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A ratiometric fluorescent sensor for zinc ions based on covalently immobilized derivative of benzoxazole
Spectrochimica Acta Part A 73 (2009) 687–693
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A ratiometric fluorescent sensor for zinc ions based on covalently immobilized derivative of benzoxazole Qiu-Juan Ma a,b , Xiao-Bing Zhang a,∗ , Xu-Hua Zhao a , Yi-Jun Gong a , Jian Tang a , Guo-Li Shen a , Ru-Qin Yu a,∗ a b
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China College of Pharmacology, Henan University of Traditional Chinese Medicine, Zhengzhou 450008, PR China
a r t i c l e
i n f o
Article history: Received 30 November 2008 Received in revised form 6 March 2009 Accepted 19 March 2009 Keywords: Fluorescent sensor Zinc ions Benzoxazole derivative Covalent immobilization Ratiometric
a b s t r a c t In the present paper, we describe the fabrication and analytical characteristics of fluorescence-based zinc ion-sensing glass slides. To construct the sensor, a benzoxazole derivative 4-benzoxazol-2 -yl-3hydroxyphenyl allyl ether (1) with a terminal double bond was synthesized and copolymerized with 2-hydroxyethyl methacrylate (HEMA) on the activated surface of glass slides by UV irradiation. In the absence of Zn2+ at pH 7.24, the resulting optical sensor emitted fluorescence at 450 nm via excited-state intramolecular proton transfer (ESIPT). Upon binding with Zn2+ , the ESIPT process was inhibited resulting in a 46 nm blue-shift of fluorescence emission. Thus, the proposed sensor can behave as a ratiometric fluorescent sensor for the selective detection of Zn2+ . In addition, the sensor shows nice selectivity, good reproducibility and fast response time. Cd2+ did not interfere with Zn2+ sensing. The sensing membrane demonstrates a good stability with a lifetime of at least 3 months. The linear response range covers a concentration range of Zn2+ from 8.0 × 10−5 to 4.0 × 10−3 mol/L and the detection limit is 4.0 × 10−5 mol/L. The determination of Zn2+ in both tap and river water samples shows satisfactory results. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Zinc is one of the most abundant transition metals in human body. It plays an important role in various biological systems such as gene expression, protein–protein interaction and neurotransmission [1,2]. In addition, zinc is also a contributing factor in many severe neurological diseases such as Alzheimer’s disease, epilepsy, cerebral ischemia and neurodegenerative disease [2]. The total concentration of zinc lies over a very broad range (in the order of nanomolar to millimolar) [3]. Though zinc is a relatively nontoxic element, it can be toxic if consumed in large enough quantities. For example, zinc is a metal pollutant of environment, significant concentrations of which may reduce the soil microbial activity causing phytotoxic effect [4,5] and which is also a common contaminant in agricultural and food wastes [6]. Thus, the determination of trace amounts of zinc is currently of great interest in many scientific fields, including medicine analyses and environmental monitoring, etc.
∗ Corresponding authors. Tel.: +86 731 8821916; fax: +86 731 8821916. E-mail addresses: [email protected] (X.-B. Zhang), [email protected] (R.-Q. Yu). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.03.023
Though a number of analytical techniques such as inductivity coupled plasma atomic emission spectroscopy (ICP-AES) [7], UV–vis spectroscopy [8], potentiometry [9] and flame atomic absorption spectrometry [10] have been used to detect trace amounts of zinc, the reliable method for zinc assay is still limited owing to its 3d10 4s0 electronic configuration not giving any spectroscopic or magnetic signals. Due to their advantages of simplicity, high sensitivity and low lost, fluorescent sensors have been developed for the determination of zinc ions in the past decades. And several fluorescent indicators for zinc ions have been reported in the previous literature [11–15]. However, from a practical point of view the immobilization of indicator dyes may be required to construct optical chemical sensors (optode). And the immobilization of indicator dyes is probably the most important factor that governs the lifetime of the sensor. Poor immobilization results in leaching and consequently no sensing. Several immobilization methods of indicator dyes have been reported including physical entrapment [16,17], electrostatic association [18,19] and covalent immobilization [20–23]. Though physical entrapment is simple, it has a drawback: the encapsulated neutral carriers are often leached from the membrane into the analyte solution, which makes the resulting sensors less durable and more toxic. The electrostatic immobilization leads to considerable binding strength between the charged dye and the oppositely charged functional group of support matrixes; however, the indicator dye can also be displaced easily
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by the sample ions or solvent molecules. Covalent immobilization might be the most efficient technique for the preparation of optical chemical sensors because it can prevent the dye exude out from the membranes resulting in a long lifetime. However, only very few chemosensors for zinc based on covalently immobilized indicator dye onto the support matrixes have been reported [24]. Searching for zinc chemosensors based on covalently immobilized indicator dyes is still an active field as well as a challenge for the analytical chemistry research effort. The photochemically initiated immobilization of indicator dyes is a relatively simple and efficient method not requiring sophisticated apparatus and with a faster speed compared with thermal polymerization. Shortreed et al. developed a fluorescent fiberoptic calcium sensor based on covalent immobilization of a fluorescent indicator [25]. Immobilization occurs via photoinitiated copolymerization of the indicator with acrylamide. Hisamoto et al. reported the optical chemical sensing of pH and water content in organic solvents based on the immobilization of indicator dyes possessing an olefin unit by UV photopolymerization [26]. Our own group has also reported some work in which indicator dyes were covalently attached onto the activated surface of glass slides by UV irradiation [27–30]. In this paper, we describe a photopolymerization method for immobilizing a benzoxazole derivative 1 for making chemical sensor for Zn2+ . In recent years, the general family of 2-(2 -hydroxyphenyl)benzazoles has been extensively studied from the viewpoint of photophysics because of its dual emission via the excitedstate intramolecular proton transfer (ESIPT) [31–35]. Moreover, some derivatives of 2-(2 -hydroxyphenyl)-benzazole have been used to sense ions as fluorescent indicators [35,36–38]. Recently, Qin et al. reported a fiber-optic fluorescence sensor for Li+ in acetonitrile which is based on the diffusion of the 2-(2 hydroxyphenyl)benzoxazole (HPBO) through a poly(vinyl chloride) (PVC) membrane into the analyte solution [39]. Zhang et al. also developed a highly selective fluorescent sensor for Cu2+ in aqueous solution based on HPBO in a PVC matrix [40]. But the proposed sensors in the literature [39,40] are both based on physical immobilization of HPBO, HPBO is apt to exude out from the membrane resulting in less durability. In a recent study, Crivat et al. proposed a zinc sensor based on indicator dye conjugated to the active surface of glass slides and the operating principle was a simple enhancement of fluorescent intensity [24]. However, in most practical applications, fluorescence intensity changes can also be caused by many other poorly quantified or variable factors such as the excitation intensity, dye concentration, and environment around the dye (pH, polarity, temperature, and so forth), this problem can be solved by using ratiometric chemosensors that exhibit a spectral shift upon binding of the cation [41]. Hence, in this work we developed a zinc(II)-selective ratiometric fluorescent sensor based on covalently immobilized derivative 1 of benzoxazole. To prepare the sensor, the zinc ion indicator HPBO was modified to include a terminal double bond and covalently immobilized on the activated surface of glass slides by UV irradiation. In the absence of Zn2+ , the sensor undergoes ESPIT process and coordination of Zn2+ inhibits the ESPIT yielding a 46 nm blue-shift of fluorescence emission. The proposed sensor exhibits excellent analytical characteristics including nice selectivity, good reproducibility and fast response time for the determination of Zn2+ in aqueous solution. Moreover, Cd2+ did not affect the detection of Zn2+ though it has similar chemical properties with Zn2+ . Covalent immobilization of the derivative 1 of benzoxazole lengthens the lifetime of the sensor. The feasibility of using the proposed sensor for the determination of Zn2+ in both tap and river water samples has been testified.
