# A Coumarin-Based Fluorescence Probe for Selective Recognition of Cu2+ Ions and Live Cell Imaging

Authors: Guangjie He; Nana Ma; Linlin Li; Chenyan Xie; Linlin Yang; Jinhe Xu; Junli Wang
Journal: Journal of Sensors
 (2019)

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## Abstract

A new fluorescence probe L was rationally designed and synthesized for the recognition of Cu2+ ions by the combination of coumarin hydrazide and 2-acetylpyrazine. The photochemical properties and selectivity of L for Cu2+ ions in a CH3CN/HEPES (3 : 2, v/v) buffer were investigated by UV-vis absorption and fluorescence emission spectra. A highly selective and sensitive response of L for Cu2+ ions over other competing metal ions was observed with limit of detection in 3 μM. The coordination stoichiometry of L to Cu2+ ions was determined to be 1 : 1 by the UV-vis absorption spectrum, the fluorescence titrations, and density functional theory (DFT) calculations. Moreover, L was applied successfully for recognition of intracellular Cu2+ ions in living cells.


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## Body

## 1. Introduction

The selective and sensitive detection for trace transition metal ions has received great attention in biological samples [1–3]. As a required element in humans, copper plays vital roles during various biochemical processes, such as cellular respiration, neurotransmission, and biosynthesis [4–7]. A copper balance is also maintained in the human body through daily diet and metabolism under normal conditions. However, excessive concentration of copper ions in the human body will cause a wide variety of diseases such as Menkes syndrome, prion disease, and Alzheimer’s disease [8–11]. Therefore, it is significant to develop a highly sensitive method for selective detection of trace Cu2+ ions.Fluorescence detection among various analytical methods is attracting more attention due to its high selectivity, short response time, and great potential for live cell imaging [12–15]. By the virtue of these distinct advantages, fluorescence bioimaging technology has been applied successfully in living cells [16–18]. Typical fluorescent sensors have been documented involving inorganic composites, such as quantum dots @MOFs, or organic fluorophores such as photochromic diarylethene derivatives, coumarin derivatives, rhodamine B-based fluorescent probes, and naphthalimide-rhodamine B derivative [19–27]. Although a variety of fluorescent probes for the recognition of Cu2+ ions have been reported, it is still a challenge to directly contact metal ions with fluorophore for signal transduction.Due to their strong fluorescence emission, efficient cell permeation, large Stokes shift, and a structure that can be easily modified, coumarin is an attractive starting material [28–33]. Besides, the carbonyl group of coumarin can take part in the coordination with metal ions. The introduction of a hydrazide chain at 3-position of coumarin could both increase the intramolecular charge transfer and provide coordination sites for metal ions via carbonyl oxygen and amide nitrogen [34–36]. In order to obtain a new efficient probe, we designed a coumarin derivative for selective recognition of Cu2+ ions (Scheme 1). Electron donors of the N,N-diethyl group were introduced at the 7-position to modulate intramolecular charge transfer. L exhibited remarkable fluorescence quenching response to Cu2+ ions due to the paramagnetic nature of Cu2+ ions and/or photo-induced electron transfer effect. Density functional theory (DFT) calculations confirm the four-coordination configuration of Cu2+ ions in complex L-Cu2+. Moreover, this probe was successfully applied for the recognition of Cu2+ ions in living cells.Scheme 1
Synthetic route of L and the presumable structure of complexL-Cu2+.


