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Hydrothermal synthesis of functionalized CdS nanoparticles and their application as fluorescence probes in the determination of uracil and thymine
Journal of Luminescence 132 (2012) 244–249
Contents lists available at SciVerse ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Hydrothermal synthesis of functionalized CdS nanoparticles and their application as fluorescence probes in the determination of uracil and thymine Yaxiang Lu a, Li Li a, Yaping Ding a,n, Fenfen Zhang a, Yaping Wang a, Weijun Yu b a b
Department of Chemistry, Shanghai University, Shanghai 200444, PR China Instrumental Analysis and Research Center, Shanghai University, Shanghai 200444, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2011 Received in revised form 6 August 2011 Accepted 12 August 2011 Available online 22 August 2011
A novel, sensitive, and convenient method for the determination of uracil and thymine by functionalized CdS nanoparticles (NPs) was proposed. CdS NPs were prepared by hydrothermal process and modified with thioglycollic acid (TGA) in aqueous solution. The fluorescence intensity of functionalized CdS NPs was quenched in the presence of uracil or thymine. Under optimal conditions, the relative fluorescence intensity (F0/F) was proportional to the concentration in the range of 9.0 10 6– 1.0 10 4 mol/L for uracil (r¼ 0.9985) and 8.8 10 7–1.5 10 4 mol/L for thymine (r ¼0.9960). The corresponding detection limits were 9.6 10 7 mol/L and 3.2 10 7 mol/L, respectively. In addition, the possible quenching mechanism was also discussed. Crown Copyright & 2011 Published by Elsevier B.V. All rights reserved.
Keywords: CdS nanoparticles Fluorescence probe Uracil Thymine
1. Introduction Semiconductor nanoparticles (NPs) or quantum dots (QDs) are nanoscale spherical particles with size-dependent tunable luminescent emission [1]. The size dependence of their properties results from quantum confinement of electron and hole carriers at dimensions smaller than the bulk Bohr exciton radius [2]. Owing to the quantum confinement effects, they are endowed with unique optical and electronic properties unlike the bulk materials, such as broad excitation spectra, high excitation cross-sections, excellent resistance to photobleaching, and narrow and symmetric emission bands, which can span the light spectrum from the ultraviolet to the infrared region [3–5]. In general, the key to develop QDs as tools in biological systems is to achieve their water solubility, biocompatibility, and photostability [6]. The surface modified quantum dots possess these characters, which are usually capped with some appropriate hydrophilic functional reagents used as modifying agents, such as L-cysteine, thioglycollic acid etc. [7–9]. Since the first demonstration of water soluble QDs as bioassay fluorophores, they have been widely used in bioanalytics such as bioimaging, biolabelling, biodetection, and clinical diagnosis [10–12].
n
Corresponding author. Tel.: þ86 021 66134734; fax: þ86 21 66132797. E-mail address: [email protected] (Y. Ding).
Uracil and thymine are important bases of RNA and DNA, respectively. They play vital roles in the storage of genetic information and protein biosynthesis [13]. The abnormal changes of these bases in organisms suggest the deficiency and mutation of the immunity system and may indicate the presence of various diseases [13,14]. Hence, the quantitative analysis of uracil and thymine is very essential and has great significance to bioscience and clinical iatrology. There have been many methods reported to detect uracil and thymine, such as gas chromatography tandem mass spectrometry [15], high-performance liquid chromatography [16–18], capillary electrophoresis [19], monoclonal antibodies [20], capillary zone electrophoresis [21], electrochemical analysis [22], fluorescence spectroscopy [23], etc. However, these methods mentioned above have some drawbacks such as environment unfriendly solvents, long analysis time, low sensitivity, limits of analysts, expensive instrumentation, and the tedious procedures [14,24]. Compared with the above-mentioned methods, the fluorescence probes method is obviously simpler, faster in the experimental processes, and more sensitive for different fluorescence emission wavelengths corresponding to different molecular structures. In this paper, CdS NPs were synthesized with hydrothermal synthesis and functionalized with TGA to obtain CdS fluorescence probes. The combination of functionalized CdS NPs with different biomolecules could make some difference in fluorescence intensity and/or fluorescence emission wavelength between each other. Under optimum conditions, the fluorescence of functionalized CdS
0022-2313/$ - see front matter Crown Copyright & 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2011.08.022
Y. Lu et al. / Journal of Luminescence 132 (2012) 244–249
245
NPs could be quenched by uracil and thymine, respectively. The quenching extent of the fluorescence intensity was proportional to the concentrations of the two pyrimidines. Based on this observation, a simple, convenient, and sensitive fluorescence probes method for the quantitative determination of uracil and thymine has been established.
2. Experimental section
3. Results and discussion 3.1. TEM and XRD images of the prepared CdS nanoparticles The morphology and dimension size distribution of CdS NPs were studied by transmission electron microscopy (TEM) as shown in Fig. 1. The typical TEM image (Fig. 1a) of CdS NPs exhibits that these NPs are monodispersed, and the shape of them is close to regular spherical NPs. The histogram of NPs dimension size is
80%
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Cumulative distribution curve of particles number percentage
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Equivalent circle diameter / nm Fig. 1. (a) TEM image of CdS NPs and (b) particle size of histograms of CdS NPs.
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2.3. Determination of uracil and thymine with functionalized nano-CdS fluorescence probes
[111]
200
Intensity (a.u.)
For determining the concentration of uracil and thymine, the resulting solutions were used to monitor the change of fluorescence emission intensity by means of spectrofluorometer. 1 mL of the as-prepared functionalized CdS NPs solution and a series of known volumes of uracil or thymine standard solutions were added into a set of dry 25 mL calibrated flasks. The solutions were diluted to the volume with phosphate buffer solution (PBS) and mixed thoroughly. The mixtures were kept for a while in the dark prior to making the fluorescence measurements at room temperature. For emission measurements the excitation wavelength was fixed at 263 nm, and the fluorescence intensity was measured at 291 nm for uracil and 289 nm for thymine, respectively. The excitation and emission slit widths were 5 nm each. The scanning bound was from 273 to 510 nm.
