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Colorimetric detection of melamine during the formation of gold nanoparticles
Biosensors and Bioelectronics 26 (2011) 2574–2578
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Colorimetric detection of melamine during the formation of gold nanoparticles Zhijiao Wu, Hong Zhao ∗ , Ying Xue, Qian Cao, Jie Yang, Yujian He, Xiangjun Li, Zhuobin Yuan College of Chemistry and Chemical Engineering, Graduate University of Chinese Academy of Sciences, 19A YuQuan Road, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 17 August 2010 Received in revised form 19 October 2010 Accepted 9 November 2010 Available online 10 December 2010 Keywords: Melamine Pyrocatechol-3,5-disodiumsulfonate Colorimetric detection
a b s t r a c t A sensitive and simple colorimetric method for determination of melamine was reported based on the seedless production of gold nanoparticles (Au-NPs). Au-NPs were synthesized by using pyrocatechol3,5-disodiumsulfonate (PD) as reducer without adding nanoparticles seeds and stabilizing agent. PD can form intramolecular hydrogen-bonding in solution by adjacent sulfurnate and ␣-phenolic hydroxyl group, resulting in its weak reducing capacity and thus the synthesis of Au-NPs was slow. While in the presence of melamine, PD reacted with melamine through hydrogen-bonding. Therefore, the intramolecular hydrogen-bonding of PD was interrupted by melamine, and the ␣-phenolic hydroxyl group was free to reduce Au3+ , hence, the synthesis of Au-NPs was accelerated. Especially, the presence of melamine led to a shift in the surface plasmon bond and a color change of Au-NPs from green to yellow. Results showed that the absorbance ratio (A436 /A600 ) was linear with the logarithm of melamine concentration in the range of 4.8 × 10−9 to 1.6 × 10−6 M with a correlation coefficient of 0.9949. The detection limit (3) obtained by UV–vis spectrum was 6.4 × 10−10 M (i.e., 0.08 ppb). The proposed method was applied successfully to the determination of melamine in pretreated liquid milk products, and the recoveries were from 93% to 107%. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Growing attentions have been attracted to detect melamine since the outbreak of renal failure and associated deaths in cats and dogs in the USA in early 2007, particularly, the 2008 melaminetainted milk powder incident in China. Melamine is a triazine heterocyclic organic chemical raw material, its combination with other triazine, cyanuric acid, results in insoluble crystals in the kidneys of cats and infant (Brown et al., 2007). Melamine is not approved by the U.S. Food and Drug Administration or Chinese government as a food additive in animal feeds or human food. Due to its high nitrogen level (66% nitrogen by mass), melamine increases the protein concentration obviously when the Kjeldahl method is used for protein analysis (Wu et al., 2009; Mauer et al., 2009). Thus, melamine was illegally added to the protein-rich foods by unethical manufactures. Therefore, there is an increasing demand for effective and reliable methods to detect melamine. The currently available techniques for detecting melamine are as following: high performance liquid chromatography (HPLC) (Ehling and Tefera, 2007), mass spectrum (MS) (Yang et al., 2009; Tang et al., 2009), gas chromatography/mass spectrum (GC/MS) (Yokley et al., 2000; Zhu et al., 2009), high performance liquid chromatography/mass spectrum (HPLC/MS) (Kim et al., 2008), capillary zone electrophore-
∗ Corresponding author. Fax: +86 10 88256092. E-mail address: [email protected] (H. Zhao). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.11.007
sis/mass spectrum (CE/MS) (Cook et al., 2005; Vo et al., 2008), surface enhanced raman spectroscopy (SERS) (Lin et al., 2008) and enzyme-linked immunosorbent assay (ELISA) (Garber, 2008; Wang et al., 2010). However, they are limited by complicated preconcentration, time-consuming steps and high-cost instruments. Hence, it is urgent to develop fast, low-cost, highly sensitive and selective sensors to determine trace melamine in real samples. Recently, Au-NPs-based colorimetric sensors have been attracted great attention because of their unique properties such as color, biocompatibility, stability and high extinction coefficients. They have been used for the detection of some species such as 2,4,6-trinitrotoluene (Jiang et al., 2008), glucose (Jiang et al., 2010), dopamine (Zhang et al., 2010), cysteine (Wang et al., 2008), and lead ions (Yoosaf et al., 2007; Huang et al., 2010). Meanwhile, there are a few reports about the colorimetric detection of melamine based on the fact that Au-NPs are induced to aggregate by interparticle crosslinking (Ai et al., 2009; Chi et al., 2010; Li et al., 2010; Kuang et al., 2010; Qi et al., 2010; Qin et al., 2009; Wei et al., 2010). We had previously investigated colorimetric detection of melamine by nonaggregation-based Au-NPs as a probe, in which the synthesis of Au-NPs was hindered by melamine (Cao et al., 2010). Herein, we report a novel method that the presence of melamine accelerates the synthesis of Au-NPs and changes Au-NPs from aggregation to dispersion (Fig. 1). PD was used to reduce Au3+ ion. However, the synthesis of Au-NPs was slow because of intramolecular hydrogen-bonding formed by adjacent sulfonate and ␣-hydroxyl group in PD in solution (Dong et al., 2003). And PD
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Fig. 1. (A) Schematic illustration of possible procedure of synthesis and aggregation of PD-capped-NPs. (B) The process that melamine accelerated the generation of Au-NPs.
