Highly Selective Fluorescent Probe for the Detection of Copper (II) and Its Application in Live Cell Imaging

The development of fluorescent methods for the detection of metal ions is of great importance due to their diverse environmental and biological roles. Herein, a rhodamine 6G-based off-on fluorescent probe (L1) with a t-butyl pyrrole moiety as the recognition site was designed and synthesized. Photophysical studies show that L1 exhibits excellent sensitivity and selectivity towards Cu2+ to other metal ions in neutral acetonitrile aqueous media. Mechanism studies suggest that the recognition process may associate with a Cu2+ promoted hydrolysis reaction of L1. Furthermore, L1 has been successfully applied in fluorescence imaging of Cu2+ ion in living cells.


Introduction
e exploration of detection methods for environmentally and biologically important metal ions is of great interest to researchers currently [1,2]. Among these metal ions, Cu 2+ receives great attention because copper is the third most abundant essential metal (after iron and zinc) in the human body and plays an important role in a variety of physiological processes. For example, copper is integrated into various proteins and metalloenzymes that perform basic metabolic functions [3]. Copper deficiency could produce osteoporosis, hyperthyroidism, and coronary heart disease [4]. However, excessive accumulation of copper can cause central nervous system damage and increase the risk of neurodegenerative diseases such as Alzheimer's, Parkinson's, Menken's, and Wilson's diseases [5][6][7][8]. Hence, development of sensitive and selective analysis methods for copper ion, especially those that could be utilized in bioimaging, is of great importance in the aspects of understanding the complex physiological functions of copper in the human body.
Traditionally, inductively coupled plasma atomic emission spectrometry (ICP-AES), atomic absorption spectroscopy (AAS), and inductively coupled plasma mass spectrometry (ICP-MS) are used to analyze copper ions [9][10][11]. However, these methods require sophisticated instruments and complicated and time-consuming processes of sample preparation. us, a simple and rapid detection method for quantifying copper ions is necessary. Fluorescence methods, with the advantages of sensitivity, simple operation, and real-time monitoring with fast response time, have been widely used in the detection of metal ions [12][13][14][15][16][17][18][19][20][21]. Due to the paramagnetic nature, Cu 2+ was usually detected through fluorescence quenching of chemical sensors [22,23], which may result in false-positive results and lesssensitive detection. Among the developed fluorophores, rhodamine derivatives, due to their excellent photophysical properties, such as large absorption coefficients, high fluorescence quantum yields, and long absorption and emission wavelengths, have attracted great attention from researchers [24][25][26][27][28][29]. In addition, it is well known that the fluorescence emission behaviors of rhodamine derivatives could be adjusted through a spirolactam ring-opening reaction. Spirocycle derivatives of rhodamine are colorless and nonfluorescent due to their nonconjugated structure. However, opening of the spirolactam ring, usually caused by metal ions, will produce intense fluorescence emission and a pink color change. Based on this spirolactam/ring-opened amide equilibrium of rhodamine, through introducing proper recognition ligands, researchers have developed many turn-on fluorescent sensors for metal ions [30][31][32][33][34][35][36][37][38][39][40].
Taking into account the criteria mentioned above, we herein incorporated a t-butyl pyrrole moiety into the rhodamine fluorophore to form a turn-on fluorescent probe L1 for the detection of Cu 2+ . e molecular structure of L1 was verified by 1 H NMR, 13 C NMR, and MS spectra. With the addition of Cu 2+ to L1 in a neutral CH 3 CN/H 2 O solution, metal-triggered ring-opening reaction of the spirolactam in L1 took place, resulting in sensitive colorimetric response and fluorescence emission. Mechanism studies suggest that the detection process may associate with a Cu 2+ promoted hydrolysis reaction of L1. Furthermore, fluorescence microscopy experiments demonstrated that L1 could be used to image Cu 2+ in living cells.

General Materials and Apparatus.
All chemicals are purchased commercially and used directly without further purification. Deionized water was used throughout, and the pH was adjusted using diluted sodium hydroxide solution or hydrochloric acid. e pH value was measured with a Rex pHS-3E pH meter. Silica gel (200-300 mesh) was used for column chromatography. NMR spectra were obtained with a 600 MHz Bruker spectrometer, and tetramethylsilane (TMS) was used as the internal standard. High-resolution mass spectra were measured on an Agilent LCMS 6500 spectrometer. Absorbance spectra were measured on a Shimadzu UV-3101PC spectrometer. Measurements of fluorescence spectra were performed on a Shimadzu RF-5301PC spectrometer. Both excitation and emission slit widths were set at 3 nm. All experiments were operated at about 298 K.

