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

Journal of Biological Engineering volume 12, Article number: 17 (2018) Cite this article

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.

Highlighting points

  • 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.

  • Label-free Electrochemical Sensor was developed to quantify ROS produced in cells exposed to UVR.

  • The anti-oxidation efficacy of tocopherol on melanoma 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 [3,4,5], which may cause cellular oxidative stress injury that is believed to be one of the key 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 [10,11,12]. 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 [14,15,16].Among the many ROS species that have been studied, O2•− is one of the principal radical species [13, 17,18,19]. 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 time-consuming, 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 biosensors have become promising candidates for real-time 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-doped multi-walled carbon nanotubes (KMWNTs)-1-butyl-3-methylimidazoliumhexa-fluorophosphate ionic liquid composite gels [20]. While hydrogen peroxide (H2O2) can directly be measured in cells growing in a 3D matrix environment [27]. These studies indicate that, with intimately coupled biological recognition elements and electrochemical transduction units, an electrochemical biosensor exhibits great potential for facilitating understanding of biological process. Also, the use of a small-volume sample allows expensive reagents, particular for rare clinical biopsy samples, to be conserved and makes the analysis more cost-effective.

In this study, we have designed and developed a label-free electrochemical sensor that can measure the generation of O2•− from UVR exposed melanoma A375 cells, though other ROS species e.g. H2O2 has not been totally excluded. To demonstrate the electrochemical analytical power of this sensor, the protective effects of model anti-oxidant (α-tocopherol) was studied in melanoma A375 cells. The specific objectives to be addressed were [1] to measure generation of O2•− from melanoma A375 cells following exposure to UVR alone i.e., UV, UVA and UVB; [2] to quantify the production of O2•− from cells pre-treated with α-tocopherol following UVR irradiation; [3] to compare the data from electrochemical measurement, the cell-survival assay and conventional ROS fluorescence staining therefore establish the potential use of the label-free electrochemical method for high-throughput screening of antioxidants.

Methods

Materials

The human melanoma cell A375 (purchased from ATCC) were maintained in RPMI 1640 medium (Gibco®) supplemented with 10% foetal calf serum (FCS, Gibco®) with 100 U mL-1 penicillin and 100 U mL-1 streptomycin at 37 °C in a humidified 5%CO2 incubator. α-Tocopherol was purchased from Sigma-Aldrich. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 2-(4-amidino¬phenyl)-6-indole carbamidine dihydrochloride (DAPI), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and superoxide dismutase (SOD) were purchased from Beyotime Biotechnology (Beijing, China). All other chemicals were purchased from Sigma-Aldrich and were used without further purification, unless otherwise indicated. All solutions were prepared with deionized (DI) water produced by a PURELAB flex system (ELGA Corporation).

Apparatus

UV irradiation

Cells were irradiated with a broad-spectrum UV lamp (MUA-165, Japan). The proportion of UVA and UVB in delivered UV light from this lamp is around 20:1, a ratio close to solar light at mid-day [28]. The lamp exposure time was calculated using an ultraviolet radiometer (Photoelectric Instrument Factory, Beijing Normal University, China). Appropriate glass filters to block UVA, UVB were used respectively to provide fractions of UVA and UVB in the following experiments, according to previous studies [29,30,31,32,33].

Electrochemical detection setup

The experimental setup is shown in Fig. 1. A three-electrode system was employed, with a nano material-functionalised glass carbon electrode (GCE) as a working electrode (WE), an Hg/HgCl2/KCl electrode as a reference electrode (RE) and a platinum wire as a counter-electrode (CE). To avoid interference from direct UV irradiation of the electrodes, the electrodes were assembled on the side-wall of the detection well. In addition, a piece of aluminium foil with a hole was placed on top of the detection well to confine the UV light to impacting only on the cells. Electrochemical detection was carried out with an electrochemical station (CHI 760e, Chen Hua Instruments Co. Ltd., China).

Fig. 1
figure 1

Electrochemical sensor for quantifying generation of superoxide anion (O2•−) from cells during UV irradiation. UV: ultraviolet; RE: reference electrode; CE: counter electrodes; WE: working electrode

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Electrochemical detection of O2 •− released by the cells

Preparation of an O2 •− electrochemical sensor

The construction of electrochemical sensor for O2•− detection was detailed in our recent paper [34, 35]. In brief, single stranded DNA (2.1 mg) was added to MnSO4 (1.0 mL, 0.10 M) under constant stirring at 60°Cstirring until the solution became transparent. The pellet of DNA-Mn3(PO4)2 composite was recovered by centrifugation. To prepare the O2•− sensing electrode, carbon nanotube (CNT, 0.5 mg mL− 1) and DNA- DNA-Mn3(PO4)2 (0.20 M) were drop-casted on a polished glass-carbon electrode. To detect O2•−, electrodes modified with CNT@DNA-Mn3(PO4)2 composite were exposed to aliquots of KO2 and cyclic voltammetry curves were recorded. The peak current value at 0.70 V was recorded and was plotted against the concentration of KO2to draw a current-dose calibration curve.

