Potential of SERS for rapid detection of melamine and cyanuric acid extracted from milk

Melamine, a nitrogen-rich chemical, was implicated in the pet and human food recalls in 2007 and in the global food safety scares in 2008 involving milk and other milk-derived products. In this study, we investigated the feasibility of using surface-enhanced Raman spectroscopy (SERS) coupled with SERS-active gold substrates for rapid detection of trace amounts of melamine and its analogue (that is, cyanuric acid) in liquid milk. Raman signals of tested samples were significantly enhanced by SERS. The identification limit for SERS using gold substrate can reach 2 ppm of melamine in liquid milk. Partial least squares (PLS) models were established for the quantification of melamine in liquid milk by SERS: R = 0.90, RMSEP = 1.48 × 10⁻⁵. Our results demonstrate that rapid detection of melamine in milk can be achieved by SERS; while detection of cyanuric acid in milk remains a challenging task due to rapid enol-keto tautomerism of cyanuric acid. The SERS method is faster and simpler than other traditional methods, and requires minimum sample preparation. These results demonstrate that SERS could be used to detect food contaminants such as melamine in foods and food ingredients quickly and accurately.     

Detecting single Bacillus spores by surface enhanced Raman spectroscopy

Detection of Bacillus spores is of considerable importance to the food industry since they can survive standard processing procedures and sanitation treatments. Surface enhanced Raman spectroscopy (SERS) coupled with gold SERS-active substrates was used to detect and discriminate among five Bacillus spores (B. cereus ATCC 13061, B. cereus ATCC 10876, B. cereus sp., B. subtilis sp., and B. stearothermophilus sp.). Tremendously enhanced Raman signals of Bacillus spores deposited onto the gold substrates were detected, allowing the limit of detection (LOD) of SERS to reach the level of a single spore. Distinct spectral differences were observed between different Bacillus spores. Hierarchical cluster analysis (HCA) and principle component analysis (PCA) show clear data segregations at the species level between five Bacillus spores. Principle component (PC) values indicate that the Raman shift range between 900 and 1200 cm⁻¹ contributed significantly to the total data variance in the PCA results. In particular, a prominent band of dipicolinic acid (DPA) was observed at 998 cm⁻¹ and served as a biomarker for bacterial spores. This study demonstrates that SERS coupled with gold nanosubstrates is able to detect and discriminate single Bacillus spores non-destructively and with minimum sample preparation. SERS method is a promising tool for rapid, ultra-sensitive, and selective detection of bacterial spores, potentially in foods and other complex biological matrices.     

Development and evaluation of a surface-enhanced Raman scattering (SERS) method for the detection of the antioxidant butylated hydroxyanisole

Butylated hydroxyanisole (BHA) is a common antioxidant used in foods and food packaging materials, and it can migrate into the food during its supply chain. BHA has been shown to be carcinogenic, which has gained substantial attention in recent years. In this study, the feasibility of using surface-enhanced Raman scattering (SERS) coupled with SERS-active gold substrates for rapid detection of trace amounts of BHA was investigated. Raman signals of tested samples were enhanced significantly by SERS. The detection limit of the BHA solution can reach 10 μg/mL by SERS enhanced with 50 nm gold nanoparticles and in a 1.0 pH solution system. The Raman peak at 480 cm⁻¹ was used as the index of quantitative analysis, the line correlation was R ² = 0.9803. The results showed that the SERS method is useful for sensitive and selective detection of BHA. By comparing the enhanced peak features, the adsorption behavior of BHA on the surface of gold nanoparticles was analyzed in detail and it was determined that the molecule was adsorbed on the gold surface by “Ph-O(H) (Ph = phenyl)”. The benzene ring vibration increased significantly, inferring that the benzene ring is perpendicular to the surface of the gold nanoparticles.     