Procedures of Sensing Experiments.
A stock solution of probe L1 (2 × 10 −4 M) was prepared in CH 3 CN. Stock solutions of metal ions (2 × 10 −3 M) were prepared in deionized water from their chloride salts or nitrate salt (Ag + ). Working solution of L1 (10 μM, CH 3 CN/H 2 O, 1 : 1, v/v) was freshly prepared prior to spectroscopic experiments by diluting the high-concentration stock solution. In the sensing experiments, each time, a 3 mL working solution of L1 (10 μM, CH 3 CN/H 2 O, 1 : 1, v/v) was put in a quartz optical cell of 1 cm optical path length, and appropriate amounts of stock solutions of metal ions were added by a pipette. Spectral data were collected 2 min after the addition of the ions.

Synthesis of L1.
To a 100 mL flask with three necks, rhodamine 6G hydrazine (2.8 mmol), which was synthesized according to reported methods [41], and 5-tert-butylpyrrole-2-carbaldehyde (3.3 mmol) were dissolved in 30 mL methanol. After addition of 0.1 mL acetic acid, the mixture was stirred and heated to reflux for 16 h. A pale yellow solid obtained was filtered off and washed using cold methanol. e solid was dried in vacuum and further purified by column chromatography (CH 2 Cl 2 /CH 3 OH � 150/1, v/v). Yield: 88.6%. 1

