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Rapid and sensitive detection of melamine in milk with gold nanoparticles by Surface Enhanced Raman Scattering
Food Chemistry 159 (2014) 250–256
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Analytical Methods
Rapid and sensitive detection of melamine in milk with gold nanoparticles by Surface Enhanced Raman Scattering Andrea Mario Giovannozzi a,⇑, Francesca Rolle a, Michela Sega a, Maria Cesarina Abete b, Daniela Marchis b, Andrea Mario Rossi a a
Thermodynamic Division, Istituto Nazionale di Ricerca Metrologica, Strada delle Cacce, 91 10135 Torino, Italy C.Re.A.A. – National Reference Centre for the Surveillance and Monitoring of Animal Feed, c/o Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e valle D’Aosta, via Bologna 148, 10154 Torino, Italy b
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 26 November 2013 Accepted 4 March 2014 Available online 13 March 2014 Keywords: Melamine Milk SERS Raman spectroscopy Gold nanoparticles
a b s t r a c t A rapid and sensitive method to detect melamine in liquid milk based on Surface Enhanced Raman Scattering (SERS) spectroscopy is presented, exploiting the selective binding of gold nanoparticles (AuNPs) with this analyte. This interaction promotes the aggregation of the AuNPs inducing a huge enhancement of the melamine signals in the Raman spectrum due to the formation of SERS ‘‘hot spots’’. An external standard calibration method was employed for quantitative analysis and the method was validated for linearity, sensitivity, repeatability and recovery. A good linearity (R2 = 0.99) was found in the concentration range of 0.31–5.0 mg l1 in milk with a limit of detection of 0.17 mg l1. This method does not require a long extraction procedure (total analysis time can be lower than 30 min) and can be reliably used for melamine detection in milk matrix in accordance with the European law limits. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Melamine is an important industrial material that is mainly used for resin production, for thermosetting plastic and for polymer manufacturing in general (Sugita, Ishiwata, & Yoshihira, 1990). Its fame, unfortunately, came out recently because it was used as a food adulterant in milk, pet and animal feed (Brown et al., 2007; Burns, 2007). As a high rich-nitrogen molecule, melamine was intentionally added into food ingredients to produce an incorrectly high reading in the measurement of the protein content based on total nitrogen. The main concern on melamine, as a food additive, is the ability of combining with its analogues, such as cyanuric acid, leading to the formation of insoluble crystals which were responsible for kidney failures and even death in infants in China (Dobson et al., 2008; Hau, Kwan, & Li, 2009; Lam et al., 2009). Considering its potential toxicity, the Codex Alimentarius Commission has set a limit of 1 mg l1 for powder infant formula and 2.5 mg l1 for other foods and animal feed (Report on the Thirty-Third Session of the Joint FAO/WHO Food Standards Programme, 2008). Currently, gas chromatography (GC) or liquid chromatography (HPLC) coupled with mass spectrometry (MS) (Squadrone et al., 2010; Tyan, Yang, Jong, Wang, & Shiea, 2009), ⇑ Corresponding author. Tel.: +39 011 3919330; fax: +39 011 346384. E-mail address: [email protected] (A.M. Giovannozzi). http://dx.doi.org/10.1016/j.foodchem.2014.03.013 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
matrix-assisted laser desorption/ionisation MS (Tang et al., 2009), ELISA (Zhou et al., 2012) and IR spectroscopy (Mauer, Chernyshova, Hiatt, Deering, & Davis, 2009), represent the major categories of techniques for melamine detection. However, these methodologies usually require expensive instrumentations and long sample preparation procedures are needed mainly due to analyte extraction steps. Recently, several methods to detect melamine based on gold nanoparticles (AuNPs) have been developed (Ai, Liu, & Lu, 2009; Li, Li, Cheng, & Mao, 2010; Wei et al., 2010). Some of these methods were based on a colorimetric visual inspection of the nanoparticles solution colour change upon melamine interaction. Melamine interaction with modified or unmodified gold nanoparticles decreases the stability of the AuNPs provoking the formation of aggregates and inducing a shift of the surface Plasmon resonance with a consequent variation of the colour solution from red to blue, that can be easily monitored by UV/Vis absorption measurements. However, in the presence of interference substances in milk, such as other organic molecules or even positively charged ions and whose competing with melamine for AuNPs binding, a change in the AuNPs aggregation state can be seen, even in absence of the analyte and thus leading to a false positive response. In order to avoid these problems, Raman spectroscopy was used since it can provide a fingerprint of the melamine molecule in the Raman spectrum. Raman spectroscopy together with the help of gold or silver nanoparticles offers a very high sensitivity due to the Surface En-
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hanced Raman Scattering (SERS) effect that occurs when a molecule is adsorbed or grafted on a rough metallic surface. The Raman signal of the molecule can be enhanced theoretically up to a 1013 factor for a potential single molecule detection. Different methodologies for the detection of melamine based on the SERS effect were developed. Most of them were based on the fabrication of SERS substrates (Betz, Cheng, & Rubloff, 2012; Cheng & Dong, 2011; Kim, Barcelo, Williams, & Li, 2012), usually prepared by metallic nanoparticles deposition on silicon or glass or by photolithography techniques. SERS substrates demonstrated to achieve a very high sensitivity (detection limit in the lg l1 range) but they usually suffered of lack of reproducibility and homogeneity of the molecule distribution on the SERS substrate, leading to problems in the quantification. Other SERS analysis were developed in liquid, mainly based on silver nanoparticles, achieving very good results for melamine detection in milk (Zhang et al., 2010). As for gold nanoparticles, instead, only few works have been published. Lou et al. (2011) developed a very sensitive indirect method (LOD 0.1 lg l1) to detect melamine in milk by SERS using 4-mercaptopyridine-modified AuNPs. However, the linearity response of this method is between 0.5 and 100 lg l1 which might affect the practical application of this assay in routine analysis. Moreover, the melamine quantification is done by using a Raman reporter and not by the melamine itself. Another interesting work was proposed by Yazgan, Boyaci, Topcu, & Tamer (2012) who developed a rapid and sensitive method to detect melamine in milk by using spherical magnetic-core gold-shell nanoparticles and rod-shaped gold nanoparticles labelled with a Raman-active compound. They reached the limits of detection (LOD) and quantification (LOQ) of 0.38 and 1.27 mg l1, respectively. In this work we propose a simpler method (based on the use of only one type of spherical AuNPs), which guarantees high sensitivity and gives a linear response in a range of concentrations useful for practical applications. We selectively tested spherical AuNPs with different dimensions in order to have the highest SERS effect. Moreover, we tuned the AuNPs concentration to reach the linearity in the selected melamine range. For the calibration of the Raman spectrometer we used the acetonitrile (ACN) Raman band to normalise the Raman intensity of melamine, minimizing possible variations due to laser power, focal distance and environmental parameters (temperature, humidity). The method developed proved to be simple and not requiring a long extraction procedure. The total analysis time can be lower down than 30 min. 2. Material and methods 2.1. Reagents and materials Hydrogen tetrachloroaurate trihydrate (HAuCl4 3H2O P99%), trisodium citrate dihydrate (P99%), melamine (99%) and 10 nm Gold nanoparticles stabilized suspension in citrate buffer were purchased from Sigma–Aldrich (Milan, Italy). Sodium hydroxyde (NaOH 97%), Hydrochloric acid (HCl 37%), Nitric acid (HNO3 68%), absolute ethanol (99,99%) and acetonitrile (>99.5%) were obtained by Carlo Erba Reagents (Rodano, Italy). All solutions were prepared with Milli-Q quality water (18 MXcm). Liquid semi-skimmed milk used for the assays was purchased in a local supermarket in Torino, Italy. 2.2. Gold nanoparticles preparation All glassware used in the experiment was soaked in aqua regia (HCl:HNO3 3:1) and rinsed thoroughly in water and dried with nitrogen prior to use. AuNPs were synthesized according to Frens, 1973. For the preparation of 40 nm AuNPs, 5 ml of a 1% aqueous
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solution of trisodium citrate was rapidly injected into 500 ml boiling solution of HAuCl4 (0.01% v/v). The mixture was further refluxed for 10 min and then cooled to room temperature under continuous stirring and a wine-red colour solution of AuNPs was obtained. For the preparation of 80 nm AuNPs, 2.5 ml of trisodium citrate (1% water solution) was injected into 500 ml boiling solution of HAuCl4 (0.01% v/v). The above mentioned procedure was then applied. All AuNPs solutions were stored at 4 °C before use.