Determination of melamine concentrations in dairy samples

Melamine, a basic organic chemical intermediate, can cause renal failure because of the formation of insoluble melamine cyanurate crystals in the kidneys. This study examined a novel fluorescence spectrometry for the determination of trace melamine in dairy. The method was based on the inhibitory effect of melamine on the decolorizing reaction of the uncatalytic oxidation of acridine red by potassium permanganate in a sulfuric acid medium. The calibration graph was linear for melamine concentrations of 2.1 × 10^?4–1.6 mg/L, and a detection limit of 61.5 ng/L was obtained. The melamine was determined in dairy samples by the proposed method after liquid–liquid and solid phase extraction. The results correlated with the high-performance liquid chromatography. The recoveries were within the range of 99.6–103.3% for liquid milk and 98.5–111.1% for milk powder. The possible mechanism of the reaction was also investigated by ultraviolet spectra.     

Residues of melamine and cyanuric acid in milk and tissues of dairy cows fed different doses of melamine

Melamine (1,3,5-triazine-2,4,6-triamine) may be degraded into cyanuric acid and some other compounds by rumen microorganisms. This study was conducted to assess the transfer of melamine and cyanuric acid in to milk and tissues by dairy cows fed different doses of melamine. Forty mid-lactation dairy cows (157 ± 43 d in milk, 20.8 ± 1.4 kg of milk/d) were divided into 4 groups (n = 10/group) using a completely randomized design. The groups were fed the following amounts of melamine (purity ≥ 99.5%) at 0 (control), 300 [treatment (Trt) 1], 500 (Trt 2), and 1,000 (Trt 3) mg/d per cow, respectively. The trial lasted for 18 d (12-d feeding period, followed by a 6-d clearance period). Milk samples were collected from the 4 groups on d 1, 2, 3, 4, 8, 12, 13, 14, 15, and 18, and analyzed for melamine and cyanuric acid. On d 13, 3 cows from Trt 2 and Trt 3 were randomly selected and slaughtered; tissue samples including kidney, liver, mammary, bladder, gluteus medius, and longissimus dorsi were collected for melamine and cyanuric acid analyses. Milk and tissue samples were analyzed for melamine and cyanuric acid using a simultaneous liquid chromatography tandem mass spectrometry procedure. Neither melamine nor cyanuric acid was detected in concentrated feed that was being fed to the cows. In melamine-treated groups, milk melamine concentration increased quickly and reached a stable level by d 4 and was at similar levels on d 8 and 12 after the first administration of melamine. Milk melamine levels in treated groups were 0.18, 0.27, and 0.50 mg/L for Trt 1, Trt 2, and Trt 3, respectively, and were highly correlated (R² = 0.91) with melamine dosing levels. No cyanuric acid was detected in any of the milk collected from the various groups. Melamine residue levels in tissues of Trt 3 were about 2-fold higher than that in Trt 2, with the highest concentration being found in kidney. No differences in cyanuric acid levels in tissues were found between Trt 3 and Trt 2. Liver, kidney, and bladder tissues were found to contain the highest cyanuric acid levels. This study shows a relationship between dietary melamine levels and cyanuric acid levels found in tissues, which might be the result of melamine being converted to cyanuric acid by microorganisms in the rumen.     

Preparation of [13 C 3]-melamine and 13 C 3]-cyanuric acid and their application to the analysis of melamine and cyanuric acid in meat and pet food using liquid chromatography-tandem mass spectrometry

For the determination of melamine and cyanuric acid the labelled internal standards [¹³ C ₃]-melamine and [¹³ C ₃]-cyanuric acid were synthesized using the common substrate [¹³ C ₃]-cyanuric chloride by reaction with ammonia and acidified water, respectively. Standards with excellent isotopic and chemical purities were obtained in acceptable yields. These compounds were used to develop an isotope dilution liquid chromatography/mass spectrometry (LC/MS) method to determine melamine and cyanuric acid in catfish, pork, chicken, and pet food. The method involved extraction into aqueous methanol, liquid–liquid extraction and ion exchange solid phase clean-up, with normal phase high-performance liquid chromatography (HPLC) in the so-called hydrophilic interaction mode. The method had a limit of detection (LOD) of 10 µg kg^?1 for both melamine and cyanuric acid in the four foods with a percentage coefficient of variation (CV) of less than 10%. The recovery of the method at this level was in the range of 87–110% and 96–110% for melamine and cyanuric acid, respectively.     