In situ detection of production of O2 •− from UV-irradiated cells

Confluent A375 cells were seeded into 24 cell plates, at a density of 1 × 105 per well, and were incubated for 6 h. Then α-tocopherol (25 μM was chosen due to optimize concentration [33]) was added into cells and incubated for another 12 h. The cell-culture disc with adherent cells was transferred to the electrochemical detection cell (Fig. 1), to which serum free medium (2.5 mL) was added. Then the cells were exposed to UVR and the electrochemical signal arising from O2•− were recorded by CHI. Cells without anti-oxidant were measured with the same schedule as controls.

Fluorescent staining of oxygen radicals

Intracellular ROS

A375 cells were collected and seeded at a density of 1 × 105 per well in 24-well plates and were incubated overnight with or without α-tocopherol. Intracellular generation of ROS upon UV irradiation was determined with a DCFH-DA (10 mM) assay kit (Beyotime Inc. S-0033). In brief, following UVA irradiation, cells are washed twice in phosphate buffer saline (PBS) and allowed to incubate in 10 μM/L DCFH-DA (1:1000 dilution in PBS) at 37 °C for 20 min according to the manufacturer’s instructions (Beyotime Inc.). The excess DCFH-DA was rinsed off with phosphate buffer saline (PBS) (1.0 mM). The cells were then observed and imaged under a fluorescence microscope (IX-71, Olympus Corp., Tokyo, Japan).

O2 •−colorimetric assay kit

Superoxide assay kit (S-0060 Beyotime Biotechnology Beijing Inc.) was conducted following the product instructions. In brief, A375 cells were seeded (1 × 104) in 96-well plates and 80–90% confluence when treated. Now the cells were washed three times in PBS, and allowed to incubate in 10 μmol/L DCFH-DA (1:1000 dilution) at 37 °C for 3 min (Beyotime Biotechnology, Beijing Inc.). The excess DCFH-DA was rinsed off with PBS (1.0 mM). Now the cells were irradiated with respective UVR (UV, UVA or UVB) stimulus. Prepare the three wells without irradiation should be used as a blank control. Select 2 wells to add 2% SOD (Beyotime Biotechnology, Beijing Inc.) in the wells of the stimulus to verify the entire assay system. The cells were then observed and imaged under a fluorescence microscope (IX-71, Olympus Corp., Tokyo, Japan).The absorbance values taken at 450 nm were compared with the reference absorbance values at 630 nm following to manufacturer’s instructions. These data were expressed as the percentage of increase of absorbance value compared to sham controls (sham-irradiated groups).

Cell viability assay

The protective effects of α-tocopherol on UV-irradiated melanoma cell A375 were evaluated using the MTT assay. In brief, cells were seeded at a density of 1 × 104 per well in 96-well plates and incubated with or without the α-tocopherol for 12 h. Following challenge with UVR, the cells were cultured in conditional culture medium for a further 24 h. Each well then received 10 μL of MTT solution (0.5 mg mL− 1) and were incubated for 3–4 h at 37 °C. Then 100μLlysisbuffer (mixture of 2-propanol, Triton-100 and hydrochloric acid) was added to each well to dissolve the purple-coloured formazan produced by viable cells, before colorimetric analysis. The absorbance at 570 nm was measured by a microplate reader and the results were expressed as the percentage of surviving cells compared to the sham-irradiated vehicle control group.

Statistical analysis

Results are expressed as means±standard error of the mean (SEM). The data were analysed by Student’s t-test using Origin Statistic software (Origin Lab Corporation, USA). P < 0.05 was considered significant. All experiments were performed three independent times in triplicates.