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7
The prepared CdS NPs were water insoluble and bio-incompatible, so they were unable to react with biomolecules. To overcome these shortcomings, TGA was chosen to modify CdS NPs for the preparation of TGA-capped-CdS fluorescence probes. The procedure was as follows: briefly, at room temperature (around 25 1C), under vigorous stirring, 0.01 g CdS NPs were added into 150 mL NaOH solution (pH 12.0). Afterwards, the pH of the mixture solution was adjusted to 7.0–8.0 with 0.10 mol/L TGA solution, then the colloidal solution was kept in a dark place for one night. Finally, the functionalized NPs solution was taken into a 500 mL brown jar and stored at room temperature. The obtained solution was ready for the following experiments.
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2.2. The preparation of TGA-capped-CdS fluorescence probes
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In a typical procedure, a mixture of 0.2280 g CdCl2 2.5H2O and 1.000 g Na2S2O3 5H2O was put into a Teflon-lined autoclave with 30 mL capacity. Then 20 mL NH3 H2O (3.0 mol/L) solution was added into the autoclave as the stabilizing agent. The autoclave was sealed, maintained at 180 1C for 24 h. After the reaction, the autoclave was cooled down to room temperature naturally. Finally, the obtained orange precipitates were collected by centrifugation, washed under ultrasonic vibration several times with doubly distilled water and ethanol. The final products were dried at 80 1C in vacuum to obtain CdS NPs.
The number percentage of CdS nanoparticles
2.1. Synthesis of CdS nanoparticles
[220]
150 [311]
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[103]
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0 20
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50 2θ / degree
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Fig. 2. XRD pattern of the as-prepared CdS nanoparticles.
shown in Fig. 1b, indicating the diameters ranging from 4 nm to 16 nm, and the average particle diameter of 8.7 nm. The powder X-ray diffraction spectrometry (XRD) pattern in Fig. 2 reveals that the CdS NPs are crystals of hexagonal wurtzite structure, which is consistent with the JCPDS card number 41-1049. 3.2. Fluorescence spectra of unfunctionalized and functionalized CdS nanoparticles The fluorescence spectra of CdS NPs and functionalized CdS NPs are shown in Fig. 3. It can be seen that the fluorescence
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Y. Lu et al. / Journal of Luminescence 132 (2012) 244–249
150 a
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3.5. Effect of uracil and thymine concentrations
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b
0 250
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350 400 450 Wavelength (nm)
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Fig. 3. Fluorescence spectra of (a) CdS nanoparticles and (b) functionalized CdS nanoparticles.
Fluorescence Intensity
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A series of uracil or thymine solutions were added into the functionalized CdS NPs system, respectively. Changes in the fluorescence emission spectra are given in Fig. 6. It is observed that the obtained graphs of fluorescence intensity are decreased with the increasing concentration of uracil or thymine with no spectra shift. The observed fluorescence band centers at 291 nm for uracil and centers at 289 nm for thymine. Considering this remarkable quenching of fluorescence intensity, the possibility of developing a sensitive method for quantitative assay of uracil and thymine based on spectrofluorometry has been evaluated.
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3.6. Effect of foreign substances
140
In order to assess the selectivity of the proposed method, the tolerance levels of coexisting foreign substances was tested. Each foreign substance with various concentrations was mixed with 5.0 10 5 mol/L uracil or thymine prior to the detection. The tolerance concentration of some metal ions and several biomolecules for the determination is summarized in Table 1. The results show that most of the diverse substances have no obvious interference in the determination, which indicates that the method has a high selectivity. So it is possible to use this method to detect uracil and thymine in human or animal body fluid samples.
120 100 80 60
TGA-CdS TGA-CdS-Uracil
40 20
TGA-CdS-Thymine
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7 pH
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Fig. 4. Effect of pH on the fluorescence intensity of TGA-CdS nanoparticles in the absence and presence of uracil or thymine. Error bars represent standard deviation of three measurements at each pH.
intensity of the functionalized CdS NPs is slightly decreased. However, the fluorescence spectrum of functionalized CdS NPs is narrower and more symmetric than that of CdS NPs. It is also shown that the fluorescence of CdS NPs in the range from 300 to 510 nm is completely quenched and the fluorescence emission peak of functionalized NPs is red shifted compared with unfunctionalized NPs. 3.3. Effect of pH value The effect of pH in a range between 4.0 and 11.0 was studied in order to select the optimum condition for the determination of uracil and thymine with the functionalized CdS NPs. PBS (0.1 mol/L) was used as the buffer to adjust the acidity of the aqueous medium. As shown in Fig. 4, it can be seen clearly that the maximum fluorescence intensity of TGA-CdS NPs occurs when pH is 7.0, and the optimal pH is also 7.0 for both TGA-CdS–uracil and TGA-CdS–thymine systems. Therefore, the pH of 7.0 was chosen to run the assay. 3.4. Effect of reaction time and temperature The reaction time influences the fluorescence intensity of the system. Fluorescence spectrum was recorded at different time
3.7. Linearity and sensitivity Under optimal conditions mentioned above, the linear calibration equations and limits of detection for uracil and thymine were obtained. As shown in Fig. 7, there are good linear relationships between relative fluorescence intensity (DF ¼F0/F) and the concentrations (C) of uracil and thymine. The dynamic range of uracil is 9.0 10 6–1.0 10 4 mol/L with the correlation coefficient (r) 120 115 Fluorescence Intensity
Fluorescence Intensity
intervals after the addition of uracil or thymine into the solution of functionalized CdS NPs. The result (Fig. 5) shows that the two systems’ fluorescence intensity drops at first, then increases, finally drops again with increase in reaction time. The maximum fluorescence intensity is obtained when the reaction time is 3 h for uracil and 4 h for thymine. It was noted that temperature, as expected, had an effect on the fluorescence intensity of the TGA capped CdS NPs. Therefore, all analytical studies were conducted at the room temperature (25 1C).
