A New Dual-Channel Optical Signal Probe for Cu2þ Detection Based on Morin and Boric Acid Peng Wang, Bin Fang Yuan, Nian Bing Li,* Hong Qun Luo* Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
In this work we utilized the common analytical reagent morin to develop a new a dual-channel, cost-effective, and sensitive method for determination of Cu2þ. It is found that morin is only weakly fluorescent by itself, but forms highly fluorescent complexes with boric acid. Moreover, the fluorescence of complexes of morin with boric acid is quenched linearly by Cu2þ in a certain concentration range. Under optimum conditions, the fluorescence quenching efficiency was linearly proportional to the concentration of cupric ions in the range of 0.5–25 lM with high sensitivity, and the detection limit for Cu2þ was 0.38 lM. The linear range was 1–25 lM determined by spectrophotometry, and the detection limit for cupric ions was 0.8 lM. Furthermore, the mechanism of sensitive fluorescence quenching response of morin to Cu2þ is discussed. Index Headings: Cupric ion; Morin; Boric acid; Fluorescence; Spectrophotometry.
INTRODUCTION Copper has attracted much attention because it plays an important role in environmental, biological, and chemical fields of study. It is an essential trace element for both plants and animals, including humans. However, it is a significant metal pollutant due to its widespread use.1 Under overloading conditions, copper, which is a potential threat to environmental safety, exhibits high toxicity to some organisms including many bacteria and viruses. In addition, it has been reported that the cellular toxicity of cupric ions is related to some neurodegenerative diseases such as Menkes and Wilson’s diseases and Alzheimer’s disease.2 Because of the Janus-faced properties of copper in organisms, it is particularly important to develop efficient, sensitive, selective, and reliable analytical techniques for copper determination. In recent years, many analytical methods have been developed to determine copper including surface plasmon resonance, atomic absorption spectrophotometry, inductively coupled plasma optical emission spectrometry, and mass spectroscopy.36 Nevertheless, complicated preconcentration, time-consuming steps, and highcost instruments limit the application of the existing methods. Compared to these approaches, fluorescent sensing shows high sensitivity, rapid response, wide linear range, easy operation, and the ability to do the detection in a nondestructive manner.710 Furthermore, development of spectrophotometric chemosensors is the very simple, reliable, and cost-effective way of environReceived 22 July 2013; accepted 4 April 2014. * Authors to whom correspondence should be sent. E-mail: linb@swu. edu.cn, [email protected]. DOI: 10.1366/13-07233
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mental detection of Cu2þ.11 A chemosensor having dualsignaling properties of evoking a ultraviolet absorbance change, along with change in the fluorescence emission, will serve the detection purpose more precisely, and multidimensional sensing devices have received increasing attention.12,13 Herein, we employ morin as a chromophore and fluorophore for the determination of Cu2þ. Morin contains a highly conjugated aromatic system and consequently exhibits an intense and characteristic absorption spectrum, and it has been used as a reagent for fluorometric analysis of aluminum as well as for spectrophotometric analysis.14 In this article it is found that morin is only weakly fluorescent by itself, but forms highly fluorescent complexes with boric acid in phosphate buffer (PB) (pH 5.3). Moreover, the fluorescence of complexes of morin with boric acid is quenched gradually by Cu2þ in a certain concentration range. The fluorescence quenching efficiency and absorbance were both directly proportional to the concentration of cupric ions over a given range. The detection limit for cupric ions was determined to be 0.38 lM by the fluorescence method and 0.8 lM by the spectrophotometric method, respectively. Based on these results, a highly sensitive, reliable, dual-channel, cost-effective, and simple method for the assay of cupric ions was developed. The reaction mechanism of this system is also discussed.
EXPERIMENTAL Materials. Morin stock solution (5.0 3 104M) was prepared by dissolving 0.0846 g of morin (guaranteed reagent; Kunming Institute of Botany, Chinese Academy of Science, China) in methanol and diluting to the mark in a 500 mL calibrated flask. Cupric sulfate standard solution (1.0 mM) was prepared by dissolving 0.0254 g of copper sulfate pentahydrate (Chengdu Kelong Chemical Reagents Company, China) in doubly distilled water and diluting to the mark in a 100 mL calibrated flask. The working solution of cupric sulfate (0.1 mM) was prepared by appropriate dilution of the stock solution with doubly distilled water. Boric acid working solution was prepared by dissolving 3.7098 g of boric acid (Chongqing Chuandong Chemical Group Co., China) in doubly distilled water and diluting to the mark in a 100 mL calibrated flask. All solutions mentioned above were stored at 0–4 8C in a refrigerator. Britton-Robinson (BR) buffer solutions (pH 1.812.0) were prepared by mixing 0.2 M NaOH and mixture of 0.04 M H3PO4, H3BO3, and CH3COOH in proportion. Na2HPO4NaH2PO4 (PB) buffer solutions (pH 4.58.0) were prepared by mixing 66.7 mM Na2HPO4 and 66.7 mM NaH2PO4 solutions according to a
0003-7028/14/6810-1148/0 Q 2014 Society for Applied Spectroscopy
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FIG. 1. Fluorescence spectra of the reaction system. (a) Morin; (b) morin–boric acid; (c) morin–boric acid–6 lM Cu2þ; (d) morin–boric acid–10 lM Cu2þ. Concentrations of morin and boric acid, 50 lM and 0.06 M, respectively; PB buffer, pH 5.3.