2.3. Gold nanoparticles characterization AuNPs characterization was done by UV–Vis absorption measurements and by Scanning Electron Microscopy (SEM) imaging. UV–Vis absorption spectra were collected with the Evolution 60s spectrophotometer (Thermo Scientific). The surface plasma resonance peaks of AuNPs solutions were measured to be 518, 530 and 554 nm for AuNPs dimensions of 10, 40 and 80 nm, respectively. SEM characterization was carried out using a SEM FEI Quanta 3D or Inspect F in UHV mode with the SE detector. Typical settings for the imaging are: 10 kV accelerating voltage, 2.5 spot (18 pA) or 3.5 spot (30 pA), 10 mm WD.
2.4. Melamine standard solutions Melamine stock standard solution was prepared by accurately dissolving 50 mg of standard in 50 ml of Ethanol/H2O 50:50 (v/v), to reach a concentration of 1000 mg l1. Melamine standard solutions were prepared by subsequent dilutions from the stock solution in water to reach the following concentrations: 100, 20, 10, 5, 1, 0.5, 0.2, 0.1 mg l1. These pure melamine standards were used to set up the analytical procedure. Aliquots of the melamine standards were mixed in a 1:1 ratio with AuNPs stock solutions, mixed with vortex for 3 s and subsequently analysed by UV–Vis and the Raman spectrophotometer. Melamine standard solutions in negative matrix pool were also prepared for the external calibration of the Raman spectrometer, as explained in the paragraph 2.7. Consecutive dilutions were made starting from 10 mg l1 to reach the following concentrations in matrix: 1, 0.50, 0.25, 0.125, 0.063 mg l1. These solutions were mixed with AuNPs (1:1) and analysed by Raman spectroscopy to build the calibration curve.
2.5. Detection of melamine in liquid milk by SERS Aliquots of the 100 mg l1 melamine stock solution were added to milk to obtain concentrations of 0.5, 1, 3, 5 and 10 mg l1. Melamine-free milk was processed as the spiked milk and used to prepare blank samples. The extraction procedure was carried out by first adding 200 ll of 1 M HCl to 4 ml of spiked milk and vigorously mixing by vortex for 10 s. The samples were then transferred into 1.5 ml centrifuge tubes and centrifugated for 30 min at 14 000 rpm. Supernatants from the same sample were collected and filtered with a 0.22 lm PTFE filter. The pH of the filtered solution was adjusted at 4.7 by adding 60 ll of 1 M NaOH. 10 ml of pure ACN were then added inducing the precipitation of most of the proteins in solution. A final centrifugation step was carried out at 14 000 rpm for 30 min in order to remove any aggregates. 250 ll of the resulting supernatant was mixed in a 1:1 ratio with a 10-fold concentrated 40 nm AuNPs solution and immediately analysed by Raman spectroscopy. The 10-fold concentrated 40 nm AuNPs solution was obtained by centrifugating the AuNPs stock solution at 4000 rpm for 30 min and subsequently resuspending in a proper amount of water solution.
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2.6. SERS measurement SERS spectra were recorded using a Thermo Scientific DXR Raman equipped with a microscope, excitation laser source at 780 nm, a motorized microscope stage sample holder, and a charge-coupled device (CCD) detector. Spectra of samples were collected using a 20x long working distance microscope objective with a 24 mW laser power and a spectral range from 200 to 1800 cm1. The acquisition time was of 20 s with 1s exposure time. 2.7. Validation of the method The validation of the method was performed by calculating linearity, sensitivity, repeatability and mean recovery. The instrumental linearity was evaluated from four calibration curves with 5 levels of melamine concentrations in non-spiked milk extract, representative of the analysed matrix: 0.031, 0.063, 0.125, 0.25, 0.50 mg l1 corresponding in the matrix to values from 0.31 to 5.0 mg l1. The calibration, calculated as the area of the Melamine Raman band at 715 cm1 versus melamine concentration, was evaluated by Partial Least Square (PLS) method and the linearity was estimated by the determination coefficient R2. Acceptability criteria to assume the linearity of response is R2 > 0.99. The limit of detection (LOD) was experimentally detected on blank samples (n = 10) and calculated by the equation (Long and Winefordner (1983):
LOD ¼
3sb b
ð1Þ
where sb is the standard deviation of the areas of the blank samples in the Raman spectrum at 715 cm1, and b is the slope of the calibration curve. Indeed, the LOD of melamine concentration in the matrix (mg l1) was evaluated by using the standard calibration curve previously built (715 cm1 band area versus concentration in matrix). The limit of quantitation (LOQ), was estimated with the following equation:
LOQ ¼
10sb b
ð2Þ
Fig. 1. (a) UV–VIS Absorption Spectra of 40 nm AuNPs mixed with an increasing concentration of pure melamine standard solutions: (a) blank, (b) 0.05 mg l1, (c) 0.1 mg l1, (d) 0.25 mg l1, (e) 0.5 mg l1, (f) 1 mg l1, (g) 2.5 mg l1. The sketch above the spectra shows the interaction mechanism of AuNPs with the melamine molecule with the subsequently formation of aggregates together with a change of the colour solution from wine-red to blue-gray. The increased melamine concentration can be followed by the arrow on the graph. SEM images of 40 nm AuNPs before (b) and after (c) interaction with melamine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The recovery (%) was calculated by the average concentration values (n = 6) obtained for melamine spiked samples (at 1 and 3 mg l1 concentration levels corresponding to 0.1 and 0.3 mg l1 in the analysed milk extracts). Each spiked sample was analysed six times in the same day, in order to test the repeatability and the accuracy of the method. The validation parameters are summarized in Table 1. 3. Results and discussion It was reported that melamine can be added into food as an adulterant to increase its apparent protein content. Several papers proposed AuNPs as useful substrates for melamine detection since the colour of the particles changes after the interaction with the melamine in solution. AuNPs are usually fabricated by using the sodium citrate method which allows an easy tunability of particles dimensions just by changing the gold precursor salt and the sodium citrate molar ratio. The sodium citrate, indeed, works both as a reducing agent for gold nucleation and as stabilizing agent by coating the surface of the nanoparticles preventing their aggregation in solution. When the melamine is injected in the system, hydrogen bonds between the melamine amino groups and citrate ions occur, decreasing the electrostatic repulsion between individual AuNPs and finally results in the aggregation of AuNPs (see the Fig. 1). As Fig. 1a shows, the stable 40 nm AuNPs solution (wine-red colour) has a surface plasmon resonance absorption peak at
Fig. 2. Raman spectra and SERS spectra of melamine with AuNPs with different size. (a) Raman spectrum of solid melamine. SERS spectra of a 0.1 mg l1 melamine solution with AuNPs of different size: (b) 40 nm AuNPs, (c) 80 nm AuNPs and (d) 10 nm AuNPs. (e) Raman spectrum of a 1000 mg l1 melamine solution.