CdS nanoparticles functionalized CdS nanoparticles
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Fig. 5. Effect of reaction time on the fluorescence intensity of TGA-CdS–uracil and TGA-CdS–thymine systems. Error bars represent standard deviation of three measurements at each time point.
Y. Lu et al. / Journal of Luminescence 132 (2012) 244–249
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Fig. 6. Fluorescence emission spectra of (a) TGA-CdS–uracil and (b) TGA-CdS– thymine systems obtained in PBS (pH¼ 7.0) with excitation wavelength at 263 nm. Curacil (10 5 mol/L): (1) 0; (2) 0.3; (3) 0.9; (4) 3.0; (5) 5.0; (6) 7.0; (7) 9.0; (8) 10.0. Cthymine (10–5 mol/L): (1) 0; (2) 0.088; (3) 0.7; (4) 0.9; (5) 3.0; (6) 7.0; (7) 10.0; (8) 15.0.
Fig. 7. Linear graph of the relative fluorescence intensity (F0/F) versus the concentrations (C) of (a) uracil and (b) thymine.
Table 1 Test for the interference of coexisting substances. Coexisting substance
Coexisting concentration with uracil (10 7 mol/L)
Change in fluorescence intensity to uracil (%)
Coexisting concentration with thymine (10 7 mol/L)
Change in fluorescence intensity to thymine (%)
K þ , Cl NH4þ , Cl Ca2 þ , Cl Mg2 þ , SO24 Al3 þ , NO3 Zn2 þ , SO24 Glucose Citric acid Uric acid Ascorbic acid Dopamine Adenine Guanine L-alanine L-cysteine L-leucine L-tryptophan L-glutamate
1600 1000 600 100 100 300 600 200 300 500 500 150 150 1000 500 150 1000 500
2.03 3.64 5.09 þ3.01 3.39 2.27 5.83 2.59 1.72 4.20 þ3.37 1.50 4.91 þ1.33 3.37 2.67 þ2.61 þ2.95
1600 1000 600 100 100 300 600 200 500 500 500 150 100 1000 500 150 1000 1000
0.25 þ1.17 3.38 þ 0.26 2.64 3.07 0.52 1.00 2.21 4.50 2.47 1.39 3.72 4.55 þ1.01 2.46 2.24 2.04
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of 0.9985, and the dynamic range of thymine is 8.8 10 7– 1.5 10 4 mol/L with the correlation coefficient (r) of 0.9960. The fluorescence quenching of functionalized CdS NPs as a function of uracil or thymine concentrations can match the Stern–Volmer equation. The equation could be drawn as follows:
288 K 298 K 318 K
2.0
ð1Þ
F0 and F are the fluorescence intensities in the absence and presence of quencher concentration solution, respectively. C is the concentration of quencher and KSV is the Stern–Volmer quenching constant. The calibration curve established the following equation, DF ¼0.939þ0.148C (C, 10 5 mol/L) for uracil and DF ¼0.915þ0.190C (C, 10 5 mol/L) for thymine. The detection limit (3s/k) is 9.6 10 7 mol/L for uracil and 3.2 10 7 mol/L for thymine, where s is the standard deviation of blank measurements (n¼10) and k is the slope of calibration line. From the dynamic ranges and detection limits of uracil and thymine, it is very clear that this method is sensitive.
1.8 F0 / F
F0 =F ¼ 1 þKSV C
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2
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8
Concentration (10-5 mol/L) 3.8. Quenching mechanism
1.8
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A highly sensitive, selective and convenient technique for uracil and thymine analysis has been developed based on the quenching of the fluorescence of TGA-CdS NPs. CdS NPs were successfully synthesized through hydrothermal process and modified with thioglycollic acid. The fluorescence intensity of the
3
4
5
6
7
8
Fig. 8. Stern–Volmer plots of (a) uracil and (b) thymine with CdS nanoparticles at different temperatures.
Table 2 Spiked recoveries of uracil and thymine in human serum samples (n¼3). Pyrimidine
4. Conclusion
2
Concentration (10-5 mol/L)
3.9. Analytical applications To confirm the feasibility of the proposed method for real sample determination, the present method was applied to determine uracil and thymine in human serum using standard addition method. The detailed results are listed in Table 2. From Table 2, it can be seen that the values found from the real samples are identical with the expected. Moreover, relative standard deviations (R.S.D.) are lower than 4%, showing a good precision on this method, and the average recoveries are in an acceptable range of 90.34–99.21%, which indicates the reliability and practicality of this method.
298 K 308 K 318 K
2.0
F0 / F
Generally, there are three types of the fluorescence quenching mechanisms: dynamic quenching, static quenching, and the combination of the two. For a single dynamic or static quenching process, the change of fluorescence intensity (F0/F) has a linear relationship with the quenching reagent concentration (C), whereas when the quenching process is the combination of the two, the Stern Volmer curve is non-linear and has an upward bending [25]. Besides, the maximum dynamic quenching constant of biomolecules is usually less than 100 L/mol; if the quenching constant is much more than 100 L/mol, the fluorescence quenching process is static [26]. The results of our experiment show that the curve is linear (Fig. 7), and the two quenching constants are both more than 100 L/mol. Hence, the quenching mechanism is considered to be static. To convince the type of fluorescence quenching we have speculated, a systematic study in the quenching mechanism was carried out. As shown in Fig. 8, comparison of experiments at different temperatures shows important difference of the Stern–Volmer plots for both pyrimidines. Decreasing of quenching observed in association with temperature increase suggests the occurrence of static quenching [27], which is due to the formation of ground-state complex between fluorescence probes and quenchers [28]. Therefore, the quenching is concluded to be static in nature.