HPLC-MS/MS同时检测大鼠体内三聚氰胺和三聚氰酸含量

目的建立HPLC-MS/MS同时检测大鼠血浆和组织中三聚氰胺和三聚氰酸含量的方法。方法以同位素标记的三聚氰胺和三聚氰酸作为内标物,大鼠血浆和组织经二氯甲烷-乙腈前处理后,经色谱柱分离,串联离子阱质谱采用ESI源按运行时间段切换正、负离子模式进行测定。结果在0.3～75μg/ml范围内,三聚氰酸和三聚氰胺具有良好的线性(r≥0.998)。在0.9、5.0、50μg/ml的给药大鼠的血浆样本中,三聚氰酸和三聚氰胺的回收率分别为97.4%～99.4%、96.8%～98.9%,两者的变异系数为2.43%～6.35%(日内)、1.70%～4.12%(日间)和0.30%～9.17%(日内)、0.75%～3.39%(日间);在0.9、5.0、50μg/ml的给药大鼠肝组织样本中,三聚氰酸和三聚氰胺的回收率分别为90.8%～104.6%、91.7%～106.0%,对应的变异系数为3.70%～5.14%(日内)、2.84%～4.50%(日间)和1.31%～2.21%(日内)、1.12%～3.36%(日间)。结论该方法样品前处理简单,基质干扰小,分析效率高,具有良好的精密度、准确度和特异性,是进行三聚氰酸、三聚氰胺在动物体内的组织代谢和毒性研究的理想分析方法。     

Occurrence of melamine contamination in powder and liquid milk in market of Iran

Melamine contamination is known as a significant health risk and seriously considered by scientists worldwide. Cyanuric acid is one of the melamine by-products that contribute to its renal toxicity via the formation of melamine cyanurate crystals in the kidney. Therefore, co-determination of melamine and cyanuric acid in both powder and liquid milk samples has grown significantly throughout the world. High-performance LC with ultraviolet detection has been used as a simple and rapid method for the determination of melamine and cyanuric acid in powder and liquid milk samples and the results confirmed by using LC-MS/MS. In this study, nine powder milk and five liquid milk samples from different brands available in Iranian markets were chosen randomly and analysed. SPE column was used to clean up the samples. The results showed that except one brand of milk powder and liquid milk, all other samples were contaminated with melamine in the range of 1.50–30.32?μg/g for milk powder and 0.11–1.48?μg/mL for liquid milk. No cyanuric acid was detected in milk powder or liquid milk samples, which reduces the risk of melamine toxicity for consumers.     

Determination of melamine by flow injection analysis based on chemiluminescence system

In this paper, based upon the phenomenon that melamine can obviously enhance the CL signal of the luminol–H₂O₂ system in basic medium, a simple, rapid and sensitive flow injection chemiluminescence (FI-CL) method for the determination of melamine has been developed. Under the optimum conditions, the linear range for the determination of melamine was 0.2–80 μg mL⁻¹ with a detection limit of 0.12 μg mL⁻¹ calculated as proposed by IUPAC and a relative standard deviation of 3.26% for 11 solutions of 10 μg mL⁻¹ melamine on the same day. The proposed method was satisfactorily applied to determine melamine in milk-based products and satisfactory results were obtained without interferences from the sample matrix. Moreover, one assay produce takes only 25 s and the minimum sampling rate is about 120 samples h⁻¹, which indicated that the FI-CL method was suitable for high throughput and real-time melamine analysis.     