capped Au-NPs obtained by the above method were aggregated because of intermolecular hydrogen-bonding (Fig. 1A). While in the presence of melamine, the synthesis of Au-NPs was fast and the obtained Au-NPs were relatively dispersed (Fig. 1B). The reasons are as follows: as it is well known, melamine has a good hydrogen-bonding ability, and it can interact with sulfonate ligand (Zhang, 2007). Therefore, intramolecular H bond of PD was interrupted by the O· · ·HN formed between PD and melamine. The ␣-phenolic-OH was free from the intramolecular H bond, which increased the reduction reactivity of PD, and thus the reduction process of Au3+ was accelerated. In this way, colorimetric detection of melamine could be realized during the formation of the Au-NPs.
All stock solution were prepared daily with Milli-Q-purified distilled water. Before synthesis of Au-NPs, 50 L of 0.01 M PD and 50 L of different concentration of melamine were mixed and shaken for 30 min to obtain a stable solution. In 4 mL centrifuge tube, the mixed solution of 3 mL of phosphate buffer solution (0.01 M pH 7.0), 2.0 × 10−4 M HAuCl4 , 1.6 × 10−4 M PD and different concentration of melamine was shaken for 20 times with a steady rhythm and was allowed to further react for 50 min at room temperature. After that, the absorbance spectra and photographs of the solution were recorded.
2. Experimental
2.4. Instruments
2.1. Chemicals and materials
The FT-IR spectra of PD and formed Au-NPs were detected in Avatar 360 FT-IR ESP. The UV–vis spectra were recorded by a UV2550 spectrophotometer (Shimadzu, Japan) at room temperature. Au-NPs were characterized by transmission electron microscopy (TEM, T20) at 200 kV. The dynamic light scattering (DLS) measurements were carried out using the Nicomp 380 ZLS. All the pH measurements were performed with PB-10 pH meter (Sartorius, Germany).
All chemicals were of analytical reagent grade and used without further purification. Milli-Q-purified distilled water was used throughout the experiments. Melamine was purchased from Sigma (USA). Pyrocatechol-3,5-disodiumsulfonate (PD) was from Shanghai Chemical Reagent Company (Shanghai, China). HAuCl4 ·4H2 O and uracil were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Vitamin C was from Xilong Chemical Factory of Guangdong (Guangdong, China). Alanine, Glutamic, Tryptophan, Arginine and Tyrosine were from Zhongbei Linge Biotechnology Ltd. (Beijing, China). Cyanuric acid was from Alfa Aesar (USA). CaCl2 , MgCl2 , glucose, sucrose, maltose and lactose were all from Beijing Chemical Reagent Company (Beijing, China). Glycerol was from Sangon Biotech Co. Ltd. (Shanghai, China). The 0.01 M phosphate buffer (PB) solutions of pH 7.0 were prepared by mixing the stock solutions of 0.01 M NaH2 PO4 and 0.01 M Na2 HPO4 . 2.2. Sample preparation The liquid milk bought from local supermarket was pretreated according to the general procedure (Cao et al., 2009). 5 g liquid milk and 5 mL of 61 mM trichloroacetic acid were added into 35 mL of 4.9 M methanol solution. After 15 min sonication and 10 min shaking, the mixture was centrifuged at 10,000 rpm for 4 min, and the supernatant was filtrated. Then, the filtrate was concentrated to 10 mL and filtered through a 0.45 m filter membrane to obtain the samples for detection.
2.3. Experimental conditions
3. Results and discussion 3.1. Synthesis of Au-NPs Gold nanoparticles (Au-NPs) were synthesized by reducing Au3+ using PD at room temperature (20 ± 2 ◦ C) without adding any other stabilizing agents. Since H+ was involved in the oxidation–reduction process of PD, the effect of pH on the oxidation of PD was carried out at a pH range from 5.0 to 9.0 (Fig. S1, supplementary material). As shown in Fig. S1, at pH 7.0, the maximum absorption of Au-NPs (436 nm) was the largest. Therefore, pH 7.0 was selected for further experiments. The selection of PD as reducer was due to its both reducing and stabilizing groups, which makes in situ synthesis of Au-NPs possible. Obviously, the concentration of PD is very important for the formation of Au-NPs. Fig. S2 shows the UV–vis absorption spectra of the Au-NPs formed in the presence of different concentrations of PD (4.9 × 10−5 to 3.8 × 10−4 M) in 0.01 M pH 7.0 PBS. It is clear that the absorption of the plasmon band of the Au-NPs is influenced by
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Fig. 2. IR spectra of PD-modified Au-NPs (a) and PD (b). Fig. 3. UV–vis spectra and the color change of Au-NPs formed without melamine (a) and with 1.6 × 10−7 M melamine (b).