530 nm. As soon as the melamine (0.1 mg l1) was added into the AuNPs solution, it was observed that the AuNPs quickly aggre-
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Fig. 3. SERS spectra of 40 nm AuNPs with different melamine concentrations in spiked milk samples: 0, 0.5, 1, 3, 5, 10 mg l1.
gate. As a result of the aggregation, the solution colour became blue-gray and the absorbance at 530 nm decreased and a new absorption band around 700 nm showed up. Increasing the melamine concentration from 0.25 to 2.5 mg l1, the surface plasmon peak at 530 nm continued to decrease and the peak at 700 nm was shifted to higher wavelengths (from 700 to 850 nm), inducing the formation of bigger aggregates. The reaction time is very fast and a equilibrium is reached in less than 1 min. SEM analyses were also used to verify the aggregation state. In Fig. 1b AuNPs appear monodisperse and free of interactions. After the melamine addition in solution, the AuNPs form bigger aggregates (Fig. 1c). This behaviour was not only investigated for the 40 nm AuNPs but it was also tested with 10 and 80 nm AuNPs particles diameter. Melamine interaction with these nanoparticles occurred in the same way and their aggregation was clearly demonstrated by UV–VIS measurements (data not shown). However, analysing the surface plasmon resonance peaks after the aggregation induced
by melamine, it was shown that while the plasmon resonance peak is centred at around 800 nm for the 40 nm AuNPs, the resonance peaks for 10 and 80 nm AuNPs were centred at 660 nm and further than 900 nm, respectively. Since the SERS effect takes place in presence of ‘‘hot spot’’ due to the formation of gold aggregates and its enhancement efficiency can be maximised when the plasmon resonance peak of the aggregates is in resonance with the laser source (780 nm), we checked the Raman signal of the melamine using these three different AuNPs. In Fig. 2 typical Raman peaks of solid melamine at 380, 580, 678 and 983 cm1 were observed. The most intense peak at 678 cm1 is assigned to the ring-breathing II mode, which involves the in-plane deformation of the triazine ring. The second most intense peak at 983 cm1 arises from the ring-breathing mode I of the triazine ring (Koglin, Kip, & Meier, 1996). In the Raman spectrum of pure melamine dissolved in water at a concentration of 1000 mg l1 only a small peak is barely visible at
Fig. 4. SERS spectra of 40 nm AuNPs with 5 levels of melamine standard in negative matrix pool (representative of the analysed matrix): 0.031, 0.063, 0.125, 0.25, 0.50 mg l1 corresponding in the matrix to values from 0.31 to 5.0 mg l1.
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678 cm1 while the other peaks are not detected. In the SERS spectra, instead, even in presence of a small concentration of melamine (0.1 mg l1), the two main melamine peaks at 715 and 1000 cm1 are observed. These specific melamine peaks are shifted by 30 cm1. Remarkable shifts of 10 and 40 cm1 from the original melamine bands at 676 and 983 cm1 were already reported in the literature using respectively Klarite SERS substrates (Liu, Lin, & Li, 2010) and triangular shape AuNPs (Wen, Li, & Ren, 2011). It is reasonable to infer that a strong polarisation occurs at the surface of these AuNPs and where the electric field increases strongly, it can result in not only an increased enhancement factor, but also in a change of the vibrational Raman selection rules, which allows the appearance of forbidden Raman bands. As Fig. 2 clearly shows, Raman enhancement of the melamine signals only occurred when 40 and 80 nm AuNPs are used. When 10 nm AuNPs are used as SERS substrates no enhancement is detected. Moreover, comparing the most intense melamine peak at 715 cm1, 40 nm AuNPs provided the highest enhancement efficiency, probably because of the resonance coupling between the laser excitation source at 780 nm and the gold aggregates absorption band at around 800 cm1. These data are in agreement with what already reported in literature (Bell & McCourt, 2009). In order to demonstrate a practical application in the food analysis field, we decided to detect melamine in liquid raw milk and we started to develop a measurement procedure based on 40 nm AuNPs as a SERS substrate in solution. Food samples are complex matrices that are difficult to analyse because of their protein and carbohydrate content. Detecting low levels of melamine in food is not easy because melamine can be bound with the milk constituents due to its strong tendency to form hydrogen bonds. Thus, prior to the analysis, extraction of melamine from milk is a fundamental step. An acidic extraction was first carried out with hydrochloric acid (1 M) in order to precipitate caseins from milk. Further purifications steps, such as filtrations and solvent extraction using ACN, resulted to be necessary to induce proteins precipitations and to extract melamine simultaneously. Since this method of analysis is based on the covalent bonding of free amino groups in melamine with the AuNPs, the removal of any source of free amino groups is important to increase the efficiency of the detection method, reducing the interfering molecules. Moreover, the pH of the solution was adjusted to maximise the melamine-AuNPs interaction. The solution pH influences both the surface charge on AuNPs and the protonation state of melamine amino groups. Considering the pKa values of the three carboxylic moieties of the citrate ions (pKa = 3.13, 4.76 and 6.34) and the melamine pKa value around 5, setting a solution pH of 4.7 results in a reduction of repulsive forces between AuNPs and it induces the protonation of the melamine molecule. Thus, a pH value around 5 was found to be the best compromise to foster AuNPs aggregation together with their interaction with melamine and it resulted in a chemical enhancement by charge transfer complexes and/or localise surface plasmons, that yield an enhancement of the melamine Raman signal. The initial setup of the analytical procedure was performed by spiking semi-skimmed liquid milk with melamine to obtain concentrations in the range of interest for practical applications. As we already mentioned, limit value of melamine has been set in Europe at 1 mg l1 in infant formula and 2.5 mg l1 in liquid milk and food in general. Various concentrations of melamine were spiked in milk and subsequently extracted and analysed by SERS. As shown in Fig. 3, five levels (0.5, 1, 3, 5 and 10 mg l1) of melamine in liquid milk were analysed. By monitoring the highest intense melamine Raman peak at 715 cm1, it was found that the area of this peak was enhanced with increasing concentration of melamine. To optimise the method several concentrations of AuNPs were tested (data not shown) and the best results were obtained with
