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Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy ab
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Wei-Nan Lian , Jessie Shiue , Huai-Hsien Wang , Wei-Chen Hong , Po-Han Shih , Chaoe
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Kai Hsu , Ching-Yi Huang , Cheng-Rong Hsing , Ching-Ming Wei , Juen-Kai Wang & Yuh-Lin Wang a
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Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan
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Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan
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Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taipei, Taiwan
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Institute of Physics, Academia Sinica, Taipei, Taiwan
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Laboratory Division, Department of Health, Taipei City Government, Taipei, Taiwan
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Centre for Condensed Matter Sciences, National Taiwan University, Taipei, Taiwan
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Department of Physics, National Taiwan University, Taipei, Taiwan Published online: 30 Mar 2015.
To cite this article: Wei-Nan Lian, Jessie Shiue, Huai-Hsien Wang, Wei-Chen Hong, Po-Han Shih, Chao-Kai Hsu, Ching-Yi Huang, Cheng-Rong Hsing, Ching-Ming Wei, Juen-Kai Wang & Yuh-Lin Wang (2015) Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy, Food Additives & Contaminants: Part A, 32:5, 627-634 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1014867
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Food Additives & Contaminants: Part A, 2015 Vol. 32, No. 5, 627–634, http://dx.doi.org/10.1080/19440049.2015.1014867
Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy Wei-Nan Liana,b*, Jessie Shiuec,d, Huai-Hsien Wanga, Wei-Chen Hongb, Po-Han Shihb, Chao-Kai Hsue, Ching-Yi Huange, Cheng-Rong Hsinga, Ching-Ming Weia, Juen-Kai Wanga,f* and Yuh-Lin Wanga,g* a
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan; bInstitute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan; cResearch Program on Nanoscience and Nanotechnology, Academia Sinica, Taipei, Taiwan; dInstitute of Physics, Academia Sinica, Taipei, Taiwan; eLaboratory Division, Department of Health, Taipei City Government, Taipei, Taiwan; fCentre for Condensed Matter Sciences, National Taiwan University, Taipei, Taiwan; gDepartment of Physics, National Taiwan University, Taipei, Taiwan
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(Received 31 October 2014; accepted 18 January 2015) The addition of copper chlorophyll and its derivatives (Cu-Chl) to vegetable oils to disguise them as more expensive oils, such as virgin olive oils, would not only create public confusion, but also disturb the olive oil market. Given that existing detection methods of Ch-Chl in oils, such as LC-MS are costly and time consuming, it is imperative to develop economical and fast analytical techniques to provide information quickly. This paper demonstrates a rapid analytical method based on surface-enhanced Raman spectroscopy (SERS) to detect Cu-Chl in vegetable oils; the spectroscopic markers of Cu-Chl are presented and a detection limit of 5 mg kg−1 is demonstrated. The analysis of a series of commercial vegetable oils is undertaken with this method and the results verified by a government agency. This study shows that a SERS-based assessment method holds high potential for quickly pinpointing the addition of minute amounts of Cu-Chl in vegetable oils. Keywords: SERS; Raman; olive oil; copper chlorophyll
Introduction The worldwide consumption of olive oil reached over 3 million tonnes in 2013, according to the statistics provided by the International Olive Council, mostly owing to its five potential health benefits (Levent İnanç 2011): (1) lowering ‘bad’ cholesterol, (2) lowering blood pressure, (3) helping cancer prevention, (4) protecting oxidative damage and (5) helping cognitive function. Prices of high-quality olive oil are, thus, considerably higher than ordinary edible oils. In some cases, cheap oils with added flavour and colour have been detected as fake olive oils that had been labelled and sold as expensive olive oil (Mueller 2007). A later investigation amounted to a lexicon (Mueller 2012). The authenticity of olive oil has consequently been a serious issue for olive-oil trading (AparicioRuiz & Harwood 2013; Aparicio-Ruiz et al. 2013). Natural chlorophyll is believed to be one of the essential ingredients in olive oils that confers the aforementioned health benefits. Unfortunately, it is easily oxidised in air (Frankel 2010; Morales & Przybylski 2013), creating challenges in the production, storage and transportation of olive oils. Copper chlorophyll is a modification of natural chlorophyll – a chlorine pigment with a magnesium ion at the chlorine centre – with a copper ion instead. It is the chlorine pigment giving the greenish colour. However, copper chlorophyll and its derivatives (Cu-Chl) with variant peripheral
functionalities, which have better colouring ability and stability (Tumolo & Lanfer-Marquez 2012), are allowed to be used as a food colorant, listed as E141 in the European Parliament and Council Directive 94/36/EC 1994, and are listed in INS 141 in the Codex Alimentarius Commission, the international food standards organisation of the WHO and FAO of the United Nations (Sarkar & Komoroski 1992). Disguising Cu-Chl-added oils as more expensive olive oils creates serious authenticity problems (Roca et al. 2010) and therefore public distrust. The addition of Cu-Chl to oils is currently not permitted by the European Union (European Parliament and Council Directive 94/36/EC 1994). To determine whether Cu-Chl is present in olive oil is not easy. The analytical methods to determine Cu-Chl in foods are limited (Del Giovine & Fabietti 2005; Scotter et al. 2005; Roca et al. 2010; Giuliani et al. 2011; Scotter 2011; Gandul-Rojas et al. 2012). The majority of these analytical methods are based on chromatographic techniques (Gandul-Rojas et al. 2013), besides a technique based on capillary electrophoresis (Del Giovine & Fabietti 2005). Conventional analytical methods demand sophisticated pre-treatment of the oil samples, which, as a consequence, are time consuming. Therefore, a rapid method for detecting Cu-Chl in vegetable oils with high sensitivity and reliability is urgently needed.
