Analytical Biochemistry 456 (2014) 43–49
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Colorimetric detection of melamine in milk by citrate-stabilized gold nanoparticles Naveen Kumar ⇑, Raman Seth, Harish Kumar Dairy Chemistry Division, National Dairy Research Institute, Karnal 132001, Haryana, India
a r t i c l e
i n f o
Article history: Received 20 December 2013 Received in revised form 27 March 2014 Accepted 2 April 2014 Available online 13 April 2014 Keywords: Gold nanoparticles Melamine Milk Quantification Limit of detection
a b s t r a c t Here, we report a simple and sensitive colorimetric method for detection of melamine in milk using gold nanoparticles (AuNPs). AuNPs of 21-nm size were synthesized by the citrate reduction method. The method is based on the principle that the melamine causes the aggregation of AuNPs and, hence, the wine red color of AuNPs changes to blue or purple. This change in color can be visualized with the naked eye or an ultraviolet–visible (UV–Vis) spectrometer. Under optimized conditions, AuNPs are highly specific for melamine and can detect melamine down to a concentration of 0.05 mg L 1. Ó 2014 Elsevier Inc. All rights reserved.
Nanotechnology has emerged as a new tool for solving food safety-related issues during recent years. Nanoparticles have been applied in making nanosensors for the detection of foodborne pathogens, microorganisms, and other contaminants. Nanoparticle-based sensing systems provide fast and reliable detection of contaminants with lower cost compared with chromatography and immunoassay-based analytical methods. Melamine adulteration of infant formula is one recent example of food safety crisis that has raised worldwide concern about food products. Melamine is a synthetic chemical compound primarily used in the manufacture of laminates, plastics, coatings (including can coatings), commercial filters, adhesives, and dishware/kitchenware. It has high nitrogen content (66%) and, hence, is used for increasing apparent protein content of milk and other food products. The standard tests (Kjeldahl and Dumas) for estimating protein in food are unable to distinguish between nitrogen of protein and nonprotein sources and, hence, give false results. The U.S. Food and Drug Administration has issued strict guidelines for the level of melamine in food products (1 ppm in infant formula and 2.5 ppm in other food products). So, there is an urgent need to develop rapid and reliable methods for melamine detection and to validate existing methods.
⇑ Corresponding author. E-mail address: [email protected] (N. Kumar). http://dx.doi.org/10.1016/j.ab.2014.04.002 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Recently, several methods for melamine detection, including gas chromatography/mass spectrometry (MS)1 [1–3], high-performance liquid chromatography/MS [4,5], capillary zone electrophoresis/MS [6–8], and potentiometric [9] and electrochemical [10–12] methods, have been reported. However, most of these methods are time-consuming, are expensive, and need trained people. Some authors have also proposed nanotechnology-based methods [13–15]. In the current study, we aimed to synthesize gold nanoparticles (AuNPs) and use them to detect melamine in milk. The citrate-stabilized gold nanoparticles are wine red in color in the absence of melamine, whereas in the presence of melamine they change to blue because melamine causes the aggregation of nanoparticles. Materials and methods Chemicals Melamine (99%) and chloroauric acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Sodium citrate was purchased from Glaxo Laboratories (India) (Mumbai, India). Sodium hydroxide was purchased from Thermo Fisher Scientific India (Mumbai, 1 Abbreviations used: MS, mass spectrometry; AuNP, gold nanoparticle; UV, ultraviolet–visible; TEM, transmission electron microscopy; DLS, dynamic light scattering; RSD, relative standard deviation; LOD, limit of detection; LOQ, limit of quantitation.
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AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
India). Potassium hexacyanoferrate(II) trihydrate and zinc sulfate were purchased from Merck (Mumbai, India). Melamine stock solution was prepared by dissolving 10 mg of melamine in 1 L of water. All of the solvents and reagents were of analytical grade and were used without further purification. Millipore Milli-Q water (18 MO cm) was used in all of the experiments. The raw milk was obtained from the cattle yard of the university campus.
round-bottom flask with three necks and boiled on a magnetic hot plate. Then 5 ml of trisodium citrate (38.8 mM) was added rapidly into boiling chloroauric acid solution with high-speed stirring. The pale yellow color of chloroauric acid changed to a wine red color within 3 min. Stirring was continued for 15 min. The wine red AuNPs that formed were then cooled to room temperature and stored at 4 °C for further use.
