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Paper–based analytical device for detection of extracellular hydrogen peroxide and its application to evaluate drug–induced apoptosis
Electrochimica Acta 204 (2016) 128–135
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Paper–based analytical device for detection of extracellular hydrogen peroxide and its application to evaluate drug–induced apoptosis Qiuhong Wang1, Weibo Li1, Dongping Qian, Yubin Li, Ning Bao, Haiying Gu* , Chunmei Yu* School of Public Health, Nantong University, Nantong 226019, PR China
A R T I C L E I N F O
Article history: Received 29 January 2016 Received in revised form 22 March 2016 Accepted 14 April 2016 Available online 19 April 2016 Keywords: paper-based device Au nanoparticles NB4 cells hydrogen peroxide cellular cytotoxicity
A B S T R A C T
Developing cost-effective and simple analysis tools is of vital importance for practical applications in bioanalysis. Here, a disposable paper-based analytical device based on Au nanoparticles modified indium tin oxide electrode has been designed, which was applied for the reliable and non-enzymatic detection of H2O2. Due to the excellent electrocatalytic activity of Au nanoparticles, the disposable electrode exhibited favorable performance toward H2O2 reduction in the linear concentration range from 0.1 to 15 mM. The detection limit has been estimated to be 0.08 mM, which was lower than certain enzymes and other metal nanomaterials-based sensors. Because of these analytical advantages, the constructed device was used to study the extracellular H2O2 release from NB4 cells and further applied to evaluate sodium selenite induced apoptosis. The results obtained by electrochemical method are correlated well with the results of MTT assays. The developed paper-based sensor is easy-to-fabricate and portable, providing an effective platform for cellular H2O2 sensing and can be used to study the dynamic biological process involving H2O2 in biological and biomedical applications. ã 2016 Elsevier Ltd. All rights reserved.
1. Introduction Developing a rapid and accurate detection assay of hydrogen peroxide (H2O2) in biological samples is of broad interest in the fields of biology and biomedicine [1–3]. As a major reactive oxygen species in living organisms, H2O2 is produced by almost all oxidases in mitochondria and its appropriate level is essential for intracellular signaling transduction and normal cell functions [4,5]. But the excessive H2O2 quantity can induce different kinds of biological damages connected with lipid peroxidation [6], DNA damage [7] and tumor promotion [8], etc. Therefore, quantitative detection of H2O2 in cellular environment and monitoring its dynamic release process are essential to fully understand its roles in cellular physiology, and can further provide reliable diagnosis of pathological conditions. Over the past years, considerable efforts have been paid on the development of electrochemical methods for determination of H2O2 in living cells [9,10]. Enzyme-based electrochemical biosensors have received considerable attention due to their good
* Corresponding authors. Tel.: +0086 513 85012913. E-mail addresses: [email protected] (H. Gu), [email protected] (C. Yu). These authors have contributed equally to this work.
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http://dx.doi.org/10.1016/j.electacta.2016.04.073 0013-4686/ ã 2016 Elsevier Ltd. All rights reserved.