## 2. Results and Discussion

### 2.1. Spectral Responses of L to Cu2+

The specific bonding of L with Cu2+ was first examined by UV-vis absorption spectroscopy in CH3CN/HEPES (3 : 2, v/v) buffer (pH=7.4). As shown in Figure 1, the free L exhibited a maximum absorption at 442 nm. With the increase of Cu2+ concentration in 0~7.5 equivalents, the maximum absorption intensity gradually decreased and the maximum absorption wavelength exhibited a red shift phenomenon. The former could be ascribed to the π–π∗ transition of the coumarin chromophore, and the latter was attributed to a metal-to-ligand charge transfer (MLCT) caused by binding L and Cu2+ ion. Based on the isosbestic point at 458 nm, a 1 : 1 stoichiometry for the L and Cu2+ ion is confirmed by the data of Job’s plot (Figure S1a) [37–40].Figure 1
UV-vis absorption spectra of L (10μM) with addition of various concentrations of Cu2+ ions (0~7.5 equivalents) in CH3CN/HEPES (3 : 2, v/v) buffer. Inset: absorbance at 442 nm versus the concentration of Cu2+.Besides, the absorption spectra of L were measured in the presence of other potentially relevant metal cations to investigate the selectivity of L for Cu2+ ions in CH3CN/HEPES (3 : 2, v/v) buffer solutions. Upon addition of 10 equivalents of relevant metal ions such as Pb2+, K+, Hg2+, Co2+, Mn2+, Al3+, Ca2+, Cd2+, Fe3+, Ag+, Mg2+, Cr3+, Na+, Fe2+, and Zn2+, no obvious changes were observed on absorption intensity (Fig. S2). Besides, the addition of Ni2+ ions caused a relatively slight absorbance change. In contrast, when 10 equiv. of Cu2+ was added to the solution of L, a significant decrease in absorption intensity and a bathochromic shift in absorption wavelength appeared obviously, indicating the excellent selectivity of L for Cu2+ over other metal ions. The results could be contributed to the strong coordination ability of Cu2+ and its larger association constant.To calculate the fluorescent response ability of L, the fluorescence emission spectra of L (10μM) were also investigated in the CH3CN/HEPES (3 : 2, v/v) buffer solution. L showed a maximum fluorescence emission at 487 nm upon excitation at 438 nm due to the presence of coumarin chromophore (Figure 2). With the increasing concentration of Cu2+ ions, the intensity of fluorescence emission at 487 nm decreased gradually. After the addition of 65 μM Cu2+ ions, the fluorescence emission was almost quenched. About 30-fold quenching of fluorescence emission intensity at 487 nm was observed compared with that of free L. The fluorescence quenching effect of L might be attributed to the paramagnetism of Cu2+ ions and/or photo-induced electron transfer process [35, 41].Figure 2
Fluorescence emission spectra of L (10μM) upon addition of various concentrations of Cu2+ ions (0~65 μM) in CH3CN/HEPES (3 : 2, v/v) buffer solution. The excitation wavelength was 438 nm. Inset: fluorescence intensities at 487 nm of the L as a function of Cu2+ ion concentrations (0~65 μM).Moreover, the fluorescence emission intensity at 487 nm showed a good linear relationship (R2=0.99) against the concentration of Cu2+ ions in 1~65 μM (Figure 2 inset). The quenching constant value of L with Cu2+ ions was determined from the titration plots. The corrected Stern-Volmer fitting indicates the value of 4.5×104mol−1·L. And Job’s plot and the molar ratio of the fluorescence titrations also revealed a 1 : 1 binding stoichiometry (Fig. S1b). The detection limit of L for Cu2+ ions was estimated about 3 μM based on LOD LimitOfDetection=3σ/s, where σ and s represent the standard deviation of blank measurements and the slope between concentration of Cu2+ ions and fluorescence intensity, respectively [42]. Thus, the probe L shows excellent sensitivity and low detection limit for the detection of Cu2+.The fluorescence changes of L in response to various relevant species in CH3CN/HEPES (3 : 2, v/v) solutions were also studied. As shown in Figure 3, only Cu2+ ions induced a significant decrease in the fluorescence intensity, whereas very weak fluorescence variations were observed for the other metal ions such as Pb2+, K+, Hg2+, Co2+, Mn2+, Al3+, Ca2+, Cd2+, Fe3+, Ag+, Mg2+, Cr3+, Na+, Fe2+, and Zn2+ (10 equivalents). Furthermore, Ni2+ ions caused a relatively weak fluorescence quenching, which may be attributed to the low associated constant between L and Ni2+ ions. Such results indicated that L could be used for selective recognition of Cu2+ ions in the presence of other relevant species. Thus, the competing experiments were carried out to observe the selectivity for Cu2+ over other transition metal ions. The addition of Cu2+ ions still led to a fluorescence quenching of L even in the presence of competing species, suggesting that L had a good selectivity towards Cu2+ ions.Figure 3
Fluorescence intensity changes (F/F0) of L upon the addition of various metal ions in CH3CN/HEPES (3 : 2, v/v) solutions. Cyan bars represented the fluorescence response of L to various metal ions. Red bars represented the subsequent addition of Cu2+ to the above solutions. Excitation and emission were recorded at 438 nm and 487 nm, respectively.


