Beta-carotene and lutein are common carotenoids found in plant pigments. Beta-carotene and lutein are highly reactive with singlet oxygen and free radical oxygen species; their mechanism of action is theorized to provide protection against oxidative species within the body [1]. Currently, serum antioxidant levels are detected with high performance liquid chromatography (HPLC); HPLC is operationally prohibitive due to its inability to handle high throughput volumes and high labor demands [2]. Other non-invasive methods such as Raman spectroscopy have been developed to detect antioxidants [3]. Reflection spectrometry based detection of carotenoid levels in skin has been shown to correlate with serum levels of carotenoids [4]. The Raman spectroscopy technique has also been used to detect macular antioxidants [5]. However, Raman spectroscopy is unable to distinguish between individual metabolites in either scan, eye, or serum samples. A novel electrochemical antioxidant test will provide fast, high throughput analytical measurement of carotenoids in serum capable of distinguishing specific carotenoids. Previously published work has shown that lutein is electrochemically active [1]. Furthermore, good calibration is shown at high concentrations (0.5-76 uM) of lutein with a limit of detection of about 0.1 uM in tetrahydrafuran and absolute ethanol solvent with a glassy carbon working electrode, a platinum counter electrode, and an AgNO3 working electrode [1]. We replicated these experiments with great accuracy, and observed two clear oxidation peaks at 338 mV and 412 mV. We expected that beta-carotene would display similar electrochemical properties as lutein due to their similar structure. A serial dilution, 1 uM to 250 uM of beta-carotene yielded a cyclic voltammogram with two clear oxidation peaks at 256 mV and 420 mV. To further improve these sensors, the glassy carbon electrode was modified with a covalently bound cyclodextrin through an electrochemically generated free-radical reaction (Hernandez et. al 2014). When compared to the unmodified electrodes, the cyclodextrin modified electrodes showed no improvement in sensitivity using cyclic voltammetry or differential pulse voltammetry. The lutein dilution showed only one peak, unlike the two peaks noted on the lutein serial dilution with the unmodified glassy carbon electrode. Furthermore the second beta-carotene peak was significantly shifted more positive (∆500 mV). Further, we used a methylene blue mediated cyclodextrin surface, where methylene blue was preloaded into the cyclodextrin and released due to the competitive affinity interaction of the carotenoids lutein and beta-carotene. We found that the experiments depended significantly on the solvents used and control experiments. The talk will focus on appropriate control experiments and choice of solvent for accurate results. In the end, we discovered the capability to measure small concentrations of carotenoids (on the order of nM) on cyclodextrin mediated surfaces. [1] Y. Yue, Q. Liang, Y. Liao, Y. Guo, and S. Shao, “Electrooxidation behavior and electrochemistry determination method of the xanthophylls: Lutein in nonaqueous media,” J. Electroanal. Chem., vol. 682, pp. 90–94, Aug. 2012. [2] M. Vogeser and C. Seger, “A decade of HPLC–MS/MS in the routine clinical laboratory — Goals for further developments,” Clin. Biochem., vol. 41, no. 9, pp. 649–662, Jun. 2008. [3] T. R. Hata, T. A. Scholz, L. K. Pershing, I. V. Ermakov, R. W. McClane, F. Khachik, and W. Gellermann, “Non-Invasive Raman Spectroscopic Detection of Carotenoids in Human Skin,” J. Invest. Dermatol., vol. 115, no. 3, pp. 441–448, Sep. 2000. [4] W. Stahl, U. Heinrich, H. Jungmann, J. von Laar, M. Schietzel, H. Sies, and H. Tronnier, “Increased dermal carotenoid levels assessed by noninvasive reflection spectrophotometry correlate with serum levels in women ingesting Betatene.,” J. Nutr., vol. 128, no. 5, pp. 903–7, May 1998. [5] P. S. Bernstein, D.-Y. Zhao, S. W. Wintch, I. V Ermakov, R. W. McClane, and W. Gellermann, “Resonance Raman measurement of macular carotenoids in normal subjects and in age-related macular degeneration patients,” Ophthalmology, vol. 109, no. 10, pp. 1780–1787, Oct. 2002.
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Jul 1, 2023
Alisha Redelfs + 7
Open Access