suitable proportion. NaH2PO4NaOH buffer solutions (pH 4.87.4) were prepared by mixing 0.2 M NaH2PO4 and 0.2 M NaOH solutions according to a suitable proportion. All pH values of buffer solutions were adjusted using a pH meter. HPLC-grade methanol was purchased from Chengdu Kelong Chemical Reagent Company (Chengdu, China). All chemicals used in the experiments were of analytical reagent grade or the best grade commercially available, and doubly distilled water was used for all experiments. Apparatus. The fluorescence intensities and spectra were recorded on a Hitachi F-2700 fluorescence spectrophotometer (Tokyo, Japan) equipped with a xenon lamp excitation source with a 1 3 1 cm quartz cuvette. The slit width was 10 and 10 nm for excitation and emission, respectively. The photomultiplier tube voltage was set at 400 V. A Shimadzu UV-2450 spectrophotometer (Suzhou Shimadzu Instrument Co., China) was used to record the absorption spectra and measure absorbance. A KQ3200DB-type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., China) was used to wash the glass instruments. A pHS-3C pH meter (Shanghai Precision & Scientific Instrument Co., China) was used to measure the pH of the solution. Measurement Procedure. In brief, to a 10 mL calibrated flask was added 1.0 mL of Na2HPO4NaH2PO4 buffer solution, followed by the addition of 1.0 mL of 5.0 3 104M morin and 1.0 mL of 0.6 M boric acid. Then appropriate volume of Cu2þ working solution was added, and the mixture was diluted to the mark with doubly distilled water. After shaking the solution and waiting for 5 min, the fluorescence spectra of the system were recorded in the wavelength range of 475–650 nm on excitation at 455 nm, and the fluorescence intensities were detected at 545 nm with all the excitation and emission slits width of 10.0 nm. Here the fluorescence quenching efficiency [(I0 I)/I0] was employed to evaluate performance, where I0 and I are the fluorescence intensity of the morin–boric acid system in the absence and presence of Cu2þ, respectively. In addition, the ultraviolet-visible absorption spectra were recorded on the Shimadzu UV-2450 spectrophotometer. All measurements were made at room temperature.
FIG. 2. Absorption spectra for the experiment system. (a) Morin; (b) morin–boric acid; (c) morin–boric acid–6 lM Cu2þ; (d) morin–boric acid–10 lM Cu2þ. Concentrations of morin and boric acid, 50 lM and 0.06 M, respectively; PB buffer, pH 5.3.
RESULTS AND DISCUSSION Photoluminescence and Absorption Properties. Figure 1 shows the fluorescence spectra of the morin– boric acid system in the presence and absence of Cu2þ. As can be seen, morin showed very low fluorescence intensity, whereas the morin–boric acid complex exhibited strong fluorescence under the same measurement conditions. However, when the morin–boric acid complex was mixed with trace amounts of Cu2þ, the fluorescence intensity was quenched substantially. The maximum fluorescence peak was observed at 545 nm on excitation at 455 nm. Furthermore, the decrease of fluorescence intensity was directly proportional to the concentration of Cu2þ in a certain range. This method can be applied for the determination of Cu2þ. Meanwhile, the absorption spectra of the reaction system were also recorded. As shown in Fig. 2, the UV-Vis absorption spectrum of morin exhibited the typical absorption band at 385 and 261 nm. With the addition of Cu2þ, the absorbance decreased observably. A linear response of absorbance as a function of Cu2þ concentration for the reaction system was observed at 381 nm. Optimum Conditions for the Reaction. Effect of the Morin Concentration. In order to obtain high sensitivity and a wide linear range, the effect of dosage of morin on the system was examined. Generally, in the presence of quencher of a given concentration, the lower the concentration of fluorophore, the larger the change signal and thus the higher the sensitivity found.15 However, on the other hand, the signal-to-noise ratio (S/N) would be decreased when using too low a concentration of fluorophore.16 The results showed that when a large dose of morin was used, the quenching effect of diluted 6 lM cupric ions was inconspicuous, leading to a low sensitivity for the cupric ion detection. In contrast, 6 lM cupric ions could effectively quench the fluorescence of the morin–boric acid system that contained a small dosage of morin with a favorable sensitivity, but narrow linear range. Taking both sensitivity and linear range into consideration, 50 lM morin was chosen for this study. Effect of Acidity. The effect of pH on the fluorescence intensity of the reaction system was studied in a BrittonRobinson (BR) buffer, which covered the wide pH range
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FIG. 3. Effect of pH on the morin–boric acid–Cu2þ system. Concentrations of morin, boric acid, and Cu2þ: 50 lM, 0.06 M, and 20 lM, respectively.
from 1.8 to 11.9, NaH2PO4-NaOH buffer, and NaH2PO4Na2HPO4 (PB) buffer. The results are shown in Fig. 3. It was revealed that the optimum pH value range in BR buffer for the system was 5.05.7. If the pH value was higher or lower than the optimum pH value, it caused a marked decrease in the (I0 I)/I0. This is probably due to the fact that at low pH, morin may exist as the protonized molecule, and the kinetics of the response were very low.14,17 But when the measurement solution has a higher pH value, cupric ions formed deposition compounds, resulting in the decrease of the response to morin. In addition, we found that the optimum pH value range was 4.45.6 in the PB buffer and 4.85.7 in the NaH2PO4-NaOH buffer, respectively. Considering that the optimum pH range of the PB buffer was the widest among the three buffers, and the (I0 I)/I0 value was relatively higher, we selected the pH 5.3 PB buffer to control the pH of the reaction system using 1.0 mL of the PB buffer. Effect of the Concentration of Boric Acid. The effect of boric acid concentration was investigated ranging from 0 to 0.18 M. The results showed that the fluorescence quenching efficiency [(I0 I)/I0] increased with increasing boric acid concentration until the concentration of boric acid reached 0.06 M. However, the fluorescence quenching efficiency decreased when the concentration of boric acid exceeded 0.06 M. The gradual enhancement of fluorescence quenching efficiency was due to the formation of highly fluorescent complexes of boric acid with morin.18 In contrast, excessive boric acid can form complexes with cupric ions,19 which will decrease the effective concentration of cupric ions. Therefore, 0.06 M boric acid was used here. Reaction Time and Stability. Under optimum conditions, the reaction time and stability were studied by determining the fluorescence intensity of the morin– boric acid–Cu2þ system every 2 min for 180 min immediately after mixing. The results showed that the fluorescence intensity of the reaction system decreased rapidly and reached a plateau in less than 2 min, indicating that cupric ions reacted promptly with the morin–boric acid complexes. Hence this reaction may be used for ‘‘fast’’ sensing of Cu2þ. The order in which reagents were added was also investigated in the following sequence: (1) buf-
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FIG. 4. Fluorescence spectra of the reaction system. Concentrations of morin and boric acid: 50 lM and 0.06 M, respectively; concentrations of cupric ions (from 1 to 13): 0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 15.0, 20.0, and 25.0 lM, respectively; PB buffer, pH 5.3. Inset: The normalized curves between the [(I0 I)/I0] and various concentrations of cupric ions.