Detection of herbicides in drinking water by surface-enhanced Raman spectroscopy coupled with gold nanostructures

This study aimed to use surface-enhanced Raman spectroscopy (SERS) to detect herbicide residues (atrazine and arsenic trioxide) in drinking water. Gold-coated nanosubstrates were used in the SERS measurements to acquire enhanced Raman signals of herbicides in drinking water. Compared with the control, characteristic patterns of SERS spectra were distinguishable for atrazine at 3 ppb and arsenic trioxide at 1 ppb. Partial least squares analysis was used to develop quantitative models for detection of two herbicides in drinking water and calibration curves were plotted with R² of 0.988 and 0.991 for atrazine and arsenic trioxide, respectively. The study of limit of detection (LOD) demonstrates that at 99.86 % confidence interval, SERS can detect both herbicides at 0.1 ppm in drinking water. Satisfactory recoveries were obtained for samples with concentration at and higher than the LOD (92.3–119.3 % for atrazine and 88.2–102.1 % for arsenic trioxide). These results demonstrate that SERS coupled with gold nanostructures holds great potential for rapid detection of herbicide residues and other chemical contaminants in drinking water.     

Electrochemical sensor based on Prussian blue nanorods and gold nanochains for the determination of H<Subscript>2</Subscript>O<Subscript>2</Subscript>

In this paper, a novel electrochemical sensor for the detection of H₂O₂ was proposed based on gold nanochains and prussian blue nanorods (PB@MWCNTs), which were synthesized with multiwall carbon nanotubes (MWCNTs) as a template. With the introduction of MWCNTs and the gold nanochains, the proposed system shows synergy among the Prussian blue (PB), MWCNTs, and the gold nanochains, which amplifies the H₂O₂ sensitivity greatly. A linear range from 1.75 × 10⁻⁶ to 1.14 × 10⁻³ M with a detection limit of 0.5 × 10⁻⁶ M (S/N = 3) and a high sensitivity 300 μA mM⁻¹ cm⁻² for H₂O₂ detection is obtained. Moreover, the sensor exhibits good repeatability and stability.     

SERS Detection of Insecticide Amitraz Residue in Milk Based on Au@Ag Core-Shell Nanoparticles

A simple, rapid, and environmentally friendly surface-enhanced Raman scattering (SERS) method was developed for the determination of trace amitraz in milk with the use of silver-coated gold nanoparticles (Au@Ag NPs) as enhancing reagent. The normal Raman and SERS spectra of amitraz were analyzed, and the peaks were assigned by density functional theory. The morphology of Au@Ag NPs was characterized and confirmed by transmission electron microscopy, scanning electron microscopy, and ultraviolet-visible spectroscopy. The SERS effects of Au@Ag NPs were investigated, including the types of solvents for dissolving amitraz, the volume ratio of the Au@Ag NPs and amitraz, and the concentration of aggregating agent (NaCl) for aggregate Au@Ag NPs. Results show that ethanol exerts the least interference on the SERS spectrum of amitraz and is more environmentally sound than methanol. The strongest SERS signal appeared when the volume ratio of Au@Ag NPs and amitraz was 2:1. Moreover, the strongest SERS signals appeared when the concentration of NaCl was 0.025 mol L^?1 because of appropriate aggregation. Under the optimum conditions, the concentration of amitraz presents a good linear relationship with Raman intensity (723 cm^?1) with a linear range of 9.77 × 10^?4~2.93 × 10^?2 g L^?1. The detected recoveries of amitraz in milk were between 81.7 and 100.5% with a relative standard deviation (RSD) of 2.61~5.51%.     

Feasibility of colloidal silver SERS for rapid bacterial screening

This study reports the feasibility of citrate-reduced colloidal silver surface-enhanced Raman scattering (SERS) for differentiating three important food borne pathogens, E. coli, Listeria, and Salmonella. FT-Raman and SERS spectra of both silver colloids and colloid-K₃PO₄ mixtures were collected and analyzed to evaluate the reproducibility and stability of silver colloids fabricated in a batch-production process. The results suggest that the reproducibility of the colloids over the batch process is high and that their binding effectiveness remains consistent over a 60-day storage period. Two specific SERS bands at 712 and 390 cm⁻¹ were identified and used to develop simple 2-band ratios for differentiating E. coli-, Listeria-, and Salmonella-colloid mixtures with a 100% success. These results indicate that colloidal silver SERS technique may be a practical alternative method suitable for routine and rapid screening of E. coli, Listeria, and Salmonella bacteria.     

