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

Authors: Zhihao Guo; Xiuji Wang; Pei Wei; Yihua Gao; Qin Li
Journal: Journal of Analytical Methods in Chemistry
 (2019)

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## Abstract

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.


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## Body

## 1. Introduction

The exploration of detection methods for environmentally and biologically important metal ions is of great interest to researchers currently [1, 2]. Among these metal ions, Cu2+ 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 [5–8]. 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 [9–11]. However, these methods require sophisticated instruments and complicated and time-consuming processes of sample preparation. Thus, 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 [12–21]. Due to the paramagnetic nature, Cu2+ was usually detected through fluorescence quenching of chemical sensors [22, 23], which may result in false-positive results and less-sensitive 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 [24–29]. 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 [30–40].Taking into account the criteria mentioned above, we herein incorporated at-butyl pyrrole moiety into the rhodamine fluorophore to form a turn-on fluorescent probe L1 for the detection of Cu2+. The molecular structure of L1 was verified by 1H NMR, 13C NMR, and MS spectra. With the addition of Cu2+ to L1 in a neutral CH3CN/H2O 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 Cu2+ promoted hydrolysis reaction of L1. Furthermore, fluorescence microscopy experiments demonstrated that L1 could be used to image Cu2+ in living cells.


## 2. Materials and Methods

### 2.1. 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. The 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.


### 2.2. Procedures of Sensing Experiments

A stock solution of probeL1 (2 × 10−4 M) was prepared in CH3CN. 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, CH3CN/H2O, 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, CH3CN/H2O, 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.


### 2.3. 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. The solid was dried in vacuum and further purified by column chromatography (CH2Cl2/CH3OH = 150/1, v/v). Yield: 88.6%. 1H NMR (600 MHz, DMSO-d6), δ (ppm): 10.93 (s, 1H, CNHC), 8.16 (s, 1H, N=CH), 7.84 (d, J = 12 Hz, 1H, ArH), 7.50 (m, 2H, ArH), 6.94 (d, J = 12 Hz, 1H, ArH), 6.30 (s, 2H, ArH), 6.18 (s, 2H, ArH), 6.05 (d, J = 6 Hz, 1H, CCHCH), 5.77 (d, J = 6 Hz, 1H, CCHCH), 5.04 (t, J = 6 Hz, 2H, CH3CH2NH), 3.12 (q, J = 12 Hz, 4H, CH3CH2), 1.85 (s, 6H, ArCH3), 1.20 (m, 15H, CH3CH2, CCH3). 13C NMR (150 MHz, DMSO-d6), δ (ppm): 163.12, 151.96, 150.64, 147.66, 146.61, 141.63, 133.12, 128.41, 128.31, 126.64, 126.50, 123.24, 122.60, 118.11, 112.32, 105.03, 104.64, 95.91, 65.00, 37.44, 31.38, 29.98, 16.96, 14.13. HRMS: m/z calculated for M+, C35H39N5O2, 561.3104. Found: 562.3192 (M + 1).


### 2.4. Cell Culture and Fluorescence Imaging

The human breast adenocarcinoma cells MCF7 were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C under an atmosphere containing 5% CO2. Before the experiments, the MCF7 cells were washed with PBS and then incubated with 10 μM L1 at 37°C for 30 min. After removal of excess probe and washed with PBS, the MCF7 cells were incubated with 20 μM CuCl2 for another 30 min. The MCF7 cells were rinsed with PBS again and live-cell imaging was conducted using an EVOS FL Auto microscope.


## 2.1. 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. The 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.


## 2.2. Procedures of Sensing Experiments

A stock solution of probeL1 (2 × 10−4 M) was prepared in CH3CN. 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, CH3CN/H2O, 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, CH3CN/H2O, 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.