Uracil
Thymine
Spiked (10–6 mol/L)
Found (10–6 mol/L)
Average recovery (%)
RSD (%)
25 50 70
24.52 49.60 66.31
98.02 99.21 94.66
1.76 1.53 2.18
25 50 100
23.55 45.17 96.29
94.21 90.34 96.29
3.05 2.23 2.76
functionalized CdS NPs was significantly quenched with the presence of trace uracil or thymine. Moreover, interferences derived from many metal ions and some biomolecules that possibly coexist with uracil and thymine can be negligible. Our results show that the present method is comparable with other methods in terms of sensitivity, simplicity, and practicality, which are very important for the analysis of biological samples. Valuable information emanating from these studies can also help us design fluorescence probes for detection of other biomolecules. The CdS fluorescence probes are expected to have more extensive applications in analytical biochemistry.
Y. Lu et al. / Journal of Luminescence 132 (2012) 244–249
Acknowledgments This research is supported by the National Natural Science Foundation of China (no. 0975066), the Nano-Foundation of Science and Techniques Commission of Shanghai Municipality (no. 0952nm01500), the Leading Academic Discipline Project of Shanghai Municipal Education Commission (no. J50102), and the Graduate Innovation Foundation of Shanghai University (no. SHUCX112027). References [1] C.B. Murray, D.J. Noms, M.G. Bawendi, J. Am. Chem. Soc. 115 (1993) 8706. [2] B.M. Lingerfelt, H. Mattoussi, E.R. Goldman, J.M. Mauro, G.P. Anderson, Anal. Chem. 75 (2003) 4043. [3] X.J. Feng, Q.K. Shang, H.J. Liu, W.L. Wang, Z.D. Wang, J.Y. Liu, J. Lumin. 130 (2010) 648. [4] V.V. Breus, C.D. Heyes, G.U. Nienhaus, J. Phys. Chem. C 111 (2007) 18589. [5] M. Koneswaran, R. Narayanaswamy, Sensors Actuators B 139 (2009) 91. [6] H.Y. Fan, E.W. Leve, C. Scullin, J. Gabaldon, D. Tallant, S. Bunge, T. Boyle, M.C. Wilson, J. Brinker, Nano Lett. 5 (2005) 645. [7] C.Q. Zhu, D.H. Zhao, J.L. Chen, Y.X. Li, L.Y. Wang, L. Wang, Y.Y. Zhou, S.J. Zhuo, Y.Q. Wu, Anal. Bioanal. Chem. 378 (2004) 811. ¨ ¨ [8] M. Gao, S. Kirstein, H. Mohwald, A.L. Rogach, A. Kornowski, A. Eychmuller, H. Weller, Phys. Chem. B 102 (1998) 8360. ¨ [9] N. Gaponik, D.V. Talapin, A. Rogach, A. Eychmuller, H. Weller, Nano Lett. 2 (2002) 803.
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[10] K.E. Sapsford, I.L. Medintz, J.P. Golden, J.R. Deschamps, H.T. Uyeda, H. Mattoussi, Langmuir 20 (2004) 7720. [11] A.M. Dennis, G. Bao, Nano Lett. 8 (2008) 1439. [12] C.S. Xing, Y.L. Xin, P.Y. Li, L. Hong, Y.W. Zuo, J. Colloid Interface Sci. 311 (2007) 400. [13] Q. Shen, X.M. Wang, J. Electroanal. Chem. 632 (2009) 149. [14] H.Y. Liu, G.F. Wang, J.S. Hu, D.L. Chen, W. Zhang, B. Fang, J. Appl. Polym. Sci. 107 (2008) 3173. [15] U. Hofmanna, M. Schwab, S. Seefried, C. Marx, U.M. Zanger, M. Eichelbaum, ¨ Murdter, J. Chromatogr. B 791 (2003) 371. ˇ [16] R. Svobait e_ , I. Solassol, F. Pinguet, L. Ivanauskas, J. Bre s, F.M.M. Bressolle, Clin. Chem. 54 (2008) 1463. [17] R. De´porte, M. Amiand, A. Moreau, C. Charbonnel, L. Campion, J. Chromatogr. B 834 (2006) 170. [18] G.R. Silva, F.A. Condessa, G.A. Pianetti, E.A. Nunan, L.M.M. Campos, Quim. Nova 31 (2008) 1686. [19] Y.X. Gong, S.P. Li, P. Li, J.J. Liu, Y.T. Wang, J. Chromatogr. A 1055 (2004) 215. [20] T. Honda, H. Inagawa, M. Fukushima, A. Moriyama, G.I. Soma, Clin. Chim. Acta 322 (2002) 59. O. Giardini, [21] C. Salerno, P.D. Eufemia, M. Celli, R. Finocchiaro, C. Crifo, J. Chromatogr. B 734 (1999) 175. [22] C. Tang, U. Yogeswaran, S.M. Chen, Anal. Chim. Acta 636 (2009) 19. ¨ H.Y. Wang, J. Instrum. Anal. 27 [23] M. Zhang, H. Zhao, M. Zhang, N.N. Yue, Q.L. Lu, (2008) 859. [24] T. Shibata, Y.K. Shin, Y.F. Jun, T. Kabashima, M. Kai, Anal. Chim. Acta 674 (2010) 234. [25] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic/Plenum Press, New York, 1999. [26] Z.X. Yang, Y.Y. Zhang, Q. Yang, S.Z. Yao, Chin. J. Appl. Chem. 24 (2007) 858. [27] A. Papadopoulou, R.J. Green, R.A. Frazier, J. Agric. Food Chem. 53 (2005) 158. [28] D. Silva, C.M. Cortez, J. Cunha-Bastos, S.R.W. Louro, Toxicol. Lett. 147 (2004) 53.