*Corresponding authors. Emails: [email protected], [email protected] and [email protected] © 2015 Taylor & Francis
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In contrast to chromatographic methods, Raman spectroscopy, relying on a fingerprint vibrational signature for identification, holds advantages as an analytical tool: (1) fast detection and (2) it only requires a minute sample in comparison with other vibrational spectroscopic technique such as infrared absorption spectroscopy. Raman spectroscopy has been applied to various fields related to the safety and quality of food, such as for examination of microorganism contamination on food surfaces and to determine the nutritional parameters in dairy products (Yang & Ying 2011). Furthermore, the discovery of surface-enhanced Raman scattering (SERS) has been demonstrated to show a high potential for assaying molecules of extremely low concentration (Kneipp et al. 2006). Compared with metallic colloids, which have commonly been used to enhance Raman signals (Grzelczak & LizMarzań 2013), 2D and quasi-2D SERS enhancers portray much greater SERS stability, because they confer an array of identical metallic nanostructures that are firmly fixated to a certain specific pattern in order to induce stable ‘hot spots’ via plasmonic couplings between adjacent nanostructures (Kleinman et al. 2013). Two archetypal examples are triangular nanodisk arrays arranged in hexagonal patterns, made by nanosphere lithography (Willets & Duyne 2007), and nanoparticles embedded in hexagonally packed nanochannels of anodic aluminium oxide (Wang et al. 2006). In particular, the latter case, invented by our team represents a SERS-active substrate that confers readily tuneable plasmon resonance (Biring et al. 2008), which has been employed to detect biocides (Wang et al. 2011) and bacteria (Liu et al. 2009). Finally, performing SERS measurements under resonant excitation conditions – the excitation wavelength is coincident with a certain absorption band of the analyte, dubbed as surface-enhanced resonance Raman scattering (SERRS) – would further enhance the Raman signal as well as highlighting pertinent vibrational modes for commodious analysis. The application of SERS for food safety has attracted great attention in recent decade (Ellis et al. 2012; Craig et al. 2013), ranging from pathogenic microorganisms to contamination and adulteration (including additives, antibiotics, drugs and hormones). Early work using SERS to identify Cu-Chl was reported in 1988, in which a related SERS spectrum was used (Hildebrandt & Spiro 1988). Using SERS to investigate Cu-Chl in solution at various pH values has recently been reported (House & Schnitzer 2008). The present paper presents a rapid method for detecting Cu-Chl in vegetable oils using a SERS technique that requires no sample pre-treatment. This method is significantly quicker and easier than other, currently available techniques, and is particularly suitable for pre-screening purposes when detecting Cu-Chl in a large amount of suspicious oil samples. This method was validated by analysing Cu-Chl that had been spiked into three
vegetable oil bases: soybean, olive and sunflower oils. In total, 23 anonymous commercial oils were analysed by this method. The results were compared with values reported by the official government agency. Materials and methods Since a commercial Cu-Chl sample consists primarily of copper pyropheophytin a and some copper pheophytin a and b, the former was chosen as the reference molecule in this study. Such a choice raises a logical question: can SERS spectra of oils with a different amount of copper pyropheophytin α be used as references to help identify the commercial Cu-Chl in an oil? We note that copper pheophytin α and β bear an identical chlorine centre with different alkyl chains and anchor points. The reported four prominent peaks in SERS spectrum of pyropheophytin α were assigned to originate from the vibrational modes of the porphyrin ring (Hildebrandt & Spiro 1988). These peaks, which are relevant to our study, are sensitive to the ligand interaction with the core copper ion, and are thus expected to be less sensitive to the difference in the attached alkyl groups. We have also conducted ab initio calculations of the Raman polarisabilities of the three copper chlorophyll derivatives and found the four vibrational modes are almost identical, further supporting the choice of pyropheophytin α as the reference molecule. Chemicals and standards Both copper pyropheophytin α (approximately 97% in purity, Frontier-Scientific, West Logan, UT, USA) and magnesium chlorophyll a (≥ 85% in purity, SigmaAldrich, St. Louis, MO, USA) were purchased in powder form and used without further purification. For measuring the SERS and normal Raman of copper pyropheophytin α, the powder was first dissolved in ethanol (99.9% in purity) with a concentration of 500 mg kg−1 and then dried on a substrate. For SERS measurements of the spiked oils, both copper pyropheophytin α and magnesium chlorophyll α were dissolved in three oil bases (soybean, olive and sunflower oils; Sigma-Aldrich) at different concentrations. Typically, 1–2 µl samples were directly deposited on fresh SERS-active substrates or glass slides with a micropipette. The ethanol droplets quickly evaporated, leaving the solutes inside a circular area with a diameter of approximately 3 mm, while the oil droplets remained during the measurements. Fabrication of SERS-active substrates The fabrication procedure of the SERS-active substrates used in this study was reported previously (Wang et al. 2006; Liu et al. 2009). The SERS substrates used in this study was be purchased from MA-tek Inc. (see
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http://www.ma-tek.com/). Briefly, a 100 nm Al film was deposited on a glass slide by sputtering. The Al-deposited glass slide was then anodised in sulfuric acid (0.3 M) at 3° C with a bias voltage of 21 V in order to create a 2D hexagonally packed array of nanochannels with an average inter-channel spacing of 53 nm. The nanochannels were widened by wet chemical etching, yielding an average channel diameter of 45 nm. An electrochemical plating procedure was then employed to grow Ag nanoparticles into the nanochannels in order to form an array of nanoparticles with an average length of 60 nm. Individual SERS-active substrates, after being rinsed with deionised water, were immediately vacuum-sealed in plastic bags, with minimum release of volatile organic compounds, for storage and shipping. The substrate was drawn from the vacuum-sealed bag just before use in order to minimise environmental contamination. Equipment Raman measurements were performed with a commercial Raman microscope (LabRAM HR800, Horiba, Kyoto, Japan). A HeNe laser, emitting at 632.8 nm, served as the excitation light source. The laser beam, after spatial filtering, was focused by a 100× objective lens (numeric aperture = 0.9) to the dried surface of the ethanol samples and to the bottom surface of the oil droplets. The scattered radiation was collected in a backward direction with the same objective lens, transmitted through a long-pass filter to minimise the Rayleigh scattering at the excitation wavelength and, finally, sent to an 80 cm spectrometer with a liquid nitrogen-cooled charge-coupled device (CCD). The spectral accuracy and precision were calibrated to be < 7 and 0.1 cm−1, respectively. The signal accumulation time was in the 1–5 s range, depending on the acquired signal strength. The recorded Raman spectra were subsequently analysed with a homemade algorithm to remove cosmic rays, high-frequency noise and the background baseline. Results and discussion To obtain a basic SERS spectrum of copper pyropheophytin α, a standard Raman measurement conducted on a 500 mg kg−1 copper pyropheophytin α in ethanol was acquired using a SERS substrate; the result is shown as the solid line in Figure 1. To avoid possible signals from unknown molecules in an oil, ethanol was used as the solvent for this basic SERS test instead of the oil. For comparison, the normal Raman spectrum obtained under the same Raman measurement conditions (laser power and signal integration time) from the same sample deposited on a glass slide is shown as the dotted line in Figure 1. The normal Raman signal is about 10% of the SERS counterpart, indicating that it is harder to detect copper pyropheophytin α with normal Raman spectroscopy.