2. Experimental 2.1. Reagents and chemicals 2,4-Dihydroxybenzaldehyde (98%) and 3-(trimethoxysilyl) propyl methacrylate (TSPM) (98%) were purchased from Acros Organics. Barium manganate (90%) was acquired from Alfa Aesar. 2-Hydroxyethyl methacrylate (HEMA), benzoin ethyl ether, benzophenone, and triethanolamine used in the membrane matrix preparation were obtained from Shanghai Chemicals. Doubly distilled water was used throughout all experiments. Before being used, toluene was subjected to simple distillation at atmospheric pressure from sodium and stored over 4 Å molecular sieves. Except when specified, other chemicals were of analytical reagent grade and used without further purification or treatment. A stock solution of 1 × 10−2 mol/L Zn2+ was prepared by dissolving Zn(OAc)2 ·2H2 O in doubly distilled water. The stock solution of Zn2+ was diluted to lower concentrations of 4 × 10−3 to 1 × 10−5 mol/L stepwise. The wide pH range solutions were prepared by adjustment of 0.05 mol/L Tris–HCl solution with HCl or NaOH solution. 2.2. Apparatus All fluorescence measurements were taken on a Hitachi F4500 Fluorescence Spectrometer (Tokyo, Japan) with excitation slit set at 5.0 nm and emission at 5.0 nm. The light source is a 150 W Xe lamp and the detector is a R928F red-sensitive photomultiplier tube. All fluorescence measurements were performed under ambient temperature at 25 ◦ C. The measurements of pH were carried out on a Mettler-Toledo Delta 320 pH meter (Shanghai, China). A 50 W ultraviolet lamp at 253.7 nm (model ZF-2, Shanghai Analytical Instruments, Shanghai, China) was used for photopolymerization. 1 H NMR spectra were recorded on an INOVE400 (Varian) spectrometer. MS spectra were obtained on a GC-17A, QP-5000 (Shimadzu) spectrometer. Data processing was performed on a Pentium IV computer with software of SigmaPlot. 2.3. Synthesis of fluorescence carrier The synthetic procedure for fluorescence carrier 4-benzoxazol2 -yl-3-hydroxyphenyl allyl ether (1) is shown in Scheme 1. Synthesis of 4-formyl-3-hydroxyphenyl allyl ether (2). To a mixture of 2,4-dihydroxybenzaldehyde (1 g, 7.2 mmol) and NaHCO3 (0.6125 g, 7.2 mmol) in 20 mL of N,N-dimethylformamide (DMF) was added dropwise 0.625 mL (7.2 mmol) of allyl bromide and reaction mixture was heated at 70 ◦ C for 48 h. After the solvent was evaporated under reduced pressure, 50 mL of H2 O was added to the mixture. The resulting precipitate was filtered and dissolved in 25 mL of CH2 Cl2 . The organic layer was dried over Na2 SO4 (anhydrous), filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using petroleum ether/dichloromethane (7:2, V/V) as eluent to afford compound 2 as a yellow oil. Yield: 0.8339 g (65%). MS (EI) m/z: 178.0. Synthesis of 3-hydroxy-4-[(2 -hydroxy-phenylimino)-methyl] phenyl allyl ether (3). A mixture of compound 2 (0.1782 g, 1 mmol) and 2-aminophenol (0.1091 g, 1 mmol) in 25 mL of EtOH was heated under reflux for 18 h. The reaction mixture was cooled to room temperature and concentrated in vacuo. The yellow solid residue was recrystallized from ether providing Schiff base 3 as a yellow solid. Yield: 0.1880 g (70%). Synthesis of 4-benzoxazol-2 -yl-3-hydroxyphenyl allyl ether (1). A mixture of compound 3 (0.1346 g, 0.5 mmol) and BaMnO4 (0.5125 g, 2 mmol) in 15 mL of toluene was heated at 80 ◦ C for 24 h. The reaction mixture was cooled to room temperature and
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Scheme 1. Synthesis of 4-benzoxazol-2 -yl-3-hydroxyphenyl allyl ether (1). (a) Allyl bromide, NaHCO3 , N,N-dimethylformamide, 70 ◦ C, 48 h, 65%; (b) 2-aminophenol, ethanol, reflux, 18 h, 70%; (c) BaMnO4 , toluene, 80 ◦ C, 24 h, 18%.
filtered. The filtrate was concentrated in vacuo affording a black residue, which was purified by silica gel column chromatography using petroleum ether/dichloromethane (8:1, V/V) as eluent to provide compound 1 as a white solid. Yield: 0.0241 g (18 %). 1 H NMR (400 MHz, CDCl3 ), ı (ppm): 4.588 (d, 2H, J = 6.8 Hz, –OCH2 –), 5.328 (dd, 1H, J = 1.2 Hz, 1.6 Hz,
), 5.447 (dd, 1H, J = 1.2 Hz, 1.2 Hz,
), 6.019–6.115 (m, 1H, CH–), 6.591 (dd, 1H, J = 1.6 Hz, 8.8 Hz, 6-H), 6.63 (d, 1H, J = 1.6 Hz, 2-H), 7.308–7.375 (m, 2H, 5-H, 4 -H), 7.554–7.577 (m, 1H, 5 -H), 7.665–7.687 (m, 1H, 6 -H), 7.910 (d, 1H, J = 8.4 Hz, 7 -H), 11.625 (s, 1H, OH). MS (EI) m/z: 267.0. 2.4. Silanization of the glass slides The microscope glass slides were cut to dimension of approximately 13 mm × 13 mm and the silanization was carried out as reported [24,30,42,43] with some modifications. First, the glass slides were cleaned with piranha solution (98% H2 SO4 :30% H2 O2 = 7:3, by volume) heated at 70 ◦ C for 20 min to remove particulate matter and organic impurities. These slides were rinsed with deionized water for several times and dried by nitrogen flow. Second, the glass slides were immersed in 1 M NaOH solution for 20 min at 80 ◦ C, thoroughly washed with distilled water and spectrophotometric grade CH3 OH and dried under nitrogen. The glass surface treatment with NaOH solution resulted in a high concentration of hydroxyl group and facilitated the subsequent reaction with TSPM. Finally, a solution of TSPM was prepared by mixing 0.2 mL of TSPM, 2 mL of 0.2 mol/L HAc–NaAc (pH 3.6), and 8 mL of water. The silanization reaction was implemented by submerging the treated glass slides into the TSPM solution for 2 h at room temperature. The silanized slides were subsequently rinsed with distilled water and dried in a nitrogen atmosphere. 2.5. Preparation of the optode membrane The optode membrane solution was prepared by mixing 2.9 mL of HEMA, 10 mg of compound 1, 45 mg of benzoin ethyl ether, 0.12 g benzophenone, and 0.2 mL of triethanolamine, then the solution was cast onto a dust-free poly(tetrafluoroethylene) (PTFE) plate. Silanized glass plates were placed over the droplets, and
UV radiation (253.7 nm, 50 W) was directed from 10 cm onto the membrane matrixes for about 1 h in a nitrogen atmosphere. After UV irradiation, the glass plates with the polymer membrane were completely washed with distilled water and methanol to take off any unreacted species using a supersonic cleaner until no leaching of the compound 1 was observed. The optode membranes prepared as above were dried and stored in a refrigerator until used. 2.6. Fluorescence measurements The fluorescence intensity of optode membrane was measured at excitation wavelength of 323 nm with the emission wavelength varied over 340–620 nm. The glass plate with the sensing membrane was fixed in a quartz cuvette which was filled with the sample solution. The sensor membrane was equilibrated with the sample solution to obtain a stable fluorescence signal. After each measurement, the fluorescence intensity of the sensing membrane was recovered by adding the blank solution through the quartz cuvette prior to the next measurement. 3. Results and discussion 3.1. Synthesis of fluorescent carrier 1 and its covalent immobilization on sensor surface In order to covalently immobilize HPBO on the glass surface, it is an alternative to introduce a polymerizable group such as a double bond in the compound HPBO and in the quartz glass surface, respectively. In our work, allyl bromide was used to react with 2,4dihydroxybenzaldehyde and the hydrogen of 4-position hydroxyl was replaced by allyl group. Consequently, the fluorescent carrier 1 made from compound 2 via condensation and oxidize in two steps includes a terminal double. TSPM was taken as the pre-treatment agent to introduce a polymerizable vinyl group onto cleaned quartz glass plate surface. Under UV radiation, compound 1 with a terminal double bond reacts with the surface double bond of the modified glass slides. The photopolymerization was initiated by using benzoin ethyl ether and benzophenone as photo initiators possessing a synergistic action. And in this case HEMA and compound 1 were used as the monomer. Triethanolamine was used to stop oxygen’s inhibition during the membrane curing process. In order to avoid
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Fig. 1. Fluorescence emission spectra of the optode membrane exposed to various concentration of Zn2+ : 0, 8 × 10−5 , 2 × 10−4 , 6 × 10−4 , 8 × 10−4 , 10−3 , 2 × 10−3 , 4 × 10−3 mol/L from a to h. These spectra were measured in 0.05 M Tris–HCl buffer (pH 7.24). The excitation wavelength was 323 nm.