Contents lists available at SciVerse ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Hydrothermal synthesis of functionalized CdS nanoparticles and their application as fluorescence probes in the determination of uracil and thymine Yaxiang Lu a, Li Li a, Yaping Ding a,n, Fenfen Zhang a, Yaping Wang a, Weijun Yu b a b
Department of Chemistry, Shanghai University, Shanghai 200444, PR China Instrumental Analysis and Research Center, Shanghai University, Shanghai 200444, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2011 Received in revised form 6 August 2011 Accepted 12 August 2011 Available online 22 August 2011
A novel, sensitive, and convenient method for the determination of uracil and thymine by functionalized CdS nanoparticles (NPs) was proposed. CdS NPs were prepared by hydrothermal process and modified with thioglycollic acid (TGA) in aqueous solution. The fluorescence intensity of functionalized CdS NPs was quenched in the presence of uracil or thymine. Under optimal conditions, the relative fluorescence intensity (F0/F) was proportional to the concentration in the range of 9.0 10 6– 1.0 10 4 mol/L for uracil (r¼ 0.9985) and 8.8 10 7–1.5 10 4 mol/L for thymine (r ¼0.9960). The corresponding detection limits were 9.6 10 7 mol/L and 3.2 10 7 mol/L, respectively. In addition, the possible quenching mechanism was also discussed. Crown Copyright & 2011 Published by Elsevier B.V. All rights reserved.
Keywords: CdS nanoparticles Fluorescence probe Uracil Thymine
1. Introduction Semiconductor nanoparticles (NPs) or quantum dots (QDs) are nanoscale spherical particles with size-dependent tunable luminescent emission [1]. The size dependence of their properties results from quantum confinement of electron and hole carriers at dimensions smaller than the bulk Bohr exciton radius [2]. Owing to the quantum confinement effects, they are endowed with unique optical and electronic properties unlike the bulk materials, such as broad excitation spectra, high excitation cross-sections, excellent resistance to photobleaching, and narrow and symmetric emission bands, which can span the light spectrum from the ultraviolet to the infrared region [3–5]. In general, the key to develop QDs as tools in biological systems is to achieve their water solubility, biocompatibility, and photostability [6]. The surface modified quantum dots possess these characters, which are usually capped with some appropriate hydrophilic functional reagents used as modifying agents, such as L-cysteine, thioglycollic acid etc. [7–9]. Since the first demonstration of water soluble QDs as bioassay fluorophores, they have been widely used in bioanalytics such as bioimaging, biolabelling, biodetection, and clinical diagnosis [10–12].
n
Corresponding author. Tel.: þ86 021 66134734; fax: þ86 21 66132797. E-mail address: [email protected] (Y. Ding).
Uracil and thymine are important bases of RNA and DNA, respectively. They play vital roles in the storage of genetic information and protein biosynthesis [13]. The abnormal changes of these bases in organisms suggest the deficiency and mutation of the immunity system and may indicate the presence of various diseases [13,14]. Hence, the quantitative analysis of uracil and thymine is very essential and has great significance to bioscience and clinical iatrology. There have been many methods reported to detect uracil and thymine, such as gas chromatography tandem mass spectrometry [15], high-performance liquid chromatography [16–18], capillary electrophoresis [19], monoclonal antibodies [20], capillary zone electrophoresis [21], electrochemical analysis [22], fluorescence spectroscopy [23], etc. However, these methods mentioned above have some drawbacks such as environment unfriendly solvents, long analysis time, low sensitivity, limits of analysts, expensive instrumentation, and the tedious procedures [14,24]. Compared with the above-mentioned methods, the fluorescence probes method is obviously simpler, faster in the experimental processes, and more sensitive for different fluorescence emission wavelengths corresponding to different molecular structures. In this paper, CdS NPs were synthesized with hydrothermal synthesis and functionalized with TGA to obtain CdS fluorescence probes. The combination of functionalized CdS NPs with different biomolecules could make some difference in fluorescence intensity and/or fluorescence emission wavelength between each other. Under optimum conditions, the fluorescence of functionalized CdS
0022-2313/$ - see front matter Crown Copyright & 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2011.08.022
Y. Lu et al. / Journal of Luminescence 132 (2012) 244–249
245
NPs could be quenched by uracil and thymine, respectively. The quenching extent of the fluorescence intensity was proportional to the concentrations of the two pyrimidines. Based on this observation, a simple, convenient, and sensitive fluorescence probes method for the quantitative determination of uracil and thymine has been established.
2. Experimental section
3. Results and discussion 3.1. TEM and XRD images of the prepared CdS nanoparticles The morphology and dimension size distribution of CdS NPs were studied by transmission electron microscopy (TEM) as shown in Fig. 1. The typical TEM image (Fig. 1a) of CdS NPs exhibits that these NPs are monodispersed, and the shape of them is close to regular spherical NPs. The histogram of NPs dimension size is
80%
60%
70%
50%
60%
40%
50%
30%
40% 30%
20%
20%
21
0%
16
10%
0%
12
10%
Cumulative distribution curve of particles number percentage
90%
Equivalent circle diameter / nm Fig. 1. (a) TEM image of CdS NPs and (b) particle size of histograms of CdS NPs.
250
2.3. Determination of uracil and thymine with functionalized nano-CdS fluorescence probes
[111]
200
Intensity (a.u.)
For determining the concentration of uracil and thymine, the resulting solutions were used to monitor the change of fluorescence emission intensity by means of spectrofluorometer. 1 mL of the as-prepared functionalized CdS NPs solution and a series of known volumes of uracil or thymine standard solutions were added into a set of dry 25 mL calibrated flasks. The solutions were diluted to the volume with phosphate buffer solution (PBS) and mixed thoroughly. The mixtures were kept for a while in the dark prior to making the fluorescence measurements at room temperature. For emission measurements the excitation wavelength was fixed at 263 nm, and the fluorescence intensity was measured at 291 nm for uracil and 289 nm for thymine, respectively. The excitation and emission slit widths were 5 nm each. The scanning bound was from 273 to 510 nm.
70%
7
The prepared CdS NPs were water insoluble and bio-incompatible, so they were unable to react with biomolecules. To overcome these shortcomings, TGA was chosen to modify CdS NPs for the preparation of TGA-capped-CdS fluorescence probes. The procedure was as follows: briefly, at room temperature (around 25 1C), under vigorous stirring, 0.01 g CdS NPs were added into 150 mL NaOH solution (pH 12.0). Afterwards, the pH of the mixture solution was adjusted to 7.0–8.0 with 0.10 mol/L TGA solution, then the colloidal solution was kept in a dark place for one night. Finally, the functionalized NPs solution was taken into a 500 mL brown jar and stored at room temperature. The obtained solution was ready for the following experiments.