Spectrophotometric determination of melamine in milk by rank annihilation factor analysis based on pH gradual change-UV spectral data

A new spectrophotometric method has been developed in this paper to determine melamine in milk by applying rank annihilation factor analysis (RAFA) based on pH gradual change-UV spectral data (pH-spectra). In the proposed method, the spectra of the sample solutions at different pH data points were recorded and the pH-spectra bilinear data matrix was generated. Based on these data, the RAFA was then applied to calculate the concentration of melamine in milk. The experiments have been conducted and the results were satisfactory. Under the optimised conditions, linearity of the proposed method was in the range of 0.04–4.0 μg mL⁻¹ for calibration samples, and 0.04–3.5 μg mL⁻¹ for the mixed solutions of melamine with the background milk components. The detection limit (DL) was 12 ng mL⁻¹. The relative predictive error (RPEs) and root mean square error of prediction (RMSEP) of applying RAFA were 0.91% and 0.0151, respectively.     

On-site detection of sub-mg/kg melamine mixed in powdered infant formula and chocolate using sharp-edged gold nanostar substrates

We report a facile method for sample preparation and sensitive on-site detection of melamine in powdered infant formula and chocolate using Raman spectroscopy on sharp-edged gold nanostars (AuNSs). The aggregation of AuNSs by sodium chloride (1.2 M) facilitates the more sensitive detection of melamine in comparison with spherical gold nanoparticles (AuNPs). Density functional theory quantum mechanical calculations were performed to determine the energetic stability on gold cluster atoms. Our spectroscopic data indicated that AuNSs are an efficient platform for detecting melamine in food mixtures. The detection limits of melamine in powdered infant formula and chocolate were found to be ~0.1 mg/kg and ~1 mg/kg, respectively, on AuNPs, whereas they were observed to be ~0.01 mg/kg and ~0.1 mg/kg, respectively, on AuNSs. Using a handheld Raman spectrometer, a sub-mg/kg detection of melamine in both powdered infant formula and chocolate could be achieved within a few minutes.     

Production of single-chain Fv antibodies against melamine from a rabbit phage display library for the development of a melamine immunoassay

A rabbit phage display library of single chain Fv (scFv) antibodies was constructed to screen specific scFv antibodies against melamine. Rabbits were immunized with melamine immunogens prepared via glutaraldehyde method. Two recombinant scFv antibodies with high specificity for melamine, termed A9 and B4, were isolated via library panning. SDS-PAGE analysis showed that the soluble A9-scFv and B4-scFv antibodies were expressed successfully and the scFvs showed good sensitivity with an IC₅₀ of 12.64 and 10.32 μM to melamine, respectively. The cross-reactivity of 2 scFv antibodies with cyanuric acid and chloromycetin were below 1%. The high similarity of 2 scFv nucleotide sequences indicated that they may originate from 1 B cell clone that had undergone somatic diversification. As a consequence, the conjugation synthesis was successful and the selected scFvs could be used for sensitive detection of melamine in foods and feeds by immunoassay.     

Visual detection of melamine in raw milk using gold nanoparticles as colorimetric probe

We report the development of a simple and rapid colorimetric detection method for melamine in raw milk using gold nanoparticles as probe. This assay relies upon the distance-dependent optical properties of gold nanoparticles. In neutral media, melamine could rapidly induce the aggregation of gold nanoparticles, thereby resulting in red-to-blue (or purple) colour change. The concentration of melamine in raw milk can be determined by monitoring with the naked eye or a UV–vis spectrometer. The present limit of detection for melamine is 0.4 mg/L. The method is rather simple, and the whole process including sample pretreatment takes only 12 min at room temperature. The merits (such as simplicity, rapidity, low cost and visual colorimetry) make the proposed method specially useful for on-site screening melamine levels well below the current safety limit in raw milk.     