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Figure 1. Normal Raman (dotted curve) and surface-enhanced Raman spectra (SERS) (solid curve) of copper pyropheophytin α.
Furthermore, the SERS signal is represented by a characteristic spectral pattern with four major peaks located at 752, 990, 1140 and 1230 cm−1, as indicated in Figure 1 (grey bars). The SERS spectrum shown in Figure 1 is in good agreement with the SERS spectrum of copper chlorophyllin – a copper chlorophyll derivative without a long phytol chain – obtained when excited by a 647 nm laser, as reported by Hildebrandt and Spiro (1988). Table 1 lists the recognised peaks from their SERS spectra. The four major peaks were assigned as follows: 752 cm−1 δ(CaCbEt), 990 cm−1 δ(CbEt) and δ(CaNCa), 1140 cm−1 δ(CbEt) and δ(CaN), and 1230 cm−1 δ(CbH) and δ(CmH) (Hildebrandt & Spiro 1988). Notably, the use of the excitation wavelength at 632.8 nm in Raman measurements renders the Raman scattering process of Cu-Chl resonantly excited (resonance Raman spectroscopy), because the excitation radiation resides within its Q band, ranging from 550 to 650 nm, for the π–π* transition of the metallochlorin ring (Hildebrandt & Spiro 1988). Resonance Raman spectroscopy of chlorophyll has previously been studied in detail (Boldt et al. 1987). In comparison, the SERS signal obtained with excitation wavelengths of 532 and 785 nm (not shown here) are significantly smaller. With the acquired SERS spectrum of copper pyropheophytin α, the next step is to perform SERS measurements of copper pyropheophytin α-added oils, whose colours can be adjusted by controlling their copper pyropheophytin α concentrations. In this study, we have chosen three oils as the oil bases: pure soybean, sunflower and olive oils, which were purchased from Sigma-Aldrich. For comparison, the SERS measurement of these unadulterated oils, as well as those after the addition of 500 ppm magnesium chlorophyll α, was also conducted. The results are shown in Figure 2. As shown, the SERS spectra of the three pure oils (dotted lines) have similar (El-Abassy et al. 2009) characteristics, with two main peaks at 1302
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Table 1. Raman peaks (in cm−1) identified from the surfaceenhanced Raman spectroscopy (SERS) spectra of copper pyropheophytin α and magnesium chlorophyll a. Copper pyropheophytin α
Magnesium chlorophyll a
Dry
In oil
In oil
461 522 570
463 522 569
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595 624 656
653
750
752 789
923 992 1111 1140 1186 1230
922 990
666 750 834 926 1005
1140 1183 1230 1240
1266 1286 1335 1346 1385 1446 1469 1510 1560
1284
1298
1342
1509 1558
1474 1514
Note: Numbers in bold are the prominent peaks that give high intensity.
and 1440 cm−1. As the spectra obtained from the pristine oils are clearly different from the SERS spectra of copper pyropheophytin α-added oils (solid lines in Figure 2), they
would not interfere in the SERS identification of samples with copper pyropheophytin α and magnesium chlorophyll α dissolved in the oils. The SERS spectra of the three oils containing 500 mg kg−1 of copper pyropheophytin α and magnesium chlorophyll α are shown in Figure 2, as solid lines and dashed lines, respectively. The recognised Raman peaks from the two chlorophylls in the oils are listed in Table 1. Except for some small peaks, the SERS spectra of copper pyropheophytin α under dry conditions and in oils are in good agreement. Similar to the results obtained from copper pyropheophytin α in ethanol (Figure 1), four major peaks were observed, but the intensities of the peaks at 752 and 990 cm−1 are further enhanced in oil, indicated by grey bars in Figure 2, particularly the peak at 752 cm−1, which has the highest intensity, suggesting that these particular SERS peaks can be used as markers to detect the presence of copper pyropheophytin α in vegetable oil. Moreover, the characteristic peaks of copper pyropheophytin α and magnesium chlorophyll α are drastically different, denoting that the SERS technique can be used to differentiate copper pyropheophytin α-added oil from nascent olive oils in which magnesium chlorophyll α was the only natural chlorophyll compound. The second spiked oil test was performed on oils with different concentrations of copper pyropheophytin α in order to determine the lowest detection limit. Figure 3 shows the SERS spectra of copper pyropheophytin α dissolved in the three oils, with concentrations of 0, 0.5, 5, 50 and 500 mg kg−1, as well as their corresponding portrayed colour images. Three inferences can be made from these results. Firstly, the intensity of the two low-frequency prominent peaks at 752 and 990 cm−1 increases monotonically with the copper pyropheophytin α concentration,
Figure 2. Normalised SERS spectra of magnesium chlorophyll a (dashed curves) and copper pyropheophytin α (solid curves) dissolved in (a) soybean oil, (b) olive oil and (c) sunflower oil, each with a concentration of 500 ppm. The corresponding spectra of the pristine oils (dotted curves) are shown for comparison. The two prominent peaks at 752 and 990 cm−1 of copper pyropheophytin α are marked with vertical grey bands.
