fluorimetric probe for selective detection of Cu2+ and application in living cell imaging

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117631

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Indole-based colori/fluorimetric probe for selective detection of Cu2þ and application in living cell imaging Yongxin Chang a, Bai Li a, Huihui Mei a, Li Yang a, Kuoxi Xu a, *, Xiaobin Pang b, ** a Institute of Functional Organic Molecular Engineering, Engineering Laboratory for Flame Retardant and Functional Materials of Henan Province, College of Chemistry and Chemical Engineering, Henan University, Kaifeng, 475004, China b Institute of Pharmacy, Henan University, Kaifeng, Henan, 475004, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 February 2019 Received in revised form 15 July 2019 Accepted 6 October 2019 Available online 9 October 2019

A highly sensitive and selective indole-based probe IHT exhibited obvious color change from colorless to violet easily detected by naked eye as well as ‘turn on’ fluorescence response to Cu2þ ion at physiological pH condition. The detection limit was determined to be as low as 8.93  108 M, which was much lower than drinking water permission concentrations by the United States Environmental Protection Agency. The 1:2 binding mechanism was well confirmed by fluorescence titration, Job’s plot, HRMS, IR analysis and DFT calculations. Furthermore, the probe IHT was successfully used for fluorescence imaging of Cu2þ ion in living cells. © 2019 Elsevier B.V. All rights reserved.

Keywords: Colori/fluorimetric probe Turn on response Cu2þ DFT calculations Cell imaging

1. Introduction Copper ion, as the third most abundant transition-metal in human body, plays a crucial role in biological progress, such as essential micronutrient for many of biological systems as cocomponent of the metalloenzyme [1]. However, the excess of copper ion in organisms, can cause a series of neurodegenerative diseases, such as Menkes, Wilson [2,3] Alzheimer’s and Parkinson’s [4], and generate disturbed cellular metabolism reactive oxygen species [5,6]. The U.S. Environmental Protection Agency (U.S. EPA) recommends the permissible level of 1.3 ppm (about 2.00  105 M) for copper ion in drinking water [7]. So, it is highly important to develop the efficient method for detecting and monitoring copper ion in the environment and in physiology systems [8,9]. Among the various analytical methods for the detection of metal ions [10e13], the selectively fluorescence probe technology possesses the advantages of high sensitivity, real-time detection, low cost, and simplicity for implementation [14e16]. Especially, those “turn-on” copper ion probes have attracted much more attention, due to their nearly no interference from the background and intrinsic paramagnetic property [17e20]. Thus, many

* Corresponding author. ** Corresponding author. E-mail address: [email protected] (K. Xu). https://doi.org/10.1016/j.saa.2019.117631 1386-1425/© 2019 Elsevier B.V. All rights reserved.

researchers have focused on the development of high selectively probes for easy detection of copper ion fluorimetrically [21], and simultaneously accompanied with the ‘naked-eyed’ detectable color changes [22,23]. In this study, a Schiff base fluorescence probe 1,5-bis(indole-3-carbaldehyde)thiocarbohydrazone (IHT) based on indole was reported (Scheme 1). The probe IHT displayed an efficient fluorescent highly selective and sensitive detection of copper ion and accompanied by an obvious color change from colorless to violet. The interaction ratio, the binding constant, the detection limit and the effects pH of probe IHT were well studied by HRMS, IR, UVevis and fluorescence titration experiments.

2. Experimental 2.1. Materials and instrumental methods Unless otherwise mentioned, all the materials were obtained from commercial suppliers and were used without further purification. Melting points were determined on a MEL-TEMPII melting point apparatus. 1H and 13C NMR spectra were recorded on Bruker AV 400 MHz instruments using DMSO‑d6 as solvent and TMS as an internal standard. Chemical shifts are expressed in d units and coupling constants in Hz. UV-vis spectra were recorded on a PerkinElmer Lamda-25 UV-vis spectrometer. Fluorescence spectra were recorded on a F-7000 FL fluorescence

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Scheme 1. The synthetic route of probe IHT.