Results and discussion

Quantification of UV-inducedO2 •− in melanoma cells by label-free electrochemical sensor

To evaluate the feasibility of using an electrochemical method to quantify production of O2•− induced by UV irradiation from melanoma cells, an electrochemical sensor was fabricated and characterised. Initially, cyclic voltammetry (CV) was performed with PBS to characterize the CNT@DNA-Mn3(PO4)2 functionalised electrochemical sensor. KO2 was used as the source of O2•− as reported in literature [19]. The peak current of the oxidative state/stress at 0.7 V increases when KO2 was added to PBS (blue line inset of Fig. 2a). The production of superoxide anions (O2•−) is linked with diverse range of biochemical phenomenon leading to various oxidative stress mediated pathologies. The electrochemical determination of superoxide ion is solely depending on SOD enzyme, hence check it’s reproducibility as well as not a cost effective, and advancement in nanosciences solved certain limitations with this procedure. Previously we demonstrated that Mn3(PO4)2 nanosheets which are a biomimetic enzyme were template-synthesized with DNA and further assembled on carbon nanotubes (CNTs) that form nanocomposite sheets (DNA-Mn3(PO4)2-CNT) [34]. The dismutation of superoxide ion was catalyzed by Mn3(PO4)2 sheets, but the CNTs allows quick shifts of electrons to maximize its sensitivity, specificity and reproducibility for O2•− detection [34].

Fig. 2
figure 2

Calibration of CNT@DNA-Mn3(PO4)2 modified glass-carbon electrode for measurement of O2•− and electrochemical characterization of O2•− released by cells following UVR irradiation. a Cyclic voltammetry (CV) curves measured with PBS and PBS plus different concentrations of KO2 (nM). a: 0, b: 5, c: 10, d: 20, e: 40, f: 60, g: 80, h: 100, i: 150, j: 200; inset: CV curves measured with PBS (black), PBS plus KO2 (150 nM, blue), and KO2 solution added with SOD (300 U mL− 1, red). b Calibration curve for serial concentrations of O2•−. c O2•− CV curves of cells under irradiation, a. UV, b. UVA, c. UVB, d. Sham-irradiation control. d Histogram of peak current changes compared to sham-irradiated cells (black column, n = 3) and generation of O2•− upon irradiation with UV as quantified by the O2•−assay kit: increase of absorbance at 450 nm (grey column, n = 3). SOD: superoxide dismutase. UV (10.5 J cm− 2 = 105 kJ m− 2), UVA (10 J cm− 2 = 100 kJ m− 2), UVB (0.5 J cm− 2 = 5 kJ m− 2)

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The increased peak current gradually decreased to PBS level after the introduction of SOD to decompose/neutralize O2•− (red line inset of Fig. 2a). Different concentrations of KO2 were added to vary the amount of O2•− and the peak current at 0.7 V was recorded that was plotted against the concentration of O2•−. The corresponding calibration curve of current response vs. O2•− concentration showed a sensitivity of 70 nA nM-1 (Fig. 2b).The CV curves showed that, the change in current (μA) is associated with changing in the production of O2•− at different UV treatments (UV, UVA, UVB) and in sham-irradiated control (Fig. 2c). Furthermore, to verify the electrochemical-sensing efficacy, the O2•− assay kit was used to measure O2•− generated from melanoma cells challenged with UVR; a similar increase was observed in both methods. Incubation of cells with SOD eliminatesincreasedO2•− in the culture medium, while the electrochemical sensing measures a similar pattern of generation of UVR-induced O2•−as did conventional fluorescent staining for O2•− (Fig. 2d), the former with less consumption of sample and shorter experimental time. These suggested that, the electrochemical sensor provides a possibility of recording production of O2•− in groups of A-375 cells facing different doses of UVR, without disturbing the cellular metabolism.

Measurement of UV-irradiation induced O2 •− and its diminution with α-tocopherol by using label-free electrochemical sensor

To demonstrate the feasibility of electrochemical sensor as an analytical tool to evaluate the anti-oxidative capability of compounds, we pre-treated UVR-challenged cells with an antioxidant compound (α-tocopherol). The electrochemical sensor was used for direct measurement of O2•− generated from A375 cells pre-treated with α-tocopherol followed UV irradiation. In addition free-radical-scavenging effects of the antioxidant α-tocopherol in A375 cells given UVA, UVB and bulk UV were also investigated with the same electrochemical sensor. For this purpose, the normalized current change at 0.7 V was calculated in sham-irradiated cells as a control by using following equation.

$$ \boldsymbol{\Delta}\ \mathbf{C}\mathbf{urrent}\%=\left[\left(\mathbf{Ci}-\mathbf{C}\mathbf{0}\right)/\mathbf{C}\mathbf{0}\right]\times \mathbf{100}\% $$
(1)

Where Ci is current value measured upon UV irradiation, C0 is current value of sham control (sham-irradiated).

