ࡱ> 9 RjbjbjPl8888dl$ ey8V@J pѣaL  8 J0xJFluorescence Enhancement System of Tb-N-(2-Pyridinyl)ketoacetamide-Et3N-Zn and Its Application  Fangfang Dang Weisheng Liu* Jiangrong Zheng Department of Chemistry and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China *Author for correspondence, E-mail: liuws@lzu.edu.cn Abstract A sensitive fluorescence enhancement system was developed for the determination of zinc (a!). The fluorescence intensity of the Tb-N-(2-Pyridinyl)ketoacetamide (PKA) system was greatly enhanced by the addition of triethylamine (Et3N) and zinc nitrate in the methanol solution. The excitation and emission wavelengths were 329 nm and 546 nm, respectively. Under optimal conditions, the fluorescence intensities varied linearly with the concentration of Zn2+ in the range of 8.010-7-5.010-6 M with a detection limit of 9.910-8 M.. The interferences of some substances were described. This method was applied to the determination of amounts of Zn2+ in soybean, rice, and wheat, respectively. The results showed that the proposed procedure is a high selective, simple, and rapid method to the determination of Zn2+ ion. The mechanism of fluorescence enhancement was also studied. Keywords: N-(2-Pyridinyl)ketoacetamide terbium(III) zinc(II) fluorescence enhancement 1 Introduction In recent years, there has been a growing need for developing highly sensitive and selective probes for the detection of metal ions in biological and environmental samples. A variety of divalent metal ions are known to be involved in the structural, catalytic, and regulatory aspects of the biological system, and some such metal ions serve as prognostics of certain human diseases [1]. For example, Cu2+, Zn2+, and Fe2+ have been found to be involved in aggregating -amyloid peptides during the onset of Alzheimer s disease [2]. However, due to the lack of metal ion specific probes, the relative contribution of one type of metal ion versus the other in causing the disease is not clearly understood. The inability to differentiate among different types of divalent metal ions in biological samples has been one of the major impediments in the area of bio-analytical chemistry. Although there has been some success in detection of biologically significant metal ions by developing fluorescence probes (e.g., fura-2 for Ca2+), most of the probes exhibit cross-reactivities for other metal ions [3]. This is not surprising since both physical and electronic properties of these metal ions are not too disparate, and they tend to exhibit comparable binding affinities with their cognate chelating agents. Due to diversity in functional roles of Zn2+ in biological system (viz., DNA synthesis, apoptosis, structural motifs in proteins, enzyme co-factors, etc.), we became interested in the determination of Zn2+. In this endeavor, we noted that several methods have been described for the determination of Zn2+ using detection techniques such as molecular spectrophotometry [4 7], atomic absorption spectrometry [8 10], and chemiluminescence s analysis [11]. However, most of these methods are lacking in sensitivity and require pretreatment of the samples and sometimes, a long incubation period. To date, there has been no report on using the  - diketonate ligands-terbium complex as a fluorescence probe for the determination of Zn2+. These lanthanide chelates have characteristics of the large Stokes shift, narrow emission bands and long lifetime hence to avoid potential background fluorescent emission interferences from the biological matrix. Presently, we have designed a series of polyfunctional ligands having both selective ability to coordinate transition metal ions and good luminescence properties with lanthanide ions, by providing proper conjugate absorption groups suitable for energy transfer, which could be used as a luminescent device. In the present work, we synthesized N-(2-Pyridinyl) ketoacetamide (PKA, Fig.1) containing a -diketonate configuration. As elaborated below, its Tb3+ complex emits intrinsic fluorescence of Tb3+ when excited at 329 nm in methanol solution. After Zn2+ was introduced into the above solution, the emission peak at 546 nm was enhanced strongly. We investigated the possibility of the enhancement of the Tb3+ sensitized fluorescence by Zn2+. Experimental results indicate that the enhancement comes from the intermolecular energy transfer in ternary complex. The other reagents such as La3+, Lu3+, Gd3+, Y3+, Al3+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Cd2+, Na+, and K+ had not the same effect as Zn2+ had and some of them even quenched the fluorescence of Tb3+. According to this character, a new fluorescence method with high sensitivity and selectivity is established for the determination of Zn2+ using PKA-Tb3+ as a fluorescent probe. This method is easily carried out, affords good precision and accuracy and has been successfully applied to the determination of Zn2+ in food samples.  