fer!morin!boric acid!cupric ions, (2) buffer!boric acid!morin!cupric ions, and (3) buffer!cupric ions!boric acid!morin. The results showed that the first of these was the optimum. PB buffer, morin, boric acid, and cupric ions were added successively, and the fluorescence quenching efficiency reached a maximum rapidly and retained stability. Considering that the order in which the other reagents were added resulted in almost the same fluorescence quenching efficiency as the first order, but needed a relatively long time to reach the reaction equilibrium, the first order was chosen as the best sequence for adding the reagents. Calibration Curve and Detection Limit. Under optimum conditions, the fluorescence quenching efficiency [(I0 I)/I0] of the experimental system was determined at the maximum fluorescence emission wavelength. The fluorescence spectra of the system with different concentrations of cupric ions are shown in Fig. 4. The fluorescence emission intensities were measured at 545 nm. A calibration curve of [(I0 I)/I0] against cupric ion concentration was constructed (Fig. 4, inset). The linear regression equation was [(I0 I)/I0] = 0.106 þ 0.035 c (c, lM) with a correlation coefficient (R) of 0.996 in the cupric ion concentration range of 0.525.0 lM. The detection limit was 0.38 lM, which was given by 3r/k, where 3 is the factor at the 95% confidence level of detection, r is the standard deviation of the 11 blank determinations, and k is the slope of the calibration curve. The UV-Vis absorption spectra of the system were also obtained under optimum conditions. As shown in Fig. 5, the absorption peaks of morin–boric acid complex were at 381 and 261 nm. With the addition of Cu2þ, the absorbance decreased observably, and the absorption band observed at 381 nm was red-shifted to 401 nm. This indicated that cupric ions interacted with morin–boric acid complex and formed a complex. In addition, a calibration curve of absorbance (A) against cupric ion concentration in a certain range was constructed (Fig. 5, inset). The linear regression equation was A = 0.803 0.164 c (c, 3 105M) with correlation coefficient (R) of 0.995 in the cupric ion concentration range of 0.102.5 3 105M. The detection
TABLE II. Effects of foreign substances ([Cu2þ] = 20 lM). Tolerable Relative Tolerable Relative Coexisting concentration error Coexisting concentration error substance (lM) (%) substance (lM) (%) Ni2þ Ca2þ Naþ Mn2þ Kþ Mg2þ Agþ Ba2þ Pb2þ FIG. 5. Absorption spectra for the experiment system. Concentrations of morin and boric acid: 50 and 0.06 M, respectively; concentrations of cupric ions (from 1 to 10): 0, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 20.0, and 25.0 lM, respectively; PB buffer, pH 5.3. Inset: The calibration curve for cupric ions.
SO42 NO3 Cl CH3COO I NO2 NH4þ Zn2þ Cd2þ
1500 6000 2500 300 100 1750 3000 150 160
10.0 9.9 8.8 8.7 9.1 8.1 10.0 6.7 9.4
TABLE III. Results for the determination of cupric ions in tap water samples. Sample
limit was 8.0 3 107M based on 3r per slope. Hence, the spectrophotometric method could be applied to determine cupric ions. It is clear that fluorimetry is a sensitive method compared to absorptiometry analysis. Table I shows a comparison of this method for the detection of cupric ions with some other methods. These results indicate that our proposed approach has a relatively low detection limit and wide linear range and will be a valuable tool for the determination of cupric ions. Effects of Foreign Substances. The effects of interferents on the fluorescence intensity with 20 lM cupric ions were investigated by adding various metal ions under optimum conditions. The results are summarized in Table II, from which it can be seen that some common metal ions hardly affect the determination of cupric ions under the permission error. Therefore, our proposed approach has good selectivity and can be applied to the determination of tap water samples. Analytical Application. In order to validate the performance of the proposed method for real samples analysis, the proposed method was applied to the analysis of cupric ions in tap water samples. The tap water was collected after discharging the tap water for about 10 min, and then the obtained test sample was filtrated through a 0.2 lm filter membrane. The filter liquor was boiled for 5 min to remove chlorine. An appropriate volume of the treated sample solution was taken for recovery analysis. The results of real samples by standard addition method are summarized in Table
6.7 9.9 8.8 8.7 5.9 9.2 7.6 8.9 8.8
100 3000 2500 150 2000 1000 120 100 150
1 2 3 a
Added (lM)
Founda (lM)
Recovery (%)
RSD (%)
1.0 5.0 10
1.03 4.98 10.4
103.0 99.6 104.0
2.7 3.2 3.5
Mean value of three experiments.
III. It can be seen that the recoveries of cupric ions for individual samples varied between 99.6 and 104.0%. The relative standard deviation (RSD), which is the ratio of the standard deviation to the mean, for the three samples were between 2.7 and 3.5%. Therefore, the method can be applied for the determination of cupric ions in real samples with reasonable precision. Reaction Mechanism. Figure 1 shows the fluorescence spectra of the morin–boric acid system with or without Cu2þ. As can be seen, morin showed very low fluorescence intensity, while boric acid was added to the morin solution and the mixture showed strong fluorescence under the same measurement conditions. This observation is attributed to the formation of morin–boric acid complexes.18 The absorption band of morin exhibits
TABLE I. Comparison of sensitivities of this work with other methods for the determination of cupric ions.