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Figure 3. (colour online) Normalised SERS spectra of copper pyropheophytin α dissolved in (a) soybean oil, (b) olive oil and (c) sunflower oil, each with concentrations of 0, 0.5, 5, 50 and 500 ppm. Their photographs are also shown to reference the colouring effects.
whereas no clear trend can be deduced for the two highfrequency peaks at 1140 and 1230 cm−1, probably because they overlap with the normal Raman peaks of the oils. Secondly, the lowest detection limit of copper pyropheophytin α in these oils, based on the SERS technique, is 5 mg kg−1, as shown in Figure 3(a)–(c), below which the two prominent peaks at 1302 and 1440 cm−1 of oils become prevailing. Thirdly, it generally requires a high concentration of copper pyropheophytin α (above 50 mg kg−1) dissolved in the oils to create colouration of the oil that is thick enough to cheat customers, as shown in Figure 3; therefore, the sensitivity of the SERS technique demonstrated here should be sufficient for identifying CuChl in oils for practical purposes. These three conclusions from the spiked oil test establish the foundation of using the SERS technique as a rapid method for detecting CuChl in vegetable oils. We applied the technique described above to test 23 commercial vegetable oil samples for the presence of CuChl, and we then compared the results with those reported by the Food and Drug Administration in Taiwan (TFDA) using LC-MS/MS. Among these samples, 21 oils showed recognisable SERS signals (Figure 4), two of which are undetermined (Figure 5) and discussed. Figure 4 shows the SERS spectra of the 21 commercial oils (solid lines), along with the SERS spectrum of copper chlorophyll (dotted lines) for comparison. Fourteen oil specimens with the two identified markers of the SERS spectrum of
copper pyropheophytin α – the Raman peaks at 752 and 990 cm−1, shown in Figure 4(a) – are regarded as being positive for the presence of Cu-Chl in the oils. The results are 100% in agreement with the results reported by the TFDA. Seven oil samples are regarded as negative based on their portrayed SERS spectra, as show in Figure 4(b), as the two markers are not recognisable, which is also consistent with the reported results by the TFDA. The successful ratio of identification is 21/23. This result demonstrates that the SERS technique is suitable for prescreening Cu-Chl in vegetable oils without requiring sample pre-treatment. Among the 23 commercial samples tested, in two of them we could not determine the presence of Cu-Chl, owing to the strong background signal in the Raman spectrum, as shown in Figure 5. The strong background signal likely originates from fluorescence. In some expensive olive oils, the main fluorescence contribution is attributed to chlorophyll (Kyriakidis & Skarkalis 2000; GarcíaGonzález et al. 2013). The 632.8 nm excitation at the Qband of chlorophyll produces fluorescence, ranging from 650 to 700 nm, corresponding to the Raman shift in the 400–1600 cm−1 range. As a consequence, the observed maximal background at around 1000 cm−1 (Figure 5) can be ascribed to that fluorescence. It is, thus, possible that these two samples contain higher chlorophyll contents, resulting in the high fluorescence signal shown in the SERS spectra. In the case of Cu-Chl in oils, although the
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Figure 4. Normalised SERS spectra of commercial vegetable oil samples that were identified as being (a) positive or (b) negative for the presence of Cu-Chl. The normalised SERS spectrum of copper pyropheophytin α (dotted curves) is shown for comparison. The two prominent peaks at 752 and 990 cm−1 of copper pyropheophytin α are marked with vertical grey bands.
absorption and fluorescence spectra at the Q band of CuChl are identical to that of magnesium chlorophyll α, its fluorescence quantum yield is about two orders of magnitude smaller, owing to a stronger spin-orbit perturbation induced by the presence of the paramagnetic Cu ion (Fernandez & Becker 1959), making its SERS signal comparatively dominant and, therefore, facilitating the SERS detection. The displayed SERS spectra in Figure 5, after background removal, are more similar to the SERS spectra of magnesium chlorophyll α dissolved in oils, as shown Figure 2, although their signal-to-noise ratios are not high enough for affirmative identification. Conclusions
Figure 5. Normalised SERS spectra of two commercial vegetable oil samples (solid curves) that exhibit a strong background signal. The SERS spectrum of copper pyropheophytin α (dotted curve) is shown for comparison.
A SERS technique for detecting the presence of copper chlorophyll in vegetable oils is reported. This new method does not require any sample pre-treatment, thus the testing time is significantly reduced relative to conventional chemical analysis methods. The reported SERS technique was validated in three copper pyropheophytin α-spiked vegetable oils, with a detection limit of 5 ppm. The testing of commercial vegetable oils using this new method is very accurate, and the results are consistent with those reported by the government agency with a 21/23 successful ratio of identification. This new technique is simple and quick, making it suitable for pre-screening
Food Additives & Contaminants: Part A purposes. Our work also suggests that the application of the SERS technique for rapid detection of trace molecules in food is possible. Acknowledgements Technical support from NanoCore, the Core Facilities for Nanoscience and Nanotechnology, Academia Sinica and from Materials Analysis Technology Inc. in Taiwan is acknowledged.
Funding
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This work was partly supported by the Ministry of Science and Technology [grant number MOST 103-2628-M-001-002] and the Academia Sinica in Taiwan.