Preparation of citrate-stabilized AuNPs
Sample extraction and detection of melamine
All of the glassware used in preparation of AuNPs was dipped in freshly prepared aqua regia (HNO3/HCl, 1:3) and rinsed thoroughly with water. Aqua regia is highly corrosive and must be handled with care; otherwise, an explosion, skin burns, or eye/respiratory tract irritation may result. One should work under a fume hood while dealing with aqua regia, and before disposal it should be neutralized. AuNPs were prepared by the citrate reduction method as described by Grabar and coworkers [16] with some modifications. Chloroauric acid (100 ml, 1 mM) was placed in a 250-ml
Milk proteins (e.g., casein) may interfere in melamine detection using AuNPs; therefore, samples were treated before analysis to precipitate the protein and remove fat. The sample extraction procedure is described here. A 10-ml raw milk sample was taken in a centrifuge tube. Then 2.5 ml of potassium hexacyanoferrate(II) trihydrate (3.6% aq.) solution was added into the sample and vortexed for 1 min. Next 2.5 ml of zinc sulfate (7.2% aq.) solution was added and vortexed for 1 min. The mixture was then centrifuged at 10,000 rpm for 5 min. The clear supernatant was taken
Fig.1. Absorption spectra of AuNPs in the absence of melamine (red line) and in the presence of melamine (blue line). Experimental conditions: 700 ll of AuNPs + 400 ll of H2O (red line) and 700 ll of AuNPs + 400 ll of melamine (blue line). The level of the addition of melamine was 0.5 mg/L, and the incubation time was 15 min. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig.2. TEM images of AuNPs in the absence of melamine (dispersed) and in the presence of melamine (aggregated).
AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
45
Fig.3. (A) Size distribution of AuNPs (average size = 21 nm). (B) Size distribution of AuNPs on the addition of 1 ppm melamine (average size = 53 nm).
Fig.4. Schematic representation of melamine detection and visual color change of AuNPs after the addition of melamine. The inset shows a photograph of visual color change of AuNPs on the addition of 1 mg/L melamine.
in another centrifuge tube and adjusted to pH 8.0 with 1 M NaOH. It was filtered through a 0.22-lm filter, and the filtrate was used for colorimetric detection of melamine using AuNPs. Then 400 ll of sample filtrate was mixed with 700 ll of AuNPs and absorption
spectra were recorded to carry out the recovery study. Spiked milk samples were made by the addition of stock melamine solution at different concentrations (0, 0.2, 0.4, 0.8, and 1.2 mg/L) and extracted following the described procedure.
46
AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
Instrumentation Absorption spectra were recorded on a SPECORD 200 ultraviolet–visible (UV–Vis) spectrophotometer (Analytikjena, Germany) at room temperature. Transmission electron microscopy (TEM) measurements were made on a TECNAI F20 high-resolution transmission electron microscope (FEI, Holland) operated at an accelerating voltage of 200 kV. The samples for TEM characterization were prepared by placing a drop of colloidal solution on carbon-coated copper grid and dried at room temperature. The sizes of nanoparticles were determined on a Malvern Zetasizer (version 7.03, Malvern Instruments, UK) equipped with a 633-nm He–Ne laser. Dynamic light scattering was used to measure particle size. This technique measures the diffusion of nanoparticles moving under Brownian motion and converts this to size. The pH measurements were carried out on a digital pH meter (Electronic Corporation of India). The centrifugation was performed on a Kubota 6800 centrifuge (Kubota, Japan). The photographs were taken with a Samsung SL-502 digital camera. Results and discussion Characterization of AuNPs AuNPs were prepared using trisodium citrate as reducing agent as well as stabilizing agent. Citrate-stabilized AuNPs have a nega[A]
tive charge on the surface [16]. Colloidal solution of AuNPs has a wine red color with a strong absorbance peak around 523 nm (Fig. 1). The wine red color of AuNPs indicated that the nanoparticles were well dispersed, and on aggregation caused by melamine they changed to blue color with an absorbance peak around 640 nm. This was confirmed by TEM images of AuNPs with and without melamine (Fig. 2). The dynamic light scattering (DLS) results determined the average size of AuNPs as 21 ± 2 nm, which on aggregation changed to 53 nm, as shown in Fig. 3.
Principle of melamine detection using AuNPs Fig. 4 describes the basic idea of melamine detection. The principle of melamine detection is based on the surface stabilization of AuNPs. AuNPs are stabilized by negatively charged citrate ions. The negatively charged citrate ions form an electrostatic layer on AuNPs and keep the nanoparticles separated and stable in aqueous solution. However, the addition of a trace amount of melamine causes the destabilization and aggregation of nanoparticles, which increases with the increase in concentration of melamine. Melamine has three amine groups (–NH2) that interact with AuNPs through the ligand exchange with negatively charged citrate ions. This aggregation results in a red-to-blue color change, and a new absorption peak around 640 nm appeared (Figs. 1 and 4). The new absorption peak is attributed to electric dipole–dipole interac-
A
1.2
B Absorpon rao (A640/A523)
1.0
0.8
0.6
0.4
0.2
0.0 0
2
4
6
8
10
12
14
16
Time (min) Fig.5. (A) Time-dependent change in absorption spectra of AuNPs in the presence of melamine (0.5 mg/L). (B) Effects of reaction time on the absorption ratio (A640/A523) (n = 3).