Method Fluorimetry Fluorimetry Spectrophotometry Fluorimetry Fluorimetry Spectrophotometry Fluorimetry
Linear range (lM)
Detection limit (lM)
Reference
Not given 0.53.0 Not given 10300 114 1–25 0.525
15.6 0.02 1.0 3.42 Not reported 0.8 0.38
7 10 11 12 13 This work This work
FIG. 6. The most stable structure of morin–cupric ion complex.
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FIG. 7. The possible reaction mechanism of the system.
a hypsochromic shift after introduction of boric acid. This phenomenon can further verify the formation of the morin–boric acid complex. However, when the morin– boric acid complex was mixed with trace amounts of Cu2þ, the fluorescence intensity was quenched significantly. It was noted that in the spectral profile the maximum emission peaks did not change. Therefore, the decrease in fluorescence was possibly due to the release of boric acid from the complex when cupric ions were added. The decomposition of morin–boric acid was due to more efficient chelation of morin with cupric ions. The results of the different reagent addition orders also confirmed that the chelation of morin with cupric ions was stronger than that of morin with boric acid. The molar ratio method was used to determine the stoichiometric ratio for the reaction between morin and cupric ions, and the result suggested the 2:1 (morin: Cu2þ) binding model, which was in agreement with the reported literature.20,21 Using Gaussian 09, density functional theory calculations at the level of B3LYP/631g* were employed to optimize the results, and a frequency calculation was used to verify the stable structures of the morin–cupric ion complex. The most stable structure of the relational compound is shown in Fig. 6. The morin reacted with cupric ions to form a planar coordination complex, and this structure has the lowest energy. According to the discussion above, the reaction mechanism of the system was presumed as shown in Fig. 7.
CONCLUSIONS In this study the interaction between a morin–boric acid complex and cupric ions by fluorimetry and absorption spectrophotometry has been investigated. The influence of some reaction conditions on the fluorescence quenching efficiency [(I0 I)/I0] was investigated. Under optimum conditions, [(I0 I)/I0] and absorbance were proportional to the cupric ion concentration in the range of 0.525.0 and 1.025.0 lM, respectively. This work has the following advantages: (1) A dual-channel analysis approach has been successfully used to detect cupric ions with a low detection limit; (2) the two methods exhibit reasonable precision and accuracy; and (3) the two methods are cost-effective and relatively simple for the assay of cupric ions. The reaction mechanism of this system was also investigated.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21273174, No. 20975083) and the Municipal Science Foundation of Chongqing City (No. CSTC-2013jjB00002). 1. Y.M. Li, X.L. Zhang, B.C. Zhu, J. Xue, Z. Zhu, W.H. Tan. ‘‘A Simple but Highly Sensitive and Selective Colorimetric and Fluorescent Probe for Cu2þ in Aqueous Media’’. Analyst. 2011. 136(6): 1124-1128. 2. H.Y. Cao, W.B. Shi, J.X. Xie, Y.M. Huang. ‘‘Highly Sensitive and Selective Fluorescent Assay for Quantitative Detection of Divalent Copper Ion in Environmental Water Samples’’. Anal. Methods. 2011. 3(9): 2102-2107. 3. S. Lee, I. Choi, S. Hong, Y.I. Yang, J. Lee, T. Kang, J. Yi. ‘‘Highly Selective Detection of Cu2þ Utilizing Specific Binding Between CuDemetallated Superoxide Dismutase 1 and the Cu2þ Ion Via Surface Plasmon Resonance Spectroscopy’’. Chem. Commun. 2009. 41: 6171-6173. 4. F.Z. Xie, X.C. Lin, X.P. Wu, Z.H. Xie. ‘‘Solid Phase Extraction of Lead (II), Copper (II), Cadmium (II) and Nickel (II) Using Gallic acidModified Silica Gel Prior to Determination by Flame Atomic Absorption Spectrometry’’. Talanta. 2008. 74(4): 836-843. 5. M. Faraji, Y. Yamini, S. Shariati. ‘‘Application of Cotton as a Solid Phase Extraction Sorbent for On-Line Preconcentration of Copper in Water Samples Prior to Inductively Coupled Plasma Optical Emission Spectrometry Determination’’. J. Hazard. Mater. 2009. 166(2–3): 1383-1388. 6. R. Matschat, J. Hinrichs, H. Kipphardt. ‘‘Application of Glow Discharge Mass Spectrometry to Multielement Ultra-Trace Determination an Ultrahigh-Purity Copper and Iron: A Calibration Approach Achieving Quantification and Traceability’’. Anal. Bioanal. Chem. 2006. 386(1): 125-141. 7. D. Zhang, M. Wang, M.M. Chai, X.P. Chen, Y. Ye, Y.F. Zhao. ‘‘Three Highly Sensitive and Selective Colorimetric and Off-On Fluorescent Chemosensors for Cu2þ in Aqueous Solution’’. Sens. Actuators, B. 2012. 168: 200-206. 8. L.L. Sun, D. Hao, W.L. Shen, Z.S. Qian, C.Q. Zhu. ‘‘Highly Sensitive Fluorescent Sensor for Copper (II) Based on Amplified Fluorescence Quenching of a Water-Soluble NIR Emitting Conjugated Polymer’’. Microchim. Acta. 2012. 177(3–4): 357-364. 9. S.J. Lai, X.J. Chang, C. Fu. ‘‘Cadmium Sulfide Quantum Dots Modified by Chitosan as Fluorescence Probe for Copper (II) Ion Determination’’. Microchim. Acta. 2009. 165(1–2): 39-44. 10. Y.X. Yang, W.L. Gao, R.L. Sheng, W.L. Wang, H. Liu, W.M. Yang, T.Y. Zhang, X.T. Zhang. ‘‘Rhodamine-Based Derivatives for Cu2þ Sensing: Spectroscopic Studies, Structure-Recognition Relationships and Its Test Strips’’. Spectrochim. Acta, Part A. 2011. 81(1): 1420. 11. Y.J. Song, K.G. Qu, C. Xu, J.S. Ren, X.G. Qu. ‘‘Visual and Quantitative Detection of Copper Ions Using Magnetic Silica Nanoparticles Clicked on Multiwalled Carbon Nanotubes’’. Chem. Commun. 2010. 46(35): 6572-6574. 12. Z.H. Xu, L.K. Zhang, R. Guo, T.C. Xiang, C.Z. Wu, Z. Zheng, F.L. Yang. ‘‘A Highly Sensitive and Selective Colorimetric and Off-On Fluorescent Chemosensor for Cu2þ Based on Rhodamine B Derivative’’. Sens. Actuators. B. 2011. 156(2): 546-552. 13. Z.Q. Hu, X.M. Wang, Y.C. Feng, L. Ding, H.Y. Lu. ‘‘Sulfonyl Rhodamine Hydrazide: A Sensitive and Selective Chromogenic
14.