The Δ Current % was plotted against irradiation energy dose of UV fractions. A higher change in current indicated a higher generation of O2•−when compared with sham-irradiated control. The changes of production of O2•− in melanoma A375 cells irradiated with UVs (UV, UVA, UVB) was comparatively measured by the electrochemical sensor (Fig. 3a-c). Different UV doses (0–105 kJ m− 2) induced the production of O2•−, that altered the currents by 13.1% (50 kJ m− 2) to that of the sham. Tocopherol (25 μM) treatment to UV-challenged cells reduces the production of O2•− to 8.2% (50.25 kJ m− 2) when compared to UVs-irradiation only (Fig. 3a). Similarly, pre-treatment of cells with tocopherol efficiently decreased (8.5%) the production of O2•− induced by a UVA (100 kJ m− 2) dose to 1.6% compared to sham (Fig. 3b). In contrast, pre-treatment of cells with α-tocopherol followed by UVB irradiation, showed little effects on the production of O2•− (Fig. 3c).

Fig. 3
figure 3

a-c Extracellular O2•− quantified by electrochemical method in melanoma A375 cells. A375 cells were exposed to UV (A), UVA (b) and UVB (c); Non-pretreatment: black line; α-tocopherol pre-treatment: blue line. O2•− generation during post-UVs irradiation quantified by electrochemical sensing: non-treatment (d) α-tocopherol pretreatment (e) (n = 3),* denotes P < 0.05,** denotes P < 0.01

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The electrochemical sensor is not only able to quantify the generation of O2•− at acute phase following UV-irradiation, but also enabled quantification at post-UVR challenge period. After 60 min. Following UVR, in melanoma A375 cells O2•− generated current was decreased to half (8.5%) than at zero time point (17%), while at 120 min. The change in electric current faded to the background level (Fig. 3d).

The effect of UV on O2•− production are not the sum of the total effects of UVA and UVB, the production of O2•− from cells challenged by various UV doses (0-105 kJ m− 2, Fig. 3a) is lower than the sum of changes induced by UVA (Fig. 3b) plus UVB (Fig. 3c). Next, we found that α-tocopherol plays a distinct role and lessen the generation of O2•− in pre-treated A375 cells followed by UV irradiation, however, UVA and UVB showed different capacities to trigger the generation of O2•−in melanoma A-375 cells.

The pre-treatment of α-tocopherol reduced the UVA and UVB induced O2•− generation by almost 85% and 20%, respectively (Fig. 3b,c). This difference may due to the generation of H2O2, or interference of some other unknown reason yet, hence need further optimisation of the method. Furthermore, antioxidant tocopherol showed different behaviour against UVA and UVB induced O2•− is due to the difference in wavelength energy. In addition to variable UV wavelength effects, antioxidants are distinct chemical units with different modes of action for their effects and possess a unique biochemical profile hence making it different from other related compounds. Based on the results of electrochemical sensor, we speculated that α-tocopherol showed different protective properties in melanoma cells exposed to different UV wavelengths. In general, the electrochemical sensor is able to characterize the O2•− production, as cellular oxidative status is one of the indicators.

Prediction of protective effect of α-tocopherol on cell survival through electrochemical sensing

To analyse the effect of α-tocopherol on cell survival following UVR, A375 cells were exposed to different doses of UV, UVA and UVB, and the anti-oxidant effect of α-tocopherol was assessed by calculating the cell survival rate by using following equation.

$$ \mathrm{Cell}\ \mathrm{survival}\%=\left[\left({\mathrm{A}}_{\mathrm{T}}\hbox{--} \mathrm{Ac}\right)/\mathrm{Ac}\right]\times 100\% $$
(2)

Where AT is the absorbance value of UV-irradiated cells and Ac is absorbance value of sham-irradiated cells. This calculation, gives survival of cells under UV irradiation is normalized to the sham or control cells. The α-tocopherol had significant cytoprotective effects on melanoma A375 cells, and it enhanced cell survival (18%) following UV light exposure (50.25 kJ m− 2 = UVA 50 kJ m− 2 + UVB 0.25 kJ m− 2) (Fig. 4a). During studies of UVA-induced cell damage, α-tocopherol appeared to be more cytoprotective as indicated by the survival rates of pre-treated (92.3%) compared with control melanoma A375 cells (80%), with 10% marked and significant difference in reduction of cell survival (Fig. 4b). On the other hand, UVB induced cell viability loss (72%) was significantly repaired by α-tocopherol pre-treatment (84%) compared with control, with 12% of protection revival (Fig. 4c).