the formed sensing membrane peeling off the quartz surface, the time of UV irradiation should be kept not shorter than 1 h. 3.2. Spectral characteristics Fig. 1 shows the fluorescence emission spectra of the optode membrane exposed to a solution containing various concentration of Zn2+ , which were recorded with ex = 323 nm in 0.05 M Tris–HCl buffer (pH 7.24). As can be seen from Fig. 1, the fluorescence emission spectra of optode membrane are sensitive to Zn2+ . With the stepwise addition of Zn2+ to optode membrane, the fluorescence emission of optode membrane at 404-nm bands increases while that at 450 nm decreases. An isoemission point appears at near 433 nm. In the presence of excess Zn2+ , a 46 nm blue-shift of flu-
Fig. 2. Fluorescence ratio I404 nm /I450 nm of optode membrane as a function of Zn2+ concentration in 0.05 M Tris–HCl buffer (pH 7.24). The excitation wavelength was 323 nm.
orescence emission from 450 to 404 nm was observed. From Fig. 1, one can see that the emission intensity ratio at 404 and 450 nm of the optode membrane enhanced with the increasing zinc concentration, which constituted the basis for the determination of zinc concentration with the optical sensor by the fluorescence ratiometric method. In 0.05 M Tris–HCl buffer solution (pH 7.24), the emission spectrum of the optode membrane exhibits two maxima at 362 and 450 nm, respectively. Possible mechanism accounting for the phenomenon is ESIPT process [35], in which the largely Stokes-shifted band at 450 nm can be attributed to ESIPT emission of the keto tautomer and the intensity of short wavelength fluorescence at 362 nm is the normal emission intensity (Scheme 2). In the presence of excess Zn2+ the proton transfer process is disrupted by the coordinated zinc ion, resulting in the tautomer emission at 450 nm
Scheme 2. Excited-state intramolecular proton transfer (ESIPT) process for the proposed sensor.
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Fig. 3. Effect of pH on I404 nm /I450 nm of the optode membrane in the absence (closed circles) or presence (open circles) of 6.0 × 10−4 mol/L Zn2+ . All data were obtained at various pH values (pH 2.00–9.19) and the excitation wavelength was 323 nm.
disappearing and the maximum emission at 404 nm appearing (Scheme 2). The maximum emission at 404 nm upon completely coordinating with Zn2+ possibly arises from the normal emission of the zinc-complex. 3.3. Principle of operation and the basis of quantitative assay Fig. 2 shows the dependence of emission intensity ratios between 404 and 450 nm (I404 nm /I450 nm ) on the concentration of Zn2+ in 0.05 M Tris–HCl buffer solution (pH 7.24). As shown in Fig. 2, I404 nm /I450 nm increased linearly with the concentration of Zn2+ . The linear response of I404 nm /I450 nm toward log CZn 2+ was obtained in zinc concentration range of 8.0 × 10−5 to 4.0 × 10−3 mol/L, which provided the quantitative base for the determination of zinc concentration. The ratiometric calibration line can be expressed by the following equation: I404 nm = 4.1700 + 0.9158 log CZn 2+ (R = 0.9872) I450 nm
(1)
Here I404 nm and I450 nm are the fluorescence emission intensity of optode membrane actually measured at a given metal concentration at 404 and 450 nm, respectively. CZn 2+ represents the concentration of zinc ion added. The detection limit obtained for Zn2+ was 4.0 × 10−5 mol/L (defined as three times standard deviation of blank solution). The curve fitting of relative fluorescence intensity supported the formation of a 1:1 complex of the benzoxazole derivative in the optode membrane and zinc cation in 0.05 M Tris–HCl buffer solution (pH 7.24), binding constant being estimated to be K = 3.0 × 103 M−1 according to the literature [41,44]. 3.4. Effect of pH The effects of pH on the fluorescence intensity of optode membrane in the absence or presence of 6 × 10−4 mol/L Zn2+ were examined at a pH range from 2.00 to 9.19 (Fig. 3). As can be seen from Fig. 3, the optode membrane shows good fluorescence response to Zn2+ over a wide pH range. The optode membrane has no noticeable fluorescence sensing ability to Zn2+ at pH value lower than 3.73, which might be assigned to the complete protonation of phenolic oxygen of the optode membrane when pH values of solution lower than 3.73 resulting in a no-coordination ability of Zn2+ [35]. However, the optode membrane shows satisfactory Zn2+ -sensing ability in the range of pH from 3.73 to 9.19. This phenomenon might be caused by deprotonation of the hydroxyl group of receptor in
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Fig. 4. Metal ion selectivity of optode membrane. All data were obtained at pH 7.24, (0.05 M Tris/HCl) and were expressed as the fluorescence ratio (I404 nm /I450 nm ). The excitation wavelength was 323 nm. The concentration of ions added to optode membrane was 6.0 × 10−4 M for Zn2+ , Ag+ and Pb2+ , and 10−3 M for all remaining ions. Black bars: different metal ions were added. Gray bars: different metal ions in the presence of Zn2+ were added.