### 2.2. Density Functional Theory (DFT) Calculations of Cu2+ Binding L

From a mechanistic viewpoint, the unique selectivity for the Cu2+ ion was attributed to various factors, including the suitable coordination conformation of the Schiff-based receptor, the nitrogen (oxygen)-binding affinity character with the Cu2+ ion, and the deprotonation ability of L. The fluorescence quenching of L by Cu2+ ion could be ascribed to the paramagnetism of Cu2+ ions and/or photo-induced electron transfer process. Density functional theory (DFT) calculations were used to determine the possible binding mode of Cu2+ to L. The geometry structures of L and metal complexes L-Cu2+ and L′-Cu2+ with all real frequency have been obtained using B3LYP/6–31G(d)/SDD level (the SDD basis set was used for transition metal Cu ion). The optimized geometries (and selected parameters) and electronic energies of metal complexes are shown in Figure 4. The result suggests that L-Cu2+ is more stable than L′-Cu2+ by 23.1 kcal/mol. Thus, the binding model between Cu2+ and L should be L-Cu2+. Cartesian coordinates were shown in Scheme S1. The results indicated that copper ions coordinated with the ketonic oxygen atom, pyrazine nitrogen atom, and imine nitrogen (C=N) in L molecule. This coordination configuration always appeared in the other similar complexes [29, 43].Figure 4
Optimized structures (and selected parameters) of possible complexL-Cu2+ and L′-Cu2+.


### 2.3. Effect of pH on the Performance of L for Cu2+

Because the fluorescence property of L for the detection of Cu2+ ions may be affected by pH value, the fluorescence responses of L (10 μM) to pH value were investigated by fluorescence spectroscopy with and without Cu2+, respectively. As shown in Figure 5, the fluorescence emission intensity of the free L and complex L-Cu2+ was almost independent of pH value in 4.0~10.0. Besides, the addition of Cu2+ causes a fluorescence quenching of L. Therefore, L could be applied for the recognition of Cu2+ over a wide pH range.Figure 5
Variation of fluorescence intensity of L and its detection of Cu2+ ions with pH value at room temperature.


### 2.4. Live Cell Imaging

Live cell imaging with a fluorescence probe had important significance and application [44–46]. The recognizing ability of L toward Cu2+ ions was examined in living cells by using an inverted fluorescence microscope. The clear green fluorescence was observed from the intracellular region in human hepatocellular carcinoma HepG2 after incubation with L (10 μM) for 2.5 hours in 0.01 M PBS buffer solution (Figure 6(a)). However, when the HepG2 cells were further treated with 6 equivalents of Cu2+ for 30 minutes, the green fluorescence intensity of L was nearly quenched (Figure 6(b)). During the whole experiment process (about 3 hours), the cells were visualized without obvious side effects. These results manifested that fluorescent L could be used for the recognition of copper ions within biological samples.Figure 6
Inverted fluorescence imaging of HepG2 cells incubated with L (10μM) for 2.5 hours (a) and then further incubated with 6 equivalents of Cu2+ ions for 30 minutes (b). The corresponding bright field images of HepG2 cells incubated with L (10 μM) (c) and then further incubated with 6 equivalents of Cu2+ ions (d).
(a)
(b)
(c)
(d)