Fig. 4
figure 4

Survival of cells pre-treated by α-tocopherol under UV irradiation with melanoma A375 cells. These survival rates were calculated using sham-irradiated cell as reference, (n = 3). (a) UV dose (50 kJ m−2) decreases cell survival to 68.8% with α-tocopherol recurrence of 86.5% cell viability. (b) Equivalent UVA dose (100 kJ m−2) gave cell viability loss to 80.6% which is recovered by α-tocopherol to 92.3% cell viability. (c) Finally the equivalent UVB dose (2.5 kJ m−2) given cell viability of 72.1% that finally recovered by α-tocopherol to 84.2% viable cells. Non-pretreatment: black line; α-tocopherol pre-treatment: blue line

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Collectively, MTT cell-survival assay showed that α-tocopherol is effective in diminishing UV, UVA and UVB-induced damage in melanoma A375 cells, this is similar to results given by electrochemical sensing and colorimetric assay for O2•− levels (Fig. 3a-e). Interpreting the survival rate (Fig. 4a-c) of cells pre-treated with α-tocopherol followed by UV irradiation caused the generation of O2•− (Fig. 3a-c) may efficiently harbour the oxidative stress and lessen it promptly. The different cytoprotective efficacy of α-tocopherol against UVA- or UVB-induced cell death was decreased, thus could be specified as the distinct capability of α-tocopherol to eliminate UV fraction induced oxidative stress, since UVA is the major oxidative stress causing wavelength in UV.

Comparison of methodologies towards UV-induced cellular oxidative stress

The MTT assay was conducted to evaluate the loss of viability induced by UV irradiation. It monitors the overall cell growth over a period of time, thus showing its strength in evaluating the long-term effect of a treatment on cells [34, 35]. A large body of work, spanning more than four decades, demonstrates fluorescent probe labelling for characterisation of changes of intracellular ROS [36, 37].

However, it is also clear that this probe-labelling method measures transient fluxes of oxidants only with great difficulty [38, 39]. Most importantly, the labelling of cells challenges the possibility to characterize the changes in situ of cells during a time period in responding to a treatment. The merits of label-free electrochemical sensing have been discussed in the literature [27, 34, 40] (Table 1). To confirm the feasibility of electrochemical sensing as an analytical tool to determine the anti-oxidative capability of unknown compounds, we performed conventional O2•− fluorescent staining with a standard DCFH-DA fluorescent staining protocol that measures intracellular ROS from cells after exposure to UV light. It is clear from the results that UV irradiation can induce an increase of the intensity of the signal for green fluorescence. Figure 5a presents the quantitative changes of ROS (O2•−) measured by fluorescent staining of three independent methods. The fluorescence signal is gradually increased in response to increasing UV dose and the maximum signal is given by cells irradiated with 105 kJ m− 2 UV light. Increases of intracellular ROS following UV irradiation visualized in this study are well in line with previously reported studies [17, 23] and our present electrochemical sensing results. Further increase of UV energy (21 J cm− 2 = 210 kJ m− 2) does not give further increased ROS fluorescence signal, perhaps due to significant cell death (Data no shown).

Table 1 Comparison of methods used to study effect of UV irradiation induced oxidative stress
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Fig. 5
figure 5

Fluorescent characterisation of intracellular ROS production and DNA damage in UV irradiated A375 cells. a Fluorescent microscopy images of DCFH-DA staining and histogram of fluorescent intensity of DCFH-DA staining quantified by Image J, n = 3. b Generation of ROS (O2•− major) from A375 cells quantified O2•− colorimetric assay kit, n = 3

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The protective efficacy of anti-oxidant α-tocopherol was assessed and much attention was paid to the UV dose of 105 kJ m− 2, i.e. [UVA (10 J cm− 2) + UVB (0.5 J cm− 2)]. The O2•− assay kit was used to measure the colorimetric changes induced by O2•− in a 96-well plate experimental setting. The results showed that the anti-oxidant can reduce the generation of O2•− induced by UV in A375 cells. Different anti-oxidation efficacies of the model anti-oxidant are observed in melanoma A375 cells against UVA and UVB exposure (Fig. 5b). It is encouraging to see that standard labelling techniques and electrochemical quantification depict a response pattern of UV irradiation and the anti-oxidant effects of α-tocopherol which are well correlated. In addition, electrochemical sensing revealed the oxidation status, which can be used to predict UV-induced ROS efflux and ultimately cell damage. The fast and easy experimental procedures permitted us to test the efficacy of anti-oxidant α-tocopherol against UVA and UVB in melanoma cells [33, 40]. We demonstrate the potential of electrochemical methods for high throughput screening of potential anti-oxidant compounds, although further improvements in the signal-to-noise ratio are needed. We believe that electrochemical sensing would be a potent and economical candidate to provide meaningful complementary information for standard fluorescent staining experiments.

