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Analyst View Article Online PAPER View Journal | View Issue Toward food analytics: fast estimation of lycopene and β-carotene content in tomatoes based on Cite this: Analyst, 2016, 141, 4447 surface enhanced Raman spectroscopy (SERS)† Andreea Ioana Radu,a,b Oleg Ryabchykov,a,b Thomas Wilhelm Bocklitz,a,b Uwe Huebner,b Karina Weber,a,b Dana Cialla-May*a,b and Jürgen Poppa,b Carotenoids are molecules that play important roles in both plant development and in the well-being of mammalian organisms. Therefore, various studies have been performed to characterize carotenoids’ pro- perties, distribution in nature and their health benefits upon ingestion. Nevertheless, there is a gap regard- ing a fast detection of them at the plant phase. Within this contribution we report the results obtained regarding the application of surface enhanced Raman spectroscopy (SERS) toward the differentiation of two carotenoid molecules (namely, lycopene and β-carotene) in tomato samples. To this end, an e-beam lithography (EBL) SERS-active substrate and a 488 nm excitation source were employed, and a relevant Creative Commons Attribution-NonCommercial 3.0 Unported Licence. simulated matrix was prepared (by mixing the two carotenoids in defined percentages) and measured. Received 16th February 2016, Next, carotenoids were extracted from tomato plants and measured as well. Finally, a combination of Accepted 12th May 2016 principal component analysis and partial least squares regression (PCA-PLSR) was applied to process the DOI: 10.1039/c6an00390g data, and the obtained results were compared with HPLC measurements of the same extracts. A good www.rsc.org/analyst agreement was obtained between the HPLC and the SERS results for most of the tomato samples. β-carotene absorption and conversion to retinol partially This article is licensed under a Introduction depends on the individual’s vitamin A availability. Moreover, For the last several centuries, the scientific focus has been according to different published statistics the necessary intake directed toward characterizing the functioning and necessities of vitamin A based on dietary sources of animal origin (e.g., – Open Access Article. Published on 12 May 2016. Downloaded 9/26/2021 9:55:03 PM. of the body. Moreover, currently, the role different molecules fatty fish, liver and eggs) is often not reached.9 11,13 Further have, their pathway upon ingestion and their daily intake on, out of the 6 carotenoids detectable in the human plasma, necessity is being documented. Among others, carotenoids lycopene was found to have the highest efficiency as an have attracted much attention because of their large antioxidant capable of neutralizing reactive oxygen species – bioavailability1 4 and their important roles in the mammalian (ROS)5,14,15 and reducing both cell-division at the G0–G1 cell – organism.5 10 Nevertheless, it has been found that only 50 out cycle phase and insulin-like growth of mitogens in various – of more than 600 different known carotenoids1 4 are actually cancer cell lines.5,16,17 There are, however, also negative effects present in the human diet, and only 5–6 of them are detectable of excessive carotenoid uptake in combination with smoking in human plasma (α- and β-carotene, β-cryptoxanthin, lyco- and alcohol drinking.9,18 It is, accordingly, important from a pene, lutein and zeaxanthin).5,11 The functions each of these health point of view to have a balanced dietary regime. Still, to carotenoids play in the body range from pro-vitamin A activity achieve such a regime, information regarding the quality and and antioxidant activity to radical scavenging. For instance, composition of the food is needed. all-trans-β-carotene is the only carotenoid capable of oxidative The golden standard in carotenoid analysis is high per- cleavage into two all-trans-retinal molecules, and this process formance liquid chromatography (HPLC).19,20 The drawbacks appears to have a feedback regulation property.2,8,11,12 That is, of this method are high costs and limited specificity (because of co-elution).21 Thus, there is potential for alternative analyti- cal methods. Among others, Raman spectroscopy and surface aFriedrich Schiller University Jena, Institute of Physical Chemistry and Abbe Center enhanced Raman spectroscopy (SERS) were tested for analyz- – of Photonics, Helmholtzweg 4, 07745 Jena, Germany ing mixtures of carotenoids in various matrices.22 26 However, b Leibniz Institute of Photonic Technology Jena, Albert-Einstein-Str. 9, 07745 Jena, previous Raman studies have failed to obtain sufficient differ- Germany. E-mail: [email protected] †Electronic supplementary information (ESI) available. See DOI: 10.1039/ entiation between the two carotenoids at lower concentrations, c6an00390g with HPLC coupled with UV-VIS or MS detection remaining This journal is © The Royal Society of Chemistry 2016 Analyst,2016,141, 