the region of higher pH value resulting in the formation of zinccomplex [35]. In the presence of 6.0 × 10−4 mol/L Zn2+ I404 nm /I450 nm of the optode membrane decreased with reducing pH value at pH 8.01–9.19, which might be ascribed to the formation of partial precipitation of Zn(OH)2 under strong basic condition resulting in the decrease of actual concentration of Zn2+ in the sample solution. Taking into consideration the sensitivity, response speed and application in real samples, a 0.05 M Tris/HCl buffer solution at pH 7.24 was chosen as optimum experimental condition. 3.5. Selectivity The fluorescence responses of optode membrane to various cations and its selectivity for Zn2+ are illustrated in Fig. 4. The concentration of Zn2+ was fixed at 6.0 × 10−4 mol/L in 0.05 M Tris–HCl buffer solution (pH 7.24). Cations were added as chlorides, nitrates, acetate and sulfates. As can be seen from the black bars in Fig. 4, I404 nm /I450 nm significantly enhanced upon the addition of Zn2+ and slightly increased upon binding to Cu2+ , Co2+ and Ni2+ . Moreover, the addition of other cations did not affect I404 nm /I450 nm of the optode membrane. Cu2+ , Co2+ and Ni2+ cannot shift the spectrum of optode membrane in the same way as Zn2+ , but they quenched the emission intensity at both 405 and 450 nm resulting in slight enhancement of I404 nm /I450 nm . Though Cd2+ has similar chemical properties compared with Zn2+ , the optode membrane shows no response to Cd2+ . In order to further test the interference for other common cations on the determination of Zn2+ , a competition experiment was performed in which a zinc solution in the presence of other metal ions was added to the fluorescence cuvette with optode membrane (white bars in Fig. 4). The competition experiments show no significant variation in the emission intensity ratio (I404 nm /I450 nm ) except Cu2+ , Co2+ and Ni2+ . Thus, the proposed sensor in this paper exhibits good selectivity for Zn2+ over other common cations except Cu2+ , Co2+ and Ni2+ . Fortunately, these three cations would have little influence in living system, since they exist at very low concentrations [45]. In environmental analysis, the possible interference of Cu2+ , Co2+ and Ni2+ on the determination of Zn2+ could be effectively circumvented by adding an appropriate concentration of Na2 S2 O3 solution. Thus, the proposed sensor in this paper could be useful for biological and environmental applications. Singh et al. reported a chemosensor for Cu2+ and Fe3+ based on Quantum Dot-Schiff base conjugate but the Schiff base itself
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3.6. Reproducibility, reversibility and response time
Fig. 5. Fluorescence emission spectra of monomer 1 (1.0 × 10−5 mol/L) in the presence of different metal ions. The concentration of ions added to monomer 1 was 0.1 mM for Zn2+ , Ag+ and Pb2+ , and 1 mM for all remaining ions. Measured at 25 ◦ C in CH3 CN–H2 O (1:1, V/V) and 0.05 M Tris–HCl buffer (pH 7.24). The excitation wavelength was 323 nm.
The reproducibility and reversibility of optode membrane were studied by exposing the sensor to a 0.05 M Tris–HCl buffer solution of pH 7.24, 6 × 10−4 and 2 × 10−3 mol/L zinc solutions in turn. Fig. 6 exhibits the emission intensity ratios between 404 and 450 nm (I404 nm /I450 nm ) upon switching from one solution to another. The relative standard deviations in I404 nm /I450 nm calculated from five measurements of 6 × 10−4 and 2 × 10−3 mol/L zinc solutions were found to be 1.34 and 1.02%, respectively. The relative standard deviations in I404 nm /I450 nm from ten measurements of blank buffer solution is estimated as 0.16%. All these results show satisfactory reproducibility and reversibility of the optode membrane. The response time of the optode membrane depends on the thickness of membrane and the zinc concentration. The thickness of the optode membrane, fabricated as described above, is about 50 m. The response time is 2 and 3 min for 6 × 10−4 and 2 × 10−3 mol/L zinc solutions, respectively. Moreover, it was found that the recovering time actually remained the same in despite of switching from low to high or the opposite. The recovering time was about 80 s. 3.7. Short-time stability and lifetime The short-time stability of the sensor was tested by exposing the sensor to the 6 × 10−4 mol/L zinc solution in a period of 7 h. The fluorescence emission spectrum was recorded at interval of 30 min. The relative standard error of 1.47% for I404 nm /I450 nm was obtained for this solution. The fluorescence response remains stable even after 2 months’ usage. Covalent immobilization effectively prevented leaching of the dye from the optode membrane into solution and remarkably improved the lifetime of sensor. A newly prepared sensor can be used at least 3 months. 3.8. Preliminary analytical application
Fig. 6. Reproducibility and reversibility for the proposed sensor. The measurement was carried out by exposing the sensor to a blank buffer solution of pH 7.24 and zinc solutions of different concentration in turn: (a) blank solution; (b) 6 × 10−4 mol/L Zn2+ ; (c) 2 × 10−3 mol/L Zn2+ . The excitation wavelength was 323 nm.
displayed no selectivity [46]. Thus, the selectivity of monomer 1 in CH3 CN–H2 O (1: 1, V/V) solution was investigated by titration of monomer 1 with various metal ions as a control experiment in this work (Fig. 5). As shown in Fig. 5, monomer 1 shows good selectivity for Zn2+ over other common cations except Fe3+ , Cu2+ , Co2+ and Ni2+ . Therefore, the immobilization of monomer 1 on an activated surface of glass slides did not alter its selectivity in this study.
In order to examine the applicability of the proposed method in practical sample analysis, the sensor was applied in the determination of zinc ion in both tap and river water samples. The river water samples obtained from Xiang River were simply filtrated. And the tap and river water samples showed that no zinc ion was present in them. All these water samples were spiked with standard Zn2+ solutions at different concentration levels and then analyzed with proposed sensor. Results are shown in Table 1. One can see that the recovery study of spiked Zn2+ determined by the sensor shows satisfactory results. The present sensor seems useful for the determination of Zn2+ in real samples. 4. Conclusion In summary, a ratiometric fluorescent sensor for zinc ions based on covalently immobilized derivative of benzoxazole has been developed. During fabricating the sensor, a new dye monomer 1 with a terminal double bond was copolymerized with 2-
Table 1 Determination of Zn2+ in tap and river water samples with the sensor. Sample
Zn2+ spiked (mol/L)
Recovery (%) – 103.0 98.0 – 104.6 102.0
Tap water
1 2 3
8 × 10−5 1 × 10−4
Not detected (8.25a ± 0.24b ) × 10−5 (0.98a ± 0.01b ) × 10−4
River water
1 2 3
0 8 × 10−5 −4 1 × 10
Not detected (8.37a ± 0.11b ) × 10−5 (1.02a ± 0.02b ) × 10−4
a b
Mean values of the three determinations. Standard deviation.