Conclusions

The results showed, in melanoma A375 cells electrochemical sensing both UVA and UVB -induced production of ROS e.g. O2•−. The potential use of in UV (UV, UVA, UVB) involved biological research is enhanced, since the electrochemical sensor quantified O2•− levels in medium could be correlated with the cell survival ability under the protection of anti-oxidant α-tocopherol as well. The electrochemical changes occurred with O2•− production, can be seen with this label-free method correlated with intracellular fluorescent staining as well. It highlighted the potential of the electrochemical method for high-throughput screening of anti-oxidant under UV irradiation, and for investigation of their related mechanisms.

Abbreviations

DCFH-DA:

2′,7′-dichlorodihydrofluorescein diacetate

H2O2 :

Hydrogen peroxide

kJ m− 2 :

Kilo Jouls per square meter

MTT:

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

O2•− :

Superoxide anion

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

UVA:

Ultraviolet A

UVB:

Ultraviolet B

UVR:

Ultraviolet radiation

References

  1. Tyrrell RM. Modulation of gene expression by the oxidative stress generated in human skin cells by UVA radiation and the restoration of redox homeostasis. Photochem Photobiol Sci. 2012;11:135–47.

    Article  Google Scholar 

  2. Godar DEUV. doses worldwide. Photochem Photobiol. 2005;81:736–49.

    Article  Google Scholar 

  3. Liebel F, Kaur S, Ruvolo E, Kollias N, Southall MD. Irradiation of skin with visible light induces reactive oxygen species andmatrix-degrading enzymes. J Invest Dermatol. 2012;132:1901–7.

    Article  Google Scholar 

  4. Armstrong BK, Kricker A. The epidemiology of UV induced skincancer. J Photochem Photobiol B. 2001;63:8–18.

    Article  Google Scholar 

  5. Dizdaroglu M, Jaruga P, Birincioglu M, Rodriguez H. Free radical-induced damage to DNA: mechanisms and measurementFree. RadBiol Med. 2002;32:1102–15.

    Google Scholar 

  6. Zhang Y, Mattjus P, Schmid PC, Dong Z, Zhong S, Ma WY, Brown RE, Bode AM, Schmid HH, Dong Z. Involvement of the acid sphingomyelinasepathway in UVA-induced apoptosis. J Biol Chem. 2001;276:11775–82.

    Article  Google Scholar 

  7. Berwick M, Lachiewicz A, Pestak C, Thomas N. Solar UV exposure and mortality from skin tumors. Advanc Experiment Med Biol. 2008;624:117–24.

    Article  Google Scholar 

  8. Tyrrell RM, Pidoux M. Singlet oxygen involvement in the inactivation of cultured human fibroblasts by UVA (334 nm, 365 nm) and near-visible (405 nm) radiations. Photochem Photobiol. 1998;49:407–12.

    Article  Google Scholar 

  9. Duthie MS, Kimber I, Dearman RJ, Norval M. Differential effects of UVA1 and UVB radiation on Langerhans cell migration in mice. J Photochem Photobiol B. 2000;57:123–31.

    Article  Google Scholar 

  10. Herrling T, Jung K, Fuchs J. Measurements of UV-generated free radicals/reactive oxygen species (ROS) in skin. Spectrochim Acta Part A. 2006;63:840–5.

    Article  Google Scholar 

  11. Krutmann J. The interaction of UVA and UVB wavebands with particular emphasis on signalling. Prog Biophys Mol Biol. 2006;92:105–7.

    Article  Google Scholar 

  12. Syed DN, Afaq F, Mukhtar H. Differential activation of signalling pathways by UVA and UVB radiation in normal human epidermalkeratinocytes. Photochem Photobiol. 2012;88:1184–90.

    Article  Google Scholar 

  13. Petersen AB, Gniadecki R, Vicanova J, Thorn T, Wulf HC. Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes. J Photochem Photobiol B. 2000;59:123–31.

    Article  Google Scholar 

  14. Wolfle U, EsserPR S-HB, Martin SF, Lademann J, Schempp CM. UVB-induced DNA damage, generation of reactive oxygen species, and inflammation are effectively attenuated by the flavonoid luteolinin vitro and in vivo. Free Rad Biol Med. 2011;50:1081–93.