the concentration of PD. The absorption at 436 nm is very weak at lower concentrations of PD and becomes more intense and sharp with the PD concentration increasing. However, the change is very small when the concentration is over 2.4 × 10−4 M. We used two Au-NPs obtained by different PD concentrations (1.6 × 10−4 M PD and 2.4 × 10−4 M PD) to study the effect of Au-NPs on the relevant optical absorption change, as a foundation for molecular recognition and analytical detection of melamine. The experimental results showed that compared with Au-NPs obtained by larger PD concentration (2.4 × 10−4 M PD), those obtained by 1.6 × 10−4 M PD were more sensitive to melamine. Thus 1.6 × 10−4 M PD was chosen for further experiments. To obtain the optimum conditions, the reaction time (10–80 min) was also examined in details (Fig. S3). Fig. S3 shows the absorbance of Au-NPs at 436 nm that was recorded at different time intervals. Obviously, the absorbance increased rapidly during 50 min and much slower over the next 30 min, indicating that the synthesis of Au-NPs almost completed within 50 min under this condition. Therefore, 50 min was selected as reaction time. The prepared Au-NPs were characterized by FT-IR spectroscopy. The FT-IR spectrum of PD and formed Au-NPs are shown in Fig. 2. In curve a, the disappearance of the bands in region 1272–1129 cm−1 in PD and a comparatively low intensity of the band in 1044 cm−1 which are the characteristic of S O stretching vibration of sulfurnate indicated that the binding of the sulfurnate moiety to the nanoparticle surface. The band at 3494 cm−1 in curve b corresponding to –OH stretch vibrations from pure PD was disappeared after reaction with Au3+ ion and a broad absorption band was observed in region 3010–3730 cm−1 , which is the contribution of the phenolicOH oxidation in the formation of Au-NPs. Furthermore, it was noticed that the peaks at 1589, 1469, 1436 and 1378 cm−1 in curve b which attribute to aromatic C C and phenolic C–O vibrations disappeared in curve a, indicating that most of PD were oxidized. The above results indicated that Au-NPs could be formed by reducer PD.
formation of Au-NPs from aggregation to dispersion, which was consistent with the TEM images (Fig. 4). Fig. S4 displays the color change during Au-NPs synthesis in the absence and presence of melamine (1.6 × 10−7 M) at different time. Compared Fig. S4A to Fig. S4B, there is a significant color change of system ‘b’ (with 1.6 × 10−7 M melamine) than that of system ‘a’ (without melamine), indicating the reaction in system ‘b’ was faster than that in system ‘a’. Compared Fig. S4C with Fig. S4D, the obvious color change of system ‘a’ than that of system ‘b’ suggested that the synthesis of Au-NPs in system ‘a’ is slower than that in system ‘b’, that is to say the presence of melamine accelerated the synthesis of Au-NPs. As depicted in Fig. 1A, there is an intramolecular H bond formed by adjacent sulfurnate and ␣phenolic-OH in PD in water solution, thus the synthesis of Au-NPs was relatively slow. On the other hand, some free PD molecules were attached to the surface of Au-NPs through sulfurnate. Hydrogen bonding interactions between neighboring –OH (free PD) and O groups (oxidized PD) resulted in the aggregations of Au-NPs. In the presence of melamine, the intramolecular H bond of PD was interrupted by the O· · ·HN formed between PD and melamine (Fig. 1B). Therefore, the ␣-phenolic-OH was free from intramolecular H bond, which increased the reactivity of PD, and the formation of Au-NPs was accelerated. Meanwhile, Au-NPs were relatively dispersed, which might be ascribed to steric hindrance induced by binding of melamine to PD. To further prove the conclusions above, the particle size of AuNPs formed in presence and absence of melamine was measured by dynamic light scattering (DLS). DLS is an analytical tool used routinely for measuring the hydrodynamic sizes of nanoparticles and colloids in a liquid environment. As shown in Table S1, the mean particle size decreased in the presence of melamine, in accordance with the results of UV–vis spectrum (Fig. 3).
3.2. Effect of melamine on Au-NPs synthesis
3.3. Colorimetric detection of melamine
The ultimate goal of this study is to detect melamine through the synthesis of Au-NPs, hence the influence of melamine on Au-NPs synthesis was investigated. As shown in Fig. 3, the color of the Au-NPs solution was green without melamine and yellow with melamine (1.6 × 10−7 M) in the same reaction conditions. The absorption spectrum of AuNPs exhibited a large change in the presence of melamine: the absorbance peak at 600 nm disappeared, indicative of the trans-
To investigate this simple assay for the direct colorimetric visualization of melamine, quantitative analysis was performed by adding different concentration of melamine into the growth solution and monitoring the absorption peak and the color changes of the system. Results showed that the color of the solution gradually changed from green to yellow after reaction for 50 min (Fig. 5A). The absorbance of Au-NPs at 600 nm (A600 ) gradually decreased with the increasing concentration of melamine. We used the absorbance
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Fig. 5. (A) Absorbance spectra of the Au-NPs generated in the presence of different concentrations of melamine: (a) 0, (b) 8.0 × 10−9 , (c) 1.6 × 10−8 , (d) 8.0 × 10−8 , (e) 1.6 × 10−7 , (f) 8.0 × 10−7 , and (g) 1.6 × 10−6 M. All systems include HAuCl4 , 2.0 × 10−4 M, and PD, 1.6 × 10−4 M, in 0.01 M PBS. Spectra were recorded after a fixed time interval of 50 min. Inset: photographic images of the Au-NPs formed in the presence of different concentrations of melamine. (Labels correspond to the curve labels in the spectra.) (B) Plot of A436 /A600 against the logarithm of melamine concentrations. Error bars show the standard deviations of measurements taken from three independent experiments. Fig. 4. TEM images of the Au-NPs formed in the absence (A) and presence (B) of 1.6 × 10−7 M melamine.