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Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy ab
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Wei-Nan Lian , Jessie Shiue , Huai-Hsien Wang , Wei-Chen Hong , Po-Han Shih , Chaoe
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Kai Hsu , Ching-Yi Huang , Cheng-Rong Hsing , Ching-Ming Wei , Juen-Kai Wang & Yuh-Lin Wang a
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Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan
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Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan
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Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taipei, Taiwan
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Institute of Physics, Academia Sinica, Taipei, Taiwan
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Laboratory Division, Department of Health, Taipei City Government, Taipei, Taiwan
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Centre for Condensed Matter Sciences, National Taiwan University, Taipei, Taiwan
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Department of Physics, National Taiwan University, Taipei, Taiwan Published online: 30 Mar 2015.
To cite this article: Wei-Nan Lian, Jessie Shiue, Huai-Hsien Wang, Wei-Chen Hong, Po-Han Shih, Chao-Kai Hsu, Ching-Yi Huang, Cheng-Rong Hsing, Ching-Ming Wei, Juen-Kai Wang & Yuh-Lin Wang (2015) Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy, Food Additives & Contaminants: Part A, 32:5, 627-634 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1014867
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Food Additives & Contaminants: Part A, 2015 Vol. 32, No. 5, 627–634, http://dx.doi.org/10.1080/19440049.2015.1014867
Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy Wei-Nan Liana,b*, Jessie Shiuec,d, Huai-Hsien Wanga, Wei-Chen Hongb, Po-Han Shihb, Chao-Kai Hsue, Ching-Yi Huange, Cheng-Rong Hsinga, Ching-Ming Weia, Juen-Kai Wanga,f* and Yuh-Lin Wanga,g* a
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan; bInstitute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan; cResearch Program on Nanoscience and Nanotechnology, Academia Sinica, Taipei, Taiwan; dInstitute of Physics, Academia Sinica, Taipei, Taiwan; eLaboratory Division, Department of Health, Taipei City Government, Taipei, Taiwan; fCentre for Condensed Matter Sciences, National Taiwan University, Taipei, Taiwan; gDepartment of Physics, National Taiwan University, Taipei, Taiwan
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(Received 31 October 2014; accepted 18 January 2015) The addition of copper chlorophyll and its derivatives (Cu-Chl) to vegetable oils to disguise them as more expensive oils, such as virgin olive oils, would not only create public confusion, but also disturb the olive oil market. Given that existing detection methods of Ch-Chl in oils, such as LC-MS are costly and time consuming, it is imperative to develop economical and fast analytical techniques to provide information quickly. This paper demonstrates a rapid analytical method based on surface-enhanced Raman spectroscopy (SERS) to detect Cu-Chl in vegetable oils; the spectroscopic markers of Cu-Chl are presented and a detection limit of 5 mg kg−1 is demonstrated. The analysis of a series of commercial vegetable oils is undertaken with this method and the results verified by a government agency. This study shows that a SERS-based assessment method holds high potential for quickly pinpointing the addition of minute amounts of Cu-Chl in vegetable oils. Keywords: SERS; Raman; olive oil; copper chlorophyll
Introduction The worldwide consumption of olive oil reached over 3 million tonnes in 2013, according to the statistics provided by the International Olive Council, mostly owing to its five potential health benefits (Levent İnanç 2011): (1) lowering ‘bad’ cholesterol, (2) lowering blood pressure, (3) helping cancer prevention, (4) protecting oxidative damage and (5) helping cognitive function. Prices of high-quality olive oil are, thus, considerably higher than ordinary edible oils. In some cases, cheap oils with added flavour and colour have been detected as fake olive oils that had been labelled and sold as expensive olive oil (Mueller 2007). A later investigation amounted to a lexicon (Mueller 2012). The authenticity of olive oil has consequently been a serious issue for olive-oil trading (AparicioRuiz & Harwood 2013; Aparicio-Ruiz et al. 2013). Natural chlorophyll is believed to be one of the essential ingredients in olive oils that confers the aforementioned health benefits. Unfortunately, it is easily oxidised in air (Frankel 2010; Morales & Przybylski 2013), creating challenges in the production, storage and transportation of olive oils. Copper chlorophyll is a modification of natural chlorophyll – a chlorine pigment with a magnesium ion at the chlorine centre – with a copper ion instead. It is the chlorine pigment giving the greenish colour. However, copper chlorophyll and its derivatives (Cu-Chl) with variant peripheral
functionalities, which have better colouring ability and stability (Tumolo & Lanfer-Marquez 2012), are allowed to be used as a food colorant, listed as E141 in the European Parliament and Council Directive 94/36/EC 1994, and are listed in INS 141 in the Codex Alimentarius Commission, the international food standards organisation of the WHO and FAO of the United Nations (Sarkar & Komoroski 1992). Disguising Cu-Chl-added oils as more expensive olive oils creates serious authenticity problems (Roca et al. 2010) and therefore public distrust. The addition of Cu-Chl to oils is currently not permitted by the European Union (European Parliament and Council Directive 94/36/EC 1994). To determine whether Cu-Chl is present in olive oil is not easy. The analytical methods to determine Cu-Chl in foods are limited (Del Giovine & Fabietti 2005; Scotter et al. 2005; Roca et al. 2010; Giuliani et al. 2011; Scotter 2011; Gandul-Rojas et al. 2012). The majority of these analytical methods are based on chromatographic techniques (Gandul-Rojas et al. 2013), besides a technique based on capillary electrophoresis (Del Giovine & Fabietti 2005). Conventional analytical methods demand sophisticated pre-treatment of the oil samples, which, as a consequence, are time consuming. Therefore, a rapid method for detecting Cu-Chl in vegetable oils with high sensitivity and reliability is urgently needed.