2 Experimental 2.1 Apparatus Elemental analyses were determined on an Elementar Vario EL analyzer. IR-spectra were measured on Nicolet Nexus 670 FT-IR using KBr pellets in the range of 4004000 cm-1. The 1H NMR spectra were recorded on a Bruker AM 200 spectrometer using CDCl3 as solvent and Me4Si as internal reference. Fluorescence measurements were performed on a Hitachi F-4500 spectrophotometer equipped with quartz curettes of 1 cm path length. The excitation slit width was 2.5 nm and the emission one was 2.5nm. 2.2 Reagents Analytical-reagent grade chemicals were used without further purification. A stock solution of Tb3+ (110-4 M) was prepared by dissolving the desired amount of Tb4O7 (99.99% purity) in concentrated nitric acid, evaporating to near dryness and diluting with relevant organic solvents. A stock PKA solution (510-4 M) was prepared by adding the appropriate amount of PKA to 50mL relevant organic solvents. The Et3N stock solution (110-3 M) and Zn2+ stock solution (510-4 M) were prepared by dissolving the desired amount of Et3N or zinc nitrate in relevant organic solvents. 2.3 Synthesis of Ligand PKA The ligand was prepared by the reaction of 2-aminopyridine with ethyl acetoacetate, as described previously [12]. Anal. Calcd. for C9H10N2O2 (%): C, 60.67; H, 5.61; N, 15.73. Found: C, 60.58; H, 5.62; N, 15.71. 1H NMR (ppm), : 2.31 (s, 3H), 3.61 (s, 2H, exchangeable), 7.07 (dt, 1H), 7.71 (dt, 1H), 8.21 (bd, 1H), 8.32 (dt, 1H), 9.68 (bs, 1H). IR (cm-1) in KBr pellet:  (CO) =1720 and  (NH) =1685. M.p. =109  110 !. 2.4 General procedure To a 10mL test tube, 1.4 mL of 110-4 M Tb3+, 0.3 mL of 510-4 M PKA, 0.6 mL of 110-3 M Et3N, and an appropriate amount of Zn2+ solution were added in that order. Then the mixture was diluted to 5 mL with relevant organic solvents. The fluorescence intensity of blank and sample solution, F0 and F1, were measured at 546 nm keeping the excitation wavelength at 329 nm. The F values (=F1"F0) were obtained. 3 Results and Discussion 3.1 Spectral characteristics The fluorescence spectra of Tb-PKA, Tb-PKA-Et3N and Tb-PKA-Et3N-Zn systems in methanol solutions were systematically studied (Figure 2). After Et3N and Zn2+ was introduced in the Tb-PKA system, the fluorescence intensity of Tb-PKA system (Figure 2, curve 1) was successively enhanced from more than 2-fold (Figure 2, curve 2) to 6-fold (Figure 2, curve 3). Figure 2 showed the fluorescence intensity  Figure 2: The Emission Spectra of (1) Tb-PKA, (2) Tb-PKA-Et3N and (3) Tb-PKA-Et3N-Zn Systems In Methanol Solutions. Conditions: Tb3+, 2.810-5 M; PKA, 3.010-5 M; Et3N, 1.210-4 M; Zn2+, 1.210-5 M. enhancement at 546 nm was stronger than that at 490 nm, so the peak at 546 nm was chosen to detect the fluorescence intensity throughout all the experiments. At the same time, the emission spectra of other different systems were also systematically studied. Under the same experiment conditions, the Et3N-Zn, Tb -Et3N, Tb -Et3N-Zn systems had no emission peak at 546 nm. It indicated that increasing fluorescence of the Tb-PKA system took place only in the presence of Et3N and Zn2+. 3.2 Effect of solvents  Figure 3: The Influence of Solvents on the Relative Fluorescence Intensity of the Tb-PKA-Et3N-Zn System. 1 CH3OH, 2 CH3CN, 3 C2H5OH, 4 THF. Conditions: Tb3+, 2.810-5 M; PKA, 3.010-5 M; Et3N, 1.210-4 M; Zn2+, 1.210-5 M. The influence of the solvents on fluorescence intensity was investigated on the above experiment conditions. The results were shown in Figure 3. It can be seen that the enhancing effect of organic solvents in the Tb-PKA-Et3N-Zn system is arranged in the order CH3OHCH3CN>C2H5OHTHF. The permittivity () values of these solvents are 37.5 (CH3CN), 32.6 (CH3OH), 24.3 (C2H5OH), and 7.0 (THF), respectively. The order of F in the system is in agreement with the  values of these solvents that contain oxygen atoms except for methanol. We consider the reason that methanol can sensitize the fluorescence of Tb-PKA-Et3N-Zn system is