spectrometer. Mass spectra (MS) were measured by a Liquid Chromatography-Ion Trap Mass spectrometer (Bruker amaZon SL). Cell experiments were applied on an inverted fluorescence microscope (Leica DMI4000B, Germany). Density functional theory (DFT) calculations with B3LYP/6-31g were carried out by using the Gaussian 09 package [24]. 2.2. Synthesis of probe IHT The probe IHT was prepared on the basis of the route according to previous report [25](Scheme 1). A solution of indole-3carboxaldehyde (0.68g, 4.6 mmol) in methanol (20 mL) was added drop-wise to a methanol (10 mL) solution of thiocarbohydrazide (0.21g, 2.0 mmol). After 6 h reflux under N2 atmosphere, off pale yellow precipitate come out which was wash with cold ethanol to give yield of 89% (0.76 g). Mp. 241.3e242.2  C, IR (KBr): 3434, 1612, 1539, 746 cm1, 1H NMR, d 11.60 (s, 2H), d 11.41 (s, 1H), d10.96 (s, 1H), 8.44 (s, 4H), 7.86 (s, 2H), d7.46 (d, J¼8.0 Hz, 2H), d7.24 (d, J¼8.0 Hz, 2H), d7.20 (d, J¼8.0 Hz, 2H). 13C NMR, d 173.57, 145.09, 141.70, 137.50, 131.13, 130.51, 124.79, 123.26, 121.17, 112.34. ESI-MS, m/z for C19H16N6S([MþH]þ): calcd: 361.1230, found: 361.1231 (Fig. S1- Fig. S4). 3. Results and discussion 3.1. UV-vis and colorimetric detection of Cu2þion UVevis titrations were conducted to investigate the interaction of the probe IHT with common important metal ions (Li, Naþ, Kþ, Agþ, Mg2þ, Ca2þ, Mn2þ, Ni2þ, Zn2þ, Cd2þ, Pb2þ, Hg2þ, Co2þ, Cu2þ, Fe2þ, Fe3þ, Cr3þ) to a mixed aqueous medium (CH3CN/H2O¼1:2, v/

Fig. 1. UV-Vis absorption spectra of probe IHT upon the addition of Cu2þ (0e2.45 equiv.) in a mixed aqueous medium (CH3CN/H2O¼1:2, v/v; Tris-HCl buffer 0.05 M; pH ¼ 7.3). Insert: visible color change under ambient light (left to right: free IHT, IHT þ Cu2þ).

v; Tris-HCl buffer 0.05 M; pH ¼ 7.3) of IHT (3.33  105 M) at 298 K. The UVevis studies of the probe was found to be disturbed only in the presence of Cu2þ in contrast to other common metal ions. Fig. 1 showed the UVevis spectral changes of IHT during the titration with Cu2þ ion. Upon incremental addition of Cu2þ (0e2.45 equiv.) to the solution of IHT, the strong absorbance peak at the lmax of 348 disappeared gradually, while a new band at 392 nm formed and developed. This phenomenon should be owing to Cu2þ coordination-enhanced LMCT effect [26]. At the same time, the colorless of the solution became violet (Fig. 1 inset). Meanwhile, the clear isosbestic point was observed at 370 nm, indicating the formation of stable complex. 3.2. Fluorescence detection of Cu2þ ion The fluorescence spectrum of IHT (3.33  105 M) in different solvent systems upon excitation at 320 nm showed in Fig S5. IHT in acetonitrile system showed the high emission intensity. As the result, the sensing behaviors of IHT in this work were focused on the observation in this solvent systems. The binding behavior of the probe IHT (3.33  105 M) was investigated by fluorescence titrations in a mixed aqueous medium, which was consistent well with the UVevis titrations. Fig. 2 displayed the changes of fluorescence emission of probe IHT in the presence of various metal ions. Free probe IHT displayed a weak fluorescence emission at 453 nm. Upon addition of Cu2þ ion, fluorescence intensity increased significantly. However, other tested metal ions induced no significant changes in fluorescence spectra. This fluorescence ‘turn-on’ response and slight red shift of probe IHT caused by Cu2þ should be attributed to the CHEF (chelation enhanced fluorescence) mechanism and ICT (internal charge transfer) processes. The formation of IHT-Cu2þ complex resulted in the deprotonation of the amide conjugated to fluorophores leading to the greatly enhanced electrondonating ability of the N atom. This change can affect the electronic

Fig. 2. Fluorescence emission spectra (lex ¼ 320 nm) of probe IHT (3.33  105 M) in the presence of different metal ions (10 equiv.) in a mixed aqueous medium (CH3CN/ H2O¼1:2, v/v; Tris-HCl buffer 0.05 M; pH ¼ 7.3).