4447–4455 | 4447 View Article Online Paper Analyst – the better option for reliable food analytics.27 29 Here, we (hexamethyldisilazane – HMDS) and a 260 nm thick positive present the first results of using SERS to differentiate between tone electron beam resist ‘AR6200.09’ (ALLRESIST GmbH) lycopene and β-carotene in tomatoes at different ripening were spun on the wafer. Further on, the resist was baked for stages. However, at this stage of the research, no advantage of 3 min at 150 °C on a hotplate, and a 10 nm gold layer was the potential the method has towards being non-invasive was evaporated on top of the resist. The electron beam exposure, applied. Instead, a significant, but small amount of the full- which was performed by using the unique character projec- food-batch production sample was taken apart, and an estab- tion-based electron beam technique,31 of the shaped beam lished extraction protocol was used to obtain analyte solutions writer SB350OS (from Vistec Electron Beam GmbH) resulted in that were measured by both SERS and HPLC. Regarding the the formation of 48 chips per wafer (5 × 10 mm2). Each of the SERS measurements, we designed a step-by-step experimental obtained chips contains 4 gratings with a size of 1 × 1 mm2 for procedure that reaches towards developing a possible protocol the SERS investigations. The exposure and the removing of the for analyzing carotenoids from tomatoes by employing a rela- gold layer were followed by the development of the resist in an tively simple spectroscopic technique in combination with a AR 600-546 developer for 60 s and the IPA rinsing for 30 s. statistical analytical tool. For this, the first step consisted of Next, the etching into the fused silica surface was performed the characterization of the pure carotenoids. Next, different with a CHF3–SF6–ICP etching process (Inductively Coupled mixtures of β-carotene/lycopene solutions were prepared and Plasma – ICP) by using an ICP power of 300 W. The etch depth measured to create a database and a statistical model that of the 2D gratings with a period of 436 nm is approximately could be used for analysis of food extract samples. The avail- 100 nm. Last, the residual resist was removed using an oxygen able literature providing information about the amounts of the plasma, and the wafer was separated into single chips. two carotenoids present in natural products was consulted to Silver films were deposited freshly (at the beginning of decide on the actual mixtures. Finally, a tomato extraction pro- every measurement day) by means of thermal evaporation at − tocol was applied, and the tomato-extracts were measured and an oil-free background pressure in the lower 10 7 mbar range. Creative Commons Attribution-NonCommercial 3.0 Unported Licence. analyzed by applying the already existing statistical model. To For this, the chips were mounted line of sight to the evapor- verify the results of the proposed SERS approach, all tomato ation boat to let the vapor strike the substrate normal to the samples were also measured by HPLC. surface. High-purity 99.999% silver granules were used as raw material. The thickness as well as the deposition rate was con- trolled in situ using a quartz microbalance. The thickness of Experimental the silver layer was 40 nm. A scanning electron microscopy (SEM) image of the measuring fields used throughout the Chemicals and reagents experiments is presented in Fig. 1. The image was obtained All reagents were of analytical or HPLC reagent grade. Lyco- using a JEOL JSM-6700F system. This article is licensed under a pene (≥90% pure), β-carotene (≥95% pure) and 2,6-di-tert- butyl-4-methylphenol (BHT, ≥99% pure) were purchased from Sample preparation Sigma Aldrich (Steinheim, Germany). Methanol (≥99.5% pure) For all of the experiments discussed in this study a mixed Open Access Article. Published on 12 May 2016. Downloaded 9/26/2021 9:55:03 PM. was purchased from Carl Roth (Karlsruhe, Germany). Tetra- solvent of methanol and THF stabilized with 0.1% BHT (1 : 1, hydrofuran (THF, ≥99.9% pure) was purchased from Merck v/v) was used. KGaA (Darmstadt, Germany). For the concentration dependent SERS measurements of Cherry tomatoes at different ripening stages were provided the two analytes, stock solutions of 106 μM β-carotene and by local producers from the area of Jena, Germany. First, a lycopene were prepared by dissolving the appropriate analyte series of tomatoes (series A) exhibiting different degrees of quantities in MeOH/THF. Measuring solutions were prepared ripeness (yellow to red) were taken from the same tomato by dilution in MeOH/THF immediately prior to use. Final con- plant. The tomatoes were immediately frozen and stored at centrations of 106, 90, 74, 58, 42, 26, 10, 9, 7.4, 5.8, 4.2, 2.6 −20 °C until analysis. A second series of four tomatoes (all of and 1 μM were obtained for both analytes.

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