*Corresponding authors. Emails: [email protected], [email protected] and [email protected] © 2015 Taylor & Francis
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In contrast to chromatographic methods, Raman spectroscopy, relying on a fingerprint vibrational signature for identification, holds advantages as an analytical tool: (1) fast detection and (2) it only requires a minute sample in comparison with other vibrational spectroscopic technique such as infrared absorption spectroscopy. Raman spectroscopy has been applied to various fields related to the safety and quality of food, such as for examination of microorganism contamination on food surfaces and to determine the nutritional parameters in dairy products (Yang & Ying 2011). Furthermore, the discovery of surface-enhanced Raman scattering (SERS) has been demonstrated to show a high potential for assaying molecules of extremely low concentration (Kneipp et al. 2006). Compared with metallic colloids, which have commonly been used to enhance Raman signals (Grzelczak & LizMarzań 2013), 2D and quasi-2D SERS enhancers portray much greater SERS stability, because they confer an array of identical metallic nanostructures that are firmly fixated to a certain specific pattern in order to induce stable ‘hot spots’ via plasmonic couplings between adjacent nanostructures (Kleinman et al. 2013). Two archetypal examples are triangular nanodisk arrays arranged in hexagonal patterns, made by nanosphere lithography (Willets & Duyne 2007), and nanoparticles embedded in hexagonally packed nanochannels of anodic aluminium oxide (Wang et al. 2006). In particular, the latter case, invented by our team represents a SERS-active substrate that confers readily tuneable plasmon resonance (Biring et al. 2008), which has been employed to detect biocides (Wang et al. 2011) and bacteria (Liu et al. 2009). Finally, performing SERS measurements under resonant excitation conditions – the excitation wavelength is coincident with a certain absorption band of the analyte, dubbed as surface-enhanced resonance Raman scattering (SERRS) – would further enhance the Raman signal as well as highlighting pertinent vibrational modes for commodious analysis. The application of SERS for food safety has attracted great attention in recent decade (Ellis et al. 2012; Craig et al. 2013), ranging from pathogenic microorganisms to contamination and adulteration (including additives, antibiotics, drugs and hormones). Early work using SERS to identify Cu-Chl was reported in 1988, in which a related SERS spectrum was used (Hildebrandt & Spiro 1988). Using SERS to investigate Cu-Chl in solution at various pH values has recently been reported (House & Schnitzer 2008). The present paper presents a rapid method for detecting Cu-Chl in vegetable oils using a SERS technique that requires no sample pre-treatment. This method is significantly quicker and easier than other, currently available techniques, and is particularly suitable for pre-screening purposes when detecting Cu-Chl in a large amount of suspicious oil samples. This method was validated by analysing Cu-Chl that had been spiked into three
vegetable oil bases: soybean, olive and sunflower oils. In total, 23 anonymous commercial oils were analysed by this method. The results were compared with values reported by the official government agency. Materials and methods Since a commercial Cu-Chl sample consists primarily of copper pyropheophytin a and some copper pheophytin a and b, the former was chosen as the reference molecule in this study. Such a choice raises a logical question: can SERS spectra of oils with a different amount of copper pyropheophytin α be used as references to help identify the commercial Cu-Chl in an oil? We note that copper pheophytin α and β bear an identical chlorine centre with different alkyl chains and anchor points. The reported four prominent peaks in SERS spectrum of pyropheophytin α were assigned to originate from the vibrational modes of the porphyrin ring (Hildebrandt & Spiro 1988). These peaks, which are relevant to our study, are sensitive to the ligand interaction with the core copper ion, and are thus expected to be less sensitive to the difference in the attached alkyl groups. We have also conducted ab initio calculations of the Raman polarisabilities of the three copper chlorophyll derivatives and found the four vibrational modes are almost identical, further supporting the choice of pyropheophytin α as the reference molecule. Chemicals and standards Both copper pyropheophytin α (approximately 97% in purity, Frontier-Scientific, West Logan, UT, USA) and magnesium chlorophyll a (≥ 85% in purity, SigmaAldrich, St. Louis, MO, USA) were purchased in powder form and used without further purification. For measuring the SERS and normal Raman of copper pyropheophytin α, the powder was first dissolved in ethanol (99.9% in purity) with a concentration of 500 mg kg−1 and then dried on a substrate. For SERS measurements of the spiked oils, both copper pyropheophytin α and magnesium chlorophyll α were dissolved in three oil bases (soybean, olive and sunflower oils; Sigma-Aldrich) at different concentrations. Typically, 1–2 µl samples were directly deposited on fresh SERS-active substrates or glass slides with a micropipette. The ethanol droplets quickly evaporated, leaving the solutes inside a circular area with a diameter of approximately 3 mm, while the oil droplets remained during the measurements. Fabrication of SERS-active substrates The fabrication procedure of the SERS-active substrates used in this study was reported previously (Wang et al. 2006; Liu et al. 2009). The SERS substrates used in this study was be purchased from MA-tek Inc. (see
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http://www.ma-tek.com/). Briefly, a 100 nm Al film was deposited on a glass slide by sputtering. The Al-deposited glass slide was then anodised in sulfuric acid (0.3 M) at 3° C with a bias voltage of 21 V in order to create a 2D hexagonally packed array of nanochannels with an average inter-channel spacing of 53 nm. The nanochannels were widened by wet chemical etching, yielding an average channel diameter of 45 nm. An electrochemical plating procedure was then employed to grow Ag nanoparticles into the nanochannels in order to form an array of nanoparticles with an average length of 60 nm. Individual SERS-active substrates, after being rinsed with deionised water, were immediately vacuum-sealed in plastic bags, with minimum release of volatile organic compounds, for storage and shipping. The substrate was drawn from the vacuum-sealed bag just before use in order to minimise environmental contamination. Equipment Raman measurements were performed with a commercial Raman microscope (LabRAM HR800, Horiba, Kyoto, Japan). A HeNe laser, emitting at 632.8 nm, served as the excitation light source. The laser beam, after spatial filtering, was focused by a 100× objective lens (numeric aperture = 0.9) to the dried surface of the ethanol samples and to the bottom surface of the oil droplets. The scattered radiation was collected in a backward direction with the same objective lens, transmitted through a long-pass filter to minimise the Rayleigh scattering at the excitation wavelength and, finally, sent to an 80 cm spectrometer with a liquid nitrogen-cooled charge-coupled device (CCD). The spectral accuracy and precision were calibrated to be < 7 and 0.1 cm−1, respectively. The signal accumulation time was in the 1–5 s range, depending on the acquired signal strength. The recorded Raman spectra were subsequently analysed with a homemade algorithm to remove cosmic rays, high-frequency noise and the background baseline. Results and discussion To obtain a basic SERS spectrum of copper pyropheophytin α, a standard Raman measurement conducted on a 500 mg kg−1 copper pyropheophytin α in ethanol was acquired using a SERS substrate; the result is shown as the solid line in Figure 1. To avoid possible signals from unknown molecules in an oil, ethanol was used as the solvent for this basic SERS test instead of the oil. For comparison, the normal Raman spectrum obtained under the same Raman measurement conditions (laser power and signal integration time) from the same sample deposited on a glass slide is shown as the dotted line in Figure 1. The normal Raman signal is about 10% of the SERS counterpart, indicating that it is harder to detect copper pyropheophytin α with normal Raman spectroscopy.