## 2.3. 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. The solid was dried in vacuum and further purified by column chromatography (CH2Cl2/CH3OH = 150/1, v/v). Yield: 88.6%. 1H NMR (600 MHz, DMSO-d6), δ (ppm): 10.93 (s, 1H, CNHC), 8.16 (s, 1H, N=CH), 7.84 (d, J = 12 Hz, 1H, ArH), 7.50 (m, 2H, ArH), 6.94 (d, J = 12 Hz, 1H, ArH), 6.30 (s, 2H, ArH), 6.18 (s, 2H, ArH), 6.05 (d, J = 6 Hz, 1H, CCHCH), 5.77 (d, J = 6 Hz, 1H, CCHCH), 5.04 (t, J = 6 Hz, 2H, CH3CH2NH), 3.12 (q, J = 12 Hz, 4H, CH3CH2), 1.85 (s, 6H, ArCH3), 1.20 (m, 15H, CH3CH2, CCH3). 13C NMR (150 MHz, DMSO-d6), δ (ppm): 163.12, 151.96, 150.64, 147.66, 146.61, 141.63, 133.12, 128.41, 128.31, 126.64, 126.50, 123.24, 122.60, 118.11, 112.32, 105.03, 104.64, 95.91, 65.00, 37.44, 31.38, 29.98, 16.96, 14.13. HRMS: m/z calculated for M+, C35H39N5O2, 561.3104. Found: 562.3192 (M + 1).


## 2.4. Cell Culture and Fluorescence Imaging

The human breast adenocarcinoma cells MCF7 were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C under an atmosphere containing 5% CO2. Before the experiments, the MCF7 cells were washed with PBS and then incubated with 10 μM L1 at 37°C for 30 min. After removal of excess probe and washed with PBS, the MCF7 cells were incubated with 20 μM CuCl2 for another 30 min. The MCF7 cells were rinsed with PBS again and live-cell imaging was conducted using an EVOS FL Auto microscope.