100%
4
2.2. The preparation of TGA-capped-CdS fluorescence probes
80%
0
In a typical procedure, a mixture of 0.2280 g CdCl2 2.5H2O and 1.000 g Na2S2O3 5H2O was put into a Teflon-lined autoclave with 30 mL capacity. Then 20 mL NH3 H2O (3.0 mol/L) solution was added into the autoclave as the stabilizing agent. The autoclave was sealed, maintained at 180 1C for 24 h. After the reaction, the autoclave was cooled down to room temperature naturally. Finally, the obtained orange precipitates were collected by centrifugation, washed under ultrasonic vibration several times with doubly distilled water and ethanol. The final products were dried at 80 1C in vacuum to obtain CdS NPs.
The number percentage of CdS nanoparticles
2.1. Synthesis of CdS nanoparticles
[220]
150 [311]
100
[100] [101]
50
[103]
[200]
0 20
30
40
50 2θ / degree
60
70
80
Fig. 2. XRD pattern of the as-prepared CdS nanoparticles.
shown in Fig. 1b, indicating the diameters ranging from 4 nm to 16 nm, and the average particle diameter of 8.7 nm. The powder X-ray diffraction spectrometry (XRD) pattern in Fig. 2 reveals that the CdS NPs are crystals of hexagonal wurtzite structure, which is consistent with the JCPDS card number 41-1049. 3.2. Fluorescence spectra of unfunctionalized and functionalized CdS nanoparticles The fluorescence spectra of CdS NPs and functionalized CdS NPs are shown in Fig. 3. It can be seen that the fluorescence
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Y. Lu et al. / Journal of Luminescence 132 (2012) 244–249
150 a
100
3.5. Effect of uracil and thymine concentrations
50
b
0 250
300
350 400 450 Wavelength (nm)
500
55 0
Fig. 3. Fluorescence spectra of (a) CdS nanoparticles and (b) functionalized CdS nanoparticles.
Fluorescence Intensity
180
A series of uracil or thymine solutions were added into the functionalized CdS NPs system, respectively. Changes in the fluorescence emission spectra are given in Fig. 6. It is observed that the obtained graphs of fluorescence intensity are decreased with the increasing concentration of uracil or thymine with no spectra shift. The observed fluorescence band centers at 291 nm for uracil and centers at 289 nm for thymine. Considering this remarkable quenching of fluorescence intensity, the possibility of developing a sensitive method for quantitative assay of uracil and thymine based on spectrofluorometry has been evaluated.
160
3.6. Effect of foreign substances
140
In order to assess the selectivity of the proposed method, the tolerance levels of coexisting foreign substances was tested. Each foreign substance with various concentrations was mixed with 5.0 10 5 mol/L uracil or thymine prior to the detection. The tolerance concentration of some metal ions and several biomolecules for the determination is summarized in Table 1. The results show that most of the diverse substances have no obvious interference in the determination, which indicates that the method has a high selectivity. So it is possible to use this method to detect uracil and thymine in human or animal body fluid samples.
120 100 80 60
TGA-CdS TGA-CdS-Uracil
40 20
TGA-CdS-Thymine
3
4
5
6
7 pH
8
9
10
11
12
Fig. 4. Effect of pH on the fluorescence intensity of TGA-CdS nanoparticles in the absence and presence of uracil or thymine. Error bars represent standard deviation of three measurements at each pH.
intensity of the functionalized CdS NPs is slightly decreased. However, the fluorescence spectrum of functionalized CdS NPs is narrower and more symmetric than that of CdS NPs. It is also shown that the fluorescence of CdS NPs in the range from 300 to 510 nm is completely quenched and the fluorescence emission peak of functionalized NPs is red shifted compared with unfunctionalized NPs. 3.3. Effect of pH value The effect of pH in a range between 4.0 and 11.0 was studied in order to select the optimum condition for the determination of uracil and thymine with the functionalized CdS NPs. PBS (0.1 mol/L) was used as the buffer to adjust the acidity of the aqueous medium. As shown in Fig. 4, it can be seen clearly that the maximum fluorescence intensity of TGA-CdS NPs occurs when pH is 7.0, and the optimal pH is also 7.0 for both TGA-CdS–uracil and TGA-CdS–thymine systems. Therefore, the pH of 7.0 was chosen to run the assay. 3.4. Effect of reaction time and temperature The reaction time influences the fluorescence intensity of the system. Fluorescence spectrum was recorded at different time
3.7. Linearity and sensitivity Under optimal conditions mentioned above, the linear calibration equations and limits of detection for uracil and thymine were obtained. As shown in Fig. 7, there are good linear relationships between relative fluorescence intensity (DF ¼F0/F) and the concentrations (C) of uracil and thymine. The dynamic range of uracil is 9.0 10 6–1.0 10 4 mol/L with the correlation coefficient (r) 120 115 Fluorescence Intensity
Fluorescence Intensity
intervals after the addition of uracil or thymine into the solution of functionalized CdS NPs. The result (Fig. 5) shows that the two systems’ fluorescence intensity drops at first, then increases, finally drops again with increase in reaction time. The maximum fluorescence intensity is obtained when the reaction time is 3 h for uracil and 4 h for thymine. It was noted that temperature, as expected, had an effect on the fluorescence intensity of the TGA capped CdS NPs. Therefore, all analytical studies were conducted at the room temperature (25 1C).
CdS nanoparticles functionalized CdS nanoparticles
200
110 105 100 95 90 85
TGA-CdS-Uracil TGA-CdS-Thymine
0
50
100
150
250 200 Time (min)
300
350
400
Fig. 5. Effect of reaction time on the fluorescence intensity of TGA-CdS–uracil and TGA-CdS–thymine systems. Error bars represent standard deviation of three measurements at each time point.