0
Zn2+ recovered (mol/L)
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hydroxyethyl methacrylate (HEMA) by UV photopolymerization and covalently immobilized on the surface of the modified quartz glass plate. The sensor shows remarkable analytical characteristics including fast response and recovery time, sufficient reproducibility, high sensitivity and selectivity for the determination of zinc in aqueous media. Moreover, its lifetime was also satisfactory since the leaching of dye from optode membrane was prevented by covalent immobilization. The fluorescence emission of the sensor at 404-nm bands increases while that at 450-nm decreases upon the addition of zinc and emission intensity ratios between 404 and 450 nm (I404 nm /I450 nm ) are selective for Zn2+ . The proposed sensor can be applied to the quantification of Zn2+ with a linear range covering from 8.0 × 10−5 to 4.0 × 10−3 mol/L and the detection limit is 4.0 × 10−5 mol/L. The proposed sensor has been used for the determination of Zn2+ in both tap and river water samples and shows satisfactory results. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos. 20505008, 20675028, 20775023 and 20435010), “973” National Key Basic Research Program of China (2007CB310500), and Hunan Natural Science Foundation (07JJ3025). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A ratiometric fluorescent sensor for zinc ions based on covalently immobilized derivative of benzoxazole Qiu-Juan Ma a,b , Xiao-Bing Zhang a,∗ , Xu-Hua Zhao a , Yi-Jun Gong a , Jian Tang a , Guo-Li Shen a , Ru-Qin Yu a,∗ a b
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China College of Pharmacology, Henan University of Traditional Chinese Medicine, Zhengzhou 450008, PR China
a r t i c l e
i n f o
Article history: Received 30 November 2008 Received in revised form 6 March 2009 Accepted 19 March 2009 Keywords: Fluorescent sensor Zinc ions Benzoxazole derivative Covalent immobilization Ratiometric
a b s t r a c t In the present paper, we describe the fabrication and analytical characteristics of fluorescence-based zinc ion-sensing glass slides. To construct the sensor, a benzoxazole derivative 4-benzoxazol-2 -yl-3hydroxyphenyl allyl ether (1) with a terminal double bond was synthesized and copolymerized with 2-hydroxyethyl methacrylate (HEMA) on the activated surface of glass slides by UV irradiation. In the absence of Zn2+ at pH 7.24, the resulting optical sensor emitted fluorescence at 450 nm via excited-state intramolecular proton transfer (ESIPT). Upon binding with Zn2+ , the ESIPT process was inhibited resulting in a 46 nm blue-shift of fluorescence emission. Thus, the proposed sensor can behave as a ratiometric fluorescent sensor for the selective detection of Zn2+ . In addition, the sensor shows nice selectivity, good reproducibility and fast response time. Cd2+ did not interfere with Zn2+ sensing. The sensing membrane demonstrates a good stability with a lifetime of at least 3 months. The linear response range covers a concentration range of Zn2+ from 8.0 × 10−5 to 4.0 × 10−3 mol/L and the detection limit is 4.0 × 10−5 mol/L. The determination of Zn2+ in both tap and river water samples shows satisfactory results. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Zinc is one of the most abundant transition metals in human body. It plays an important role in various biological systems such as gene expression, protein–protein interaction and neurotransmission [1,2]. In addition, zinc is also a contributing factor in many severe neurological diseases such as Alzheimer’s disease, epilepsy, cerebral ischemia and neurodegenerative disease [2]. The total concentration of zinc lies over a very broad range (in the order of nanomolar to millimolar) [3]. Though zinc is a relatively nontoxic element, it can be toxic if consumed in large enough quantities. For example, zinc is a metal pollutant of environment, significant concentrations of which may reduce the soil microbial activity causing phytotoxic effect [4,5] and which is also a common contaminant in agricultural and food wastes [6]. Thus, the determination of trace amounts of zinc is currently of great interest in many scientific fields, including medicine analyses and environmental monitoring, etc.
∗ Corresponding authors. Tel.: +86 731 8821916; fax: +86 731 8821916. E-mail addresses: [email protected] (X.-B. Zhang), [email protected] (R.-Q. Yu). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.03.023
Though a number of analytical techniques such as inductivity coupled plasma atomic emission spectroscopy (ICP-AES) [7], UV–vis spectroscopy [8], potentiometry [9] and flame atomic absorption spectrometry [10] have been used to detect trace amounts of zinc, the reliable method for zinc assay is still limited owing to its 3d10 4s0 electronic configuration not giving any spectroscopic or magnetic signals. Due to their advantages of simplicity, high sensitivity and low lost, fluorescent sensors have been developed for the determination of zinc ions in the past decades. And several fluorescent indicators for zinc ions have been reported in the previous literature [11–15]. However, from a practical point of view the immobilization of indicator dyes may be required to construct optical chemical sensors (optode). And the immobilization of indicator dyes is probably the most important factor that governs the lifetime of the sensor. Poor immobilization results in leaching and consequently no sensing. Several immobilization methods of indicator dyes have been reported including physical entrapment [16,17], electrostatic association [18,19] and covalent immobilization [20–23]. Though physical entrapment is simple, it has a drawback: the encapsulated neutral carriers are often leached from the membrane into the analyte solution, which makes the resulting sensors less durable and more toxic. The electrostatic immobilization leads to considerable binding strength between the charged dye and the oppositely charged functional group of support matrixes; however, the indicator dye can also be displaced easily
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by the sample ions or solvent molecules. Covalent immobilization might be the most efficient technique for the preparation of optical chemical sensors because it can prevent the dye exude out from the membranes resulting in a long lifetime. However, only very few chemosensors for zinc based on covalently immobilized indicator dye onto the support matrixes have been reported [24]. Searching for zinc chemosensors based on covalently immobilized indicator dyes is still an active field as well as a challenge for the analytical chemistry research effort. The photochemically initiated immobilization of indicator dyes is a relatively simple and efficient method not requiring sophisticated apparatus and with a faster speed compared with thermal polymerization. Shortreed et al. developed a fluorescent fiberoptic calcium sensor based on covalent immobilization of a fluorescent indicator [25]. Immobilization occurs via photoinitiated copolymerization of the indicator with acrylamide. Hisamoto et al. reported the optical chemical sensing of pH and water content in organic solvents based on the immobilization of indicator dyes possessing an olefin unit by UV photopolymerization [26]. Our own group has also reported some work in which indicator dyes were covalently attached onto the activated surface of glass slides by UV irradiation [27–30]. In this paper, we describe a photopolymerization method for immobilizing a benzoxazole derivative 1 for making chemical sensor for Zn2+ . In recent years, the general family of 2-(2 -hydroxyphenyl)benzazoles has been extensively studied from the viewpoint of photophysics because of its dual emission via the excitedstate intramolecular proton transfer (ESIPT) [31–35]. Moreover, some derivatives of 2-(2 -hydroxyphenyl)-benzazole have been used to sense ions as fluorescent indicators [35,36–38]. Recently, Qin et al. reported a fiber-optic fluorescence sensor for Li+ in acetonitrile which is based on the diffusion of the 2-(2 hydroxyphenyl)benzoxazole (HPBO) through a poly(vinyl chloride) (PVC) membrane into the analyte solution [39]. Zhang et al. also developed a highly selective fluorescent sensor for Cu2+ in aqueous solution based on HPBO in a PVC matrix [40]. But the proposed sensors in the literature [39,40] are both based on physical immobilization of HPBO, HPBO is apt to exude out from the membrane resulting in less durability. In a recent study, Crivat et al. proposed a zinc sensor based on indicator dye conjugated to the active surface of glass slides and the operating principle was a simple enhancement of fluorescent intensity [24]. However, in most practical applications, fluorescence intensity changes can also be caused by many other poorly quantified or variable factors such as the excitation intensity, dye concentration, and environment around the dye (pH, polarity, temperature, and so forth), this problem can be solved by using ratiometric chemosensors that exhibit a spectral shift upon binding of the cation [41]. Hence, in this work we developed a zinc(II)-selective ratiometric fluorescent sensor based on covalently immobilized derivative 1 of benzoxazole. To prepare the sensor, the zinc ion indicator HPBO was modified to include a terminal double bond and covalently immobilized on the activated surface of glass slides by UV irradiation. In the absence of Zn2+ , the sensor undergoes ESPIT process and coordination of Zn2+ inhibits the ESPIT yielding a 46 nm blue-shift of fluorescence emission. The proposed sensor exhibits excellent analytical characteristics including nice selectivity, good reproducibility and fast response time for the determination of Zn2+ in aqueous solution. Moreover, Cd2+ did not affect the detection of Zn2+ though it has similar chemical properties with Zn2+ . Covalent immobilization of the derivative 1 of benzoxazole lengthens the lifetime of the sensor. The feasibility of using the proposed sensor for the determination of Zn2+ in both tap and river water samples has been testified.