a 10 folds concentrated AuNPs solution which guarantees a linear detection response of the melamine Raman signal in the selected concentrations range. As shown in Fig. 3, the melamine peaks corresponding to each concentration of melamine in the spiked samples are well separated and the method gives good responses in the concentration range studied. Moreover, since the final extraction of melamine from the matrix is done in ACN and the solvent concentration is the same for every analysed sample, the area of the ACN peak at 922 cm1 was established as a internal reference to normalise the area of the peak at 715 cm1 and to correct the Raman signal in order to eliminate the effects of the matrix and other factors, such as environmental parameters (temperature, humidity) or instrumental settings like the focal distance. An external calibration procedure was chosen for the melamine quantification in milk. Taking into account the dilution factor for the melamine concentration from the starting matrix through the extraction procedure, five levels of melamine concentrations (0.031, 0.063, 0.125, 0.25 and 0.50 mg l1) were chosen for the cal-
Fig. 5. Calibration curves of melamine standards in negative matrix pool obtained by plotting the normalised area of melamine Raman band at 715 cm1 vs. melamine concentration. (a) Four calibration curves for intra-day analysis and PLS linear fit with equation and determination coefficient. (b) Average values with error bars calculated from the calibration curves in (Fig. 5a).
A.M. Giovannozzi et al. / Food Chemistry 159 (2014) 250–256 Table 1a Recovery of melamine from spiked milk samples.
a b
Concentration of melamine in milk extracts (mg l1)
Average Raman band area (a.u.)
Amount founda (mg l1)
Mean Recovery (%) (n = 6)
RSDb (%)
0.1 0.3
0.8317 2.0288
0.10 ± 0.01 0.29 ± 0.01
99.9 96.3
9.6 3.8
Average value from six determinations for each concentration. Relative standard deviation of mean recovery (RSD (%) = (SD/mean) 100).
Table 1b Intra-day repeatability of the method. Analysesa
Spiked concentration in milk extracts (mg l1)
Amount found (mg l1)
Precision as RSDb (%)
Accuracyc (bias%)
Intra-day (n = 6)
0.1
0.10 ± 0.01
9.6
0.1
0.3
0.29 ± 0.01
3.8
3.7
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for melamine detection. SERS analysis can be also performed with a motorized sample holder stage together with a 96 wells plate in order to make the method automatic and to reduce the total volume of the analysis. The whole extraction procedure can be carried out in less than 30 min without substantially affecting the sensitivity of the methodology and the reliability of the quantification step. Acknowledgements The present work has been supported by EMRP project ‘‘ SurfChem’’. EMRP is jointly founded by the EMRP participating countries within EURAMET and the European Union. Part of this work was carried out by NanoFacility Piemonte, INRiM, a laboratory supported by Compagnia di San Paolo (Italy). References
a
Average of six determinations for each sample. Relative standard deviation (RSD (%) = (SD/mean) 100) of individual measurements. c Bias% = (mean concentrationreal concentration)/real concentration 100). b
ibration curve, which correspond to melamine values range of 0.31–5.0 mg l1 in the milk samples. Melamine-free milk was used to prepare the blank samples, processing it in the same way as the spiked milk. SERS spectra of melamine standard solutions in milk extracts are shown in Fig. 4. The method was validated for linearity, sensitivity, recovery and repeatability. A linear regression was found between the normalised Raman signal at 715 cm1 and melamine concentration with a good determination coefficient (R2) of 0.99 (Fig. 5a-b). According to Eqs. (1) and (2) the LOD and LOQ were 0.017 and 0.057 mg l1 respectively in the milk extracts which correspond to values of 0.17 mg l1 (LOD) and 0.57 mg l1 (LOQ) in the milk matrix. Our method is then suitable for melamine quantification in the concentration range of 0.57–5.0 mg l1 in milk matrix in accordance with the European law limits of 1 and 2.5 mg l1 in dairy products for infants and and other food and animal feed, respectively. Mean recovery was determined in milk samples spiked at 1 and 3 mg l1 concentration levels (corresponding to 0.1 and 0.3 mg l1 in the analysed milk extracts) and it was found to be 99.9% and 96.3% (Table 1a). The intra-day repeatability and accuracy of the method were determined for milk spiked samples with 1 and 3 mg l1 melamine. Repeatability is expressed as relative standard deviation (RSD%) and accuracy is explained by computing bias%. The results for intra-day measurements are shown in Table 1b. The RSD values were 9.6% and 3.8% for 1 and 3 mg l1 spiked samples, respectively. It can be concluded that the method had good repeatability (intraday) and accuracy for melamine detection. 4. Conclusions A sensitive and rapid method to detect melamine in liquid milk was developed by using AuNPs and Raman spectroscopy. Melamine in liquid milk is able to promote the formation of AuNPs aggregates which behave as ‘‘Raman hot spots’’ and enhance the melamine Raman signal allowing melamine detection in the mg l1 range. SERS is emerging as a new technique for analytical methods that can be suitable for high throughput screening analysis and could become a valid alternative to the classical analytical methodologies based on GC or HPLC mass spectrometry. The method here developed is sensitive, fast and it can be applied in routine analysis
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Wei, F., Lam, R., Cheng, S., Lu, S., Ho, D., & Li, N. (2010). Rapid detection of melamine in whole milk mediated by unmodified gold nanoparticles. Applied Physics Letters, 96(13).. Wen, Z.-Q., Li, G., & Ren, D. (2011). Detection of trace melamine in raw materials used for protein pharmaceutical manufacturing using surface-enhanced Raman spectroscopy (SERS) with gold nanoparticles. Applied Spectroscopy, 65(5), 514–521. . Yazgan, N. N., Boyaci, I. H., Topcu, A., & Tamer, U. (2012). Detection of melamine in milk by surface-enhanced Raman spectroscopy coupled with magnetic and Raman-labeled nanoparticles. Analytical and Bioanalytical Chemistry, 403(7), 2009–2017. . Zhang, X.-F., Zou, M.-Q., Qi, X.-H., Liu, F., Zhu, X.-H., & Zhao, B.-H. (2010). Detection of melamine in liquid milk using surface-enhanced Raman scattering spectroscopy. Journal of Raman Spectroscopy, 41(12), 1655–1660. . Zhou, Y., Li, C.-Y., Li, Y.-S., Ren, H.-L., Lu, S.-Y., Tian, X.-L., Hao, Y.-M., Zhang, Y.-Y., Shen, Q.-F., Liu, Z.-S., Meng, X.-M., & Zhang, J.-H. (2012). Monoclonal antibody based inhibition ELISA as a new tool for the analysis of melamine in milk and pet food samples. Food Chemistry, 135(4), 2681–2686. .