## 3. Results and Discussion

CompoundL1 was easily synthesized from rhodamine 6G through a two-step reaction (Scheme 1). After purification by column chromatography (CH2Cl2/CH3OH = 150/1, v/v), L1 was obtained in an 88.6% yield. The molecular structure was verified by 1H NMR, 13C NMR, and MS spectra (Figures S1–S3). The 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 CH3CN/H2O 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 13C NMR spectra was observed, which further supported this estimation [42].Scheme 1
Synthesis of probeL1.The absorption spectrum ofL1 (10 μM) in neutral CH3CN/H2O (1 : 1, v/v) solution is shown in 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 Cu2+ was added, a new peak was detected at 525 nm. With the increase of Cu2+ 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 Cu2+ ion. As shown in Figure 1, with the increasing concentration of Cu2+, the absorption value of L1 (10 μM) was found to increase linearly in the range of 2 μM–14 μM at 525 nm. The selective sensory studies of L1 (10 μM) in neutral CH3CN/H2O solution were then extended to other metal ions (Cr3+, Al3+, Fe3+, Ca2+, Ba2+, Co2+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Pd2+, Zn2+, Ag+, Li+, K+, Na+, and Sn4+). When 1 equiv metal ions were added into the relevant solution, only Cu2+ could induce a purple color and an obvious increase of absorbance at 525 nm (Figure 2). Al3+ exhibited weak absorption response, and the other metal ions showed almost no absorbance increase in the same condition. The results indicated that L1 showed high selectivity towards Cu2+ in the detection of metal ions.Figure 1
Absorbance spectra ofL1 (10 μM) containing different concentrations of Cu2+ (0–18 μM) in CH3CN/H2O solution (1 : 1, v/v). Inset: the absorbance at 525 nm as a function of Cu2+ concentration (2–14 μM).Figure 2
Absorbance spectra ofL1 (10 μM) with addition of 10 μM various metal ions in CH3CN/H2O solution (1 : 1, v/v).The fluorescence sensing behavior ofL1 for Cu2+ in neutral CH3CN/H2O solution (10 μM, 1 : 1, v/v) was also investigated. When no Cu2+ 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 Cu2+ 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. The fluorescence intensity at 545 nm of L1 (10 μM) was linear with the concentration of Cu2+ in the range of 2 μM–26 μM, and the detection limit of Cu2+ was calculated as 0.38 μM (S/N = 3). The fluorescence quantum yield was calculated to be 0.19 in the presence of Cu2+ ion (26 μM) by using rhodamine 6G as the standard (Φf = 0.94 in ethanol) [43]. The selectivity of L1 towards Cu2+ over other cations was satisfactory. On excitation at 500 nm, no obvious spectral change of L1 (10 μM, CH3CN/H2O, 1 : 1, v/v) was observed with the addition of Cr3+, Al3+, Fe3+, Ca2+, Ba2+, Co2+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Pd2+, Zn2+, Ag+, Li+, K+, Na+, and Sn4+ (10 μM). In contrast, more than 500-fold fluorescence intensity increase was detected when the same amount of Cu2+ ion was added (Figure 4). In order to further evaluate the selectivity of L1 towards Cu2+ among other metal ions, the interference experiments were investigated. As shown in Figure 5, no obvious changes were observed in the Cu2+-induced fluorescence emission of L1 when comparing the spectra data obtained in the presence and absence of other metal ions. These results indicated that L1 could be used as a selective Cu2 fluorescent sensor without interference from other metal ions.Figure 3
Fluorescence spectra ofL1 (10 μM) containing different concentrations of Cu2+ (0–26 μM) in CH3CN/H2O solution (1 : 1, v/v). Excitation was performed at 500 nm. Inset: fluorescence intensity at 545 nm as a function of Cu2+ concentration (2–26 μM).Figure 4
Fluorescence spectra ofL1 (10 μM) with addition of 10 μM various metal ions in CH3CN/H2O solution (1 : 1, v/v). Excitation was performed at 500 nm.Figure 5
Fluorescence intensity at 545 nm ofL1 (10 μM) with addition of 10 μM Cu2+ in the absence (blank) and presence of various metal ions (50 μM) in CH3CN/H2O solution (1 : 1, v/v). (1) blank, (2) Ag+, (3) Al3+, (4) Ba2+, (5) Ca2+, (6) Co2+, (7) Cr3+, (8) Fe2+, (9) Fe3+, (10) Hg2+, (11) K+, (12) Li+, (13) Mg2+, (14) Mn2+, (15) Na+, (16) Ni2+, (17) Pd2+, (18) Sn4+, and (19) Zn2+. Excitation was performed at 500 nm.The influence of solution pH on the fluorescence response towards Cu2+ 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 Cu2+, fluorescence change of L1 was observed with different fluorescence enhancement efficiency under different pH values (Figure S5). A marked fluorescence response towards Cu2+ was achieved in a pH range from 7 to 8. These results indicated that L1 could be used as a fluorescent probe for the detection of Cu2+ in physiological pH conditions.The effects of reaction media for the detection of Cu2+ by L1 were also studied. As shown in Figure S6, in the presence of 1 equiv. Cu2+, 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. The response time of the detection system on fluorescence emission was studied to evaluate the sensitivity of L1 towards Cu2+. After addition of 1 equiv. Cu2+, 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). These results indicated that L1 was sensitive for the detection of Cu2+ 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 Cu2+ and L1, excess (10 equiv) Na2EDTA was added to the solution of L1 in neutral CH3CN/H2O solution (10 μM, 1 : 1, v/v) containing Cu2+ ion (10 μM). No obvious decrease in the fluorescent intensity or color change was observed after the addition, indicating that the detection of Cu2+ ion by L1 was an irreversible process (Figure S8). HRMS experiments were carried out to analyze the reaction products of Cu2+ and L1. The peak at m/z = 415.2025 was ascribed to rhodamine 6G (M + 1; m/z calculated for M+, C26H26N2O3, 414.1943), indicating rhodamine 6G as a final product (Figure S9). Moreover, the addition of Cu2+ into the solution of L1 in pure CH3CN resulted in no fluorescent emission or color change. Based on these experimental results, a mechanism involving Cu2+-promoted redox hydrolysis of L1 was proposed (Scheme 2), which was similar to that of the sensing towards Cu2+ by rhodamine B hydrazide [44].Scheme 2
The proposed reaction mechanism ofL1 with Cu2+.For further studying the practical application ofL1 in the detection of Cu2+, fluorescence imaging experiments were carried out in MCF7 cells. The 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 CuCl2 at the same conditions for another 30 min, strong fluorescence in the MCF7 cells was observed (Figure 6(b)). These results suggest that L1 could pass through the cell membrane and be used for the detection of Cu2+ in living cells. The 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.Figure 6
(a) Fluorescence image of MCF7 cells incubated withL1 (10 μM) for 30 min at 37°C. (b) Fluorescence image of MCF7 cells incubated with L1 (10 μM) for 30 min and then incubated with Cu2+ (20 μM) for another 30 min at 37°C. (c) Brightfield image of cells shown in (b). (d) The overlay image of b and c.
(a)
(b)
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## 4. Conclusion

In summary, a new colorimetric and fluorescent probeL1 for Cu2+ 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 Cu2+ detection in acetonitrile aqueous media with a low detection limit. Moreover, fluorescence bioimaging experiments confirmed that L1 could be used to detect intracellular Cu2+ in living cells.



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*Source: 8130767-2019-05-19.xml*