ratio A436 /A600 to quality the concentration of melamine in this system. A linear correlation existed between the ratio A436 /A600 and the logarithm of melamine concentration in the range from 4.8 × 10−9 to 1.6 × 10−6 M with a correlation coefficient of 0.9949 (Fig. 5B). Theoretical detection limits defined as 3 of the proposed method was 6.4 × 10−10 M (i.e., 0.08 ppb) obtained by UV–vis spectrum. Relative standard deviation for measurement of 4.8 × 10−8 M melamine for 10 independent determinations was 4.2%, indicating that the proposed method has good reproducibility. In addition, the results obtained above were compared with those of recently reported colorimetric detection of melamine based on the fact that Au-NPs are induced to aggregate by inter-particle cross-linkers (color changed from red to blue). The advantages of the proposed method are as following: First, the proposed colorimetric detection of melamine in this study is realized during the formation of Au-NPs, i.e., the preparation of Au-NPs and melamine detection could be carried out simultaneously at room temperature. Second, the presence of melamine accelerated the synthesis of Au-NPs by interrupting the intramolecular hydrogen-
bonding of the reducer PD. Third, the presence of melamine made Au-NPs changed from aggregation to dispersion (color changed from green to yellow). Last, the proposed method has a lower detection limit (Table S2). Fig. 6 shows the absorbance ratio A436 /A600 caused by melamine and other interferences. Obviously, melamine caused the largest absorbance ratio A436 /A600 of formed Au-NPs, indicating that the synthesis of Au-NPs was influenced by melamine most effectively. In addition, uracil gave a larger ratio A436 /A600 than other interferences did, which demonstrated that its presence could affect the determination of melamine to some degree. 3.4. Analytical applications The detection of the melamine in pretreated liquid milk was carried out. 50 L of the pretreated sample was added into 200 L of different concentration of melamine, and then the mixture was shaken for 20 min to get a stable solution. Then the other steps were the same as those above mentioned. As shown in Fig. S5, different color was observed with different melamine concentrations. The recoveries of 6.4 × 10−8 , 12.8 × 10−8 , 63.9 × 10−8 and 12.8 × 10−7 M of melamine were 107%, 93%, 95% and 106%, respec-
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analytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences (Grant no. SKLEAC2010005), National Sci-Tech Major Special Item for Water Pollution Control and Management (Grant no. 2009ZX07527-007-003), and the National Natural Science Foundation of China (Grant nos. 20877099 and 20775088). We are grateful to Dr. Wensheng Lu at ICCAS for his helpful discussion. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2010.11.007. References
Fig. 6. Absorbance ratio A436 /A600 of Au-NPs in the presence of melamine or other interferences (the concentrations of melamine and other interferences were 1.6 × 10−6 M). Spectra were recorded after a fixed time interval of 50 min. Error bars show the standard deviations of measurements taken from three independent experiments.