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AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
1.2
tion and coupling between plasmons of neighboring particles in the aggregates [17].
A
Optimization of assay conditions The current colorimetric method is based on the melamineinduced aggregation of AuNPs and the resulting change in absorption spectra of AuNPs. The aggregation of AuNPs can be influenced by factors such as media pH and reaction time. Therefore, we investigated these parameters for making this method highly stable and reproducible. The pH of media is an important factor influencing the stability of AuNPs. When AuNPs are prepared following the reduction of tetrachloroauric acid with trisodium citrate, the pH of colloidal AuNPs solution is approximately 6.5. AuNPs are best stable near neutral pH. When the pH of filtrate from the milk sample was highly acidic or basic, it caused the abrupt color change of AuNPs even in the absence of melamine. When the pH of filtrate was adjusted to 8.0 using 1 M NaOH, no color change was observed in the absence of melamine, whereas the presence of trace melamine caused a significant color change of AuNPs. Thus, the pH of media was kept at 8.0 for all experiments. We also investigated the interaction time required for optimum color change of AuNPs in the presence of melamine. For this, a change in absorption spectra of AuNPs after the addition of melamine (0.5 mg/L) was observed, and the absorption ratio (A640/ A523) was plotted against time (Fig. 5). Fig. 5A shows that there is a gradual increase in absorption peak around 640 nm with time that became constant after 15 min. It is assumed that all of the melamine molecules have been exhausted and that no further aggregation of AuNPs occurred. Therefore, the results were taken after 15 min.
Absorpon rao (A640/A523)
1.0 0.8 0.6
y = 0.4571x + 0.1042 R² = 0.9753
0.4 0.2 0.0 0
0.5
1
1.5
2
2.5
Concentraon (mg/L)
B
Calibration curve and sensitivity Standard melamine solutions were prepared by dissolving the known concentration of melamine in water. AuNPs were used to detect the melamine in standard melamine solution under the above optimized conditions, and the calibration curve were drawn by plotting the absorbance ratio (A640/A523) against the melamine concentration (Fig. 6). The calibration curve was linear over a concentration range of 0.1 to 2 mg/L with the regression equation y = 0.4571x + 0.1042 and R2 of 0.975. There was a gradual increase in the absorption value of A640/A523 with an increase in the concentration of melamine. Matrix effect To study the matrix effect, a blank milk sample was extracted and the filtrate was spiked at seven different concentrations of melamine (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 ppm). Melamine was analyzed using AuNPs, and matrix-matched calibration curves were drawn. The slopes of matrix-matched calibration curves and solvent calibration curves were compared by means of a Student’s t test (see Fig. S2 in online Supplementary material). No significant difference was obtained between the slopes of the two curves (solvent and matrix matched), with the calculated t value (2.179) being lower than the tabulated t value (2.919) with a probability of 95%. Therefore, the calibration curve obtained in solvent (water) was used to carry out the recovery study in milk samples. Interference study To examine the specificity of the AuNPs-based melamine detection method, we investigated the interference of common adulterants and preservatives on detection of melamine (1 mg/L). This
Fig.6. (A) Calibration curve of melamine detection. (B) UV–Vis absorption spectra of AuNPs in the presence of different concentrations of melamine (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 ppm) (n = 3).
was evaluated by monitoring the change in the absorption ratio of AuNPs in the presence of melamine and other substances (Fig. 7). Results showed that the presence of selected adulterants and preservatives does not interfere in melamine detection. We also investigated the possible interference of selected amino acids, lactose, galactose, and amine-bearing compounds on melamine detection using AuNPs. Results are shown in Fig. S1 of the supplementary material. It was found that the presence of amino acids, lactose, galactose, and diphenylamine did not cause any interference in melamine detection, but the presence of aminecontaining compounds, namely o-phenylenediamine dihydrochloride and p-phenylenediamine, caused a change in the absorption ratio (A640/A523) of AuNPs. Analysis of melamine in real samples To validate the analytical performance of AuNPs to detect melamine in milk samples, different amounts of melamine were added in milk samples. The spiked milk samples were extracted and analyzed for qualitative and quantitative estimation of melamine under optimized conditions. No distinguishable color change of AuNPs was observed on the addition of filtrate from control samples, whereas a red-to-purple/blue color change was observed on the addition of filtrate from melamine-spiked samples and confirmed by the change in absorption spectra (Fig. 8). Above a concentration of 1 mg/L, the change in color of AuNPs was very
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AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
1.0
Absorpon rao (A640/A523)
0.8
0.6
0.4
0.2
0.0
Melamine
Glucose
Urea
Formalin
H2O2
Sucrose
Nitrate
Dextrose
Fig.7. Selectivity of the AuNPs toward melamine versus other tested adulterants and preservatives. The concentration of melamine is 1 ppm, whereas that of all the other substances is 100 ppm (n = 3).