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17.
and Fluorescent Chemodosimeter for Copper Ion in Aqueous Media’’. Dyes Pigm. 2011. 88(3): 257-261. L.A. Saari, W.R. Seitz. ‘‘Immobilized Morin as Fluorescence Sensor for Determination of Aluminum (III)’’. Anal. Chem. 1983. 55(4): 667670. X.D. Lou, L. Qiang, J.G. Qin, Z. Li. ‘‘A New Rhodamine-Based Colorimetric Cyanide Chemosensor: Convenient Detecting Procedure and High Sensitivity and Selectivity’’. ACS Appl. Mater. Interfaces. 2009. 1(11): 2529-2535. Y.Q. Dong, R.X. Wang, G.L. Li, C.Q. Chen, Y.W. Chi, G.N. Chen. ‘‘Polyamine-Functionalized Carbon Quantum Dots as Fluorescent Probes for Selective and Sensitive Detection of Copper Ions’’. Anal. Chem. 2012. 84(14): 6220-6224. Y.M. Temerk, M.S. Ibrahim, M. Kotb. ‘‘Voltammetric and Spectroscopic Studies on Binding of Antitumor Morin, Morin-Cu Complex
18.
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and Morin-b-Cyclodextrin with DNA’’. Spectrochim. Acta, Part A. 2009. 71(5): 1830-1836. L. Pszonicki, W. Tkacz. ‘‘Complexes of Morin and Quercetin with Boric Acid and Oxalic Acid in Acetic Acid Medium. Fluorimetric Determination of Boron’’. Anal. Chim. Acta. 1976. 87(1): 177-184. K. Shiozawa. Wood Preservative Composition, Process for Treating Wood with the Same, Wood Treated with the Same. US Patent 5478598. Filed 1994. Issued 1995. J.E. Brown, H. Khodr, R.C. Hider, C.A. Rice-Evans. ‘‘Structural Dependence of Flavonoid Interactions with Cu2þ Ions: Implications for Their Antioxidant Properties’’. Biochem. J. 1998. 330(3): 11731178. W.S. Liao, F.Y. Wu, Y.M. Wu, X.J. Wang. ‘‘Highly Sensitive Spectrofluorimetric Determination of Cysteine by Cu2þ-Morin Complex’’. Microchim. Acta. 2008. 162(1–2): 147-152.
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In this work we utilized the common analytical reagent morin to develop a new a dual-channel, cost-effective, and sensitive method for determination of Cu2þ. It is found that morin is only weakly fluorescent by itself, but forms highly fluorescent complexes with boric acid. Moreover, the fluorescence of complexes of morin with boric acid is quenched linearly by Cu2þ in a certain concentration range. Under optimum conditions, the fluorescence quenching efficiency was linearly proportional to the concentration of cupric ions in the range of 0.5–25 lM with high sensitivity, and the detection limit for Cu2þ was 0.38 lM. The linear range was 1–25 lM determined by spectrophotometry, and the detection limit for cupric ions was 0.8 lM. Furthermore, the mechanism of sensitive fluorescence quenching response of morin to Cu2þ is discussed. Index Headings: Cupric ion; Morin; Boric acid; Fluorescence; Spectrophotometry.
INTRODUCTION Copper has attracted much attention because it plays an important role in environmental, biological, and chemical fields of study. It is an essential trace element for both plants and animals, including humans. However, it is a significant metal pollutant due to its widespread use.1 Under overloading conditions, copper, which is a potential threat to environmental safety, exhibits high toxicity to some organisms including many bacteria and viruses. In addition, it has been reported that the cellular toxicity of cupric ions is related to some neurodegenerative diseases such as Menkes and Wilson’s diseases and Alzheimer’s disease.2 Because of the Janus-faced properties of copper in organisms, it is particularly important to develop efficient, sensitive, selective, and reliable analytical techniques for copper determination. In recent years, many analytical methods have been developed to determine copper including surface plasmon resonance, atomic absorption spectrophotometry, inductively coupled plasma optical emission spectrometry, and mass spectroscopy.36 Nevertheless, complicated preconcentration, time-consuming steps, and highcost instruments limit the application of the existing methods. Compared to these approaches, fluorescent sensing shows high sensitivity, rapid response, wide linear range, easy operation, and the ability to do the detection in a nondestructive manner.710 Furthermore, development of spectrophotometric chemosensors is the very simple, reliable, and cost-effective way of environReceived 22 July 2013; accepted 4 April 2014. * Authors to whom correspondence should be sent. E-mail: linb@swu. edu.cn, [email protected]. DOI: 10.1366/13-07233
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mental detection of Cu2þ.11 A chemosensor having dualsignaling properties of evoking a ultraviolet absorbance change, along with change in the fluorescence emission, will serve the detection purpose more precisely, and multidimensional sensing devices have received increasing attention.12,13 Herein, we employ morin as a chromophore and fluorophore for the determination of Cu2þ. Morin contains a highly conjugated aromatic system and consequently exhibits an intense and characteristic absorption spectrum, and it has been used as a reagent for fluorometric analysis of aluminum as well as for spectrophotometric analysis.14 In this article it is found that morin is only weakly fluorescent by itself, but forms highly fluorescent complexes with boric acid in phosphate buffer (PB) (pH 5.3). Moreover, the fluorescence of complexes of morin with boric acid is quenched gradually by Cu2þ in a certain concentration range. The fluorescence quenching efficiency and absorbance were both directly proportional to the concentration of cupric ions over a given range. The detection limit for cupric ions was determined to be 0.38 lM by the fluorescence method and 0.8 lM by the spectrophotometric method, respectively. Based on these results, a highly sensitive, reliable, dual-channel, cost-effective, and simple method for the assay of cupric ions was developed. The reaction mechanism of this system is also discussed.
