Journal of Sensors
Volume 2019 (2019), Article ID 2814947, 7 pages
https://doi.org/10.1155/2019/2814947

Research Article

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

Guangjie He,¹ Nana Ma,² Linlin Li,¹ Chenyan Xie,¹ Linlin Yang,¹ Jinhe Xu,¹ and Junli Wang³

¹Xinxiang Key Laboratory of Forensic Science Evidence, School of Forensic Medicine, Xinxiang Medical University, Jinsui Road No. 601, Xinxiang, 453003 Henan Province, China
²School of Chemistry and Chemical Engineering, Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, Henan Key Laboratory of Boron Chemistry and Advanced Energy Materials, Henan Normal University, East of Construction Road No. 46, Xinxiang, 453007 Henan Province, China
³College of Chemistry and Chemical Engineering, Xinxiang University, Jinsui Road No. 191, Xinxiang, 453003 Henan Province, China

Correspondence should be addressed to Guangjie He; guangjiehe@163.com, Linlin Yang; yanglin1989728@163.com, and Junli Wang; wangxiaojun2010@163.com

Received 13 May 2019; Accepted 14 September 2019; Published 30 October 2019

Academic Editor: Hai-Feng Ji

Copyright © 2019 Guangjie He et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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, ) 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.

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 complex L-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, ) buffer (). 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 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 (10 μM) with addition of various concentrations of Cu²⁺ ions (0~7.5 equivalents) in CH₃CN/HEPES (3 : 2, ) 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, ) 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 (10 μM) were also investigated in the CH₃CN/HEPES (3 : 2, ) 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 (10 μM) upon addition of various concentrations of Cu²⁺ ions (0~65 μM) in CH₃CN/HEPES (3 : 2, ) 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 () 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 ·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 , where and 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, ) 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 () of L upon the addition of various metal ions in CH₃CN/HEPES (3 : 2, ) 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 complex L-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.

(a)

(b)

(c)

(d)

Figure 6: Inverted fluorescence imaging of HepG2 cells incubated with L (10 μM) 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).

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.

Data Availability

The data used to support the findings of this study are included within the article and supplementary information file(s).

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 21801215 and 21371148), the Natural Science Foundation of Henan Province (No. 182300410309), the Key Scientific and Technological Project of Henan Province (No. 182102310648), the Key Research Project of Higher Education Institutions of Henan Province (No. 18A150044), and the National College Students’ Innovation and Entrepreneurship Training Program (No. 201610472020).

Supplementary Materials

The synthesis process of probe L and specific test conditions for spectra and cell imaging reported in this article have been deposited in supplementary material. Figure S1: Job’s plot fitting of probe L with Cu²⁺. Figure S2: UV-vis absorption spectra of probe L upon addition of various metal ions. Scheme S1: the Cartesian coordinate data of complex L-Cu²⁺ and L-Cu²⁺. Figure S3, S4, and S5: the NMR and MS spectra of probe L. (Supplementary Materials)

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