Article pubs.acs.org/JAFC
Feasibility of Surface-Enhanced Raman Spectroscopy for Rapid Detection of Aflatoxins in Maize Kyung-Min Lee,*,† Timothy J. Herrman,† Yordanos Bisrat,‡ and Seth C. Murray§ †
Office of the Texas State Chemist, Texas A&M AgriLife Research, Texas A&M University System, College Station, Texas 77841, United States ‡ Materials Characterization Facility, Texas A&M University, College Station, Texas 77843, United States § Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas 77843, United States S Supporting Information *
ABSTRACT: Rapid and sensitive surface-enhanced Raman spectroscopy (SERS) for aflatoxin detection was employed for development of the models to classify and quantify aflatoxin levels in maize at concentrations of 0 to 1,206 μg/kg. Highly effective SERS substrate (Ag nanosphere) was prepared and mixed with a sample extract for SERS measurement. Strong Raman bands associated with aflatoxins and changes in maize kernels induced by aflatoxin contamination were observed in different SERS spectroscopic regions. The k-nearest neighbors (KNN) classification model yielded high classification accuracy and lower prediction error with no misclassification of contaminated samples as aflatoxin negative. The multiple linear regression (MLR) models showed a higher predictive accuracy with stronger correlation coefficients (r = 0.939−0.967) and a higher sensitivity with lower limits of detection (13−36 μg/kg) and quantitation (44−121 μg/kg) over other quantification models. Paired sample t test exhibited no statistically significant difference between the reference values and the predicted values of SERS in most chemometric models. The proposed SERS method would be a more effective and efficient analytical tool with a higher accuracy and lower constraints for aflatoxin analysis in maize compared to other existing spectroscopic methods and conventional Raman spectroscopy. KEYWORDS: aflatoxin, surface-enhanced Raman spectroscopy (SERS), nanostructure, chemometrics, food safety
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INTRODUCTION Aflatoxins as secondary metabolites produced by the Aspergillus strains have been causing a major problem for agriculture in many countries. Aflatoxins in contaminated food and feed products are carcinogens as recognized by the International Agency for Research on Cancer (IARC) as well as other health hazards for animals and humans, leading to substantial economic losses to relevant industries.1 Like fumonisins and other mycotoxins, maximum levels for aflatoxins in grains and oilseeds destined for animal and human consumptions have been set by the Food and Drug Administration (FDA) and European Commission (EC).2 The aflatoxin detection methods currently available in laboratory and nonlaboratory sites such as high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) are highly selective, very accurate, and precise with good detection limits.3 However, these methods are not practical and convenient for a real-time response due to long analysis time, low cost-effectiveness, analysis complexity, and requirement of trained personnel. Therefore, numerous studies have been continuously conducted to develop rapid, simple, and inexpensive methods with high sensitivity and specificity for early detection and identification of aflatoxins. A method employing portable equipment and nonhazardous solvents for aflatoxin analysis would be more suitable for application at the sites where the samples are collected.4 Spectroscopic methods are very promising and excellent tools for screening of aflatoxin contaminated samples because © XXXX American Chemical Society
they are inherently rapid, specific, and possibly partially or completely computerized. Among spectroscopic methods, Raman spectroscopy is valuable due to its great possibilities and advantages in detecting mycotoxins in grains and oilseeds.5−7 Compared to other spectroscopic methods used for mycotoxin analysis such as near-infrared spectroscopy (NIR) and Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy can provide well-resolved bands of mycotoxin molecules and allow acquisition of spectra in aqueous environments. However, conventional Raman spectroscopy has a small cross-section of Raman scattering compared to that of Rayleigh scattering, resulting in lack of sensitivity and thus requiring a longer collection time for improvement of spectral quality. Surface-enhanced Raman spectroscopy (SERS) has been considered as one of the most promising techniques to greatly improve the sensitivity of conventional Raman spectroscopy. SERS is an advanced technique that uses nanostructural surface phenomena to enhance a weak inelastic-scattering Raman effect of molecules adsorbed on and/or at the vicinity of metal particles like silver, gold, and copper.4,8,9 SERS can measure samples with great sensitivity and rapidity to provide comprehensive information, even on orientation of single Received: February 18, 2014 Revised: April 28, 2014 Accepted: April 28, 2014
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dx.doi.org/10.1021/jf500854u | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
resultant mixture was filtered through the filter paper (Whatman #1). The sample extract was directly used for SERS measurement. Preparation of SERS Substrate. Ag nanosphere (NS) for SERS measurements was prepared with slight modifications to the previous methods.10,19,20 In brief, 0.5 g of AgNO3, 10 mL of deionized water, 0.8 g of sodium oleate, 1 mL of oleic acid, and 5 mL of ethanol were added to a 20 mL glass vial in order under agitation. The glass vial was air-tightly sealed and placed in the oven at 150 °C for 24 h. Afterward the system was cooled down to room temperature. The products inside the vial were washed with ethanol three times and dried to form Ag nanocrystals (NC) at the bottom. The collected 4 mg of AgNC was added to 1 mL of cyclohexane. This solution was added to a solution of 28 mg of sodium dodecyl sulfate (SDS) in 1 mL of deionized water. The system containing AgNC was subjected to ultrasonic treatment for emulsification for about 1 h and subsequently heated at 70 °C with stirring until the cyclohexane was removed to manufacture AgNSs. HPLC Analysis of Aflatoxins. Fifty grams of ground maize sample was shaken for 1 h at 200 rpm for sample extraction using a solvent of methanol:water (70:30, v/v). The resultant mixture was filtered through the folded filter paper (Whatman #1). Ten milliliters of the filtered extract was mixed with 20 mL of deionized water and 1 g of sodium chloride and filtered to obtain the clean sample extract for loading onto an Aflatest immunoaffinity column (Vicam, Watertown, MA). The eluate from the column though washing with water and methanol was diluted with 1 mL of HPLC water and injected into a Waters 2695 HPLC system (Waters, Milford, MA). The HPLC system was equipped with a Spherisorb 4.6 × 150 mm, C18 column connected with Waters Spherisorb guard column 4.6 × 10 mm. The mobile phase consisted of a mixture of water:acetonitrile:methanol (3:1:1), flowing at a rate of 1.0 mL/min. Empower software (Milford, MA) was used to obtain and analyze chromatographic data. The mixture of standard aflatoxins B1, B2, G1, and G2 was prepared to build a standard calibration curve for quantification of aflatoxins in ground maize samples. SERS Measurement. The mixture of AgNSs and the extract at the ratio of 1:1 (v/v) in volume was prepared. A 300 μL volume of the mixture was placed on 96 wells for SERS measurements in quadruplicate using spectroscopy with a RamanStation 400F (PerkinElmer, Beaconsfield, Buckinghamshire, U.K.). The Raman system was interfaced with the Spectrum (v. 6.3) software for data acquisition and analysis and consisted of a 256 × 1024 pixel CCD detector and a near-infrared laser source of 785 nm wavelength, with 175 mW output power focused on the sample. The softwarecontrolled motorized sample stage was operated at
