Synthesis, characterization and fluorescent ... - Wiley Online Library

Research Article Received: 14 February 2009,

Revised: 21 April 2009,

Accepted: 20 June 2009,

Published online in Wiley Online Library: 27 August 2009

(wileyonlinelibrary.com) DOI 10.1002/bio.1165

Synthesis, characterization and fluorescent properties of cerium(III) glutathione complex Guo-Cheng Han and You-Nian Liu* ABSTRACT: The cerium (III) glutathione complex was synthesized by the redox reaction of cerium (IV) with glutathione reduced (GSH) in aqueous solution. The Job-plots indicate an ML (L = GSSG) stoichiometry of the complex. The fluorescent properties of the compound were investigated. The as-prepared complex showed the characteristic maximum emission spectra of Ce(III) at 350 nm (lex = 255 nm). The fluorescence results show that the Ce(IV) ions are first reduced to Ce(III), and then form Ce(III) complex after reacting with GSH. The complex was characterized by element analysis and FT-IR spectra; the stability of the complex was analyzed by cyclic voltammeters and DSC-TG as well. Finally, Ce(IV) was successfully employed to determine the concentrations of GSH in the presence of GSSG, in which the fluorescence intensities are proportional to the concentrations of GSH in the range of 1–100 nM with the detection limit of 0.05 nM of GSH, without interference from the presence of GSSG. Copyright © 2009 John Wiley & Sons, Ltd. Keywords: glutathione reduced (GSH); cerium glutathione complex; fluorescent properties

Introduction

Luminescence 2010; 25: 389–393

Experimental Reagents and apparatus Ce(SO4)2·4H2O (AR) was purchased from Jinke Fine Chemical Research Institute, China. GSH (purity, 99%) was purchased from Scientific Research Special, Japan. All the aqueous solutions were prepared using deionized water with a resistivity of 18.2 MΩ cm−1 collected from a Millipore Simplicity 185 System (Millipore Co., Billerica, MA, USA). All other chemical reagents were used as received. The Ce(III) ion was determined by EDTA titration using xylenolorange as an indicator. Carbon, nitrogen, sulfur and hydrogen were determined using an Elementar Vario EL. FT-IR spectra of all compounds were recorded on an Avatar360 FT-IR Fourier transform spectrometer in KBr pellets. The fluorescence spectra were conducted on an F-2500 luminescence spectrometer with a xenon lamp as the light source (wavelength from 220 to 700 nm, excitation and emission slit widths 2.5 nm). All spectra were recorded at a temperature of 23 ± 2°C. All solution electrochemical measurements were carried out on a model CHI660B voltammetric analyzer at room temperature (23 ± 2°C). Na2SO4 was employed as supporting electrolyte, gold electrode was the working electrode (diameter 3 mm). The gold electrode was pol-

* Correspondence to: You-Nian Liu, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, People’s Republic of China. E-mail: [email protected] College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, People’s Republic of China

Copyright © 2009 John Wiley & Sons, Ltd.

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Lanthanide complexes have been intensively investigated for their good luminescent properties, which enable them to be promising in applications from biological assays to display devices.[1,2] More and more scientists are attracted to designing the organized molecular architectures containing trivalent lanthanide ions working as efficient luminescent materials.[3,4] In particular, these luminescent lanthanide complexes are used as fluorescence probes for analytical applications due to their unique luminescence characteristics, such as narrow spectral width, long luminescence lifetime and large Stokes shift.[5–9] Recently, there has been an increased interest in exploration of the structure functions of bimolecular reactions with lanthanide complexes.[10,11] However, lanthanide ions in aqueous solution are weakly fluorescing probes in bimolecular systems, due to their poor molar absorptivities and low quantum yields. Thus, the analytical sensitivity is not sufficiently based on lanthanide ions as fluorescence probe.[12–14] Fortunately, the fluorescence of lanthanides has been proved to be enhanced by ligand sensitization with appropriately chosen ligands.[15,16] Glutathione is a bioactive tripeptide, which is broadly present in living systems in both the reduced (GSH) and oxidized (GSSG) forms, and plays key physiological roles.[17] The concentrations and the ratio of GSH and GSSG are influenced by stress factors such as oxidative stress and infections. Thus, evaluation of GSH levels in the presence of GSSG may provide valuable information regarding the oxidative stress a biological system is experiencing. [18] Several methods, such as high-performance liquid chromatography,[19] electrochemistry[20] and spectrofluorimetry,[21] have already been developed for determining GSSG and total GSH (GSH and GSSG) levels. In the spectrofluorimetric determination of GSH, some fluorescent reagents, for instance 3-maleimidylbenzanthrone,[22] N-{p-[2-(6-dimethylamino)benzofuranyl]phenyl}maleimide[23] and 7-fluorobenzo-2-oxa-1,3diazole-4- sulfonate,[24] have been proposed.

In this article, we report the synthesis of Ce(III) (GSSG), and a rapid and sensitive spectrofluorimetric method for GSH in the presence of GSSG in aqueous solutions using the unique fluorescent properties of the complex.