[A] 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 400
450
500
550
600
650
Absorbance
700
750
800
[nm]
Fig.8. UV–Vis spectra of AuNPs in the presence of melamine (0, 0.2, 0.4, 0.8, and 1.2 mg/L). The inset shows a photograph of visual color change of AuNPs.
rapid, but at lower concentrations it became slow; so, the results were taken after 15 min. Recoveries and precision data are shown in Table 1. The recoveries of melamine from spiked milk samples varied from 95 to 105%, with an average recovery ± standard deviation (n = 3) of 99.8 ± 4.42. The relative standard deviations (RSDs) ranged from 1.28 to 10.53% (n = 3). In general, the method accuracy Table 1 Recovery and precision data for milk samples spiked with melamine. Added (mg/L)
Found (mg/L)
Recovery (%)
RSD (%)
0.2 0.4 0.8 1.2
0.19 ± 0.02 0.42 ± 0.04 0.78 ± 0.01 1.22 ± 0.07
95.0 105.0 97.5 101.7
10.53 9.52 1.28 5.74
Note. Data are presented as means ± standard deviations (n = 3).
and precision were very good, with recoveries greater than 95% and RSDs less than 11%. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated as 3 and 10 times, respectively, the standard deviations of the intercepts of the calibration curves divided by the slopes of the calibration curves [18]. Under the optimized conditions, the current method exhibited an LOD of 0.05 mg/L and an LOQ of 0.16 mg/L in milk samples; which is much lower than the permitted level of melamine by the Food and Drug Administration.
Conclusion In this work, we have demonstrated an AuNPs-based colorimetric method for detection of melamine in milk. Melamine is able to
AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
cause the aggregation of AuNPs and the subsequent color change from red to blue/violet. The change in color of AuNPs can be visualized with the naked eye without the use of any sophisticated instrument. The method is highly sensitive for melamine and able to detect melamine down to a concentration of 0.05 mg/L.
[7]
[8]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.04.002.
[9]
[10]
References [11] [1] X.M. Xu, Y.P. Ren, Y. Zhu, Z.X. Cai, J.L. Han, B.F. Huang, Y. Zhu, Direct determination of melamine in dairy products by gas chromatography/mass spectrometry with coupled column separation, Anal. Chim. Acta 650 (2009) 39–43. [2] H. Miao, S. Fan, P.P. Zhou, L. Zhang, Y.F. Zhao, Y.N. Wu, Determination of melamine and its analogues in egg by gas chromatography–tandem mass spectrometry using an isotope dilution technique, Food Addit. Contam. A 27 (2010) 1497–1506. [3] M. Ibáñez, J.V. Sancho, F. Hernández, Determination of melamine in milk-based products and other food and beverage products by ion-pair liquid chromatography–tandem mass spectrometry, Anal. Chim. Acta 649 (2009) 91–97. [4] X. Xia, S.Y. Ding, X.W. Li, X. Gong, S.X. Zhang, H.Y. Jiang, J.C. Li, J.Z. Shen, Validation of a confirmatory method for the determination of melamine in egg by gas chromatography–mass spectrometry and ultra- performance liquid chromatography–tandem mass spectrometry, Anal. Chim. Acta 651 (2009) 196–200. [5] M.S. Fligenzi, E.R. Tor, R.H. Poppenga, L.A. Aston, B. Puschner, Simultaneous determination and confirmation of melamine and cyanuric acid in animal feed by zwitterionic hydrophilic interaction chromatography and tandem mass spectrometry, Rapid Commun. Mass Spectrom. 21 (2007) 4027–4032. [6] R. Muñiz-Valencia, S.G. Ceballos-Magaña, D. Rosales-Martinez, R. GonzaloLumbreras, A. Santos-Montes, A. Cubedo-Fernandez-Trapiella, R.C. Izquierdo-
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Hornillos, Method development and validation for melamine and its derivatives in rice concentrates by liquid chromatography: application to animal feed samples, Anal. Bioanal. Chem. 392 (2008) 523–531. C.W. Klampfl, L. Andersen, M. Haunschmidt, M. Himmelsbach, W. Buchberger, Analysis of melamine in milk powder by CZE using UV detection and hyphenation with ESI quadrupole/TOF MS detection, Electrophoresis 30 (2009) 1743–1746. Y. Wen, H. Liu, P. Han, Y. Gao, F. Luan, X. Li, Determination of melamine in milk powder, milk, and fish feed by capillary electrophoresis: a good alternative to HPLC, J. Sci. Food Agric. 