    Article  Google Scholar 

  15. Yasui H, Hakozaki T, Date A, Yoshii T, Sakurai H. Real-time chemiluminescent imaging and detection of reactive oxygen species generated in the UVB-exposed human skin equivalent model. Biochem Biophys Res Comm. 2006;347:83–8.

    Article  Google Scholar 

  16. Peus D, Vasa RA, Meves A, Pott M, Beyerle A, Squillace K, Pittelkow MR. H2O2 is an important mediator of UVB-inducedEGF-receptor phosphorylation in cultured keratinocytes. J Invest Dermat. 1998;110:966–71.

    Article  Google Scholar 

  17. Rezvani HR, Mazurier F, Cario-Andre M, Pain C, Ged C, Taieb A, de Verneuil H. Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes. J Biolog Chem. 2006;281:17999–8007.

    Article  Google Scholar 

  18. Fridovich I. Superoxide anion radical (O2 .-), superoxide dismutases, and relatedmatters. J Biol Chem. 1997;272:18515–7.

    Article  Google Scholar 

  19. Komarov DA, Slepneva IA, Glupov VV, Khramtsov VV. Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase. Free Rad Res. 2005;39:853–8.

    Article  Google Scholar 

  20. Li XR, Wang B, Xu JJ, Chen HY. In vitro detection of superoxide anions released from cancer cells based on potassium-dopedcarbon nanotubes ionicliquid composite gels. Nanoscale. 2011;3:5026–33.

    Article  Google Scholar 

  21. Suschek CV, Mahotka C, Schnorr O, Kolb-BachofenV. UVB radiation-mediated expression of inducible nitric oxide synthase activity and the augmenting role of co-induced TNF-alpha in human skin endothelial cells. J Invest Dermat. 2004;123:950–7.

    Article  Google Scholar 

  22. Lee ER, Kim JH, Kang YJ, Cho SG. The anti-apoptotic and anti-oxidant effect of eriodictyol on UV-induced apoptosis in keratinocytes. Biolog Pharm Bull. 2007;30:32–7.

    Article  Google Scholar 

  23. Tada M, Kohno M, Niwano Y. Scavenging or quenching effect of melanin on superoxide anion and singlet oxygen. J ClinBiochemNutrit. 2010;46:224–8.

    Google Scholar 

  24. Grange PA, Chereau C, Raingeaud J, Nicco C, Weill B, Dupin N, Batteux F. Production of superoxide anions by keratinocytes initiates P acnes-induced inflammation of the skin. PLoS Pathog. 2009;5:e1000527.

    Article  Google Scholar 

  25. Korshunov S, Imlay JA. Detection and quantification of superoxide formed within the periplasm of Escherichia coli. J Bacter. 2006;188:6326–34.

    Article  Google Scholar 

  26. Herrling T, Zastrow L, Fuchs J, Groth N. Electron spin resonance detection of UVA-induced free radicals. Skin Pharm ApplSkin Physiol. 2002;15:381–3.

    Article  Google Scholar 

  27. Shi ZZ, Wu XS, Gao LX, Tian YL, Yu L. Electrodes/paper sandwich devices for in situ sensing of hydrogen peroxide secretion from cells growing in gels-in-paper 3 dimensional matrix. Analyt Methods. 2014;6:4446–54.

    Article  Google Scholar 

  28. Balasaraswathy P, Kumar U, Srinivas CR, Nair S. UVA and UVB in sunlight, optimal utilization of UV rays in sunlight for phototherapy. Ind J Dermat, Vener Leprol. 2002;68:198–201.

    Google Scholar 

  29. Zhong JL, Yang L, Lu F, Xiao H, Xu R, Wang L, Zhu F, Zhang Y. UVA, UVB and UVC induce differential response signalling pathways converged on the eIF2α phosphorylation. Photochem Photobiol. 2011;87:1092–104.

    Article  Google Scholar 

  30. Tian FF, Zhang FF, Lai XD, Wang LJ, Yang L, Wang X, Singh G, Zhong JL. Nrf2-mediated protection against UVA radiation in human skin keratinocytes. Biosci Trends. 2011;5:23–9.

    Article  Google Scholar 

  31. Zhong JL, Raval C, Edwards GP, Tyrrell RM. A role for Bach1 and HO-2 in suppression of basal and UVA-induced HO-1 expression in human keratinocytes. Free Rad Biol Med. 2010;48:196–206.