2. Experimental 2.1. Reagents and chemicals 2,4-Dihydroxybenzaldehyde (98%) and 3-(trimethoxysilyl) propyl methacrylate (TSPM) (98%) were purchased from Acros Organics. Barium manganate (90%) was acquired from Alfa Aesar. 2-Hydroxyethyl methacrylate (HEMA), benzoin ethyl ether, benzophenone, and triethanolamine used in the membrane matrix preparation were obtained from Shanghai Chemicals. Doubly distilled water was used throughout all experiments. Before being used, toluene was subjected to simple distillation at atmospheric pressure from sodium and stored over 4 Å molecular sieves. Except when specified, other chemicals were of analytical reagent grade and used without further purification or treatment. A stock solution of 1 × 10−2 mol/L Zn2+ was prepared by dissolving Zn(OAc)2 ·2H2 O in doubly distilled water. The stock solution of Zn2+ was diluted to lower concentrations of 4 × 10−3 to 1 × 10−5 mol/L stepwise. The wide pH range solutions were prepared by adjustment of 0.05 mol/L Tris–HCl solution with HCl or NaOH solution. 2.2. Apparatus All fluorescence measurements were taken on a Hitachi F4500 Fluorescence Spectrometer (Tokyo, Japan) with excitation slit set at 5.0 nm and emission at 5.0 nm. The light source is a 150 W Xe lamp and the detector is a R928F red-sensitive photomultiplier tube. All fluorescence measurements were performed under ambient temperature at 25 ◦ C. The measurements of pH were carried out on a Mettler-Toledo Delta 320 pH meter (Shanghai, China). A 50 W ultraviolet lamp at 253.7 nm (model ZF-2, Shanghai Analytical Instruments, Shanghai, China) was used for photopolymerization. 1 H NMR spectra were recorded on an INOVE400 (Varian) spectrometer. MS spectra were obtained on a GC-17A, QP-5000 (Shimadzu) spectrometer. Data processing was performed on a Pentium IV computer with software of SigmaPlot. 2.3. Synthesis of fluorescence carrier The synthetic procedure for fluorescence carrier 4-benzoxazol2 -yl-3-hydroxyphenyl allyl ether (1) is shown in Scheme 1. Synthesis of 4-formyl-3-hydroxyphenyl allyl ether (2). To a mixture of 2,4-dihydroxybenzaldehyde (1 g, 7.2 mmol) and NaHCO3 (0.6125 g, 7.2 mmol) in 20 mL of N,N-dimethylformamide (DMF) was added dropwise 0.625 mL (7.2 mmol) of allyl bromide and reaction mixture was heated at 70 ◦ C for 48 h. After the solvent was evaporated under reduced pressure, 50 mL of H2 O was added to the mixture. The resulting precipitate was filtered and dissolved in 25 mL of CH2 Cl2 . The organic layer was dried over Na2 SO4 (anhydrous), filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using petroleum ether/dichloromethane (7:2, V/V) as eluent to afford compound 2 as a yellow oil. Yield: 0.8339 g (65%). MS (EI) m/z: 178.0. Synthesis of 3-hydroxy-4-[(2 -hydroxy-phenylimino)-methyl] phenyl allyl ether (3). A mixture of compound 2 (0.1782 g, 1 mmol) and 2-aminophenol (0.1091 g, 1 mmol) in 25 mL of EtOH was heated under reflux for 18 h. The reaction mixture was cooled to room temperature and concentrated in vacuo. The yellow solid residue was recrystallized from ether providing Schiff base 3 as a yellow solid. Yield: 0.1880 g (70%). Synthesis of 4-benzoxazol-2 -yl-3-hydroxyphenyl allyl ether (1). A mixture of compound 3 (0.1346 g, 0.5 mmol) and BaMnO4 (0.5125 g, 2 mmol) in 15 mL of toluene was heated at 80 ◦ C for 24 h. The reaction mixture was cooled to room temperature and
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Scheme 1. Synthesis of 4-benzoxazol-2 -yl-3-hydroxyphenyl allyl ether (1). (a) Allyl bromide, NaHCO3 , N,N-dimethylformamide, 70 ◦ C, 48 h, 65%; (b) 2-aminophenol, ethanol, reflux, 18 h, 70%; (c) BaMnO4 , toluene, 80 ◦ C, 24 h, 18%.
filtered. The filtrate was concentrated in vacuo affording a black residue, which was purified by silica gel column chromatography using petroleum ether/dichloromethane (8:1, V/V) as eluent to provide compound 1 as a white solid. Yield: 0.0241 g (18 %). 1 H NMR (400 MHz, CDCl3 ), ı (ppm): 4.588 (d, 2H, J = 6.8 Hz, –OCH2 –), 5.328 (dd, 1H, J = 1.2 Hz, 1.6 Hz,
), 5.447 (dd, 1H, J = 1.2 Hz, 1.2 Hz,
), 6.019–6.115 (m, 1H, CH–), 6.591 (dd, 1H, J = 1.6 Hz, 8.8 Hz, 6-H), 6.63 (d, 1H, J = 1.6 Hz, 2-H), 7.308–7.375 (m, 2H, 5-H, 4 -H), 7.554–7.577 (m, 1H, 5 -H), 7.665–7.687 (m, 1H, 6 -H), 7.910 (d, 1H, J = 8.4 Hz, 7 -H), 11.625 (s, 1H, OH). MS (EI) m/z: 267.0. 2.4. Silanization of the glass slides The microscope glass slides were cut to dimension of approximately 13 mm × 13 mm and the silanization was carried out as reported [24,30,42,43] with some modifications. First, the glass slides were cleaned with piranha solution (98% H2 SO4 :30% H2 O2 = 7:3, by volume) heated at 70 ◦ C for 20 min to remove particulate matter and organic impurities. These slides were rinsed with deionized water for several times and dried by nitrogen flow. Second, the glass slides were immersed in 1 M NaOH solution for 20 min at 80 ◦ C, thoroughly washed with distilled water and spectrophotometric grade CH3 OH and dried under nitrogen. The glass surface treatment with NaOH solution resulted in a high concentration of hydroxyl group and facilitated the subsequent reaction with TSPM. Finally, a solution of TSPM was prepared by mixing 0.2 mL of TSPM, 2 mL of 0.2 mol/L HAc–NaAc (pH 3.6), and 8 mL of water. The silanization reaction was implemented by submerging the treated glass slides into the TSPM solution for 2 h at room temperature. The silanized slides were subsequently rinsed with distilled water and dried in a nitrogen atmosphere. 2.5. Preparation of the optode membrane The optode membrane solution was prepared by mixing 2.9 mL of HEMA, 10 mg of compound 1, 45 mg of benzoin ethyl ether, 0.12 g benzophenone, and 0.2 mL of triethanolamine, then the solution was cast onto a dust-free poly(tetrafluoroethylene) (PTFE) plate. Silanized glass plates were placed over the droplets, and
UV radiation (253.7 nm, 50 W) was directed from 10 cm onto the membrane matrixes for about 1 h in a nitrogen atmosphere. After UV irradiation, the glass plates with the polymer membrane were completely washed with distilled water and methanol to take off any unreacted species using a supersonic cleaner until no leaching of the compound 1 was observed. The optode membranes prepared as above were dried and stored in a refrigerator until used. 2.6. Fluorescence measurements The fluorescence intensity of optode membrane was measured at excitation wavelength of 323 nm with the emission wavelength varied over 340–620 nm. The glass plate with the sensing membrane was fixed in a quartz cuvette which was filled with the sample solution. The sensor membrane was equilibrated with the sample solution to obtain a stable fluorescence signal. After each measurement, the fluorescence intensity of the sensing membrane was recovered by adding the blank solution through the quartz cuvette prior to the next measurement. 3. Results and discussion 3.1. Synthesis of fluorescent carrier 1 and its covalent immobilization on sensor surface In order to covalently immobilize HPBO on the glass surface, it is an alternative to introduce a polymerizable group such as a double bond in the compound HPBO and in the quartz glass surface, respectively. In our work, allyl bromide was used to react with 2,4dihydroxybenzaldehyde and the hydrogen of 4-position hydroxyl was replaced by allyl group. Consequently, the fluorescent carrier 1 made from compound 2 via condensation and oxidize in two steps includes a terminal double. TSPM was taken as the pre-treatment agent to introduce a polymerizable vinyl group onto cleaned quartz glass plate surface. Under UV radiation, compound 1 with a terminal double bond reacts with the surface double bond of the modified glass slides. The photopolymerization was initiated by using benzoin ethyl ether and benzophenone as photo initiators possessing a synergistic action. And in this case HEMA and compound 1 were used as the monomer. Triethanolamine was used to stop oxygen’s inhibition during the membrane curing process. In order to avoid
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Fig. 1. Fluorescence emission spectra of the optode membrane exposed to various concentration of Zn2+ : 0, 8 × 10−5 , 2 × 10−4 , 6 × 10−4 , 8 × 10−4 , 10−3 , 2 × 10−3 , 4 × 10−3 mol/L from a to h. These spectra were measured in 0.05 M Tris–HCl buffer (pH 7.24). The excitation wavelength was 323 nm.