G.-C. Han and Y.-N. Liu ished to mirror with 0.05 μm a-Al2O3 slurry on microcloth pads and sonicated in distillated water and absolute ethanol for 2 min each, and the electrode was then dried under a stream of nitrogen. Pt wire was the counter electrode, and Ag–AgCl–3.0 M KCl was used as the reference electrode. IR compensation was applied. For each of the complex solutions five replicate measurements were recorded at the same scan rate (100 mV s−1) over a potential range of 0–1600 mV. DSC-TG measurement was obtained on a Netzsch STA449C from 20 to 750°C. Preparation of cerium (III) glutathione complex Ce(SO4)2·4H2O (0.4 g, 1 mmol) was dissolved in distilled water, then transferred to a flask. The solution was stirred and heated until the temperature arrived at 60°C. GSH aqueous solution (20 ml, 0.1 M) was then added to the flask dropwise, and stirred continuously for 4 h at 60°C. After the reaction, the mixed solution was concentrated by evaporation. Precipitate was obtained by adding ethanol to the above concentrated mixture. The precipitate was filtered and washed by distilled water three times. Yielded 0.65 g (63.2%) of the desired product cerium glutathione complex as a buff solid by dried at 80°C in an oven. Element analysis for complex Ce(GSSG)·2H2O (C20H36N6O14S2Ce): Calcd C, 30.38; H, 4.81; N, 10.63; S, 8.10 and Ce, 17.72%. Found: C, 30.40; H, 4.79; N, 10.58; S, 8.11 and Ce, 17.71%. Cerium glutathione complex is slightly soluble in water, rather than in organic solvents, such as methanol, acetone, THF, DMF and acetonitrile.

appeared at 350 nm excited at 255 nm, which was attributed to the 5d–4f transitions of cerium (III).[25–28] Accordingly, we can confirm that Ce(IV) is reduced by GSH and Ce(III) glutathione complex is formed, and the efficient energy transfer from ligand GSH to centre cerium (III) ions is achieved. As an f 0 lanthanide ion, Ce(IV) is well known to participate in redox reactions which are induced by low-energy ligand-tometal charge transfer (LMCT) excitation.[29] However, the Ce(IV) aqueous solution was found to be nonfluorescent in the absence of the ligand in the experiment. On the contrary, the emission at lmax = 350 nm appears in the solution after addition of GSH. The result is similar to the work of Cui and coworkers,[30] in which they found that rhodamine 6G is oxidized by Ce(IV) to form the excited-state Ce(III), and then energy is transferred from Ce(III) to rhodamine 6G to form the excited-state rhodamine 6G, which emits its characteristic radiation. In order to determine the ratio of ligand to metal ion, we performed titration, in which ligand was added to a metal solution and the fluorescent intensities were recorded (see Fig. 2). The Job-plot experimental results suggest that the fluorescent intensities vary with the molar ratios of metal ion to GSH, and a maximum fluorescent intensity is reached at a mole fraction of about 0.667(Ce : GSH = 1 : 2), indicating an ML (L = GSSG) stoichiometry of the complex. This result is consistent with elemental analysis. The possible mechanism of the reaction can be described as the following equation: + Ce (IV ) + 2GSH Ce (III)( GSSG) + 2H

Results and discussion Fluorescent properties of the cerium glutathione complex The excitation and emission spectra of cerium glutathione complex in aqueous solutions are shown in Fig. 1. We suggested that GSH is firstly oxidized by Ce(IV) and then produces the Ce(III) glutathione complex. The excitation spectra detected with the emission at 350 nm shows several peaks; the maximum is appeared at 255 nm with a shoulder at 240 nm, which is consistent with the L–S splitting (2FJ = 5/2, 7/2) of Ce(III) ion in the ground state. In addition, there were two weak excitation bands at 226 and 295 nm. The maximum of the fluorescence bands

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Figure 1. Excitation (a) and emission (b) spectra of Ce(III)(GSSG) complex in aqueous solutions.

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To further confirm the reduction of Ce(IV), glutathione oxidized (GSSG) was employed as a ligand to coordinate with Ce(IV). No luminescent emission originated from the complex, due to the formation of the Ce(IV) complex of GSSG, rather than Ce(III) GSSG complex.

FT-IR spectra The IR spectrum of the free ligand GSH shows bands at 1 713 and 1 074 cm−1, which are attributed to the stretch vibration of the carbonyl group (C=O) of amide group

Figure 2. Results of a Job-type titration experiment indicating a Ce(III)(GSSG) complex.