## 2.1. Spectral Responses of L to Cu2+

The specific bonding of L with Cu2+ was first examined by UV-vis absorption spectroscopy in CH3CN/HEPES (3 : 2, v/v) buffer (pH=7.4). As shown in Figure 1, the free L exhibited a maximum absorption at 442 nm. With the increase of Cu2+ concentration in 0~7.5 equivalents, the maximum absorption intensity gradually decreased and the maximum absorption wavelength exhibited a red shift phenomenon. The former could be ascribed to the π–π∗ transition of the coumarin chromophore, and the latter was attributed to a metal-to-ligand charge transfer (MLCT) caused by binding L and Cu2+ ion. Based on the isosbestic point at 458 nm, a 1 : 1 stoichiometry for the L and Cu2+ ion is confirmed by the data of Job’s plot (Figure S1a) [37–40].Figure 1
UV-vis absorption spectra of L (10μM) with addition of various concentrations of Cu2+ ions (0~7.5 equivalents) in CH3CN/HEPES (3 : 2, v/v) buffer. Inset: absorbance at 442 nm versus the concentration of Cu2+.Besides, the absorption spectra of L were measured in the presence of other potentially relevant metal cations to investigate the selectivity of L for Cu2+ ions in CH3CN/HEPES (3 : 2, v/v) buffer solutions. Upon addition of 10 equivalents of relevant metal ions such as Pb2+, K+, Hg2+, Co2+, Mn2+, Al3+, Ca2+, Cd2+, Fe3+, Ag+, Mg2+, Cr3+, Na+, Fe2+, and Zn2+, no obvious changes were observed on absorption intensity (Fig. S2). Besides, the addition of Ni2+ ions caused a relatively slight absorbance change. In contrast, when 10 equiv. of Cu2+ was added to the solution of L, a significant decrease in absorption intensity and a bathochromic shift in absorption wavelength appeared obviously, indicating the excellent selectivity of L for Cu2+ over other metal ions. The results could be contributed to the strong coordination ability of Cu2+ and its larger association constant.To calculate the fluorescent response ability of L, the fluorescence emission spectra of L (10μM) were also investigated in the CH3CN/HEPES (3 : 2, v/v) buffer solution. L showed a maximum fluorescence emission at 487 nm upon excitation at 438 nm due to the presence of coumarin chromophore (Figure 2). With the increasing concentration of Cu2+ ions, the intensity of fluorescence emission at 487 nm decreased gradually. After the addition of 65 μM Cu2+ ions, the fluorescence emission was almost quenched. About 30-fold quenching of fluorescence emission intensity at 487 nm was observed compared with that of free L. The fluorescence quenching effect of L might be attributed to the paramagnetism of Cu2+ ions and/or photo-induced electron transfer process [35, 41].Figure 2
Fluorescence emission spectra of L (10μM) upon addition of various concentrations of Cu2+ ions (0~65 μM) in CH3CN/HEPES (3 : 2, v/v) buffer solution. The excitation wavelength was 438 nm. Inset: fluorescence intensities at 487 nm of the L as a function of Cu2+ ion concentrations (0~65 μM).Moreover, the fluorescence emission intensity at 487 nm showed a good linear relationship (R2=0.99) against the concentration of Cu2+ ions in 1~65 μM (Figure 2 inset). The quenching constant value of L with Cu2+ ions was determined from the titration plots. The corrected Stern-Volmer fitting indicates the value of 4.5×104mol−1·L. And Job’s plot and the molar ratio of the fluorescence titrations also revealed a 1 : 1 binding stoichiometry (Fig. S1b). The detection limit of L for Cu2+ ions was estimated about 3 μM based on LOD LimitOfDetection=3σ/s, where σ and s represent the standard deviation of blank measurements and the slope between concentration of Cu2+ ions and fluorescence intensity, respectively [42]. Thus, the probe L shows excellent sensitivity and low detection limit for the detection of Cu2+.The fluorescence changes of L in response to various relevant species in CH3CN/HEPES (3 : 2, v/v) solutions were also studied. As shown in Figure 3, only Cu2+ ions induced a significant decrease in the fluorescence intensity, whereas very weak fluorescence variations were observed for the other metal ions such as Pb2+, K+, Hg2+, Co2+, Mn2+, Al3+, Ca2+, Cd2+, Fe3+, Ag+, Mg2+, Cr3+, Na+, Fe2+, and Zn2+ (10 equivalents). Furthermore, Ni2+ ions caused a relatively weak fluorescence quenching, which may be attributed to the low associated constant between L and Ni2+ ions. Such results indicated that L could be used for selective recognition of Cu2+ ions in the presence of other relevant species. Thus, the competing experiments were carried out to observe the selectivity for Cu2+ over other transition metal ions. The addition of Cu2+ ions still led to a fluorescence quenching of L even in the presence of competing species, suggesting that L had a good selectivity towards Cu2+ ions.Figure 3
Fluorescence intensity changes (F/F0) of L upon the addition of various metal ions in CH3CN/HEPES (3 : 2, v/v) solutions. Cyan bars represented the fluorescence response of L to various metal ions. Red bars represented the subsequent addition of Cu2+ to the above solutions. Excitation and emission were recorded at 438 nm and 487 nm, respectively.