mainly its coordination ability with Tb3+. This is the coordinating effect of solvents, which is solvate effect [13]. The present ligand containing O, O and N, O donor sets forms a caverned conformation suitable for the coordination with lanthanide ions, but this ajar cavity could not prevent absolutely the solvent molecules from entering. Together with the raising coordination abilities of CH3OH, CH3CN, C2H5OH, THF for the lanthanide ions in this system, the oscillatory motions of the entering molecules consume more energy which the ligand triple level transfer to the emitting level of the lanthanide ion. It is inferred that coordination ability of the solvent besides polarity is also an important factor in this system. Consequently, methanol was selected as an appropriate solvent. 3.3 Effect of Tb3+ concentration Figure 4 showed the influence of Tb3+ concentration on the fluorescence intensity. As the concentration of Tb3+ increased up to 2.410-5 to 3.210-5 M, maximum and constant F was observed. Above 3.210-5 M of Tb3+ concentration, F was decreased. Hence, a 2.810-5 M of Tb3+ solution was used for the subsequent work.  3.4 Effect of PKA concentration The effect of PKA concentration on the fluorescence intensity of the system was also examined (Figure 5).   Figure 6: Effect of the Concentration of Et3N on Relative Fluorescence Intensity. Conditions: Tb3+, 2.810-5 M; PKA, 3.010-5 M; Zn2+, 1.210-5 M. The influence of the amount of Et3N in the range of 0 to 1.810-4 M on the fluorescence intensity was studied. The results were shown in Figure 6. When the concentration of Et3N was controlled between 1.010-4 and 1.610-4 M, F reached a maximum. Therefore, 1.210-4 M was employed as the final Et3N concentration. 3.6 Effect of reaction time The effect of time on the fluorescence intensity was investigated. The results had shown that the solutions could be allowed to stand for at least 2 h under normal laboratory conditions. The reactions occurred rapidly at room temperature (1min. So this assay did not require crucial timing. 3.7 Effect of other substances The interference from foreign ions on the fluorescence intensity of the Tb -PKA- Et3N-Zn was tested by analyzing a standard solution containing Tb3+ 2.810-5 M, PKA 3.010-5 M, Et3N 1.210-4 M, Zn2+ 1.2 10-5 M, to which interfering species were added. The tolerance allowed in the variation of the fluorescence intensity was 10%. The results showed that 100 fold molar excess of Cd2+, 50 fold molar excess Na+, Ca2+, NO3, Cl, 30 fold molar excess Ac, 20 fold K+, I, 10 fold molar excess Ba2+, Br, 5 fold molar excess Mn2+, Ni2+. 3.8 Analytical characters The calibration graph for the determination of zinc (a!) was constructed under the optimal conditions. Excellent linearity F=128.35c - 28.82 (where c is in 10-6 M and represents the concentration of Zn2+ with a linear correlation coefficient (r) of 0.997) was obtained in the range 8.010-7-5.010-6 M. The limitation of detection (LOD) was calculated by multiplying the standard derivation of 10 blank measurements by three and dividing by the slope of the linear calibration curve as 9.910-8 M. 3.9 Recovery study and sample analysis The procedure was used for the determination of amounts of Zn2+ in soybean, rice, and wheat, respectively. The samples were pretreated for the determination of Zn2+ according to literature [14]. The results were shown in Table 1. From the results presented it can be concluded that the proposed procedure is a high selective, simple, and rapid method to the determination of Zn2+ ion. 3.10 Mechanism for the fluorescence enhancement PKA possesses an equilibrium between keto-type and enol-type as a -diketonate derivative, and base such as Et3N makes it easier enolization, which can lead to more coordination ability with metal ion [15-17], thus the characteristic fluorescence of Tb3+ is enhanced in the Tb-PKA system by Et3N. When Zn2+ was added to the Tb-PKA-Et3N system, the fluorescence of Tb3+ is more greatly enhanced. In the TbPKAEt3NZn system, it is considered that there existed simultaneously two complexes: TbPKAEt3N and ZnPKAEt3N. In order to know more details of this transfer, according to Frster theory [18, 19]:  the energy transfer efficiency Ea is calculated, where Ida and Id are the fluorescence intensities of the donor in presence of acceptor and donor in absence of acceptor, respectively. The values of Ea are listed in Table 2. From Table 2, the Ea from the complex of