Results and Discussion
Compound L1 was easily synthesized from rhodamine 6G through a two-step reaction (Scheme 1). After purification by column chromatography (CH 2 Cl 2 /CH 3 OH � 150/1, v/v), L1 was obtained in an 88.6% yield. e molecular structure was verified by 1 H NMR, 13 C NMR, and MS spectra ( Figures S1-S3). e m/z value of the molecular ion peak in the HRMS spectra was in good accordance with the accurate molecular weight with small derivation. Similar with other spirocycle derivatives of rhodamine, the solution of L1 in neutral CH 3 CN/H 2 O media was colorless and weakly fluorescent, indicating that it existed mainly in the form of spirolactam. In addition, a characteristic spirocycle carbon chemical shift at 65.0 ppm in the 13 C NMR spectra was observed, which further supported this estimation [42]. e absorption spectrum of L1 (10 μM) in neutral CH 3 Figure 1. No absorption peaks in the visible wavelength range was exhibited, suggesting that L1 existed with the structure of spirolactam. Once the solution of Cu 2+ was added, a new peak was detected at 525 nm. With the increase of Cu 2+ concentration, the intensity of the peak was gradually enhanced, which could be interpreted as the transform from a spirolactam structure to a ring-opened form of L1. Correspondingly, the color of the solution changed from colorless to purple, which help to achieve the naked-eye recognition of Cu 2+ ion. As shown in Figure 1, with the increasing concentration of Cu 2+ , the absorption value of L1 (10 μM) was found to increase linearly in the range of 2 μM-14 μM at 525 nm. e selective sensory studies of L1 (10 μM) in neutral CH 3 CN/H 2 O solution were then extended to other metal ions (Cr 3+ , Al 3+ , Fe 3+ , Ca 2+ , Ba 2+ , Co 2+ , Fe 2+ , Hg 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Pd 2+ , Zn 2+ , Ag + , Li + , K + , Na + , and Sn 4+ ). When 1 equiv metal ions were added into the relevant solution, only Cu 2+ could induce a purple color and an obvious increase of absorbance at 525 nm ( Figure 2). Al 3+ exhibited weak absorption response, and the other metal ions showed almost no absorbance increase in the same condition.
e results indicated that L1 showed high selectivity towards Cu 2+ in the detection of metal ions. e fluorescence sensing behavior of L1 for Cu 2+ in neutral CH 3 CN/H 2 O solution (10 μM, 1 : 1, v/v) was also investigated. When no Cu 2+ was added, the free L1 solution had little fluorescence at the excitation wavelength of 500 nm due to the spirocyclic form of its molecular structure. However, similar to the results of the absorption experiments, once the solution of Cu 2+ was added, an obvious increase of the fluorescence intensity at 545 nm ( Figure 3) could be detected, which could be ascribed to the generation of ring-opened conjugate structure in the rhodamine moiety. e fluorescence intensity at 545 nm of L1 (10 μM) was linear with the concentration of Cu 2+ in the range of 2 μM-26 μM, and the detection limit of Cu 2+ was calculated as 0.38 μM (S/N � 3). e fluorescence quantum yield was calculated to be 0.19 in the presence of Cu 2+ ion (26 μM) by using rhodamine 6G as the standard (Φ f � 0.94 in ethanol) [43]. e selectivity of L1 towards Cu 2+ over other cations was satisfactory. On excitation at 500 nm, no obvious spectral change of L1 (10 μM, CH 3 CN/H 2 O, 1 : 1, v/v) was observed with the addition of Cr 3+ , Al 3+ , Fe 3+ , Ca 2+ , Ba 2+ , Co 2+ , Fe 2+ , Hg 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Pd 2+ , Zn 2+ , Ag + , Li + , K + , Na + , and Sn 4+ (10 μM). In contrast, more than 500-fold fluorescence intensity increase was detected when the same amount of Cu 2+ ion was added (Figure 4). In order to further evaluate the selectivity of L1 towards Cu 2+ among other metal ions, the interference experiments were investigated. As shown in Figure 5, no obvious changes were observed in the Cu 2+ -induced fluorescence emission of L1 when comparing the spectra data obtained in the presence and absence of other metal ions. ese results indicated that L1 could be     used as a selective Cu 2 fluorescent sensor without interference from other metal ions. e influence of solution pH on the fluorescence response towards Cu 2+ of L1 was studied. As shown in Figure S4, no obvious fluorescence could be found for free L1 between pH 4.0 and 8.0, indicating that the spirolactam structure still dominated in this pH range. However, with the addition of Cu 2+ , fluorescence change of L1 was observed with different fluorescence enhancement efficiency under different pH values ( Figure S5). A marked fluorescence response towards Cu 2+ was achieved in a pH range from 7 to 8. ese results indicated that L1 could be used as a fluorescent probe for the detection of Cu 2+ in physiological pH conditions. e effects of reaction media for the detection of Cu 2+ by L1 were also studied. As shown in Figure S6, in the presence of 1 equiv. Cu 2+ , the fluorescence signal value of L1 reached the maximum when acetonitrile content was at 50%-60%. As a result, 50% aqueous acetonitrile was employed in all optical experiments. e response time of the detection system on fluorescence emission was studied to evaluate the sensitivity of L1 towards Cu 2+ . After addition of 1 equiv. Cu 2+ , the fluorescence intensity of L1 increased rapidly and reached maximum after two minutes and did not increase with the prolongation of reaction time ( Figure S7). ese results indicated that L1 was sensitive for the detection of Cu 2+ in the aqueous acetonitrile solution, and 2 min was selected as the detection time in this research.
In order to illustrate the interaction mechanism between Cu 2+ and L1, excess (10 equiv) Na 2 EDTA was added to the solution of L1 in neutral CH 3 CN/H 2 O solution (10 μM, 1 : 1, v/v) containing Cu 2+ ion (10 μM). No obvious decrease in the fluorescent intensity or color change was observed after the addition, indicating that the detection of Cu 2+ ion by L1 was an irreversible process ( Figure S8). HRMS experiments were carried out to analyze the reaction products of Cu 2+ and L1. e peak at m/z � 415.2025 was ascribed to rhodamine 6G (M + 1; m/z calculated for M + , C 26 H 26 N 2 O 3 , 414.1943), indicating rhodamine 6G as a final product ( Figure S9). Moreover, the addition of Cu 2+ into the solution of L1 in pure CH 3 CN resulted in no fluorescent emission or color change. Based on these experimental results, a mechanism involving Cu 2+ -promoted redox hydrolysis of L1 was proposed (Scheme 2), which was similar to that of the sensing towards Cu 2+ by rhodamine B hydrazide [44].
For further studying the practical application of L1 in the detection of Cu 2+ , fluorescence imaging experiments were carried out in MCF7 cells. e MCF7 cells were incubated with L1 for 30 min and washed with PBS. As shown in Figure  6(a), no intracellular fluorescence was observed in the image. However, after subsequent treatment with CuCl 2 at the same conditions for another 30 min, strong fluorescence in the MCF7 cells was observed (Figure 6(b)). ese results suggest that L1 could pass through the cell membrane and be used for the detection of Cu 2+ in living cells. e cytotoxicity tests were studied by MTT assay with the concentrations of L1 from 0 to 30 μM ( Figure S10). Experimental results showed that more than 95% of the MCF7 cells were viable after incubation with L1 for 24 h at 37°C, indicating that L1 has low cytotoxicity to cells in this dosage range.

Conclusion
In summary, a new colorimetric and fluorescent probe L1 for Cu 2+ was developed through the combination of rhodamine 6G and a pyrrole moiety. As a turn-on fluorescent probe, L1 exhibited excellent sensitivity and selectivity for Cu 2+ detection in acetonitrile aqueous media with a low detection limit. Moreover, fluorescence bioimaging experiments confirmed that L1 could be used to detect intracellular Cu 2+ in living cells.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors report no conflicts of interest.