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Analytical Methods
Rapid and sensitive detection of melamine in milk with gold nanoparticles by Surface Enhanced Raman Scattering Andrea Mario Giovannozzi a,⇑, Francesca Rolle a, Michela Sega a, Maria Cesarina Abete b, Daniela Marchis b, Andrea Mario Rossi a a
Thermodynamic Division, Istituto Nazionale di Ricerca Metrologica, Strada delle Cacce, 91 10135 Torino, Italy C.Re.A.A. – National Reference Centre for the Surveillance and Monitoring of Animal Feed, c/o Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e valle D’Aosta, via Bologna 148, 10154 Torino, Italy b
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 26 November 2013 Accepted 4 March 2014 Available online 13 March 2014 Keywords: Melamine Milk SERS Raman spectroscopy Gold nanoparticles
a b s t r a c t A rapid and sensitive method to detect melamine in liquid milk based on Surface Enhanced Raman Scattering (SERS) spectroscopy is presented, exploiting the selective binding of gold nanoparticles (AuNPs) with this analyte. This interaction promotes the aggregation of the AuNPs inducing a huge enhancement of the melamine signals in the Raman spectrum due to the formation of SERS ‘‘hot spots’’. An external standard calibration method was employed for quantitative analysis and the method was validated for linearity, sensitivity, repeatability and recovery. A good linearity (R2 = 0.99) was found in the concentration range of 0.31–5.0 mg l1 in milk with a limit of detection of 0.17 mg l1. This method does not require a long extraction procedure (total analysis time can be lower than 30 min) and can be reliably used for melamine detection in milk matrix in accordance with the European law limits. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Melamine is an important industrial material that is mainly used for resin production, for thermosetting plastic and for polymer manufacturing in general (Sugita, Ishiwata, & Yoshihira, 1990). Its fame, unfortunately, came out recently because it was used as a food adulterant in milk, pet and animal feed (Brown et al., 2007; Burns, 2007). As a high rich-nitrogen molecule, melamine was intentionally added into food ingredients to produce an incorrectly high reading in the measurement of the protein content based on total nitrogen. The main concern on melamine, as a food additive, is the ability of combining with its analogues, such as cyanuric acid, leading to the formation of insoluble crystals which were responsible for kidney failures and even death in infants in China (Dobson et al., 2008; Hau, Kwan, & Li, 2009; Lam et al., 2009). Considering its potential toxicity, the Codex Alimentarius Commission has set a limit of 1 mg l1 for powder infant formula and 2.5 mg l1 for other foods and animal feed (Report on the Thirty-Third Session of the Joint FAO/WHO Food Standards Programme, 2008). Currently, gas chromatography (GC) or liquid chromatography (HPLC) coupled with mass spectrometry (MS) (Squadrone et al., 2010; Tyan, Yang, Jong, Wang, & Shiea, 2009), ⇑ Corresponding author. Tel.: +39 011 3919330; fax: +39 011 346384. E-mail address: [email protected] (A.M. Giovannozzi). http://dx.doi.org/10.1016/j.foodchem.2014.03.013 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
matrix-assisted laser desorption/ionisation MS (Tang et al., 2009), ELISA (Zhou et al., 2012) and IR spectroscopy (Mauer, Chernyshova, Hiatt, Deering, & Davis, 2009), represent the major categories of techniques for melamine detection. However, these methodologies usually require expensive instrumentations and long sample preparation procedures are needed mainly due to analyte extraction steps. Recently, several methods to detect melamine based on gold nanoparticles (AuNPs) have been developed (Ai, Liu, & Lu, 2009; Li, Li, Cheng, & Mao, 2010; Wei et al., 2010). Some of these methods were based on a colorimetric visual inspection of the nanoparticles solution colour change upon melamine interaction. Melamine interaction with modified or unmodified gold nanoparticles decreases the stability of the AuNPs provoking the formation of aggregates and inducing a shift of the surface Plasmon resonance with a consequent variation of the colour solution from red to blue, that can be easily monitored by UV/Vis absorption measurements. However, in the presence of interference substances in milk, such as other organic molecules or even positively charged ions and whose competing with melamine for AuNPs binding, a change in the AuNPs aggregation state can be seen, even in absence of the analyte and thus leading to a false positive response. In order to avoid these problems, Raman spectroscopy was used since it can provide a fingerprint of the melamine molecule in the Raman spectrum. Raman spectroscopy together with the help of gold or silver nanoparticles offers a very high sensitivity due to the Surface En-
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hanced Raman Scattering (SERS) effect that occurs when a molecule is adsorbed or grafted on a rough metallic surface. The Raman signal of the molecule can be enhanced theoretically up to a 1013 factor for a potential single molecule detection. Different methodologies for the detection of melamine based on the SERS effect were developed. Most of them were based on the fabrication of SERS substrates (Betz, Cheng, & Rubloff, 2012; Cheng & Dong, 2011; Kim, Barcelo, Williams, & Li, 2012), usually prepared by metallic nanoparticles deposition on silicon or glass or by photolithography techniques. SERS substrates demonstrated to achieve a very high sensitivity (detection limit in the lg l1 range) but they usually suffered of lack of reproducibility and homogeneity of the molecule distribution on the SERS substrate, leading to problems in the quantification. Other SERS analysis were developed in liquid, mainly based on silver nanoparticles, achieving very good results for melamine detection in milk (Zhang et al., 2010). As for gold nanoparticles, instead, only few works have