Y. Lu et al. / Journal of Luminescence 132 (2012) 244–249
2.6
160
2.4
140 0 0.3 0.9 3.0 5.0 7.0 9.0 10.0
100 80
2.2 2.0 F0 / F
Fluorescence Intensity
120
60
1.8 1.6 1.4
40
1.2
20
1.0
0 275
247
285
280
290 295 Wavelength (nm)
300
305
0.8
310
2
0
4
6
8
10
Concentration (10-5 mol/L)
160
4.0
140
100 80
3.0 F0 / F
Fluorescence Intensity
3.5
0 0.088 0.7 0.9 3.0 7.0 10.0 15.0
120
60
2.5 2.0
40
1.5
20 0 270
1.0 275
280
285
290 295 300 Wavelength (nm)
305
310
315
0
2
4
6
8
10
12
14
16
Concentration (10-5 mol/L)
Fig. 6. Fluorescence emission spectra of (a) TGA-CdS–uracil and (b) TGA-CdS– thymine systems obtained in PBS (pH¼ 7.0) with excitation wavelength at 263 nm. Curacil (10 5 mol/L): (1) 0; (2) 0.3; (3) 0.9; (4) 3.0; (5) 5.0; (6) 7.0; (7) 9.0; (8) 10.0. Cthymine (10–5 mol/L): (1) 0; (2) 0.088; (3) 0.7; (4) 0.9; (5) 3.0; (6) 7.0; (7) 10.0; (8) 15.0.
Fig. 7. Linear graph of the relative fluorescence intensity (F0/F) versus the concentrations (C) of (a) uracil and (b) thymine.
Table 1 Test for the interference of coexisting substances. Coexisting substance
Coexisting concentration with uracil (10 7 mol/L)
Change in fluorescence intensity to uracil (%)
Coexisting concentration with thymine (10 7 mol/L)
Change in fluorescence intensity to thymine (%)
K þ , Cl NH4þ , Cl Ca2 þ , Cl Mg2 þ , SO24 Al3 þ , NO3 Zn2 þ , SO24 Glucose Citric acid Uric acid Ascorbic acid Dopamine Adenine Guanine L-alanine L-cysteine L-leucine L-tryptophan L-glutamate
1600 1000 600 100 100 300 600 200 300 500 500 150 150 1000 500 150 1000 500
2.03 3.64 5.09 þ3.01 3.39 2.27 5.83 2.59 1.72 4.20 þ3.37 1.50 4.91 þ1.33 3.37 2.67 þ2.61 þ2.95
1600 1000 600 100 100 300 600 200 500 500 500 150 100 1000 500 150 1000 1000
0.25 þ1.17 3.38 þ 0.26 2.64 3.07 0.52 1.00 2.21 4.50 2.47 1.39 3.72 4.55 þ1.01 2.46 2.24 2.04
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of 0.9985, and the dynamic range of thymine is 8.8 10 7– 1.5 10 4 mol/L with the correlation coefficient (r) of 0.9960. The fluorescence quenching of functionalized CdS NPs as a function of uracil or thymine concentrations can match the Stern–Volmer equation. The equation could be drawn as follows:
288 K 298 K 318 K
2.0
ð1Þ
F0 and F are the fluorescence intensities in the absence and presence of quencher concentration solution, respectively. C is the concentration of quencher and KSV is the Stern–Volmer quenching constant. The calibration curve established the following equation, DF ¼0.939þ0.148C (C, 10 5 mol/L) for uracil and DF ¼0.915þ0.190C (C, 10 5 mol/L) for thymine. The detection limit (3s/k) is 9.6 10 7 mol/L for uracil and 3.2 10 7 mol/L for thymine, where s is the standard deviation of blank measurements (n¼10) and k is the slope of calibration line. From the dynamic ranges and detection limits of uracil and thymine, it is very clear that this method is sensitive.
1.8 F0 / F
F0 =F ¼ 1 þKSV C
2.2
1.6 1.4 1.2 1.0
2
3
5
4
6
7
8
Concentration (10-5 mol/L) 3.8. Quenching mechanism
1.8
1.6
1.4
1.2
1.0
A highly sensitive, selective and convenient technique for uracil and thymine analysis has been developed based on the quenching of the fluorescence of TGA-CdS NPs. CdS NPs were successfully synthesized through hydrothermal process and modified with thioglycollic acid. The fluorescence intensity of the
3
4
5
6
7
8
Fig. 8. Stern–Volmer plots of (a) uracil and (b) thymine with CdS nanoparticles at different temperatures.
Table 2 Spiked recoveries of uracil and thymine in human serum samples (n¼3). Pyrimidine
4. Conclusion
2
Concentration (10-5 mol/L)
3.9. Analytical applications To confirm the feasibility of the proposed method for real sample determination, the present method was applied to determine uracil and thymine in human serum using standard addition method. The detailed results are listed in Table 2. From Table 2, it can be seen that the values found from the real samples are identical with the expected. Moreover, relative standard deviations (R.S.D.) are lower than 4%, showing a good precision on this method, and the average recoveries are in an acceptable range of 90.34–99.21%, which indicates the reliability and practicality of this method.
298 K 308 K 318 K
2.0
F0 / F
Generally, there are three types of the fluorescence quenching mechanisms: dynamic quenching, static quenching, and the combination of the two. For a single dynamic or static quenching process, the change of fluorescence intensity (F0/F) has a linear relationship with the quenching reagent concentration (C), whereas when the quenching process is the combination of the two, the Stern Volmer curve is non-linear and has an upward bending [25]. Besides, the maximum dynamic quenching constant of biomolecules is usually less than 100 L/mol; if the quenching constant is much more than 100 L/mol, the fluorescence quenching process is static [26]. The results of our experiment show that the curve is linear (Fig. 7), and the two quenching constants are both more than 100 L/mol. Hence, the quenching mechanism is considered to be static. To convince the type of fluorescence quenching we have speculated, a systematic study in the quenching mechanism was carried out. As shown in Fig. 8, comparison of experiments at different temperatures shows important difference of the Stern–Volmer plots for both pyrimidines. Decreasing of quenching observed in association with temperature increase suggests the occurrence of static quenching [27], which is due to the formation of ground-state complex between fluorescence probes and quenchers [28]. Therefore, the quenching is concluded to be static in nature.