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Scheme 2. Fluorescence enhancement mechanism and proposed structure of IHTeCu2þ complex.

absorption properties of a fluorophore with an ICT excited state while its fluorescence emission properties are less affected by ICT in excited states [27]. As showed in Scheme 2, the low fluorescence intensity of probe IHT may be due to its flexibility. The free rotation of the probe IHT got restricted in presence of Cu2þ ion, and the formed complex became a more rigid than free probe, resulting in a CHEF effect [28]. With increasing the amount of Cu2þ (0e2.10 equiv.), the fluorescence intensity reached maximum with a red-shift of 7 nm from 441 to 448 nm, and then almost remained constant with increasing Cu2þ ion (Fig. 3a). The Benesi-Hildebrand plot of [1/(IeI0)] versus

[Cu2þ]2 exhibited a good linearity (R2 > 0.99) between fluorescence intensity and concentration suggested that probe IHT was useful for quantitative analysis of Cu2þ, which strongly supported the 1:2 binding model (Fig. 3b). Furthermore, the binding stoichiometry of probe IHT with Cu2þ was achieved by the fluorescence Job’s plot. The plot was recorded at maximum by continuously varied mole fraction of Cu2þ in a solution of probe IHT and total concentration of solution (1.00  104 M) retain constant. The results of 1:2 stoichiometry was obtained, which was about 0.67 fraction in the highest emission intensity (Fig. 4a). Moreover, the stoichiometry of IHT-Cu2þ was further confirmed by ESI-mass spectrometry analysis.

Fig. 3. Fluorescence titration spectra of compound I (lex ¼320 nm) in a mixed aqueous medium (CH3CN/H2O¼1:2, v/v; Tris-HCl buffer 0.05 M; pH ¼ 7.3) solution upon the addition of Cu2þ (0e2.10 equiv.); (b) Benesi-Hildebrand plot of sensor I using 2:1 stoichiometry for association between Cu2þ ion and I at 320 nm.

Fig. 4. (a) Job’s plot showing 1:2 (probe: Cu2þ) stoichiometry for Cu2þ complex formation with probe in a mixed aqueous medium (CH3CN/H2O¼1:2, v/v; Tris-HCl buffer 0.05 M; pH ¼ 7.3); (b) Calibration curves of fluorescence intensity (I) at 448 nm of probe to Cu2þ ion concentrations changes.

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Kþ which generally coexist with Cu2þ in the biological systems do not interfere in the detection of Cu2þ under the physiological conditions. Especially, the probe IHT detection behavior of Cu2þ not interfered by Fe3þ and Fe2þ in the normal case. So, the compound IHT could be considered as an excellent turn-on probe candidate for Cu2þ. 3.3. Mechanism of complexation between IHT and Cu2þ

Fig. 5. The pH’s impact of recognition ability of probe IHT.

Upon addition of 2.0 equiv. of Cu2þ, the positive-ion mass spectrum showed the formation of the [IHT-2Hþþ2Cu2þþ2NO 3 ] (m/z: 303.9633; calcd, 303.9669) (Fig.S6). From the Benesi-Hildebrand analysis, the association constant of IHT-Cu2þ was calculated as 2.63£1010 M¡2 [29]. Furthermore, the detection limit for Cu2þ was found to be 8.93  108 M (Fig. 4b) based on the equation (LOD ¼ 3d/slope) [30], which was much lower than the limit of copper of the US EPA in drinking water (~2.00  105 M) [7,31]. Meanwhile, the probe IHT exhibited low quantum yield (F¼ 0.0118) and it improved with Cu2þ ion (F ¼ 0.2905). Based on these findings, the probe IHT could be used as a sensitive probe for Cu2þ in aqueous media. Moreover, the fluorescence intensity of probe IHT in the absence and presence of Cu2þ at various pH effect on the emission response was measured in mixed aqueous medium for exploitation of practical applications of the probe. The results showed that Cu2þ can be employed in a wide pH range (Fig. 5). To further evaluate the selectivity of probe IHT for Cu2þ, the competition experiments were conducted by most of the mixtures of Cu2þ with other metal ions under the same experimental conditions. The changes of fluorescence intensity caused by most of the mixtures of Cu2þ with other metal ions was similar to that caused by Cu2þ alone (Fig. 6). Most importantly, higher concentrations (10 equiv.) of Ca2þ, Mg2þ, Naþ and