been published. Lou et al. (2011) developed a very sensitive indirect method (LOD 0.1 lg l1) to detect melamine in milk by SERS using 4-mercaptopyridine-modified AuNPs. However, the linearity response of this method is between 0.5 and 100 lg l1 which might affect the practical application of this assay in routine analysis. Moreover, the melamine quantification is done by using a Raman reporter and not by the melamine itself. Another interesting work was proposed by Yazgan, Boyaci, Topcu, & Tamer (2012) who developed a rapid and sensitive method to detect melamine in milk by using spherical magnetic-core gold-shell nanoparticles and rod-shaped gold nanoparticles labelled with a Raman-active compound. They reached the limits of detection (LOD) and quantification (LOQ) of 0.38 and 1.27 mg l1, respectively. In this work we propose a simpler method (based on the use of only one type of spherical AuNPs), which guarantees high sensitivity and gives a linear response in a range of concentrations useful for practical applications. We selectively tested spherical AuNPs with different dimensions in order to have the highest SERS effect. Moreover, we tuned the AuNPs concentration to reach the linearity in the selected melamine range. For the calibration of the Raman spectrometer we used the acetonitrile (ACN) Raman band to normalise the Raman intensity of melamine, minimizing possible variations due to laser power, focal distance and environmental parameters (temperature, humidity). The method developed proved to be simple and not requiring a long extraction procedure. The total analysis time can be lower down than 30 min. 2. Material and methods 2.1. Reagents and materials Hydrogen tetrachloroaurate trihydrate (HAuCl4 3H2O P99%), trisodium citrate dihydrate (P99%), melamine (99%) and 10 nm Gold nanoparticles stabilized suspension in citrate buffer were purchased from Sigma–Aldrich (Milan, Italy). Sodium hydroxyde (NaOH 97%), Hydrochloric acid (HCl 37%), Nitric acid (HNO3 68%), absolute ethanol (99,99%) and acetonitrile (>99.5%) were obtained by Carlo Erba Reagents (Rodano, Italy). All solutions were prepared with Milli-Q quality water (18 MXcm). Liquid semi-skimmed milk used for the assays was purchased in a local supermarket in Torino, Italy. 2.2. Gold nanoparticles preparation All glassware used in the experiment was soaked in aqua regia (HCl:HNO3 3:1) and rinsed thoroughly in water and dried with nitrogen prior to use. AuNPs were synthesized according to Frens, 1973. For the preparation of 40 nm AuNPs, 5 ml of a 1% aqueous
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solution of trisodium citrate was rapidly injected into 500 ml boiling solution of HAuCl4 (0.01% v/v). The mixture was further refluxed for 10 min and then cooled to room temperature under continuous stirring and a wine-red colour solution of AuNPs was obtained. For the preparation of 80 nm AuNPs, 2.5 ml of trisodium citrate (1% water solution) was injected into 500 ml boiling solution of HAuCl4 (0.01% v/v). The above mentioned procedure was then applied. All AuNPs solutions were stored at 4 °C before use.
2.3. Gold nanoparticles characterization AuNPs characterization was done by UV–Vis absorption measurements and by Scanning Electron Microscopy (SEM) imaging. UV–Vis absorption spectra were collected with the Evolution 60s spectrophotometer (Thermo Scientific). The surface plasma resonance peaks of AuNPs solutions were measured to be 518, 530 and 554 nm for AuNPs dimensions of 10, 40 and 80 nm, respectively. SEM characterization was carried out using a SEM FEI Quanta 3D or Inspect F in UHV mode with the SE detector. Typical settings for the imaging are: 10 kV accelerating voltage, 2.5 spot (18 pA) or 3.5 spot (30 pA), 10 mm WD.
2.4. Melamine standard solutions Melamine stock standard solution was prepared by accurately dissolving 50 mg of standard in 50 ml of Ethanol/H2O 50:50 (v/v), to reach a concentration of 1000 mg l1. Melamine standard solutions were prepared by subsequent dilutions from the stock solution in water to reach the following concentrations: 100, 20, 10, 5, 1, 0.5, 0.2, 0.1 mg l1. These pure melamine standards were used to set up the analytical procedure. Aliquots of the melamine standards were mixed in a 1:1 ratio with AuNPs stock solutions, mixed with vortex for 3 s and subsequently analysed by UV–Vis and the Raman spectrophotometer. Melamine standard solutions in negative matrix pool were also prepared for the external calibration of the Raman spectrometer, as explained in the paragraph 2.7. Consecutive dilutions were made starting from 10 mg l1 to reach the following concentrations in matrix: 1, 0.50, 0.25, 0.125, 0.063 mg l1. These solutions were mixed with AuNPs (1:1) and analysed by Raman spectroscopy to build the calibration curve.
2.5. Detection of melamine in liquid milk by SERS Aliquots of the 100 mg l1 melamine stock solution were added to milk to obtain concentrations of 0.5, 1, 3, 5 and 10 mg l1. Melamine-free milk was processed as the spiked milk and used to prepare blank samples. The extraction procedure was carried out by first adding 200 ll of 1 M HCl to 4 ml of spiked milk and vigorously mixing by vortex for 10 s. The samples were then transferred into 1.5 ml centrifuge tubes and centrifugated for 30 min at 14 000 rpm. Supernatants from the same sample were collected and filtered with a 0.22 lm PTFE filter. The pH of the filtered solution was adjusted at 4.7 by adding 60 ll of 1 M NaOH. 10 ml of pure ACN were then added inducing the precipitation of most of the proteins in solution. A final centrifugation step was carried out at 14 000 rpm for 30 min in order to remove any aggregates. 250 ll of the resulting supernatant was mixed in a 1:1 ratio with a 10-fold concentrated 40 nm AuNPs solution and immediately analysed by Raman spectroscopy. The 10-fold concentrated 40 nm AuNPs solution was obtained by centrifugating the AuNPs stock solution at 4000 rpm for 30 min and subsequently resuspending in a proper amount of water solution.
