescent emission intensity of the system was quenched (Figure 6), which suggested that the EDTA replaced the receptor P to coordinate Cu 2+ . When Cu 2+ was added to the system again, the signals were completely reproduced, and the colorless solution turned to pink. These findings indicated that P can be classified as a reversible chemosensor for Cu 2+ .

Preliminary Analytical Application
To further demonstrate the practical applicability of the probe P, a cell imaging experiment was carried out to detect Cu 2+ in living cells, and the fluorescence images of HepG2 cells were recorded before and after the addition of Cu 2+ (Figure 7). The cells were supplemented with only P in the growth medium for 30 min at 37 °C, which led to very weak fluorescence, as determined by laser scanning confocal microscopy (ex = 559 nm) (Figure 7c). In contrast, when loaded with 1 µM CuCl2 for 30 min, a bright fluorescence was detected (Figure 7a). These results suggested that probe P can penetrate the cell membrane and might be used for detecting Cu 2+ in living cells.

Method Performance Comparison
The performance of the proposed probe P was compared with the corresponding performance of some reported fluorescent probes for Cu 2+ determination, as shown in Table 1. All of the fluorescent methods present good selectivity for Cu 2+ . The fluorescence quenching methods using rhodamine derivative exhibit a bad detection limit and are not applicable for living cells [13]. Most of the rhodamine derivatives possess good fluorescent properties [14][15][16], but some of them have more or less disadvantages, such as irreversibility [14] and complicated purification [14,15]. As for the two types of enhancement probes based on rhodamine derivative, dual-function detection for Cu 2+ and ClO − are realized [17], and a reversible response in living cells is exhibited [18]; however, low yields still restrict their further applications [17,18]. Our newly developed fluorescence enhancement method presents a number of attractive analytical features, such as a wide linear range, good reversibility and reproducibility, good selectivity and wide applicability. The fluorescence probe P based on rhodamine spirolactame derivative is easy to prepare at a low cost and can be used for routine analysis of ultra-trace levels of Cu 2+ in living cells.

Conclusions
A new rhodamine B derivative was successfully characterized as a Cu 2+ -selective probe. The proposed probe has good selectivity towards Cu 2+ compared with other common metal ions. Under optimized conditions, P exhibited a dynamic response range for Cu 2+ from 1.0 × 10 −6 to 1.0 × 10 −5 M with a detection limit of 3.3 × 10 −7 M Cu 2+ in ethanol-water solution (9:1, v:v. 20 mM HEPES, pH 7.0).