Zn2+ to Tb3+ is 0.71, and Ea from PKA+Et3N to Tb3+ is raised to 0.84 in the system of TbPKAEt3NZn. This indicates that there is intermolecular energy transfer between these two complexes. Since the concentration of the zinc complex is much greater than that of the terbium complex and the distance between Zn PKAEt3N and TbPKAEt3N complexes is short enough, Tb3+ in the TbPKAEt3N can accept the energy from Zn PKAEt3N through intermolecular energy transfer in this system, resulting in the enhanced fluorescence of Tb3+ compared to the system in the absence of Zn2+. Table 1: Analytical Results of Samples SAMPLES ANALYTICAL RESULTS (g g-1) AVERAGE (g g-1) R.S.D. (%) ADDED (ga) FOUND (g g-1) RECOVERY (%) Soybean 18.5, 18.7, 19.4, 18.3, 19.2 18.8 2.5 1.00 0.975 97.5 Rice 12.8, 13.5, 12.6, 12.4, 13.1 12.9 3.4 1.00 1.033 103.3 Wheat 37.0, 37.8, 36.6, 36.9, 37.2 37.1 1.2 1.00 0.986 98.6  a The amount of Zn2+ in standard solution in 5 mL comparison tubes. Table 2: The Efficiency of Energy Transfer (Ea) SYSTEM DONOR ACCEPTOREaTbPKAEt3NZn PKAEt3NZn Tb 0.71 PKAEt3N Tb 0.84  In addition, it is found that the fluorescence intensity of the TbPKAEt3NZn system is considerably stronger than that of the TbPKAEt3N system. In the latter system the concentration of Tb3+ equals the sum of the concentrations of Tb3+ and Zn2+ in the former system. This indicates that besides the intermolecular energy transfer mentioned above, the increasing of the fluorescence quantum yield is another reason for the fluorescence enhancement of the TbPKAEt3NZn system. Because the concentration of the Zn2+ complex is much higher than that of the Tb3+ complex, each of TbPKAEt3N complex molecules is surrounded by many ZnPKAEt3N complex molecules. These surrounding complexes could act as an energy-insulating sheath, which could prevent collision with solvent molecules and decrease the energy loss of TbPKAEt3N complex, thus improving the fluorescence quantum yield. 4 Conclusion In this work, it is found that the complex of N-(2-Pyridinyl) ketoacetamide (PKA) with Tb3+ in methanol solution can emit the intrinsic fluorescence of Tb3+. When Et3N and Zn2+ are added to the above system, the fluorescence is significantly enhanced. Based on it, a method is established to determine zinc ions selectively and its detection limit reaches as low as 9.910-8 M. Its reliability is validated by detecting the amounts of zinc (a!) in the food samples and its result is satisfactory. The mechanism of fluorescence enhancement is in detail studied. In the Tb PKA Et3N Zn system, it is considered that there existed simultaneously two complexes: Tb PKA Et3N and Zn PKA Et3N. The complexes of Zn2+ not only transfer energy to the Tb3+ complexes but also increase the fluorescence quantum yield. Hence, the fluorescence of Tb3+ is significantly enhanced. Acknowledgements We are grateful to the NSFC (Grants 20371022, 20431010 and 20021001), the Specialized Research Fund for the Doctoral Program of Higher Education (200307300015), and the Key Project of the Ministry of Education of China (Grant 01170) for financial support. References [1] Eiichi K, Koike T. Recent development of zinc-fluorophores. Chem Soc Rev 1998; 27: 179 184. [2] Bush AL. The metallobiology of Alzheimer's disease. Trends Neurosci 2003; 26: 207 214. [3] (a) Walkup GK, Imperiali B. Design and Evaluation of a Peptidyl Fluorescent Chemosensor for Divalent Zinc. J Am Chem Soc 1996; 118: 3053 3054; (b) Reany O, Gunnlaugsson, T, Parker D. 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[17] Banger KK, Kornilov A, Claessen RU. The first metal complex containing a silylated -diketonate ligand: bis (2, 2,6,6-tetramethyl- 2- sila-3,5-heptanedionato) copper (II). Inorg Chem Commun 2001; 4: 496-500. [18] Clegg RM. Fluorescence resonance energy transfer. Curr Opin Biotech 1995; 6: 103110. [19] Kumar GA, Unnikrishnan NV. Energy transfer and optical gain studies of FDS: Rh B dye mixture investigated under cw laser excitation. J Photochem Photobiol A 2001; 144: 107117. &'EF_ab|~T\lmvwRTlo, /  l m | ˿˶uhhhhhhB*CJEHH*aJphB*CJEHH*aJphB*CJOJaJo(ph B*ph5B*\ph6>*B*CJ]aJph6B*CJ]aJphB*CJaJph5B*CJ\aJphB*CJaJphB*CJo(phCJ"aJ"5CJ"EHH*\aJ"56CJ"\]aJ"5CJ"\aJ"'`ab8mw m } " {rr$da$$ & Fhd^h`a$$xd]^a$$a$$a$<$$Ifl," 6`64 la$$&`#$/Ifa$ j| }  " * . > D   " ~ce\h6<mp#%)+/146:<@BFHLNRTXZ^`dfjlpqxz6CJ]aJCJEHH*aJCJaJB*CJaJo(phB*CJEHH*aJphB*CJaJph B*o(phM^amp  !"12@8:KL.2;<r u !!J!K!|!!! 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