To deeply understand the complexations mode of IHT with Cu2þ, we tried to perform 1H NMR titration experiments. However, the limited solubility of IHT at the required concentrations precluded the more detailed structural study. Thus, the Fourier transform infrared (FTIR) spectrum and DFT calculations were have been carried in the absence and presence of Cu2þ ion. FTIR spectra of the complex was compared to the free probe spectra in Fig.S4. The amide bands of IHT that appeared at 1611 and 1502 cm¡1 shifted lower frequencies (1617 and 1542 cm¡1), indicating that the amide nitrogen atoms of IHT were coordinated to Cu2þ [32]. Then, the DFT calculations were performed to further confirm the proposed mechanism of probe IHT with Cu2þ ion. In the proposed structure, the coordination of Cu2þ ion was coordinated to two nitrogen atoms and one sulfur atom of thiocarbohydrazide unit, and the distances were 2.06, 2.35, 2.88 and 2.06, 2.39, 2.94 Å, respectively (Fig. 7). The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of probe IHT and its Cu2þ complexes were also presented in Fig. 7. In the case of free IHT, the p-e of HOMO (5.18V) resided at the fluorophore units whereas the p-e of LUMO (1.23 eV) located at the skeleton of thiocarbohydrazide unit, suggesting the ICT effect occurred and thus IHT exhibited strong fluorescence character. For the complex, the p-e of HOMO (16.97 eV) mainly situated at Cu2þ ion coordination center and the conjugate skeleton whereas the p-e of LUMO (16.42 eV) located at the fluorophore units. Based on the CHEF mechanism, since the frontier molecular orbitals were mainly spread over the whole complex, the excited electrons were back to the ground state, and eventually caused the fluorescence enhancement. And the energy of the IHT-Cu2þ complex (0.55 eV) was much lower than that of free IHT (3.95 eV), suggested that the interaction of Cu2þ to IHT effectively decreases the energy gap of the complex and stabilized the system [33]. 3.4. Living cell imaging experiments Owing to the spectroscopic properties of probe IHT, it should be ideally suited to monitoring Cu2þ ion in living cells. To test this proposal, the intracellular Cu2þ ion was evaluated with fluorescence images. the living PC-12 cells were incubated in PBS buffer (pH 7.3) containing 10¡5 M of the probe for 30 min at 37  C, followed by washing the cells three times with the same buffer to remove the excess of the probe. At this stage, the cells imaging (Fig. 8a) under the bright field also indirectly indicated that the probe IHT has a low toxicity to the cells, and the cell imaging did not exhibit fluorescence emission (Fig. 8b). Then continued to incubate at 37  C for 30 min after adding of Cu2þ (20 mL, 2  105 M) ions followed by washing the cells three times with the same buffer. As shown in Fig. 8c and d, a significant blue fluorescence was observed in the intracellular. These results suggested that probe IHT was membrane permeable and could be used to detect Cu2þ ion in living cells. Therefore, the probe IHT could be supplied as a useful tool for studying the distribution and physiological activity of Cu2þ in live cells. 3.5. Comparison with recently reported probes

2þ

Fig. 6. Competitive experiments toward the fluorescence effect of Cu in presence of 10 equiv. of other metal ions in a mixed aqueous medium (CH3CN/H2O¼1:2, v/v; TrisHCl buffer 0.05 M; pH ¼ 7.3).

Some important parameters of previously reported Schiff base Cu2þ fluorescence probes showed in Table 1. Our probe IHT exhibits

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Fig. 7. Optimized structures and Energy level diagram of IHT and IHT þ Cu2þ.

Fig. 8. (a) fluorescence images of PC-12 cells incubation with 10 mM IHT for 30 min (b) Confocal fluorescence images of a (c) fluorescence images with 10 mM IHT and 20 mM Cu2þ ion for 30 min (d) Confocal fluorescence images of c.

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Table 1 Comparison of IHT with some previously reported Cu2þ probes. Entry

synthesis steps

Color change

LOD (M)

Response type

Ref.

1

Structure

2

Blue to colorless

1.8  108

Turn off

[2]

2

3

Green to colorless

2.4  106

Turn off

[3]

3

1

Yellow to colorless

4.2  107

Turn off

[4]

4

2

Yellow to colorless

1.50  106

Turn off

[5]

5

4

No

12.3 nM

Turn off

[7]

6

2

Yellow to wine red

48 mM

Turn off

[11]

7

1

Blue to colorless

13.2 nM

Turn off

[12]

8

3

No

1.9  106

Turn on

[17]

9

2

Yellow to pale orange

1.1  107

Turn on

[34]

10

2

Colorless to green

1.5  106

Turn on

[35]

11

3

Colorless to yellow

8.1  109

Turn on

[23]