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2.6. SERS measurement SERS spectra were recorded using a Thermo Scientific DXR Raman equipped with a microscope, excitation laser source at 780 nm, a motorized microscope stage sample holder, and a charge-coupled device (CCD) detector. Spectra of samples were collected using a 20x long working distance microscope objective with a 24 mW laser power and a spectral range from 200 to 1800 cm1. The acquisition time was of 20 s with 1s exposure time. 2.7. Validation of the method The validation of the method was performed by calculating linearity, sensitivity, repeatability and mean recovery. The instrumental linearity was evaluated from four calibration curves with 5 levels of melamine concentrations in non-spiked milk extract, representative of the analysed matrix: 0.031, 0.063, 0.125, 0.25, 0.50 mg l1 corresponding in the matrix to values from 0.31 to 5.0 mg l1. The calibration, calculated as the area of the Melamine Raman band at 715 cm1 versus melamine concentration, was evaluated by Partial Least Square (PLS) method and the linearity was estimated by the determination coefficient R2. Acceptability criteria to assume the linearity of response is R2 > 0.99. The limit of detection (LOD) was experimentally detected on blank samples (n = 10) and calculated by the equation (Long and Winefordner (1983):
LOD ¼
3sb b
ð1Þ
where sb is the standard deviation of the areas of the blank samples in the Raman spectrum at 715 cm1, and b is the slope of the calibration curve. Indeed, the LOD of melamine concentration in the matrix (mg l1) was evaluated by using the standard calibration curve previously built (715 cm1 band area versus concentration in matrix). The limit of quantitation (LOQ), was estimated with the following equation:
LOQ ¼
10sb b
ð2Þ
Fig. 1. (a) UV–VIS Absorption Spectra of 40 nm AuNPs mixed with an increasing concentration of pure melamine standard solutions: (a) blank, (b) 0.05 mg l1, (c) 0.1 mg l1, (d) 0.25 mg l1, (e) 0.5 mg l1, (f) 1 mg l1, (g) 2.5 mg l1. The sketch above the spectra shows the interaction mechanism of AuNPs with the melamine molecule with the subsequently formation of aggregates together with a change of the colour solution from wine-red to blue-gray. The increased melamine concentration can be followed by the arrow on the graph. SEM images of 40 nm AuNPs before (b) and after (c) interaction with melamine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The recovery (%) was calculated by the average concentration values (n = 6) obtained for melamine spiked samples (at 1 and 3 mg l1 concentration levels corresponding to 0.1 and 0.3 mg l1 in the analysed milk extracts). Each spiked sample was analysed six times in the same day, in order to test the repeatability and the accuracy of the method. The validation parameters are summarized in Table 1. 3. Results and discussion It was reported that melamine can be added into food as an adulterant to increase its apparent protein content. Several papers proposed AuNPs as useful substrates for melamine detection since the colour of the particles changes after the interaction with the melamine in solution. AuNPs are usually fabricated by using the sodium citrate method which allows an easy tunability of particles dimensions just by changing the gold precursor salt and the sodium citrate molar ratio. The sodium citrate, indeed, works both as a reducing agent for gold nucleation and as stabilizing agent by coating the surface of the nanoparticles preventing their aggregation in solution. When the melamine is injected in the system, hydrogen bonds between the melamine amino groups and citrate ions occur, decreasing the electrostatic repulsion between individual AuNPs and finally results in the aggregation of AuNPs (see the Fig. 1). As Fig. 1a shows, the stable 40 nm AuNPs solution (wine-red colour) has a surface plasmon resonance absorption peak at
Fig. 2. Raman spectra and SERS spectra of melamine with AuNPs with different size. (a) Raman spectrum of solid melamine. SERS spectra of a 0.1 mg l1 melamine solution with AuNPs of different size: (b) 40 nm AuNPs, (c) 80 nm AuNPs and (d) 10 nm AuNPs. (e) Raman spectrum of a 1000 mg l1 melamine solution.
530 nm. As soon as the melamine (0.1 mg l1) was added into the AuNPs solution, it was observed that the AuNPs quickly aggre-
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Fig. 3. SERS spectra of 40 nm AuNPs with different melamine concentrations in spiked milk samples: 0, 0.5, 1, 3, 5, 10 mg l1.
gate. As a result of the aggregation, the solution colour became blue-gray and the absorbance at 530 nm decreased and a new absorption band around 700 nm showed up. Increasing the melamine concentration from 0.25 to 2.5 mg l1, the surface plasmon peak at 530 nm continued to decrease and the peak at 700 nm was shifted to higher wavelengths (from 700 to 850 nm), inducing the formation of bigger aggregates. The reaction time is very fast and a equilibrium is reached in less than 1 min. SEM analyses were also used to verify the aggregation state. In Fig. 1b AuNPs appear monodisperse and free of interactions. After the melamine addition in solution, the AuNPs form bigger aggregates (Fig. 1c). This behaviour was not only investigated for the 40 nm AuNPs but it was also tested with 10 and 80 nm AuNPs particles diameter. Melamine interaction with these nanoparticles occurred in the same way and their aggregation was clearly demonstrated by UV–VIS measurements (data not shown). However, analysing the surface plasmon resonance peaks after the aggregation induced
by melamine, it was shown that while the plasmon resonance peak is centred at around 800 nm for the 40 nm AuNPs, the resonance peaks for 10 and 80 nm AuNPs were centred at 660 nm and further than 900 nm, respectively. Since the SERS effect takes place in presence of ‘‘hot spot’’ due to the formation of gold aggregates and its enhancement efficiency can be maximised when the plasmon resonance peak of the aggregates is in resonance with the laser source (780 nm), we checked the Raman signal of the melamine using these three different AuNPs. In Fig. 2 typical Raman peaks of solid melamine at 380, 580, 678 and 983 cm1 were observed. The most intense peak at 678 cm1 is assigned to the ring-breathing II mode, which involves the in-plane deformation of the triazine ring. The second most intense peak at 983 cm1 arises from the ring-breathing mode I of the triazine ring (Koglin, Kip, & Meier, 1996). In the Raman spectrum of pure melamine dissolved in water at a concentration of 1000 mg l1 only a small peak is barely visible at
Fig. 4. SERS spectra of 40 nm AuNPs with 5 levels of melamine standard in negative matrix pool (representative of the analysed matrix): 0.031, 0.063, 0.125, 0.25, 0.50 mg l1 corresponding in the matrix to values from 0.31 to 5.0 mg l1.