90 (2010) 2178–2182. R. Liang, R. Zhang, W. Qin, Potentiometric sensor based on molecularly imprinted polymer for determination of melamine in milk, Sens. Actuators B 141 (2009) 544–550. Q. Cao, H. Zhao, L. Zeng, J. Wang, R. Wang, X. Qiu, Y. He, Electrochemical determination of melamine using oligonucleotides modified gold electrodes, Talanta 80 (2009) 484–488. Q. Cao, H. Zhao, Y. He, N. Ding, J. Wang, Electrochemical sensing of melamine with 3,4-dihydroxyphenylacetic acid as recognition element, Anal. Chim. Acta 675 (2010) 24–28. T.H. Tsai, S. Thiagarajan, S.M. Chen, Detection of melamine in milk powder and human urine, J. Agric. Food Chem. 58 (2010) 4537–4544. L. Li, B.X. Li, D. Cheng, L.H. Mao, Visual detection of melamine in raw milk using gold nanoparticles as colorimetric probe, Food Chem. 122 (2010) 895– 900. Z.J. Wu, H. Zhao, Y. Xue, Q. Cao, J. Yang, Y.J. He, Colorimetric detection of melamine during the formation of gold nanoparticles, Biosens. Bioelectron. 26 (2011) 2574–2578. L.P. Zhang, B. Hu, J.H. Wang, Label-free colorimetric sensing of ascorbic acid based on Fenton reaction with unmodified gold nanoparticle probes and multiple molecular logic gates, Anal. Chim. Acta 717 (2012) 127–133. K.C. Grabar, R.G. Freeman, M.B. Hommer, M.J. Natan, Preparation and characterization of Au colloid monolayers, Anal. Chem. 67 (1995) 735–743. S. Basu, S.K. Ghosh, S. Kundu, S. Panigrahi, S. Praharaj, S. Pande, S. Jana, T. Pal, Biomolecule induced nanoparticle aggregation: effect of particle size on interparticle coupling, J. Colloid Interface Sci. 313 (2007) 724–734. S. Ehling, S. Tefera, I.P. Ho, High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid, Food Addit. Contam. A 24 (2007) 1319–1325.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Colorimetric detection of melamine in milk by citrate-stabilized gold nanoparticles Naveen Kumar ⇑, Raman Seth, Harish Kumar Dairy Chemistry Division, National Dairy Research Institute, Karnal 132001, Haryana, India
a r t i c l e
i n f o
Article history: Received 20 December 2013 Received in revised form 27 March 2014 Accepted 2 April 2014 Available online 13 April 2014 Keywords: Gold nanoparticles Melamine Milk Quantification Limit of detection
a b s t r a c t Here, we report a simple and sensitive colorimetric method for detection of melamine in milk using gold nanoparticles (AuNPs). AuNPs of 21-nm size were synthesized by the citrate reduction method. The method is based on the principle that the melamine causes the aggregation of AuNPs and, hence, the wine red color of AuNPs changes to blue or purple. This change in color can be visualized with the naked eye or an ultraviolet–visible (UV–Vis) spectrometer. Under optimized conditions, AuNPs are highly specific for melamine and can detect melamine down to a concentration of 0.05 mg L 1. Ó 2014 Elsevier Inc. All rights reserved.
Nanotechnology has emerged as a new tool for solving food safety-related issues during recent years. Nanoparticles have been applied in making nanosensors for the detection of foodborne pathogens, microorganisms, and other contaminants. Nanoparticle-based sensing systems provide fast and reliable detection of contaminants with lower cost compared with chromatography and immunoassay-based analytical methods. Melamine adulteration of infant formula is one recent example of food safety crisis that has raised worldwide concern about food products. Melamine is a synthetic chemical compound primarily used in the manufacture of laminates, plastics, coatings (including can coatings), commercial filters, adhesives, and dishware/kitchenware. It has high nitrogen content (66%) and, hence, is used for increasing apparent protein content of milk and other food products. The standard tests (Kjeldahl and Dumas) for estimating protein in food are unable to distinguish between nitrogen of protein and nonprotein sources and, hence, give false results. The U.S. Food and Drug Administration has issued strict guidelines for the level of melamine in food products (1 ppm in infant formula and 2.5 ppm in other food products). So, there is an urgent need to develop rapid and reliable methods for melamine detection and to validate existing methods.