Uracil
Thymine
Spiked (10–6 mol/L)
Found (10–6 mol/L)
Average recovery (%)
RSD (%)
25 50 70
24.52 49.60 66.31
98.02 99.21 94.66
1.76 1.53 2.18
25 50 100
23.55 45.17 96.29
94.21 90.34 96.29
3.05 2.23 2.76
functionalized CdS NPs was significantly quenched with the presence of trace uracil or thymine. Moreover, interferences derived from many metal ions and some biomolecules that possibly coexist with uracil and thymine can be negligible. Our results show that the present method is comparable with other methods in terms of sensitivity, simplicity, and practicality, which are very important for the analysis of biological samples. Valuable information emanating from these studies can also help us design fluorescence probes for detection of other biomolecules. The CdS fluorescence probes are expected to have more extensive applications in analytical biochemistry.
Y. Lu et al. / Journal of Luminescence 132 (2012) 244–249
Acknowledgments This research is supported by the National Natural Science Foundation of China (no. 0975066), the Nano-Foundation of Science and Techniques Commission of Shanghai Municipality (no. 0952nm01500), the Leading Academic Discipline Project of Shanghai Municipal Education Commission (no. J50102), and the Graduate Innovation Foundation of Shanghai University (no. SHUCX112027). References [1] C.B. Murray, D.J. Noms, M.G. Bawendi, J. Am. Chem. Soc. 115 (1993) 8706. [2] B.M. Lingerfelt, H. Mattoussi, E.R. Goldman, J.M. Mauro, G.P. Anderson, Anal. Chem. 75 (2003) 4043. [3] X.J. Feng, Q.K. Shang, H.J. Liu, W.L. Wang, Z.D. Wang, J.Y. Liu, J. Lumin. 130 (2010) 648. [4] V.V. Breus, C.D. Heyes, G.U. Nienhaus, J. Phys. Chem. C 111 (2007) 18589. [5] M. Koneswaran, R. Narayanaswamy, Sensors Actuators B 139 (2009) 91. [6] H.Y. Fan, E.W. Leve, C. Scullin, J. Gabaldon, D. Tallant, S. Bunge, T. Boyle, M.C. Wilson, J. Brinker, Nano Lett. 5 (2005) 645. [7] C.Q. Zhu, D.H. Zhao, J.L. Chen, Y.X. Li, L.Y. Wang, L. Wang, Y.Y. Zhou, S.J. Zhuo, Y.Q. Wu, Anal. Bioanal. Chem. 378 (2004) 811. ¨ ¨ [8] M. Gao, S. Kirstein, H. Mohwald, A.L. Rogach, A. Kornowski, A. Eychmuller, H. Weller, Phys. Chem. B 102 (1998) 8360. ¨ [9] N. Gaponik, D.V. Talapin, A. Rogach, A. Eychmuller, H. Weller, Nano Lett. 2 (2002) 803.
249
[10] K.E. Sapsford, I.L. Medintz, J.P. Golden, J.R. Deschamps, H.T. Uyeda, H. Mattoussi, Langmuir 20 (2004) 7720. [11] A.M. Dennis, G. Bao, Nano Lett. 8 (2008) 1439. [12] C.S. Xing, Y.L. Xin, P.Y. Li, L. Hong, Y.W. Zuo, J. Colloid Interface Sci. 311 (2007) 400. [13] Q. Shen, X.M. Wang, J. Electroanal. Chem. 632 (2009) 149. [14] H.Y. Liu, G.F. Wang, J.S. Hu, D.L. Chen, W. Zhang, B. Fang, J. Appl. Polym. Sci. 107 (2008) 3173. [15] U. Hofmanna, M. Schwab, S. Seefried, C. Marx, U.M. Zanger, M. Eichelbaum, ¨ Murdter, J. Chromatogr. B 791 (2003) 371. ˇ [16] R. Svobait e_ , I. Solassol, F. Pinguet, L. Ivanauskas, J. Bre s, F.M.M. Bressolle, Clin. Chem. 54 (2008) 1463. [17] R. De´porte, M. Amiand, A. Moreau, C. Charbonnel, L. Campion, J. Chromatogr. B 834 (2006) 170. [18] G.R. Silva, F.A. Condessa, G.A. Pianetti, E.A. Nunan, L.M.M. Campos, Quim. Nova 31 (2008) 1686. [19] Y.X. Gong, S.P. Li, P. Li, J.J. Liu, Y.T. Wang, J. Chromatogr. A 1055 (2004) 215. [20] T. Honda, H. Inagawa, M. Fukushima, A. Moriyama, G.I. Soma, Clin. Chim. Acta 322 (2002) 59. O. Giardini, [21] C. Salerno, P.D. Eufemia, M. Celli, R. Finocchiaro, C. Crifo, J. Chromatogr. B 734 (1999) 175. [22] C. Tang, U. Yogeswaran, S.M. Chen, Anal. Chim. Acta 636 (2009) 19. ¨ H.Y. Wang, J. Instrum. Anal. 27 [23] M. Zhang, H. Zhao, M. Zhang, N.N. Yue, Q.L. Lu, (2008) 859. [24] T. Shibata, Y.K. Shin, Y.F. Jun, T. Kabashima, M. Kai, Anal. Chim. Acta 674 (2010) 234. [25] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic/Plenum Press, New York, 1999. [26] Z.X. Yang, Y.Y. Zhang, Q. Yang, S.Z. Yao, Chin. J. Appl. Chem. 24 (2007) 858. [27] A. Papadopoulou, R.J. Green, R.A. Frazier, J. Agric. Food Chem. 53 (2005) 158. [28] D. Silva, C.M. Cortez, J. Cunha-Bastos, S.R.W. Louro, Toxicol. Lett. 147 (2004) 53.