Properties of cerium(III) glutathione complex and(C=O=C), respectively (Fig. 3). The bonding interactions between Ce(III) ion and oxygen atoms of GSH molecules can be identified by the wavenumber shift of the carbonyl band from 1713 cm−1 for the free ligand to 1638 cm−1 for the complex. Additionally, a new peak assigned to M&sbond;O stretching vibration appears at 432 cm−1 in the complex.[31] In addition, a broad band at 3360 cm−1 indicates that the water molecules exist in the complexes.[32]

increases in the electron cloud density of metal ions due to the electron donations of GSH after forming the complex. Interestingly, the oxidation current Ce(III)(GSSG) is very small, indicating that the complex can hardly be oxidized to Ce(IV), i.e. the Ce(III) complex is very stable. The electrochemical results have further proved that Ce(IV) is reduced to Ce(III) by the ligand GSH in the complexation. Thermogravimetric analyses

Electrochemical behavior of Ce(IV)/Ce(III) in Na2SO4 solution The electrochemical properties of complex (1.0 mM) were studied by cyclic voltammetry in 10 mM Na2SO4 solutions (Fig. 4). The free metal ion displays an oxidation wave at E1/2 = 916 mV vs Ag–AgCl with a peak separation of 610 mV (Fig. 4a), and the ratio of oxidative to reductive peak currents is 0.59, which agrees with the results reported by Chen[33] and Kumagai.[34] On formation of the complex (Fig. 4b), a significant negative shift in the reduction potential of more than 157 mV vs Ag–AgCl was observed. This big negative shift may be caused by the

The thermal stability of Ce(III) glutathione complex was demonstrated by TGA-DSC measurements. As shown in Fig. 5, the first decomposition occurred in the range of 20–300°C with a corresponding weight loss of 16.72%, which contributed to the loss of free water and coordinated water. The result is in agreement with the FT-IR spectrum. The second stage of decomposition was observed at 300–660°C (37.4% wt loss), which may be caused by loss of the free ligand from cerium glutathione complex. The char yield was about 44.7 wt% at 750°C. In the DSC curve (Fig. 5), the endothermic peaks of cerium glutathione complex were in the range 123–173°C, implying the cleavage of coordinated H2O. Meanwhile, the ligand decomposition appeared at the two stages of 565–606 and 726–739°C.[35] The results suggest that the decomposition temperature of Ce(III) glutathione complex is very high. The good thermal stability of the complex may be due to the fact that the M–O bond is highly polarized.[36] Determination of GSH in the presence of GSSG

Figure 3.

Infrared spectra of GSH (a) and Ce(III)(GSSG) complex (b).


Figure 5. TGA (- - -) and DSC (—) curves of the Ce(III)(GSSG) complex.



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Figure 4. Cyclic voltammograms of 1.0 mM Ce (SO4)2 (a) and 2.5 mM Ce(III)(GSSG) complex (b) in 0.01 M Na2SO4 solutions.

Finally, cerium(IV) ion was utilized as a probe to determine the GSH concentration in aqueous solution by fluorescence method. The fluorescence intensities of the solutions were achieved by addition of GSH to the Ce(IV) solution. As expected, the fluorescence intensities were proportional to the concentrations of GSH in the range 1–100 nM with the detection limit of 0.05 nM (Fig. 6). The linear relationship between fluorescence intensity I (a.u.), and the concentration c of GSH in nM, is: I = 19.50c +35.72. The correlation coefficient R is 0.99897, SD is 33.38% (n = 10) and p < 0.0001. In order to investigate the interference of GSSG, we conducted an experiment on the determination of GSH in the presence of

G.-C. Han and Y.-N. Liu

Figure 6. Emission spectra of cerium (IV) (1.0 μM) with various concentrations of GSH. Inset: plot of relative fluorescence intensity (a.u.) vs the concentration (nM) of glutathione in aqueous solutions.

Table 1. Determination of GSH in the presence of GSSGa c(GSH) (nM) 10.0

25.0

50.0

Added GSSG (nM)

Ib (Cal.)

I (Found)

0 5.00 10.0 0.0 10.0 25.0 0.0 25.0 50.0

230.7

214.3 209.6 204.5 534.4 539.7 529.4 1046.8 1038.2 1024.5

523.2

1010.7

Fluorescence responses of Ce(IV) (1.0 μM) to GSH and GSSG in aqueous solution. All data were obtained after incubation at 25°C for 6.0 min (lex/lem = 255/350 nm). b Calculated from equation I = 19.50c +35.72 (a.u.). a

GSSG. It was found that the fluorescence intensity was almost unchanged with the addition of GSSG, and the concentrations of GSH were successfully determined by Ce(IV) without interference from the presence of GSSG. The results are listed in Table 1.

Conclusion

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The as-prepared Ce(III) GSSG complex was performed by the redox reaction of Ce(IV) and GSH in aqueous solution. The Ce(III) in the complex was proved to coordinate with the ligand carbonyl groups and water molecules. The complex exhibited characteristic maximum emission spectra of Ce(III) at 350 nm (lex = 255 nm). Moreover, Ce(IV) was successfully employed for the determination of GSH in the presence of GSSG as an extrinsic fluorescent probe.


Unfortunately, the crystal structure of the complex was not obtained in the experiment, resulting in the structure of this complex and precise coordination mechanism remaining unknown. Further, we are trying to use Ce(IV) as a probe to detect the concentration of GSH in real samples of biological systems. Acknowledgments The authors are grateful for the financial support of this study by the Natural Science Foundation of China (20676153 and 20876179).

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