the formed sensing membrane peeling off the quartz surface, the time of UV irradiation should be kept not shorter than 1 h. 3.2. Spectral characteristics Fig. 1 shows the fluorescence emission spectra of the optode membrane exposed to a solution containing various concentration of Zn2+ , which were recorded with ex = 323 nm in 0.05 M Tris–HCl buffer (pH 7.24). As can be seen from Fig. 1, the fluorescence emission spectra of optode membrane are sensitive to Zn2+ . With the stepwise addition of Zn2+ to optode membrane, the fluorescence emission of optode membrane at 404-nm bands increases while that at 450 nm decreases. An isoemission point appears at near 433 nm. In the presence of excess Zn2+ , a 46 nm blue-shift of flu-
Fig. 2. Fluorescence ratio I404 nm /I450 nm of optode membrane as a function of Zn2+ concentration in 0.05 M Tris–HCl buffer (pH 7.24). The excitation wavelength was 323 nm.
orescence emission from 450 to 404 nm was observed. From Fig. 1, one can see that the emission intensity ratio at 404 and 450 nm of the optode membrane enhanced with the increasing zinc concentration, which constituted the basis for the determination of zinc concentration with the optical sensor by the fluorescence ratiometric method. In 0.05 M Tris–HCl buffer solution (pH 7.24), the emission spectrum of the optode membrane exhibits two maxima at 362 and 450 nm, respectively. Possible mechanism accounting for the phenomenon is ESIPT process [35], in which the largely Stokes-shifted band at 450 nm can be attributed to ESIPT emission of the keto tautomer and the intensity of short wavelength fluorescence at 362 nm is the normal emission intensity (Scheme 2). In the presence of excess Zn2+ the proton transfer process is disrupted by the coordinated zinc ion, resulting in the tautomer emission at 450 nm
Scheme 2. Excited-state intramolecular proton transfer (ESIPT) process for the proposed sensor.
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Fig. 3. Effect of pH on I404 nm /I450 nm of the optode membrane in the absence (closed circles) or presence (open circles) of 6.0 × 10−4 mol/L Zn2+ . All data were obtained at various pH values (pH 2.00–9.19) and the excitation wavelength was 323 nm.
disappearing and the maximum emission at 404 nm appearing (Scheme 2). The maximum emission at 404 nm upon completely coordinating with Zn2+ possibly arises from the normal emission of the zinc-complex. 3.3. Principle of operation and the basis of quantitative assay Fig. 2 shows the dependence of emission intensity ratios between 404 and 450 nm (I404 nm /I450 nm ) on the concentration of Zn2+ in 0.05 M Tris–HCl buffer solution (pH 7.24). As shown in Fig. 2, I404 nm /I450 nm increased linearly with the concentration of Zn2+ . The linear response of I404 nm /I450 nm toward log CZn 2+ was obtained in zinc concentration range of 8.0 × 10−5 to 4.0 × 10−3 mol/L, which provided the quantitative base for the determination of zinc concentration. The ratiometric calibration line can be expressed by the following equation: I404 nm = 4.1700 + 0.9158 log CZn 2+ (R = 0.9872) I450 nm
(1)
Here I404 nm and I450 nm are the fluorescence emission intensity of optode membrane actually measured at a given metal concentration at 404 and 450 nm, respectively. CZn 2+ represents the concentration of zinc ion added. The detection limit obtained for Zn2+ was 4.0 × 10−5 mol/L (defined as three times standard deviation of blank solution). The curve fitting of relative fluorescence intensity supported the formation of a 1:1 complex of the benzoxazole derivative in the optode membrane and zinc cation in 0.05 M Tris–HCl buffer solution (pH 7.24), binding constant being estimated to be K = 3.0 × 103 M−1 according to the literature [41,44]. 3.4. Effect of pH The effects of pH on the fluorescence intensity of optode membrane in the absence or presence of 6 × 10−4 mol/L Zn2+ were examined at a pH range from 2.00 to 9.19 (Fig. 3). As can be seen from Fig. 3, the optode membrane shows good fluorescence response to Zn2+ over a wide pH range. The optode membrane has no noticeable fluorescence sensing ability to Zn2+ at pH value lower than 3.73, which might be assigned to the complete protonation of phenolic oxygen of the optode membrane when pH values of solution lower than 3.73 resulting in a no-coordination ability of Zn2+ [35]. However, the optode membrane shows satisfactory Zn2+ -sensing ability in the range of pH from 3.73 to 9.19. This phenomenon might be caused by deprotonation of the hydroxyl group of receptor in
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Fig. 4. Metal ion selectivity of optode membrane. All data were obtained at pH 7.24, (0.05 M Tris/HCl) and were expressed as the fluorescence ratio (I404 nm /I450 nm ). The excitation wavelength was 323 nm. The concentration of ions added to optode membrane was 6.0 × 10−4 M for Zn2+ , Ag+ and Pb2+ , and 10−3 M for all remaining ions. Black bars: different metal ions were added. Gray bars: different metal ions in the presence of Zn2+ were added.