tively. The detection limit (3) obtained by UV–vis spectrum was 8.0 × 10−9 M (i.e., 1.0 ppb) and below the safety limit (2.5 ppm in USA and the UK; 1 ppm for infant formula in China), indicating that the proposed method was applied successfully to the determination of melamine in pretreated liquid milk products. 4. Conclusions A selective and simple colorimetric assay to detect melamine during the formation of Au-NPs was proposed in this study. At room temperature, the synthesis of the Au-NPs was realized by using PD as the reducer of Au3+ without adding any other stabilizing agents. Particularly, the preparation of Au-NPs and melamine detection could be carried out simultaneously. Concentrations of melamine could be directly monitored according to the color change of the formed Au-NPs, which made the melamine determination convenient and easy. The absorbance ratio (A436 /A600 ) was linear with the logarithm of melamine concentration in the range of 4.8 × 10−9 to 1.6 × 10−6 M with a correlation coefficient of 0.9949. The detection limit (3) obtained by UV–vis spectrum was 6.4 × 10−10 M (i.e., 0.08 ppb). The proposed method was applied successfully to the detection of melamine in the pretreated liquid milk products, and the recoveries were from 93% to 107%. Acknowledgments This work was supported by a grant from the Major State Basic Research Development Program of China (973 Program) (Grant no. 2011CB933200), the State Key Laboratory of Electro-
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Colorimetric detection of melamine during the formation of gold nanoparticles Zhijiao Wu, Hong Zhao ∗ , Ying Xue, Qian Cao, Jie Yang, Yujian He, Xiangjun Li, Zhuobin Yuan College of Chemistry and Chemical Engineering, Graduate University of Chinese Academy of Sciences, 19A YuQuan Road, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 17 August 2010 Received in revised form 19 October 2010 Accepted 9 November 2010 Available online 10 December 2010 Keywords: Melamine Pyrocatechol-3,5-disodiumsulfonate Colorimetric detection
a b s t r a c t A sensitive and simple colorimetric method for determination of melamine was reported based on the seedless production of gold nanoparticles (Au-NPs). Au-NPs were synthesized by using pyrocatechol3,5-disodiumsulfonate (PD) as reducer without adding nanoparticles seeds and stabilizing agent. PD can form intramolecular hydrogen-bonding in solution by adjacent sulfurnate and ␣-phenolic hydroxyl group, resulting in its weak reducing capacity and thus the synthesis of Au-NPs was slow. While in the presence of melamine, PD reacted with melamine through hydrogen-bonding. Therefore, the intramolecular hydrogen-bonding of PD was interrupted by melamine, and the ␣-phenolic hydroxyl group was free to reduce Au3+ , hence, the synthesis of Au-NPs was accelerated. Especially, the presence of melamine led to a shift in the surface plasmon bond and a color change of Au-NPs from green to yellow. Results showed that the absorbance ratio (A436 /A600 ) was linear with the logarithm of melamine concentration in the range of 4.8 × 10−9 to 1.6 × 10−6 M with a correlation coefficient of 0.9949. The detection limit (3) obtained by UV–vis spectrum was 6.4 × 10−10 M (i.e., 0.08 ppb). The proposed method was applied successfully to the determination of melamine in pretreated liquid milk products, and the recoveries were from 93% to 107%. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Growing attentions have been attracted to detect melamine since the outbreak of renal failure and associated deaths in cats and dogs in the USA in early 2007, particularly, the 2008 melaminetainted milk powder incident in China. Melamine is a triazine heterocyclic organic chemical raw material, its combination with other triazine, cyanuric acid, results in insoluble crystals in the kidneys of cats and infant (Brown et al., 2007). Melamine is not approved by the U.S. Food and Drug Administration or Chinese government as a food additive in animal feeds or human food. Due to its high nitrogen level (66% nitrogen by mass), melamine increases the protein concentration obviously when the Kjeldahl method is used for protein analysis (Wu et al., 2009; Mauer et al., 2009). Thus, melamine was illegally added to the protein-rich foods by unethical manufactures. Therefore, there is an increasing demand for effective and reliable methods to detect melamine. The currently available techniques for detecting melamine are as following: high performance liquid chromatography (HPLC) (Ehling and Tefera, 2007), mass spectrum (MS) (Yang et al., 2009; Tang et al., 2009), gas chromatography/mass spectrum (GC/MS) (Yokley et al., 2000; Zhu et al., 2009), high performance liquid chromatography/mass spectrum (HPLC/MS) (Kim et al., 2008), capillary zone electrophore-
∗ Corresponding author. Fax: +86 10 88256092. E-mail address: [email protected] (H. Zhao). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.11.007
sis/mass spectrum (CE/MS) (Cook et al., 2005; Vo et al., 2008), surface enhanced raman spectroscopy (SERS) (Lin et al., 2008) and enzyme-linked immunosorbent assay (ELISA) (Garber, 2008; Wang et al., 2010). However, they are limited by complicated preconcentration, time-consuming steps and high-cost instruments. Hence, it is urgent to develop fast, low-cost, highly sensitive and selective sensors to determine trace melamine in real samples. Recently, Au-NPs-based colorimetric sensors have been attracted great attention because of their unique properties such as color, biocompatibility, stability and high extinction coefficients. They have been used for the detection of some species such as 2,4,6-trinitrotoluene (Jiang et al., 2008), glucose (Jiang et al., 2010), dopamine (Zhang et al., 2010), cysteine (Wang et al., 2008), and lead ions (Yoosaf et al., 2007; Huang et al., 2010). Meanwhile, there are a few reports about the colorimetric detection of melamine based on the fact that Au-NPs are induced to aggregate by interparticle crosslinking (Ai et al., 2009; Chi et al., 2010; Li et al., 2010; Kuang et al., 2010; Qi et al., 2010; Qin et al., 2009; Wei et al., 2010). We had previously investigated colorimetric detection of melamine by nonaggregation-based Au-NPs as a probe, in which the synthesis of Au-NPs was hindered by melamine (Cao et al., 2010). Herein, we report a novel method that the presence of melamine accelerates the synthesis of Au-NPs and changes Au-NPs from aggregation to dispersion (Fig. 1). PD was used to reduce Au3+ ion. However, the synthesis of Au-NPs was slow because of intramolecular hydrogen-bonding formed by adjacent sulfonate and ␣-hydroxyl group in PD in solution (Dong et al., 2003). And PD
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Fig. 1. (A) Schematic illustration of possible procedure of synthesis and aggregation of PD-capped-NPs. (B) The process that melamine accelerated the generation of Au-NPs.