⇑ Corresponding author. E-mail address: [email protected] (N. Kumar). http://dx.doi.org/10.1016/j.ab.2014.04.002 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Recently, several methods for melamine detection, including gas chromatography/mass spectrometry (MS)1 [1–3], high-performance liquid chromatography/MS [4,5], capillary zone electrophoresis/MS [6–8], and potentiometric [9] and electrochemical [10–12] methods, have been reported. However, most of these methods are time-consuming, are expensive, and need trained people. Some authors have also proposed nanotechnology-based methods [13–15]. In the current study, we aimed to synthesize gold nanoparticles (AuNPs) and use them to detect melamine in milk. The citrate-stabilized gold nanoparticles are wine red in color in the absence of melamine, whereas in the presence of melamine they change to blue because melamine causes the aggregation of nanoparticles. Materials and methods Chemicals Melamine (99%) and chloroauric acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Sodium citrate was purchased from Glaxo Laboratories (India) (Mumbai, India). Sodium hydroxide was purchased from Thermo Fisher Scientific India (Mumbai, 1 Abbreviations used: MS, mass spectrometry; AuNP, gold nanoparticle; UV, ultraviolet–visible; TEM, transmission electron microscopy; DLS, dynamic light scattering; RSD, relative standard deviation; LOD, limit of detection; LOQ, limit of quantitation.
44
AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
India). Potassium hexacyanoferrate(II) trihydrate and zinc sulfate were purchased from Merck (Mumbai, India). Melamine stock solution was prepared by dissolving 10 mg of melamine in 1 L of water. All of the solvents and reagents were of analytical grade and were used without further purification. Millipore Milli-Q water (18 MO cm) was used in all of the experiments. The raw milk was obtained from the cattle yard of the university campus.
round-bottom flask with three necks and boiled on a magnetic hot plate. Then 5 ml of trisodium citrate (38.8 mM) was added rapidly into boiling chloroauric acid solution with high-speed stirring. The pale yellow color of chloroauric acid changed to a wine red color within 3 min. Stirring was continued for 15 min. The wine red AuNPs that formed were then cooled to room temperature and stored at 4 °C for further use.
Preparation of citrate-stabilized AuNPs
Sample extraction and detection of melamine
All of the glassware used in preparation of AuNPs was dipped in freshly prepared aqua regia (HNO3/HCl, 1:3) and rinsed thoroughly with water. Aqua regia is highly corrosive and must be handled with care; otherwise, an explosion, skin burns, or eye/respiratory tract irritation may result. One should work under a fume hood while dealing with aqua regia, and before disposal it should be neutralized. AuNPs were prepared by the citrate reduction method as described by Grabar and coworkers [16] with some modifications. Chloroauric acid (100 ml, 1 mM) was placed in a 250-ml
Milk proteins (e.g., casein) may interfere in melamine detection using AuNPs; therefore, samples were treated before analysis to precipitate the protein and remove fat. The sample extraction procedure is described here. A 10-ml raw milk sample was taken in a centrifuge tube. Then 2.5 ml of potassium hexacyanoferrate(II) trihydrate (3.6% aq.) solution was added into the sample and vortexed for 1 min. Next 2.5 ml of zinc sulfate (7.2% aq.) solution was added and vortexed for 1 min. The mixture was then centrifuged at 10,000 rpm for 5 min. The clear supernatant was taken
Fig.1. Absorption spectra of AuNPs in the absence of melamine (red line) and in the presence of melamine (blue line). Experimental conditions: 700 ll of AuNPs + 400 ll of H2O (red line) and 700 ll of AuNPs + 400 ll of melamine (blue line). The level of the addition of melamine was 0.5 mg/L, and the incubation time was 15 min. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig.2. TEM images of AuNPs in the absence of melamine (dispersed) and in the presence of melamine (aggregated).
AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
45
Fig.3. (A) Size distribution of AuNPs (average size = 21 nm). (B) Size distribution of AuNPs on the addition of 1 ppm melamine (average size = 53 nm).
Fig.4. Schematic representation of melamine detection and visual color change of AuNPs after the addition of melamine. The inset shows a photograph of visual color change of AuNPs on the addition of 1 mg/L melamine.
in another centrifuge tube and adjusted to pH 8.0 with 1 M NaOH. It was filtered through a 0.22-lm filter, and the filtrate was used for colorimetric detection of melamine using AuNPs. Then 400 ll of sample filtrate was mixed with 700 ll of AuNPs and absorption
spectra were recorded to carry out the recovery study. Spiked milk samples were made by the addition of stock melamine solution at different concentrations (0, 0.2, 0.4, 0.8, and 1.2 mg/L) and extracted following the described procedure.