the region of higher pH value resulting in the formation of zinccomplex [35]. In the presence of 6.0 × 10−4 mol/L Zn2+ I404 nm /I450 nm of the optode membrane decreased with reducing pH value at pH 8.01–9.19, which might be ascribed to the formation of partial precipitation of Zn(OH)2 under strong basic condition resulting in the decrease of actual concentration of Zn2+ in the sample solution. Taking into consideration the sensitivity, response speed and application in real samples, a 0.05 M Tris/HCl buffer solution at pH 7.24 was chosen as optimum experimental condition. 3.5. Selectivity The fluorescence responses of optode membrane to various cations and its selectivity for Zn2+ are illustrated in Fig. 4. The concentration of Zn2+ was fixed at 6.0 × 10−4 mol/L in 0.05 M Tris–HCl buffer solution (pH 7.24). Cations were added as chlorides, nitrates, acetate and sulfates. As can be seen from the black bars in Fig. 4, I404 nm /I450 nm significantly enhanced upon the addition of Zn2+ and slightly increased upon binding to Cu2+ , Co2+ and Ni2+ . Moreover, the addition of other cations did not affect I404 nm /I450 nm of the optode membrane. Cu2+ , Co2+ and Ni2+ cannot shift the spectrum of optode membrane in the same way as Zn2+ , but they quenched the emission intensity at both 405 and 450 nm resulting in slight enhancement of I404 nm /I450 nm . Though Cd2+ has similar chemical properties compared with Zn2+ , the optode membrane shows no response to Cd2+ . In order to further test the interference for other common cations on the determination of Zn2+ , a competition experiment was performed in which a zinc solution in the presence of other metal ions was added to the fluorescence cuvette with optode membrane (white bars in Fig. 4). The competition experiments show no significant variation in the emission intensity ratio (I404 nm /I450 nm ) except Cu2+ , Co2+ and Ni2+ . Thus, the proposed sensor in this paper exhibits good selectivity for Zn2+ over other common cations except Cu2+ , Co2+ and Ni2+ . Fortunately, these three cations would have little influence in living system, since they exist at very low concentrations [45]. In environmental analysis, the possible interference of Cu2+ , Co2+ and Ni2+ on the determination of Zn2+ could be effectively circumvented by adding an appropriate concentration of Na2 S2 O3 solution. Thus, the proposed sensor in this paper could be useful for biological and environmental applications. Singh et al. reported a chemosensor for Cu2+ and Fe3+ based on Quantum Dot-Schiff base conjugate but the Schiff base itself
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3.6. Reproducibility, reversibility and response time
Fig. 5. Fluorescence emission spectra of monomer 1 (1.0 × 10−5 mol/L) in the presence of different metal ions. The concentration of ions added to monomer 1 was 0.1 mM for Zn2+ , Ag+ and Pb2+ , and 1 mM for all remaining ions. Measured at 25 ◦ C in CH3 CN–H2 O (1:1, V/V) and 0.05 M Tris–HCl buffer (pH 7.24). The excitation wavelength was 323 nm.
The reproducibility and reversibility of optode membrane were studied by exposing the sensor to a 0.05 M Tris–HCl buffer solution of pH 7.24, 6 × 10−4 and 2 × 10−3 mol/L zinc solutions in turn. Fig. 6 exhibits the emission intensity ratios between 404 and 450 nm (I404 nm /I450 nm ) upon switching from one solution to another. The relative standard deviations in I404 nm /I450 nm calculated from five measurements of 6 × 10−4 and 2 × 10−3 mol/L zinc solutions were found to be 1.34 and 1.02%, respectively. The relative standard deviations in I404 nm /I450 nm from ten measurements of blank buffer solution is estimated as 0.16%. All these results show satisfactory reproducibility and reversibility of the optode membrane. The response time of the optode membrane depends on the thickness of membrane and the zinc concentration. The thickness of the optode membrane, fabricated as described above, is about 50 m. The response time is 2 and 3 min for 6 × 10−4 and 2 × 10−3 mol/L zinc solutions, respectively. Moreover, it was found that the recovering time actually remained the same in despite of switching from low to high or the opposite. The recovering time was about 80 s. 3.7. Short-time stability and lifetime The short-time stability of the sensor was tested by exposing the sensor to the 6 × 10−4 mol/L zinc solution in a period of 7 h. The fluorescence emission spectrum was recorded at interval of 30 min. The relative standard error of 1.47% for I404 nm /I450 nm was obtained for this solution. The fluorescence response remains stable even after 2 months’ usage. Covalent immobilization effectively prevented leaching of the dye from the optode membrane into solution and remarkably improved the lifetime of sensor. A newly prepared sensor can be used at least 3 months. 3.8. Preliminary analytical application
Fig. 6. Reproducibility and reversibility for the proposed sensor. The measurement was carried out by exposing the sensor to a blank buffer solution of pH 7.24 and zinc solutions of different concentration in turn: (a) blank solution; (b) 6 × 10−4 mol/L Zn2+ ; (c) 2 × 10−3 mol/L Zn2+ . The excitation wavelength was 323 nm.
displayed no selectivity [46]. Thus, the selectivity of monomer 1 in CH3 CN–H2 O (1: 1, V/V) solution was investigated by titration of monomer 1 with various metal ions as a control experiment in this work (Fig. 5). As shown in Fig. 5, monomer 1 shows good selectivity for Zn2+ over other common cations except Fe3+ , Cu2+ , Co2+ and Ni2+ . Therefore, the immobilization of monomer 1 on an activated surface of glass slides did not alter its selectivity in this study.
In order to examine the applicability of the proposed method in practical sample analysis, the sensor was applied in the determination of zinc ion in both tap and river water samples. The river water samples obtained from Xiang River were simply filtrated. And the tap and river water samples showed that no zinc ion was present in them. All these water samples were spiked with standard Zn2+ solutions at different concentration levels and then analyzed with proposed sensor. Results are shown in Table 1. One can see that the recovery study of spiked Zn2+ determined by the sensor shows satisfactory results. The present sensor seems useful for the determination of Zn2+ in real samples. 4. Conclusion In summary, a ratiometric fluorescent sensor for zinc ions based on covalently immobilized derivative of benzoxazole has been developed. During fabricating the sensor, a new dye monomer 1 with a terminal double bond was copolymerized with 2-
Table 1 Determination of Zn2+ in tap and river water samples with the sensor. Sample
Zn2+ spiked (mol/L)
Recovery (%) – 103.0 98.0 – 104.6 102.0
Tap water
1 2 3
8 × 10−5 1 × 10−4
Not detected (8.25a ± 0.24b ) × 10−5 (0.98a ± 0.01b ) × 10−4
River water
1 2 3
0 8 × 10−5 −4 1 × 10
Not detected (8.37a ± 0.11b ) × 10−5 (1.02a ± 0.02b ) × 10−4
a b
Mean values of the three determinations. Standard deviation.
0
Zn2+ recovered (mol/L)
Q.-J. Ma et al. / Spectrochimica Acta Part A 73 (2009) 687–693
hydroxyethyl methacrylate (HEMA) by UV photopolymerization and covalently immobilized on the surface of the modified quartz glass plate. The sensor shows remarkable analytical characteristics including fast response and recovery time, sufficient reproducibility, high sensitivity and selectivity for the determination of zinc in aqueous media. Moreover, its lifetime was also satisfactory since the leaching of dye from optode membrane was prevented by covalent immobilization. The fluorescence emission of the sensor at 404-nm bands increases while that at 450-nm decreases upon the addition of zinc and emission intensity ratios between 404 and 450 nm (I404 nm /I450 nm ) are selective for Zn2+ . The proposed sensor can be applied to the quantification of Zn2+ with a linear range covering from 8.0 × 10−5 to 4.0 × 10−3 mol/L and the detection limit is 4.0 × 10−5 mol/L. The proposed sensor has been used for the determination of Zn2+ in both tap and river water samples and shows satisfactory results. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos. 20505008, 20675028, 20775023 and 20435010), “973” National Key Basic Research Program of China (2007CB310500), and Hunan Natural Science Foundation (07JJ3025). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
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