capped Au-NPs obtained by the above method were aggregated because of intermolecular hydrogen-bonding (Fig. 1A). While in the presence of melamine, the synthesis of Au-NPs was fast and the obtained Au-NPs were relatively dispersed (Fig. 1B). The reasons are as follows: as it is well known, melamine has a good hydrogen-bonding ability, and it can interact with sulfonate ligand (Zhang, 2007). Therefore, intramolecular H bond of PD was interrupted by the O· · ·HN formed between PD and melamine. The ␣-phenolic-OH was free from the intramolecular H bond, which increased the reduction reactivity of PD, and thus the reduction process of Au3+ was accelerated. In this way, colorimetric detection of melamine could be realized during the formation of the Au-NPs.
All stock solution were prepared daily with Milli-Q-purified distilled water. Before synthesis of Au-NPs, 50 L of 0.01 M PD and 50 L of different concentration of melamine were mixed and shaken for 30 min to obtain a stable solution. In 4 mL centrifuge tube, the mixed solution of 3 mL of phosphate buffer solution (0.01 M pH 7.0), 2.0 × 10−4 M HAuCl4 , 1.6 × 10−4 M PD and different concentration of melamine was shaken for 20 times with a steady rhythm and was allowed to further react for 50 min at room temperature. After that, the absorbance spectra and photographs of the solution were recorded.
2. Experimental
2.4. Instruments
2.1. Chemicals and materials
The FT-IR spectra of PD and formed Au-NPs were detected in Avatar 360 FT-IR ESP. The UV–vis spectra were recorded by a UV2550 spectrophotometer (Shimadzu, Japan) at room temperature. Au-NPs were characterized by transmission electron microscopy (TEM, T20) at 200 kV. The dynamic light scattering (DLS) measurements were carried out using the Nicomp 380 ZLS. All the pH measurements were performed with PB-10 pH meter (Sartorius, Germany).
All chemicals were of analytical reagent grade and used without further purification. Milli-Q-purified distilled water was used throughout the experiments. Melamine was purchased from Sigma (USA). Pyrocatechol-3,5-disodiumsulfonate (PD) was from Shanghai Chemical Reagent Company (Shanghai, China). HAuCl4 ·4H2 O and uracil were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Vitamin C was from Xilong Chemical Factory of Guangdong (Guangdong, China). Alanine, Glutamic, Tryptophan, Arginine and Tyrosine were from Zhongbei Linge Biotechnology Ltd. (Beijing, China). Cyanuric acid was from Alfa Aesar (USA). CaCl2 , MgCl2 , glucose, sucrose, maltose and lactose were all from Beijing Chemical Reagent Company (Beijing, China). Glycerol was from Sangon Biotech Co. Ltd. (Shanghai, China). The 0.01 M phosphate buffer (PB) solutions of pH 7.0 were prepared by mixing the stock solutions of 0.01 M NaH2 PO4 and 0.01 M Na2 HPO4 . 2.2. Sample preparation The liquid milk bought from local supermarket was pretreated according to the general procedure (Cao et al., 2009). 5 g liquid milk and 5 mL of 61 mM trichloroacetic acid were added into 35 mL of 4.9 M methanol solution. After 15 min sonication and 10 min shaking, the mixture was centrifuged at 10,000 rpm for 4 min, and the supernatant was filtrated. Then, the filtrate was concentrated to 10 mL and filtered through a 0.45 m filter membrane to obtain the samples for detection.
2.3. Experimental conditions
3. Results and discussion 3.1. Synthesis of Au-NPs Gold nanoparticles (Au-NPs) were synthesized by reducing Au3+ using PD at room temperature (20 ± 2 ◦ C) without adding any other stabilizing agents. Since H+ was involved in the oxidation–reduction process of PD, the effect of pH on the oxidation of PD was carried out at a pH range from 5.0 to 9.0 (Fig. S1, supplementary material). As shown in Fig. S1, at pH 7.0, the maximum absorption of Au-NPs (436 nm) was the largest. Therefore, pH 7.0 was selected for further experiments. The selection of PD as reducer was due to its both reducing and stabilizing groups, which makes in situ synthesis of Au-NPs possible. Obviously, the concentration of PD is very important for the formation of Au-NPs. Fig. S2 shows the UV–vis absorption spectra of the Au-NPs formed in the presence of different concentrations of PD (4.9 × 10−5 to 3.8 × 10−4 M) in 0.01 M pH 7.0 PBS. It is clear that the absorption of the plasmon band of the Au-NPs is influenced by
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Fig. 2. IR spectra of PD-modified Au-NPs (a) and PD (b). Fig. 3. UV–vis spectra and the color change of Au-NPs formed without melamine (a) and with 1.6 × 10−7 M melamine (b).