    Article  Google Scholar 

  32. Yang SY, Zhou B, Wei X, Fangfang X, Nisar MF, Bian CX, Huang X, Li Y, Zhang Y, Bartsch JW, Zhong JL. Nrf2- and Bach1 may play a role in the modulation of UVA-induced oxidative stress by acetyl-11-keto-β-boswellic acid (AKBA) in skin keratinocytes. Skin Pharmacol Physiol. 2017;30:13–23.

    Article  Google Scholar 

  33. Zhong JL. UVA Mediated iron release in skin cells. In: Thesis, the University of Bath, UK, Library; 2001.

    Google Scholar 

  34. Ma XQ, Hu H, Guo CX, Yu L, Gao LX, Xie JL, Li CM. DNA-templated biomimetic enzyme sheets on carbon nanotubes to sensitively in situ detect superoxide AnionsReleased from cells. Adv Funct Mater. 2014;24:375897–903.

    Article  Google Scholar 

  35. Yu L, Gao LX, Ma XQ, Hu FX, Li CM, Lu Z. Involvement of superoxide and nitric oxide in BRAFV600E inhibitor PLX4032-induced growth inhibition of melanoma cells. Integr Biol. 2014;6:1211–7.

    Article  Google Scholar 

  36. Buch K, Peters T, Nawroth T, Sanger M, Schmidberger H, Langguth P. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT assay-a comparative study. Rad Oncol. 2012;7:1.

    Article  Google Scholar 

  37. Stockert JC, Blazquez-Castro A, Canete M, Horobin RW, Villanueva A. MTT assay for cell viability: intracellular localization of the formazan product is in lipid droplets. Acta Histochem. 2012;114:785–96.

    Article  Google Scholar 

  38. Grisham MB. Methods to detect hydrogen peroxide in living cells:possibilities and pitfalls. Comp Biochem Physiol A Mol Integr Physiol. 2013;165:429–38.

    Article  Google Scholar 

  39. Winterbourn CC. The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells. Biochimicaet Biophysica Acta. 2014;1840:730–8.

    Article  Google Scholar 

  40. Sakagami H, Satoh K, Makino Y, Kojima T, Takeda M. Effect of alpha-tocopherol on cytotoxicity induced by UV irradiation and antioxidants. Antican Res. 1997;17:2079–82.

    Google Scholar 

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Acknowledgements

We thank Prof. Rex M. Tyrrell and Prof. Threadgill (The University of Bath, UK) for useful discussion.

Funding

This work is financially supported by Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies (Chongqing, China), the National Science Foundation of China (No. 31200700, 21375108, 81271776 and81573073). Open funding, Chongqing Municipal Key laboratory of oral diseases and biomedical sciences, Biomedical Engineering of Higher Education. None of the projects are in a financial or personal relationship to this study.

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No additional data is linked with these findings. Moreover, in any query please contact author for data requests.

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  1. LiXia Gao and Chunxiang Bian contributed equally.

Authors and Affiliations

  1. College of Bioengineering & School of Life Sciences, Chongqing University, Chongqing, 400044, China

    Li Xia Gao, Chunxiang Bian, Yan Wu, Muhammad Farrukh Nisar, Shida Chen & Julia Li Zhong

  2. Institute for Clean Energy & Advanced Materials, Faculty of Materials & Energy, Southwest University, Chongqing, 400715, China

    Li Xia Gao, Chang Ming Li & Ling Yu

  3. Chongqing Municipal Key laboratory of oral diseases and biomedical sciences, Biomedical Engineering of Higher Education, Chongqing, 401147, China

    Ping Ji, Enyi Huang & Julia Li Zhong

  4. Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS University Islamabad, Lahore Campus, Lahore, 54000, Pakistan

    Muhammad Farrukh Nisar

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  1. Li Xia Gao
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Contributions

LG and CB performed the experiments and most of the lab work. MFN, SC, YL, CML, and JLZ designed the study and experimental procedures and statistical analyses. PJ, EH, MFN and JLZ drafted this study to a final form. All authors read and approved the final mansucript.

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Correspondence to Enyi Huang or Julia Li Zhong.

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LiXia Gao and Chunxiang Bian are co first-authors.

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Gao, L.X., Bian, C., Wu, Y. et al. Label-free electrochemical sensor to investigate the effect of tocopherol on generation of superoxide ions following UV irradiation. J Biol Eng 12, 17 (2018). https://doi.org/10.1186/s13036-018-0099-2

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Journal of Biological Engineering

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