46
AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
Instrumentation Absorption spectra were recorded on a SPECORD 200 ultraviolet–visible (UV–Vis) spectrophotometer (Analytikjena, Germany) at room temperature. Transmission electron microscopy (TEM) measurements were made on a TECNAI F20 high-resolution transmission electron microscope (FEI, Holland) operated at an accelerating voltage of 200 kV. The samples for TEM characterization were prepared by placing a drop of colloidal solution on carbon-coated copper grid and dried at room temperature. The sizes of nanoparticles were determined on a Malvern Zetasizer (version 7.03, Malvern Instruments, UK) equipped with a 633-nm He–Ne laser. Dynamic light scattering was used to measure particle size. This technique measures the diffusion of nanoparticles moving under Brownian motion and converts this to size. The pH measurements were carried out on a digital pH meter (Electronic Corporation of India). The centrifugation was performed on a Kubota 6800 centrifuge (Kubota, Japan). The photographs were taken with a Samsung SL-502 digital camera. Results and discussion Characterization of AuNPs AuNPs were prepared using trisodium citrate as reducing agent as well as stabilizing agent. Citrate-stabilized AuNPs have a nega[A]
tive charge on the surface [16]. Colloidal solution of AuNPs has a wine red color with a strong absorbance peak around 523 nm (Fig. 1). The wine red color of AuNPs indicated that the nanoparticles were well dispersed, and on aggregation caused by melamine they changed to blue color with an absorbance peak around 640 nm. This was confirmed by TEM images of AuNPs with and without melamine (Fig. 2). The dynamic light scattering (DLS) results determined the average size of AuNPs as 21 ± 2 nm, which on aggregation changed to 53 nm, as shown in Fig. 3.
Principle of melamine detection using AuNPs Fig. 4 describes the basic idea of melamine detection. The principle of melamine detection is based on the surface stabilization of AuNPs. AuNPs are stabilized by negatively charged citrate ions. The negatively charged citrate ions form an electrostatic layer on AuNPs and keep the nanoparticles separated and stable in aqueous solution. However, the addition of a trace amount of melamine causes the destabilization and aggregation of nanoparticles, which increases with the increase in concentration of melamine. Melamine has three amine groups (–NH2) that interact with AuNPs through the ligand exchange with negatively charged citrate ions. This aggregation results in a red-to-blue color change, and a new absorption peak around 640 nm appeared (Figs. 1 and 4). The new absorption peak is attributed to electric dipole–dipole interac-
A
1.2
B Absorpon rao (A640/A523)
1.0
0.8
0.6
0.4
0.2
0.0 0
2
4
6
8
10
12
14
16
Time (min) Fig.5. (A) Time-dependent change in absorption spectra of AuNPs in the presence of melamine (0.5 mg/L). (B) Effects of reaction time on the absorption ratio (A640/A523) (n = 3).
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AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
1.2
tion and coupling between plasmons of neighboring particles in the aggregates [17].
A
Optimization of assay conditions The current colorimetric method is based on the melamineinduced aggregation of AuNPs and the resulting change in absorption spectra of AuNPs. The aggregation of AuNPs can be influenced by factors such as media pH and reaction time. Therefore, we investigated these parameters for making this method highly stable and reproducible. The pH of media is an important factor influencing the stability of AuNPs. When AuNPs are prepared following the reduction of tetrachloroauric acid with trisodium citrate, the pH of colloidal AuNPs solution is approximately 6.5. AuNPs are best stable near neutral pH. When the pH of filtrate from the milk sample was highly acidic or basic, it caused the abrupt color change of AuNPs even in the absence of melamine. When the pH of filtrate was adjusted to 8.0 using 1 M NaOH, no color change was observed in the absence of melamine, whereas the presence of trace melamine caused a significant color change of AuNPs. Thus, the pH of media was kept at 8.0 for all experiments. We also investigated the interaction time required for optimum color change of AuNPs in the presence of melamine. For this, a change in absorption spectra of AuNPs after the addition of melamine (0.5 mg/L) was observed, and the absorption ratio (A640/ A523) was plotted against time (Fig. 5). Fig. 5A shows that there is a gradual increase in absorption peak around 640 nm with time that became constant after 15 min. It is assumed that all of the melamine molecules have been exhausted and that no further aggregation of AuNPs occurred. Therefore, the results were taken after 15 min.