the concentration of PD. The absorption at 436 nm is very weak at lower concentrations of PD and becomes more intense and sharp with the PD concentration increasing. However, the change is very small when the concentration is over 2.4 × 10−4 M. We used two Au-NPs obtained by different PD concentrations (1.6 × 10−4 M PD and 2.4 × 10−4 M PD) to study the effect of Au-NPs on the relevant optical absorption change, as a foundation for molecular recognition and analytical detection of melamine. The experimental results showed that compared with Au-NPs obtained by larger PD concentration (2.4 × 10−4 M PD), those obtained by 1.6 × 10−4 M PD were more sensitive to melamine. Thus 1.6 × 10−4 M PD was chosen for further experiments. To obtain the optimum conditions, the reaction time (10–80 min) was also examined in details (Fig. S3). Fig. S3 shows the absorbance of Au-NPs at 436 nm that was recorded at different time intervals. Obviously, the absorbance increased rapidly during 50 min and much slower over the next 30 min, indicating that the synthesis of Au-NPs almost completed within 50 min under this condition. Therefore, 50 min was selected as reaction time. The prepared Au-NPs were characterized by FT-IR spectroscopy. The FT-IR spectrum of PD and formed Au-NPs are shown in Fig. 2. In curve a, the disappearance of the bands in region 1272–1129 cm−1 in PD and a comparatively low intensity of the band in 1044 cm−1 which are the characteristic of S O stretching vibration of sulfurnate indicated that the binding of the sulfurnate moiety to the nanoparticle surface. The band at 3494 cm−1 in curve b corresponding to –OH stretch vibrations from pure PD was disappeared after reaction with Au3+ ion and a broad absorption band was observed in region 3010–3730 cm−1 , which is the contribution of the phenolicOH oxidation in the formation of Au-NPs. Furthermore, it was noticed that the peaks at 1589, 1469, 1436 and 1378 cm−1 in curve b which attribute to aromatic C C and phenolic C–O vibrations disappeared in curve a, indicating that most of PD were oxidized. The above results indicated that Au-NPs could be formed by reducer PD.
formation of Au-NPs from aggregation to dispersion, which was consistent with the TEM images (Fig. 4). Fig. S4 displays the color change during Au-NPs synthesis in the absence and presence of melamine (1.6 × 10−7 M) at different time. Compared Fig. S4A to Fig. S4B, there is a significant color change of system ‘b’ (with 1.6 × 10−7 M melamine) than that of system ‘a’ (without melamine), indicating the reaction in system ‘b’ was faster than that in system ‘a’. Compared Fig. S4C with Fig. S4D, the obvious color change of system ‘a’ than that of system ‘b’ suggested that the synthesis of Au-NPs in system ‘a’ is slower than that in system ‘b’, that is to say the presence of melamine accelerated the synthesis of Au-NPs. As depicted in Fig. 1A, there is an intramolecular H bond formed by adjacent sulfurnate and ␣phenolic-OH in PD in water solution, thus the synthesis of Au-NPs was relatively slow. On the other hand, some free PD molecules were attached to the surface of Au-NPs through sulfurnate. Hydrogen bonding interactions between neighboring –OH (free PD) and O groups (oxidized PD) resulted in the aggregations of Au-NPs. In the presence of melamine, the intramolecular H bond of PD was interrupted by the O· · ·HN formed between PD and melamine (Fig. 1B). Therefore, the ␣-phenolic-OH was free from intramolecular H bond, which increased the reactivity of PD, and the formation of Au-NPs was accelerated. Meanwhile, Au-NPs were relatively dispersed, which might be ascribed to steric hindrance induced by binding of melamine to PD. To further prove the conclusions above, the particle size of AuNPs formed in presence and absence of melamine was measured by dynamic light scattering (DLS). DLS is an analytical tool used routinely for measuring the hydrodynamic sizes of nanoparticles and colloids in a liquid environment. As shown in Table S1, the mean particle size decreased in the presence of melamine, in accordance with the results of UV–vis spectrum (Fig. 3).
3.2. Effect of melamine on Au-NPs synthesis
3.3. Colorimetric detection of melamine
The ultimate goal of this study is to detect melamine through the synthesis of Au-NPs, hence the influence of melamine on Au-NPs synthesis was investigated. As shown in Fig. 3, the color of the Au-NPs solution was green without melamine and yellow with melamine (1.6 × 10−7 M) in the same reaction conditions. The absorption spectrum of AuNPs exhibited a large change in the presence of melamine: the absorbance peak at 600 nm disappeared, indicative of the trans-
To investigate this simple assay for the direct colorimetric visualization of melamine, quantitative analysis was performed by adding different concentration of melamine into the growth solution and monitoring the absorption peak and the color changes of the system. Results showed that the color of the solution gradually changed from green to yellow after reaction for 50 min (Fig. 5A). The absorbance of Au-NPs at 600 nm (A600 ) gradually decreased with the increasing concentration of melamine. We used the absorbance
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Fig. 5. (A) Absorbance spectra of the Au-NPs generated in the presence of different concentrations of melamine: (a) 0, (b) 8.0 × 10−9 , (c) 1.6 × 10−8 , (d) 8.0 × 10−8 , (e) 1.6 × 10−7 , (f) 8.0 × 10−7 , and (g) 1.6 × 10−6 M. All systems include HAuCl4 , 2.0 × 10−4 M, and PD, 1.6 × 10−4 M, in 0.01 M PBS. Spectra were recorded after a fixed time interval of 50 min. Inset: photographic images of the Au-NPs formed in the presence of different concentrations of melamine. (Labels correspond to the curve labels in the spectra.) (B) Plot of A436 /A600 against the logarithm of melamine concentrations. Error bars show the standard deviations of measurements taken from three independent experiments. Fig. 4. TEM images of the Au-NPs formed in the absence (A) and presence (B) of 1.6 × 10−7 M melamine.