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Figure 1. Normal Raman (dotted curve) and surface-enhanced Raman spectra (SERS) (solid curve) of copper pyropheophytin α.
Furthermore, the SERS signal is represented by a characteristic spectral pattern with four major peaks located at 752, 990, 1140 and 1230 cm−1, as indicated in Figure 1 (grey bars). The SERS spectrum shown in Figure 1 is in good agreement with the SERS spectrum of copper chlorophyllin – a copper chlorophyll derivative without a long phytol chain – obtained when excited by a 647 nm laser, as reported by Hildebrandt and Spiro (1988). Table 1 lists the recognised peaks from their SERS spectra. The four major peaks were assigned as follows: 752 cm−1 δ(CaCbEt), 990 cm−1 δ(CbEt) and δ(CaNCa), 1140 cm−1 δ(CbEt) and δ(CaN), and 1230 cm−1 δ(CbH) and δ(CmH) (Hildebrandt & Spiro 1988). Notably, the use of the excitation wavelength at 632.8 nm in Raman measurements renders the Raman scattering process of Cu-Chl resonantly excited (resonance Raman spectroscopy), because the excitation radiation resides within its Q band, ranging from 550 to 650 nm, for the π–π* transition of the metallochlorin ring (Hildebrandt & Spiro 1988). Resonance Raman spectroscopy of chlorophyll has previously been studied in detail (Boldt et al. 1987). In comparison, the SERS signal obtained with excitation wavelengths of 532 and 785 nm (not shown here) are significantly smaller. With the acquired SERS spectrum of copper pyropheophytin α, the next step is to perform SERS measurements of copper pyropheophytin α-added oils, whose colours can be adjusted by controlling their copper pyropheophytin α concentrations. In this study, we have chosen three oils as the oil bases: pure soybean, sunflower and olive oils, which were purchased from Sigma-Aldrich. For comparison, the SERS measurement of these unadulterated oils, as well as those after the addition of 500 ppm magnesium chlorophyll α, was also conducted. The results are shown in Figure 2. As shown, the SERS spectra of the three pure oils (dotted lines) have similar (El-Abassy et al. 2009) characteristics, with two main peaks at 1302
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Table 1. Raman peaks (in cm−1) identified from the surfaceenhanced Raman spectroscopy (SERS) spectra of copper pyropheophytin α and magnesium chlorophyll a. Copper pyropheophytin α
Magnesium chlorophyll a
Dry
In oil
In oil
461 522 570
463 522 569
466 526
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595 624 656
653
750
752 789
923 992 1111 1140 1186 1230
922 990
666 750 834 926 1005
1140 1183 1230 1240
1266 1286 1335 1346 1385 1446 1469 1510 1560
1284
1298
1342
1509 1558
1474 1514
Note: Numbers in bold are the prominent peaks that give high intensity.
and 1440 cm−1. As the spectra obtained from the pristine oils are clearly different from the SERS spectra of copper pyropheophytin α-added oils (solid lines in Figure 2), they
would not interfere in the SERS identification of samples with copper pyropheophytin α and magnesium chlorophyll α dissolved in the oils. The SERS spectra of the three oils containing 500 mg kg−1 of copper pyropheophytin α and magnesium chlorophyll α are shown in Figure 2, as solid lines and dashed lines, respectively. The recognised Raman peaks from the two chlorophylls in the oils are listed in Table 1. Except for some small peaks, the SERS spectra of copper pyropheophytin α under dry conditions and in oils are in good agreement. Similar to the results obtained from copper pyropheophytin α in ethanol (Figure 1), four major peaks were observed, but the intensities of the peaks at 752 and 990 cm−1 are further enhanced in oil, indicated by grey bars in Figure 2, particularly the peak at 752 cm−1, which has the highest intensity, suggesting that these particular SERS peaks can be used as markers to detect the presence of copper pyropheophytin α in vegetable oil. Moreover, the characteristic peaks of copper pyropheophytin α and magnesium chlorophyll α are drastically different, denoting that the SERS technique can be used to differentiate copper pyropheophytin α-added oil from nascent olive oils in which magnesium chlorophyll α was the only natural chlorophyll compound. The second spiked oil test was performed on oils with different concentrations of copper pyropheophytin α in order to determine the lowest detection limit. Figure 3 shows the SERS spectra of copper pyropheophytin α dissolved in the three oils, with concentrations of 0, 0.5, 5, 50 and 500 mg kg−1, as well as their corresponding portrayed colour images. Three inferences can be made from these results. Firstly, the intensity of the two low-frequency prominent peaks at 752 and 990 cm−1 increases monotonically with the copper pyropheophytin α concentration,
Figure 2. Normalised SERS spectra of magnesium chlorophyll a (dashed curves) and copper pyropheophytin α (solid curves) dissolved in (a) soybean oil, (b) olive oil and (c) sunflower oil, each with a concentration of 500 ppm. The corresponding spectra of the pristine oils (dotted curves) are shown for comparison. The two prominent peaks at 752 and 990 cm−1 of copper pyropheophytin α are marked with vertical grey bands.