Absorpon rao (A640/A523)
1.0 0.8 0.6
y = 0.4571x + 0.1042 R² = 0.9753
0.4 0.2 0.0 0
0.5
1
1.5
2
2.5
Concentraon (mg/L)
B
Calibration curve and sensitivity Standard melamine solutions were prepared by dissolving the known concentration of melamine in water. AuNPs were used to detect the melamine in standard melamine solution under the above optimized conditions, and the calibration curve were drawn by plotting the absorbance ratio (A640/A523) against the melamine concentration (Fig. 6). The calibration curve was linear over a concentration range of 0.1 to 2 mg/L with the regression equation y = 0.4571x + 0.1042 and R2 of 0.975. There was a gradual increase in the absorption value of A640/A523 with an increase in the concentration of melamine. Matrix effect To study the matrix effect, a blank milk sample was extracted and the filtrate was spiked at seven different concentrations of melamine (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 ppm). Melamine was analyzed using AuNPs, and matrix-matched calibration curves were drawn. The slopes of matrix-matched calibration curves and solvent calibration curves were compared by means of a Student’s t test (see Fig. S2 in online Supplementary material). No significant difference was obtained between the slopes of the two curves (solvent and matrix matched), with the calculated t value (2.179) being lower than the tabulated t value (2.919) with a probability of 95%. Therefore, the calibration curve obtained in solvent (water) was used to carry out the recovery study in milk samples. Interference study To examine the specificity of the AuNPs-based melamine detection method, we investigated the interference of common adulterants and preservatives on detection of melamine (1 mg/L). This
Fig.6. (A) Calibration curve of melamine detection. (B) UV–Vis absorption spectra of AuNPs in the presence of different concentrations of melamine (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 ppm) (n = 3).
was evaluated by monitoring the change in the absorption ratio of AuNPs in the presence of melamine and other substances (Fig. 7). Results showed that the presence of selected adulterants and preservatives does not interfere in melamine detection. We also investigated the possible interference of selected amino acids, lactose, galactose, and amine-bearing compounds on melamine detection using AuNPs. Results are shown in Fig. S1 of the supplementary material. It was found that the presence of amino acids, lactose, galactose, and diphenylamine did not cause any interference in melamine detection, but the presence of aminecontaining compounds, namely o-phenylenediamine dihydrochloride and p-phenylenediamine, caused a change in the absorption ratio (A640/A523) of AuNPs. Analysis of melamine in real samples To validate the analytical performance of AuNPs to detect melamine in milk samples, different amounts of melamine were added in milk samples. The spiked milk samples were extracted and analyzed for qualitative and quantitative estimation of melamine under optimized conditions. No distinguishable color change of AuNPs was observed on the addition of filtrate from control samples, whereas a red-to-purple/blue color change was observed on the addition of filtrate from melamine-spiked samples and confirmed by the change in absorption spectra (Fig. 8). Above a concentration of 1 mg/L, the change in color of AuNPs was very
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AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
1.0
Absorpon rao (A640/A523)
0.8
0.6
0.4
0.2
0.0
Melamine
Glucose
Urea
Formalin
H2O2
Sucrose
Nitrate
Dextrose
Fig.7. Selectivity of the AuNPs toward melamine versus other tested adulterants and preservatives. The concentration of melamine is 1 ppm, whereas that of all the other substances is 100 ppm (n = 3).
[A] 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 400
450
500
550
600
650
Absorbance
700
750
800
[nm]
Fig.8. UV–Vis spectra of AuNPs in the presence of melamine (0, 0.2, 0.4, 0.8, and 1.2 mg/L). The inset shows a photograph of visual color change of AuNPs.
rapid, but at lower concentrations it became slow; so, the results were taken after 15 min. Recoveries and precision data are shown in Table 1. The recoveries of melamine from spiked milk samples varied from 95 to 105%, with an average recovery ± standard deviation (n = 3) of 99.8 ± 4.42. The relative standard deviations (RSDs) ranged from 1.28 to 10.53% (n = 3). In general, the method accuracy Table 1 Recovery and precision data for milk samples spiked with melamine. Added (mg/L)
Found (mg/L)
Recovery (%)
RSD (%)
0.2 0.4 0.8 1.2
0.19 ± 0.02 0.42 ± 0.04 0.78 ± 0.01 1.22 ± 0.07
95.0 105.0 97.5 101.7
10.53 9.52 1.28 5.74
Note. Data are presented as means ± standard deviations (n = 3).
and precision were very good, with recoveries greater than 95% and RSDs less than 11%. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated as 3 and 10 times, respectively, the standard deviations of the intercepts of the calibration curves divided by the slopes of the calibration curves [18]. Under the optimized conditions, the current method exhibited an LOD of 0.05 mg/L and an LOQ of 0.16 mg/L in milk samples; which is much lower than the permitted level of melamine by the Food and Drug Administration.
Conclusion In this work, we have demonstrated an AuNPs-based colorimetric method for detection of melamine in milk. Melamine is able to
AuNPs-based detection of melamine in milk / N. Kumar et al. / Anal. Biochem. 456 (2014) 43–49
cause the aggregation of AuNPs and the subsequent color change from red to blue/violet. The change in color of AuNPs can be visualized with the naked eye without the use of any sophisticated instrument. The method is highly sensitive for melamine and able to detect melamine down to a concentration of 0.05 mg/L.
[7]
[8]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.04.002.
[9]
[10]
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