EXPERIMENTAL Materials. Morin stock solution (5.0 3 104M) was prepared by dissolving 0.0846 g of morin (guaranteed reagent; Kunming Institute of Botany, Chinese Academy of Science, China) in methanol and diluting to the mark in a 500 mL calibrated flask. Cupric sulfate standard solution (1.0 mM) was prepared by dissolving 0.0254 g of copper sulfate pentahydrate (Chengdu Kelong Chemical Reagents Company, China) in doubly distilled water and diluting to the mark in a 100 mL calibrated flask. The working solution of cupric sulfate (0.1 mM) was prepared by appropriate dilution of the stock solution with doubly distilled water. Boric acid working solution was prepared by dissolving 3.7098 g of boric acid (Chongqing Chuandong Chemical Group Co., China) in doubly distilled water and diluting to the mark in a 100 mL calibrated flask. All solutions mentioned above were stored at 0–4 8C in a refrigerator. Britton-Robinson (BR) buffer solutions (pH 1.812.0) were prepared by mixing 0.2 M NaOH and mixture of 0.04 M H3PO4, H3BO3, and CH3COOH in proportion. Na2HPO4NaH2PO4 (PB) buffer solutions (pH 4.58.0) were prepared by mixing 66.7 mM Na2HPO4 and 66.7 mM NaH2PO4 solutions according to a
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FIG. 1. Fluorescence spectra of the reaction system. (a) Morin; (b) morin–boric acid; (c) morin–boric acid–6 lM Cu2þ; (d) morin–boric acid–10 lM Cu2þ. Concentrations of morin and boric acid, 50 lM and 0.06 M, respectively; PB buffer, pH 5.3.
suitable proportion. NaH2PO4NaOH buffer solutions (pH 4.87.4) were prepared by mixing 0.2 M NaH2PO4 and 0.2 M NaOH solutions according to a suitable proportion. All pH values of buffer solutions were adjusted using a pH meter. HPLC-grade methanol was purchased from Chengdu Kelong Chemical Reagent Company (Chengdu, China). All chemicals used in the experiments were of analytical reagent grade or the best grade commercially available, and doubly distilled water was used for all experiments. Apparatus. The fluorescence intensities and spectra were recorded on a Hitachi F-2700 fluorescence spectrophotometer (Tokyo, Japan) equipped with a xenon lamp excitation source with a 1 3 1 cm quartz cuvette. The slit width was 10 and 10 nm for excitation and emission, respectively. The photomultiplier tube voltage was set at 400 V. A Shimadzu UV-2450 spectrophotometer (Suzhou Shimadzu Instrument Co., China) was used to record the absorption spectra and measure absorbance. A KQ3200DB-type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., China) was used to wash the glass instruments. A pHS-3C pH meter (Shanghai Precision & Scientific Instrument Co., China) was used to measure the pH of the solution. Measurement Procedure. In brief, to a 10 mL calibrated flask was added 1.0 mL of Na2HPO4NaH2PO4 buffer solution, followed by the addition of 1.0 mL of 5.0 3 104M morin and 1.0 mL of 0.6 M boric acid. Then appropriate volume of Cu2þ working solution was added, and the mixture was diluted to the mark with doubly distilled water. After shaking the solution and waiting for 5 min, the fluorescence spectra of the system were recorded in the wavelength range of 475–650 nm on excitation at 455 nm, and the fluorescence intensities were detected at 545 nm with all the excitation and emission slits width of 10.0 nm. Here the fluorescence quenching efficiency [(I0 I)/I0] was employed to evaluate performance, where I0 and I are the fluorescence intensity of the morin–boric acid system in the absence and presence of Cu2þ, respectively. In addition, the ultraviolet-visible absorption spectra were recorded on the Shimadzu UV-2450 spectrophotometer. All measurements were made at room temperature.
FIG. 2. Absorption spectra for the experiment system. (a) Morin; (b) morin–boric acid; (c) morin–boric acid–6 lM Cu2þ; (d) morin–boric acid–10 lM Cu2þ. Concentrations of morin and boric acid, 50 lM and 0.06 M, respectively; PB buffer, pH 5.3.