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Figure 3. (colour online) Normalised SERS spectra of copper pyropheophytin α dissolved in (a) soybean oil, (b) olive oil and (c) sunflower oil, each with concentrations of 0, 0.5, 5, 50 and 500 ppm. Their photographs are also shown to reference the colouring effects.
whereas no clear trend can be deduced for the two highfrequency peaks at 1140 and 1230 cm−1, probably because they overlap with the normal Raman peaks of the oils. Secondly, the lowest detection limit of copper pyropheophytin α in these oils, based on the SERS technique, is 5 mg kg−1, as shown in Figure 3(a)–(c), below which the two prominent peaks at 1302 and 1440 cm−1 of oils become prevailing. Thirdly, it generally requires a high concentration of copper pyropheophytin α (above 50 mg kg−1) dissolved in the oils to create colouration of the oil that is thick enough to cheat customers, as shown in Figure 3; therefore, the sensitivity of the SERS technique demonstrated here should be sufficient for identifying CuChl in oils for practical purposes. These three conclusions from the spiked oil test establish the foundation of using the SERS technique as a rapid method for detecting CuChl in vegetable oils. We applied the technique described above to test 23 commercial vegetable oil samples for the presence of CuChl, and we then compared the results with those reported by the Food and Drug Administration in Taiwan (TFDA) using LC-MS/MS. Among these samples, 21 oils showed recognisable SERS signals (Figure 4), two of which are undetermined (Figure 5) and discussed. Figure 4 shows the SERS spectra of the 21 commercial oils (solid lines), along with the SERS spectrum of copper chlorophyll (dotted lines) for comparison. Fourteen oil specimens with the two identified markers of the SERS spectrum of
copper pyropheophytin α – the Raman peaks at 752 and 990 cm−1, shown in Figure 4(a) – are regarded as being positive for the presence of Cu-Chl in the oils. The results are 100% in agreement with the results reported by the TFDA. Seven oil samples are regarded as negative based on their portrayed SERS spectra, as show in Figure 4(b), as the two markers are not recognisable, which is also consistent with the reported results by the TFDA. The successful ratio of identification is 21/23. This result demonstrates that the SERS technique is suitable for prescreening Cu-Chl in vegetable oils without requiring sample pre-treatment. Among the 23 commercial samples tested, in two of them we could not determine the presence of Cu-Chl, owing to the strong background signal in the Raman spectrum, as shown in Figure 5. The strong background signal likely originates from fluorescence. In some expensive olive oils, the main fluorescence contribution is attributed to chlorophyll (Kyriakidis & Skarkalis 2000; GarcíaGonzález et al. 2013). The 632.8 nm excitation at the Qband of chlorophyll produces fluorescence, ranging from 650 to 700 nm, corresponding to the Raman shift in the 400–1600 cm−1 range. As a consequence, the observed maximal background at around 1000 cm−1 (Figure 5) can be ascribed to that fluorescence. It is, thus, possible that these two samples contain higher chlorophyll contents, resulting in the high fluorescence signal shown in the SERS spectra. In the case of Cu-Chl in oils, although the
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Figure 4. Normalised SERS spectra of commercial vegetable oil samples that were identified as being (a) positive or (b) negative for the presence of Cu-Chl. The normalised SERS spectrum of copper pyropheophytin α (dotted curves) is shown for comparison. The two prominent peaks at 752 and 990 cm−1 of copper pyropheophytin α are marked with vertical grey bands.
absorption and fluorescence spectra at the Q band of CuChl are identical to that of magnesium chlorophyll α, its fluorescence quantum yield is about two orders of magnitude smaller, owing to a stronger spin-orbit perturbation induced by the presence of the paramagnetic Cu ion (Fernandez & Becker 1959), making its SERS signal comparatively dominant and, therefore, facilitating the SERS detection. The displayed SERS spectra in Figure 5, after background removal, are more similar to the SERS spectra of magnesium chlorophyll α dissolved in oils, as shown Figure 2, although their signal-to-noise ratios are not high enough for affirmative identification. Conclusions
Figure 5. Normalised SERS spectra of two commercial vegetable oil samples (solid curves) that exhibit a strong background signal. The SERS spectrum of copper pyropheophytin α (dotted curve) is shown for comparison.
A SERS technique for detecting the presence of copper chlorophyll in vegetable oils is reported. This new method does not require any sample pre-treatment, thus the testing time is significantly reduced relative to conventional chemical analysis methods. The reported SERS technique was validated in three copper pyropheophytin α-spiked vegetable oils, with a detection limit of 5 ppm. The testing of commercial vegetable oils using this new method is very accurate, and the results are consistent with those reported by the government agency with a 21/23 successful ratio of identification. This new technique is simple and quick, making it suitable for pre-screening
Food Additives & Contaminants: Part A purposes. Our work also suggests that the application of the SERS technique for rapid detection of trace molecules in food is possible. Acknowledgements Technical support from NanoCore, the Core Facilities for Nanoscience and Nanotechnology, Academia Sinica and from Materials Analysis Technology Inc. in Taiwan is acknowledged.
Funding
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This work was partly supported by the Ministry of Science and Technology [grant number MOST 103-2628-M-001-002] and the Academia Sinica in Taiwan.
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