ratio A436 /A600 to quality the concentration of melamine in this system. A linear correlation existed between the ratio A436 /A600 and the logarithm of melamine concentration in the range from 4.8 × 10−9 to 1.6 × 10−6 M with a correlation coefficient of 0.9949 (Fig. 5B). Theoretical detection limits defined as 3 of the proposed method was 6.4 × 10−10 M (i.e., 0.08 ppb) obtained by UV–vis spectrum. Relative standard deviation for measurement of 4.8 × 10−8 M melamine for 10 independent determinations was 4.2%, indicating that the proposed method has good reproducibility. In addition, the results obtained above were compared with those of recently reported colorimetric detection of melamine based on the fact that Au-NPs are induced to aggregate by inter-particle cross-linkers (color changed from red to blue). The advantages of the proposed method are as following: First, the proposed colorimetric detection of melamine in this study is realized during the formation of Au-NPs, i.e., the preparation of Au-NPs and melamine detection could be carried out simultaneously at room temperature. Second, the presence of melamine accelerated the synthesis of Au-NPs by interrupting the intramolecular hydrogen-
bonding of the reducer PD. Third, the presence of melamine made Au-NPs changed from aggregation to dispersion (color changed from green to yellow). Last, the proposed method has a lower detection limit (Table S2). Fig. 6 shows the absorbance ratio A436 /A600 caused by melamine and other interferences. Obviously, melamine caused the largest absorbance ratio A436 /A600 of formed Au-NPs, indicating that the synthesis of Au-NPs was influenced by melamine most effectively. In addition, uracil gave a larger ratio A436 /A600 than other interferences did, which demonstrated that its presence could affect the determination of melamine to some degree. 3.4. Analytical applications The detection of the melamine in pretreated liquid milk was carried out. 50 L of the pretreated sample was added into 200 L of different concentration of melamine, and then the mixture was shaken for 20 min to get a stable solution. Then the other steps were the same as those above mentioned. As shown in Fig. S5, different color was observed with different melamine concentrations. The recoveries of 6.4 × 10−8 , 12.8 × 10−8 , 63.9 × 10−8 and 12.8 × 10−7 M of melamine were 107%, 93%, 95% and 106%, respec-
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analytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences (Grant no. SKLEAC2010005), National Sci-Tech Major Special Item for Water Pollution Control and Management (Grant no. 2009ZX07527-007-003), and the National Natural Science Foundation of China (Grant nos. 20877099 and 20775088). We are grateful to Dr. Wensheng Lu at ICCAS for his helpful discussion. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2010.11.007. References
Fig. 6. Absorbance ratio A436 /A600 of Au-NPs in the presence of melamine or other interferences (the concentrations of melamine and other interferences were 1.6 × 10−6 M). Spectra were recorded after a fixed time interval of 50 min. Error bars show the standard deviations of measurements taken from three independent experiments.
tively. The detection limit (3) obtained by UV–vis spectrum was 8.0 × 10−9 M (i.e., 1.0 ppb) and below the safety limit (2.5 ppm in USA and the UK; 1 ppm for infant formula in China), indicating that the proposed method was applied successfully to the determination of melamine in pretreated liquid milk products. 4. Conclusions A selective and simple colorimetric assay to detect melamine during the formation of Au-NPs was proposed in this study. At room temperature, the synthesis of the Au-NPs was realized by using PD as the reducer of Au3+ without adding any other stabilizing agents. Particularly, the preparation of Au-NPs and melamine detection could be carried out simultaneously. Concentrations of melamine could be directly monitored according to the color change of the formed Au-NPs, which made the melamine determination convenient and easy. The absorbance ratio (A436 /A600 ) was linear with the logarithm of melamine concentration in the range of 4.8 × 10−9 to 1.6 × 10−6 M with a correlation coefficient of 0.9949. The detection limit (3) obtained by UV–vis spectrum was 6.4 × 10−10 M (i.e., 0.08 ppb). The proposed method was applied successfully to the detection of melamine in the pretreated liquid milk products, and the recoveries were from 93% to 107%. Acknowledgments This work was supported by a grant from the Major State Basic Research Development Program of China (973 Program) (Grant no. 2011CB933200), the State Key Laboratory of Electro-
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