Label-free electrochemical sensor to investigate the effect of tocopherol on generation of superoxide ions following UV irradiation

Gao et al. Journal of Biological Engineering (2018) 12:17 https://doi.org/10.1186/s13036-018-0099-2 METHODOLOGY Open Access Label-free electrochemical sensor to investigate the effect of tocopherol on generation of superoxide ions following UV irradiation Li Xia Gao1,2, Chunxiang Bian1, Yan Wu1, Muhammad Farrukh Nisar1,4, Shida Chen1, Chang Ming Li2, Ling Yu2, Ping Ji3, Enyi Huang3* and Julia Li Zhong1,3* Abstract Background: Generation of reactive oxygen species (ROS), triggered by ultraviolet radiation (UVR), is associated with carcinogenesis of the skin. UV irradiation induced superoxide anion (O2•−) is the key ROS involved in the cellular damage. The cytoprotective efficacy of an unknown anti-oxidant compound can be evaluated by analyzing the production of O2•− from treated cells. Methods: In this study, a glass carbon electrode functionalized with nanotube@DNA-Mn3(PO4)2 composite was applied to quantitative determination of generation of highly unstable O2•− from the melanoma A375 cell line following UVR(UV, UVA and UVB). In addition, the cytoprotective efficacy of anti-oxidant α-tocopherol was evaluated by quantifying the production of O2•−. Results: The results showed that, UVR triggers generation of O2•− in melanoma A375 cells, and α-tocopherol is effective in diminishing the production of O2•− following UV irradiation. By comparing the conventional cell-survival assays results, we found that our simple and quick electrochemical sensing method can quantify O2•− generation through the biological activity of an anti-oxidant compound (α-tocopherol). Conclusion: Our label-free electrochemical quantification method for ROS (O2•− major) in cells facing UVR stress demonstrates its potential application for high-throughput screening of anti-oxidation compounds. Keywords: Skin, Electrochemical sensing, O2•−/ROS, UVR, Anti-oxidant screening
Label-free Electrochemical Sensor was developed to Highlighting points quantify ROS produced in cells exposed to UVR.
A carbon nanotube@DNA- Mn3(PO4)2compositefunctionalized glass carbon electrode was applied for quantitative determination of O2•−generation from melanoma cell A375 following UV, UVA, UVB irradiation. * Correspondence: ; LiXia Gao and Chunxiang Bian are co first-authors. LiXia Gao and Chunxiang Bian contributed equally. 3 Chongqing Municipal Key laboratory of oral diseases and biomedical sciences, Biomedical Engineering of Higher Education, Chongqing 401147, China 1 College of Bioengineering & School of Life Sciences, Chongqing University, Chongqing 400044, China Full list of author information is available at the end of the article
The anti-oxidation efficacy of tocopherol on melan- oma cells towards UV, UVA and UVB were also investigated. Background Ultraviolet (UV) irradiation represents one of the most important environmental impacts for humans and recently became prominent because of the depletion of the atmospheric ozone layer, leads to increased UV irradiation exposure by the majority of population [1, 2]. It is well documented that UVR can stimulate the production of a series of ROS [3–5], which may cause cellular oxidative stress injury that is believed to be one of the key © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gao et al. Journal of Biological Engineering (2018) 12:17 factors in carcinogenesis [6, 7]. For the most part, UV light from sunlight consists of three regions of wavelengths: UVC, UVB and UVA. UVC (100–290 nm) is absorbed by ozone (O3) in the upper atmosphere but UVB and UVA reach to earth surface, are the major fractions linked to skin diseases. UVB (290–320 nm) is absorbed mostly by the epidermis and keratinocyte DNA, while UVA (320-400 nm) is primarily oxidative in nature and penetrates more deeply into the dermal layers of the skin [8, 9]. Since UV fractions reach different biological layers of skin and lead to skin pathology via different cellular pathways. The effects and mechanisms of action initiated by UVA and UVB have been studied extensively [10–12]. For instance, Petersen et al. reported that superoxide anion (O2•−) production in HaCaT cells was probably linked to DNA damage induced by UVA [13]. UVB can also induce the formation of ROS, leading to cellular damage [14–16]. Among the many ROS species that have been studied, O2•− is one of the principal radical species [13, 17–19]. It is generated as a reduced intermediate of molecular oxygen in a variety of biological systems. It can easily form hydroxyl radical (HO•) in the presence of transition metal ions such as Fe2+ and Cu2+ [20]. In addition, the reaction between O2•−and nitric oxide (•NO) leads to the formation of highly reactive peroxynitrite (ONOO-) in the pathogenesis of atherosclerosis and neurodegenerative diseases [21]. As a consequence, efforts have been made to investigate generation of O2•− induced by UVR. Particularly, the cytoprotective efficacy of an unknown anti-oxidant compound can be evaluated by analyzing the production of O2•− from treated cells. A survey of the literature shows that the main techniques for measuring O2•− are based on probe-labelling assays. Intracellular fluorescent histochemistry [22], flow cytometry [16] and spectrofluorometric analyses [23, 24] are the most used approaches to characterize ROS such as O2•− by using fluorescent dyes such as 2′,7′dichlorofluorescein diacetate (DCFH-DA), hydroethidine and dihydrorhodamine [1, 23]. The production of extracellular O2•− can also be measured by using the ability of O2•− to reduce ferricytochrome C that was added to the cell suspension [25]. Another advanced technique is electron spin resonance (ESR)-spin trapping, was applied for determination of O2•− generated by UV-irradiated skin cells [10, 24]. Apart from the expensive equipment and complicated assay procedures, these probe-labelling approaches are timeconsuming, difficult to automate and highly prone to interference. The short lifetime of free radicals, such as O2•−, particularly demands fast response of the analytical tool to the changes in concentration to obtain sufficient signal-to-noise ratios [20, 26]. Electrochemical Page 2 of 10 biosensors have become promising candidates for realtime analysis of free radicals, since they provide the advantages of rather simpler equipment and operation protocols. Li et al. [20] found that an electrochemical biosensor can sense O2•− released from cancer cells, using potassium-do (...truncated)