RESULTS AND DISCUSSION Photoluminescence and Absorption Properties. Figure 1 shows the fluorescence spectra of the morin– boric acid system in the presence and absence of Cu2þ. As can be seen, morin showed very low fluorescence intensity, whereas the morin–boric acid complex exhibited strong fluorescence under the same measurement conditions. However, when the morin–boric acid complex was mixed with trace amounts of Cu2þ, the fluorescence intensity was quenched substantially. The maximum fluorescence peak was observed at 545 nm on excitation at 455 nm. Furthermore, the decrease of fluorescence intensity was directly proportional to the concentration of Cu2þ in a certain range. This method can be applied for the determination of Cu2þ. Meanwhile, the absorption spectra of the reaction system were also recorded. As shown in Fig. 2, the UV-Vis absorption spectrum of morin exhibited the typical absorption band at 385 and 261 nm. With the addition of Cu2þ, the absorbance decreased observably. A linear response of absorbance as a function of Cu2þ concentration for the reaction system was observed at 381 nm. Optimum Conditions for the Reaction. Effect of the Morin Concentration. In order to obtain high sensitivity and a wide linear range, the effect of dosage of morin on the system was examined. Generally, in the presence of quencher of a given concentration, the lower the concentration of fluorophore, the larger the change signal and thus the higher the sensitivity found.15 However, on the other hand, the signal-to-noise ratio (S/N) would be decreased when using too low a concentration of fluorophore.16 The results showed that when a large dose of morin was used, the quenching effect of diluted 6 lM cupric ions was inconspicuous, leading to a low sensitivity for the cupric ion detection. In contrast, 6 lM cupric ions could effectively quench the fluorescence of the morin–boric acid system that contained a small dosage of morin with a favorable sensitivity, but narrow linear range. Taking both sensitivity and linear range into consideration, 50 lM morin was chosen for this study. Effect of Acidity. The effect of pH on the fluorescence intensity of the reaction system was studied in a BrittonRobinson (BR) buffer, which covered the wide pH range
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FIG. 3. Effect of pH on the morin–boric acid–Cu2þ system. Concentrations of morin, boric acid, and Cu2þ: 50 lM, 0.06 M, and 20 lM, respectively.
from 1.8 to 11.9, NaH2PO4-NaOH buffer, and NaH2PO4Na2HPO4 (PB) buffer. The results are shown in Fig. 3. It was revealed that the optimum pH value range in BR buffer for the system was 5.05.7. If the pH value was higher or lower than the optimum pH value, it caused a marked decrease in the (I0 I)/I0. This is probably due to the fact that at low pH, morin may exist as the protonized molecule, and the kinetics of the response were very low.14,17 But when the measurement solution has a higher pH value, cupric ions formed deposition compounds, resulting in the decrease of the response to morin. In addition, we found that the optimum pH value range was 4.45.6 in the PB buffer and 4.85.7 in the NaH2PO4-NaOH buffer, respectively. Considering that the optimum pH range of the PB buffer was the widest among the three buffers, and the (I0 I)/I0 value was relatively higher, we selected the pH 5.3 PB buffer to control the pH of the reaction system using 1.0 mL of the PB buffer. Effect of the Concentration of Boric Acid. The effect of boric acid concentration was investigated ranging from 0 to 0.18 M. The results showed that the fluorescence quenching efficiency [(I0 I)/I0] increased with increasing boric acid concentration until the concentration of boric acid reached 0.06 M. However, the fluorescence quenching efficiency decreased when the concentration of boric acid exceeded 0.06 M. The gradual enhancement of fluorescence quenching efficiency was due to the formation of highly fluorescent complexes of boric acid with morin.18 In contrast, excessive boric acid can form complexes with cupric ions,19 which will decrease the effective concentration of cupric ions. Therefore, 0.06 M boric acid was used here. Reaction Time and Stability. Under optimum conditions, the reaction time and stability were studied by determining the fluorescence intensity of the morin– boric acid–Cu2þ system every 2 min for 180 min immediately after mixing. The results showed that the fluorescence intensity of the reaction system decreased rapidly and reached a plateau in less than 2 min, indicating that cupric ions reacted promptly with the morin–boric acid complexes. Hence this reaction may be used for ‘‘fast’’ sensing of Cu2þ. The order in which reagents were added was also investigated in the following sequence: (1) buf-
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FIG. 4. Fluorescence spectra of the reaction system. Concentrations of morin and boric acid: 50 lM and 0.06 M, respectively; concentrations of cupric ions (from 1 to 13): 0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 15.0, 20.0, and 25.0 lM, respectively; PB buffer, pH 5.3. Inset: The normalized curves between the [(I0 I)/I0] and various concentrations of cupric ions.
fer!morin!boric acid!cupric ions, (2) buffer!boric acid!morin!cupric ions, and (3) buffer!cupric ions!boric acid!morin. The results showed that the first of these was the optimum. PB buffer, morin, boric acid, and cupric ions were added successively, and the fluorescence quenching efficiency reached a maximum rapidly and retained stability. Considering that the order in which the other reagents were added resulted in almost the same fluorescence quenching efficiency as the first order, but needed a relatively long time to reach the reaction equilibrium, the first order was chosen as the best sequence for adding the reagents. Calibration Curve and Detection Limit. Under optimum conditions, the fluorescence quenching efficiency [(I0 I)/I0] of the experimental system was determined at the maximum fluorescence emission wavelength. The fluorescence spectra of the system with different concentrations of cupric ions are shown in Fig. 4. The fluorescence emission intensities were measured at 545 nm. A calibration curve of [(I0 I)/I0] against cupric ion concentration was constructed (Fig. 4, inset). The linear regression equation was [(I0 I)/I0] = 0.106 þ 0.035 c (c, lM) with a correlation coefficient (R) of 0.996 in the cupric ion concentration range of 0.525.0 lM. The detection limit was 0.38 lM, which was given by 3r/k, where 3 is the factor at the 95% confidence level of detection, r is the standard deviation of the 11 blank determinations, and k is the slope of the calibration curve. The UV-Vis absorption spectra of the system were also obtained under optimum conditions. As shown in Fig. 5, the absorption peaks of morin–boric acid complex were at 381 and 261 nm. With the addition of Cu2þ, the absorbance decreased observably, and the absorption band observed at 381 nm was red-shifted to 401 nm. This indicated that cupric ions interacted with morin–boric acid complex and formed a complex. In addition, a calibration curve of absorbance (A) against cupric ion concentration in a certain range was constructed (Fig. 5, inset). The linear regression equation was A = 0.803 0.164 c (c, 3 105M) with correlation coefficient (R) of 0.995 in the cupric ion concentration range of 0.102.5 3 105M. The detection
TABLE II. Effects of foreign substances ([Cu2þ] = 20 lM). Tolerable Relative Tolerable Relative Coexisting concentration error Coexisting concentration error substance (lM) (%) substance (lM) (%) Ni2þ Ca2þ Naþ Mn2þ Kþ Mg2þ Agþ Ba2þ Pb2þ FIG. 5. Absorption spectra for the experiment system. Concentrations of morin and boric acid: 50 and 0.06 M, respectively; concentrations of cupric ions (from 1 to 10): 0, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 20.0, and 25.0 lM, respectively; PB buffer, pH 5.3. Inset: The calibration curve for cupric ions.