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678 cm1 while the other peaks are not detected. In the SERS spectra, instead, even in presence of a small concentration of melamine (0.1 mg l1), the two main melamine peaks at 715 and 1000 cm1 are observed. These specific melamine peaks are shifted by 30 cm1. Remarkable shifts of 10 and 40 cm1 from the original melamine bands at 676 and 983 cm1 were already reported in the literature using respectively Klarite SERS substrates (Liu, Lin, & Li, 2010) and triangular shape AuNPs (Wen, Li, & Ren, 2011). It is reasonable to infer that a strong polarisation occurs at the surface of these AuNPs and where the electric field increases strongly, it can result in not only an increased enhancement factor, but also in a change of the vibrational Raman selection rules, which allows the appearance of forbidden Raman bands. As Fig. 2 clearly shows, Raman enhancement of the melamine signals only occurred when 40 and 80 nm AuNPs are used. When 10 nm AuNPs are used as SERS substrates no enhancement is detected. Moreover, comparing the most intense melamine peak at 715 cm1, 40 nm AuNPs provided the highest enhancement efficiency, probably because of the resonance coupling between the laser excitation source at 780 nm and the gold aggregates absorption band at around 800 cm1. These data are in agreement with what already reported in literature (Bell & McCourt, 2009). In order to demonstrate a practical application in the food analysis field, we decided to detect melamine in liquid raw milk and we started to develop a measurement procedure based on 40 nm AuNPs as a SERS substrate in solution. Food samples are complex matrices that are difficult to analyse because of their protein and carbohydrate content. Detecting low levels of melamine in food is not easy because melamine can be bound with the milk constituents due to its strong tendency to form hydrogen bonds. Thus, prior to the analysis, extraction of melamine from milk is a fundamental step. An acidic extraction was first carried out with hydrochloric acid (1 M) in order to precipitate caseins from milk. Further purifications steps, such as filtrations and solvent extraction using ACN, resulted to be necessary to induce proteins precipitations and to extract melamine simultaneously. Since this method of analysis is based on the covalent bonding of free amino groups in melamine with the AuNPs, the removal of any source of free amino groups is important to increase the efficiency of the detection method, reducing the interfering molecules. Moreover, the pH of the solution was adjusted to maximise the melamine-AuNPs interaction. The solution pH influences both the surface charge on AuNPs and the protonation state of melamine amino groups. Considering the pKa values of the three carboxylic moieties of the citrate ions (pKa = 3.13, 4.76 and 6.34) and the melamine pKa value around 5, setting a solution pH of 4.7 results in a reduction of repulsive forces between AuNPs and it induces the protonation of the melamine molecule. Thus, a pH value around 5 was found to be the best compromise to foster AuNPs aggregation together with their interaction with melamine and it resulted in a chemical enhancement by charge transfer complexes and/or localise surface plasmons, that yield an enhancement of the melamine Raman signal. The initial setup of the analytical procedure was performed by spiking semi-skimmed liquid milk with melamine to obtain concentrations in the range of interest for practical applications. As we already mentioned, limit value of melamine has been set in Europe at 1 mg l1 in infant formula and 2.5 mg l1 in liquid milk and food in general. Various concentrations of melamine were spiked in milk and subsequently extracted and analysed by SERS. As shown in Fig. 3, five levels (0.5, 1, 3, 5 and 10 mg l1) of melamine in liquid milk were analysed. By monitoring the highest intense melamine Raman peak at 715 cm1, it was found that the area of this peak was enhanced with increasing concentration of melamine. To optimise the method several concentrations of AuNPs were tested (data not shown) and the best results were obtained with
a 10 folds concentrated AuNPs solution which guarantees a linear detection response of the melamine Raman signal in the selected concentrations range. As shown in Fig. 3, the melamine peaks corresponding to each concentration of melamine in the spiked samples are well separated and the method gives good responses in the concentration range studied. Moreover, since the final extraction of melamine from the matrix is done in ACN and the solvent concentration is the same for every analysed sample, the area of the ACN peak at 922 cm1 was established as a internal reference to normalise the area of the peak at 715 cm1 and to correct the Raman signal in order to eliminate the effects of the matrix and other factors, such as environmental parameters (temperature, humidity) or instrumental settings like the focal distance. An external calibration procedure was chosen for the melamine quantification in milk. Taking into account the dilution factor for the melamine concentration from the starting matrix through the extraction procedure, five levels of melamine concentrations (0.031, 0.063, 0.125, 0.25 and 0.50 mg l1) were chosen for the cal-
Fig. 5. Calibration curves of melamine standards in negative matrix pool obtained by plotting the normalised area of melamine Raman band at 715 cm1 vs. melamine concentration. (a) Four calibration curves for intra-day analysis and PLS linear fit with equation and determination coefficient. (b) Average values with error bars calculated from the calibration curves in (Fig. 5a).
A.M. Giovannozzi et al. / Food Chemistry 159 (2014) 250–256 Table 1a Recovery of melamine from spiked milk samples.
a b
Concentration of melamine in milk extracts (mg l1)
Average Raman band area (a.u.)
Amount founda (mg l1)
Mean Recovery (%) (n = 6)
RSDb (%)
0.1 0.3
0.8317 2.0288
0.10 ± 0.01 0.29 ± 0.01
99.9 96.3
9.6 3.8
Average value from six determinations for each concentration. Relative standard deviation of mean recovery (RSD (%) = (SD/mean) 100).
Table 1b Intra-day repeatability of the method. Analysesa
Spiked concentration in milk extracts (mg l1)
Amount found (mg l1)
Precision as RSDb (%)
Accuracyc (bias%)
Intra-day (n = 6)
0.1
0.10 ± 0.01
9.6
0.1
0.3
0.29 ± 0.01
3.8
3.7
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for melamine detection. SERS analysis can be also performed with a motorized sample holder stage together with a 96 wells plate in order to make the method automatic and to reduce the total volume of the analysis. The whole extraction procedure can be carried out in less than 30 min without substantially affecting the sensitivity of the methodology and the reliability of the quantification step. Acknowledgements The present work has been supported by EMRP project ‘‘ SurfChem’’. EMRP is jointly founded by the EMRP participating countries within EURAMET and the European Union. Part of this work was carried out by NanoFacility Piemonte, INRiM, a laboratory supported by Compagnia di San Paolo (Italy). References
a
Average of six determinations for each sample. Relative standard deviation (RSD (%) = (SD/mean) 100) of individual measurements. c Bias% = (mean concentrationreal concentration)/real concentration 100). b
ibration curve, which correspond to melamine values range of 0.31–5.0 mg l1 in the milk samples. Melamine-free milk was used to prepare the blank samples, processing it in the same way as the spiked milk. SERS spectra of melamine standard solutions in milk extracts are shown in Fig. 4. The method was validated for linearity, sensitivity, recovery and repeatability. A linear regression was found between the normalised Raman signal at 715 cm1 and melamine concentration with a good determination coefficient (R2) of 0.99 (Fig. 5a-b). According to Eqs. (1) and (2) the LOD and LOQ were 0.017 and 0.057 mg l1 respectively in the milk extracts which correspond to values of 0.17 mg l1 (LOD) and 0.57 mg l1 (LOQ) in the milk matrix. Our method is then suitable for melamine quantification in the concentration range of 0.57–5.0 mg l1 in milk matrix in accordance with the European law limits of 1 and 2.5 mg l1 in dairy products for infants and and other food and animal feed, respectively. Mean recovery was determined in milk samples spiked at 1 and 3 mg l1 concentration levels (corresponding to 0.1 and 0.3 mg l1 in the analysed milk extracts) and it was found to be 99.9% and 96.3% (Table 1a). The intra-day repeatability and accuracy of the method were determined for milk spiked samples with 1 and 3 mg l1 melamine. Repeatability is expressed as relative standard deviation (RSD%) and accuracy is explained by computing bias%. The results for intra-day measurements are shown in Table 1b. The RSD values were 9.6% and 3.8% for 1 and 3 mg l1 spiked samples, respectively. It can be concluded that the method had good repeatability (intraday) and accuracy for melamine detection. 4. Conclusions A sensitive and rapid method to detect melamine in liquid milk was developed by using AuNPs and Raman spectroscopy. Melamine in liquid milk is able to promote the formation of AuNPs aggregates which behave as ‘‘Raman hot spots’’ and enhance the melamine Raman signal allowing melamine detection in the mg l1 range. SERS is emerging as a new technique for analytical methods that can be suitable for high throughput screening analysis and could become a valid alternative to the classical analytical methodologies based on GC or HPLC mass spectrometry. The method here developed is sensitive, fast and it can be applied in routine analysis
Ai, K., Liu, Y., & Lu, L. (2009). Hydrogen-bonding recognition-induced color change of gold nanoparticles for visual detection of melamine in raw milk and infant formula. Journal of the American Chemical Society, 131(27), 9496–9497.
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