## 2.2. Density Functional Theory (DFT) Calculations of Cu2+ Binding L

From a mechanistic viewpoint, the unique selectivity for the Cu2+ ion was attributed to various factors, including the suitable coordination conformation of the Schiff-based receptor, the nitrogen (oxygen)-binding affinity character with the Cu2+ ion, and the deprotonation ability of L. The fluorescence quenching of L by Cu2+ ion could be ascribed to the paramagnetism of Cu2+ ions and/or photo-induced electron transfer process. Density functional theory (DFT) calculations were used to determine the possible binding mode of Cu2+ to L. The geometry structures of L and metal complexes L-Cu2+ and L′-Cu2+ with all real frequency have been obtained using B3LYP/6–31G(d)/SDD level (the SDD basis set was used for transition metal Cu ion). The optimized geometries (and selected parameters) and electronic energies of metal complexes are shown in Figure 4. The result suggests that L-Cu2+ is more stable than L′-Cu2+ by 23.1 kcal/mol. Thus, the binding model between Cu2+ and L should be L-Cu2+. Cartesian coordinates were shown in Scheme S1. The results indicated that copper ions coordinated with the ketonic oxygen atom, pyrazine nitrogen atom, and imine nitrogen (C=N) in L molecule. This coordination configuration always appeared in the other similar complexes [29, 43].Figure 4
Optimized structures (and selected parameters) of possible complexL-Cu2+ and L′-Cu2+.


## 2.3. Effect of pH on the Performance of L for Cu2+

Because the fluorescence property of L for the detection of Cu2+ ions may be affected by pH value, the fluorescence responses of L (10 μM) to pH value were investigated by fluorescence spectroscopy with and without Cu2+, respectively. As shown in Figure 5, the fluorescence emission intensity of the free L and complex L-Cu2+ was almost independent of pH value in 4.0~10.0. Besides, the addition of Cu2+ causes a fluorescence quenching of L. Therefore, L could be applied for the recognition of Cu2+ over a wide pH range.Figure 5
Variation of fluorescence intensity of L and its detection of Cu2+ ions with pH value at room temperature.


## 2.4. Live Cell Imaging

Live cell imaging with a fluorescence probe had important significance and application [44–46]. The recognizing ability of L toward Cu2+ ions was examined in living cells by using an inverted fluorescence microscope. The clear green fluorescence was observed from the intracellular region in human hepatocellular carcinoma HepG2 after incubation with L (10 μM) for 2.5 hours in 0.01 M PBS buffer solution (Figure 6(a)). However, when the HepG2 cells were further treated with 6 equivalents of Cu2+ for 30 minutes, the green fluorescence intensity of L was nearly quenched (Figure 6(b)). During the whole experiment process (about 3 hours), the cells were visualized without obvious side effects. These results manifested that fluorescent L could be used for the recognition of copper ions within biological samples.Figure 6
Inverted fluorescence imaging of HepG2 cells incubated with L (10μM) for 2.5 hours (a) and then further incubated with 6 equivalents of Cu2+ ions for 30 minutes (b). The corresponding bright field images of HepG2 cells incubated with L (10 μM) (c) and then further incubated with 6 equivalents of Cu2+ ions (d).
(a)
(b)
(c)
(d)


## 3. Conclusion

In conclusion, a new fluorescence probe L based on coumarin hydrazide and 2-acetylpyrazine moiety was synthesized to detect trace Cu2+ ions. A 1 : 1 stoichiometry of L and Cu2+ ions was established based on Job’s plot analysis. Density functional theory (DFT) calculations confirm the four-coordination configuration of Cu2+ ions. The probe exhibits excellent stability over a wide range of pH, high selectivity, and sensitivity (detection limit of about 3 μM) for Cu2+ over other relevant transition metal ions. Further, cell imaging test demonstrated that probe L was also used for the recognition of Cu2+ ions in living cells.



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