12

1

Colorless to violet

8.9  108

Turn on

This work

2

some advantages over other probers while few parameters of other probes are better than this study. In the first place, some probes including IHT need only one step for their synthesis, while other probes need more steps for their synthesis. Minimum synthesis steps mean more economical and desirable. Moreover, among the reported probes, colorimetric fluorescence probes are excellent to detect metal ions due to the color change detection independent of expensive instruments, especially the color change from colorless

dark. Besides, the lower LOD value of the probe means better sensitivity towards metal ions. Although, our probe shows very good sensitivity in the 108 M order towards Cu2þ ion, which is quite low compared to other probes in the Table 1. This is a disadvantage of our probe. Finally, the “turn-on” type fluorescent probes were more superior to the “turn-off” type due to its better for the detection distribution in cellular conditions. Especially, the paramagnetic effect of Cu2þ ion will lead to the probe showing

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turn-off fluorescent response. Most reported probes are the “turnoff” type, few probes including our probes are “turn-on” type. 4. Conclusion In this paper, we reported a facile and low-cost probe that showed highly selectivity and sensitivity for Cu2þ ion via the CHEF and ICT mechanisms in MeCN-Tris buffered aqueous solution with different fluorescence color change. The probe offered visible color change to the naked eye for Cu2þ ion with a detection limit of 8.93  108 M. Moreover, the probe IHT was applied in fluorescence imaging of living cells and the confocal microscopy images indicated that cell-permeable IHT could visualize the changes of intracellular Cu2þ ion in living cells. Declaration of competing interest There are no conflicts to declare. Acknowledgements We thank the National Natural Science Foundation of China (No. U1404207) for financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019.117631. References € m, J. Leckner, The chemical biology of copper, Curr. Opin. Chem. [1] B.G. Malmstro Biol. 2 (1998) 286e292. [2] L. Tang, M. Cai, A highly selective and sensitive fluorescent sensor for Cu2þ and its complex for successive sensing of cyanide via Cu2þ displacement approach, Sens. Actuators, B 173 (2012) 862e867. [3] Y. Fu, C. Fan, G. Liu, S. Pu, A colorimetric and fluorescent sensor for Cu2þ and F based on a diarylethene with a 1,8-naphthalimide Schiff base unit, Sens. Actuators, B 239 (2017) 295e303. [4] Z. Guo, T. Hu, X. Wang, T. Sun, T. Li, Q. Niu, Highly sensitive and selective fluorescent sensor for visual detection of Cu2þ in water and food samples based on oligothiophene derivative, J. Photochem. Photobiol., A 371 (2019) 50e58. [5] Y. Wang, P.D. Mao, W.N. Wu, X.J. Mao, X.L. Zhao, Z.Q. Xu, Y.C. Fan, Z.H. Xu, A novel colorimetric and ratiometric fluorescent Cu2þ sensor based on hydrazone bearing 1,8-naphthalimide and pyrrole moieties, Sens. Actuators, B 251 (2017) 813e820. [6] L.M. Gaetke, C.K. Chow, Copper toxicity, oxidative stress, and antioxidant nutrients, Toxicology 189 (2003) 147e163. [7] S. Bhardwaj, N. Maurya, A.K. Singh, Chromone based fluorescent organic nanoparticles for high-precision in-situ sensing of Cu2þ and CN ions in 100% aqueous solutions, Sens. Actuators, B 260 (2018) 753e762. [8] H.S. Kumbhar, B.L. Gadilohar, G.S. Shankarling, A highly selective quinaldineeindole based spiropyran with intramolecular H-bonding for visual detection of Cu(II) ions, Sens. Actuators, B 222 (2016) 35e42. [9] L. Zong, Y. Song, Q. Li, Z. Li, A “turn-on” fluorescence probe towards copper ions based on core-substitued naphthalene diimide, Sens. Actuators, B 226 (2016) 239e244. [10] N. Mergu, V.K. Gupta, A novel colorimetric detection probe for copper(II) ions based on a Schiff base, Sens. Actuators, B 210 (2015) 408e417. [11] K.S. Mani, R. Rajamanikandan, B. Murugesapandian, R. Shankar, G. Sivaraman, M. Ilanchelian, S.P. Rajendran, Coumarin based hydrazone as an ICT-based fluorescence chemosensor for the detection of Cu2þ ions and the application in HeLa cells, Spectrochim. Acta 214 (2019) 170e176. [12] T. Sun, Y. Li, Q. Niu, T. Li, Y. Liu, Highly selective and sensitive determination of Cu2þ in drink and water samples based on a 1,8-diaminonaphthalene derived

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