Determination of melamine and its analogues in egg by gas chromatography-tandem mass spectrometry using an isotope dilution technique

A method was developed for the simultaneous determination of melamine, ammeline, ammelide, and cyanuric acid in egg using gas chromatography-tandem mass spectrometry (GC-MS/MS). The samples were first extracted by the solution of diethylamine–water–acetonitrile (10:40:50, v/v/v). Clean-up employed an ‘On Guard II’ RP cartridge, and the dried elute was derivatised using bis-(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS). Derivatised samples were analysed by GC-MS/MS using multiple-reaction monitoring (MRM) with ¹³C₃-¹⁵N₃-labelled melamine and cyanuric acid as internal standards. Blank samples of egg were spiked with the four analytes at concentration level of 0.1, 0.5, 1.0 mg kg^?1, and the intra-day and inter-day recoveries were in the range 75.7–122.5% with the relative standard deviation (RSD) from 2.6% to 22.8%. Decision limits (CCα, α = 0.01) for melamine, ammeline, ammelide, and cyanuric acid in egg samples and milk powder were 3.5–5.9 and 2.5 to 3.8 µg kg^?1, and the detection capabilities (CCβ, β = 0.05) were 4.9–8.4 and 3.6–9.5 µg kg^?1, respectively. The method was successfully applied to egg samples and milk products as well. Satisfactory results were obtained as part of the 2009 European Union melamine proficiency test.     

Melamine and cyanuric acid do not interfere with Bradford and Ninhydrin assays for protein determination

In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food resulted in the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely concentrations added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10–30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives.     

Migration of melamine from can coatings cross-linked with melamine-based resins,into food simulants and foods

Resins based on melamine-formaldehyde and related analogues such as methylolated melamine are used to cross-link coatings used inside food cans and on the metal closures of glass jars. Thirteen commercially coated cans and closures representing 80% of the European market were tested using simulants under realistic industrial heat-processing conditions for canned and jarred foods. The food simulants and the retort conditions used were 3% acetic acid for 1?h at 100°C and 10% ethanol for 1?h at 130°C. The highest migration level seen for melamine into simulant was 332?µg?kg^?1. There was no detectable migration of the melamine analogues cyanuric acid (<1?µg?kg^?1) or ammelide (<5?µg?kg^?1) from any sample. Twelve of the thirteen samples released no detectable ammeline (<5?µg?kg^?1) but the coating giving the highest release of melamine did also release ammeline at 8?µg?kg^?1 with the higher of the two process temperatures used. Migration experiments into food simulant and foods themselves were then conducted using two experimental coatings made using amino-based cross-linking resins. Coated metal panels were exposed to the food simulant 10% (v/v) aqueous ethanol and to three foodstuffs under a range of time and temperature conditions both in the laboratory and in a commercial food canning facility using proprietary time and temperature conditions. The highest migration into a food was 152?µg?kg^?1 from the first coating processed for a long time at a moderate sterilisation temperature. The highest migration into simulant was also from this coating at 220?µg?kg^?1 when processed at 134°C for 60?min, dropping to 190?µg?kg^?1 when processed at 123°C for 70?min. Migration from the second coating was quite uniformly two to three times lower under all tests. These migration results were significantly higher than the levels of melamine extractable using 95% ethanol at room temperature. The experiments show that commercial canning and retorting can be mimicked in an acceptable way using laboratory tests with an autoclave or a simple pressure cooker. The results overall show there is hydrolytic degradation of the melamine cross-linked resins to release additional melamine. There is a strong influence of the temperature of heat treatment applied with foods or simulants but only a minor influence of time of heating and only a minor influence, if any, of food/simulant acidity.