SO42 NO3 Cl CH3COO I NO2 NH4þ Zn2þ Cd2þ
1500 6000 2500 300 100 1750 3000 150 160
10.0 9.9 8.8 8.7 9.1 8.1 10.0 6.7 9.4
TABLE III. Results for the determination of cupric ions in tap water samples. Sample
limit was 8.0 3 107M based on 3r per slope. Hence, the spectrophotometric method could be applied to determine cupric ions. It is clear that fluorimetry is a sensitive method compared to absorptiometry analysis. Table I shows a comparison of this method for the detection of cupric ions with some other methods. These results indicate that our proposed approach has a relatively low detection limit and wide linear range and will be a valuable tool for the determination of cupric ions. Effects of Foreign Substances. The effects of interferents on the fluorescence intensity with 20 lM cupric ions were investigated by adding various metal ions under optimum conditions. The results are summarized in Table II, from which it can be seen that some common metal ions hardly affect the determination of cupric ions under the permission error. Therefore, our proposed approach has good selectivity and can be applied to the determination of tap water samples. Analytical Application. In order to validate the performance of the proposed method for real samples analysis, the proposed method was applied to the analysis of cupric ions in tap water samples. The tap water was collected after discharging the tap water for about 10 min, and then the obtained test sample was filtrated through a 0.2 lm filter membrane. The filter liquor was boiled for 5 min to remove chlorine. An appropriate volume of the treated sample solution was taken for recovery analysis. The results of real samples by standard addition method are summarized in Table
6.7 9.9 8.8 8.7 5.9 9.2 7.6 8.9 8.8
100 3000 2500 150 2000 1000 120 100 150
1 2 3 a
Added (lM)
Founda (lM)
Recovery (%)
RSD (%)
1.0 5.0 10
1.03 4.98 10.4
103.0 99.6 104.0
2.7 3.2 3.5
Mean value of three experiments.
III. It can be seen that the recoveries of cupric ions for individual samples varied between 99.6 and 104.0%. The relative standard deviation (RSD), which is the ratio of the standard deviation to the mean, for the three samples were between 2.7 and 3.5%. Therefore, the method can be applied for the determination of cupric ions in real samples with reasonable precision. Reaction Mechanism. Figure 1 shows the fluorescence spectra of the morin–boric acid system with or without Cu2þ. As can be seen, morin showed very low fluorescence intensity, while boric acid was added to the morin solution and the mixture showed strong fluorescence under the same measurement conditions. This observation is attributed to the formation of morin–boric acid complexes.18 The absorption band of morin exhibits
TABLE I. Comparison of sensitivities of this work with other methods for the determination of cupric ions.
Method Fluorimetry Fluorimetry Spectrophotometry Fluorimetry Fluorimetry Spectrophotometry Fluorimetry
Linear range (lM)
Detection limit (lM)
Reference
Not given 0.53.0 Not given 10300 114 1–25 0.525
15.6 0.02 1.0 3.42 Not reported 0.8 0.38
7 10 11 12 13 This work This work
FIG. 6. The most stable structure of morin–cupric ion complex.
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FIG. 7. The possible reaction mechanism of the system.
a hypsochromic shift after introduction of boric acid. This phenomenon can further verify the formation of the morin–boric acid complex. However, when the morin– boric acid complex was mixed with trace amounts of Cu2þ, the fluorescence intensity was quenched significantly. It was noted that in the spectral profile the maximum emission peaks did not change. Therefore, the decrease in fluorescence was possibly due to the release of boric acid from the complex when cupric ions were added. The decomposition of morin–boric acid was due to more efficient chelation of morin with cupric ions. The results of the different reagent addition orders also confirmed that the chelation of morin with cupric ions was stronger than that of morin with boric acid. The molar ratio method was used to determine the stoichiometric ratio for the reaction between morin and cupric ions, and the result suggested the 2:1 (morin: Cu2þ) binding model, which was in agreement with the reported literature.20,21 Using Gaussian 09, density functional theory calculations at the level of B3LYP/631g* were employed to optimize the results, and a frequency calculation was used to verify the stable structures of the morin–cupric ion complex. The most stable structure of the relational compound is shown in Fig. 6. The morin reacted with cupric ions to form a planar coordination complex, and this structure has the lowest energy. According to the discussion above, the reaction mechanism of the system was presumed as shown in Fig. 7.
CONCLUSIONS In this study the interaction between a morin–boric acid complex and cupric ions by fluorimetry and absorption spectrophotometry has been investigated. The influence of some reaction conditions on the fluorescence quenching efficiency [(I0 I)/I0] was investigated. Under optimum conditions, [(I0 I)/I0] and absorbance were proportional to the cupric ion concentration in the range of 0.525.0 and 1.025.0 lM, respectively. This work has the following advantages: (1) A dual-channel analysis approach has been successfully used to detect cupric ions with a low detection limit; (2) the two methods exhibit reasonable precision and accuracy; and (3) the two methods are cost-effective and relatively simple for the assay of cupric ions. The reaction mechanism of this system was also investigated.
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