selectivity and sensitivity [11,12]. However, the enzyme-based biosensors are limited by some serious disadvantages, such as environmental instability, high cost, and a complicated immobilization procedure. Besides, the majority of other sensors for H2O2 detection usually use a glassy carbon electrode or Au electrode as working electrodes in a standard three-electrode system [13–15]. Unfortunately, these electrodes are limited by some serious disadvantages for analyzing biological samples. For example, the surface of electrodes could be easily contaminated during studies and the electrodes should be polished physically to obtain a clean and refreshed surface for the following detection. In addition, the treatment process requires complex operation and is timeconsuming, which further hinders the practical applications of the sensors. To overcome these obstacles, simplified and disposable paper-based analytical devices could be explored as alternative to construct sensors for H2O2 detection. Paper is an attractive substrate for the development of cheap, point-of-care sensors because it is easily available, portable, disposable and biocompatible [16–19]. The first paper-based sensor was reported in 1883 [20], and since that time the field has evolved from lateral flow assays to three-dimensional origami devices [21,22]. Researchers have put significant efforts into developing highly sensitive paper-based sensors for biological analysis. Crooks’s group reported a self-powered aptamer-based
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origami paper analytical device for electrochemical detection of adenosine [23]. Noiphung et al. developed electrochemical paperbased analytical devices with integrated plasma isolation for determination of glucose from whole blood samples [24]. So far, it is believed that the paper-based electrochemical sensors could open more opportunities for real time analysis of biological analytes that requires low sample volumes. In the present work, we describe a paper-based electrochemical sensor for detection of extracellular H2O2 and its application to evaluate drug-induced apoptosis in NB4 cells. Indium tin oxide (ITO) was selected as the substrate electrode, which was further modified by Au nanoparticles (AuNPs) through electrochemical deposition. By coupling the AuNPs/ITO electrode in a paper-based analytical device, the electrochemical sensing of H2O2 can reach as low as 0.08 mM. Subsequently, the fabricated device was used to monitor H2O2 release from NB4 cells and further applied to evaluate sodium selenite induced apoptosis. The cellulose paper used here provided an effective strategy for cell-based electrochemical sensors. Living cells could be well trapped by the fiber matrix of paper and thus cells could be maintained in good conditions conveniently. In addition, the porous structure of paper allows external materials, such as stimuli, to reach cells without disturbance so that responses from cells could be well investigated. In this way, the developed paper-based sensor provided a useful platform for in vitro electrochemical sensing of H2O2 in living cells with high sensitivity, highlighting the potential application of such devices in cell biology study and drug screening. 2. Experimental 2.1. Materials and reagents Hydrogen tetrachloroaurate trihydrate (HAuCl4, 99%), ascorbic acid (AA, 99%) and sodium selenite (purity 98%) were purchased from Sigma (St. Louis, MO, USA) and used without further purification. H2O2 (30%), glucose, dopamine and urine was purchased from Shanghai Chemical Reagent Company and was freshly prepared before being used. Indium tin oxide (ITO) conductive glass (355.6 406.4 1.1 mm STN, 10 V/cm2) was purchased from Nanbo Display Technology Co. LTD (Shenzhen, China). The qualitative filter papers (Whatman No.1) were from Whatman International Ltd. (Maidstone, United Kingdom). Phosphate buffer saline (PBS, pH 7.4) containing 87.2 mM Na2HPO4, 14.1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl was used as the electrolyte. A fresh solution of H2O2 was prepared daily. Other
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chemicals were of analytical grade. All the solutions were prepared with doubly distilled water. 2.2. Design and fabrication of the disposable electrode ITO plate was cut into pieces of 20 mm 10 mm to fabricate disposable electrodes. The ITO glasses were ultrasonically cleaned sequentially with acetone, absolute ethanol, and deionized water for 5 min, respectively, and dried with nitrogen gas. Subsequently, a section of plastic adhesive tape was punched with a circle hole (4 mm diameter) and attached on the ITO surface (Scheme 1a) to provide an identical detection area. Subsequently, the Au nanoparticles (AuNPs) were electrochemically deposited on the prepared ITO glass by cyclic voltammetry scanning from 0.2 V to 1.0 V in 0.1 M KCl solution containing 0.5 mM HAuCl4 at a scan rate of 50 mV s 1 for 10 cycles (Scheme 1b). After rinsed clearly with twice-distilled deionized water, AuNPs/ITO electrode was developed and allowed to dry at room temperature. The UV–vis spectra were recorded on an UV-2450 spectrophotometer (Shimadzu, Japan). The scanning electron microscopy (SEM) images were obtained with a JSM-6510 scanning electron microscope (JEOL Ltd., Japan). Electronic dispersive spectroscopy (EDS) analysis was obtained by using a Hitachi S3400 SEM (Hitachi, Japan). 2.3. Electrochemical measurements The electrochemical experiments were performed with a CHI1230B workstation (CH Instrumentation, Shanghai, China). A three-electrode system comprised the AuNPs/ITO as the working electrode, a platinum wire as the auxiliary electrode, and Ag/AgCl as the reference electrode. Sample solutions in pH 7.4 PBS with the volume of 10 mL was dropped on the electrode region of the AuNPs/ ITO (Scheme 1c). Then a piece of Whatman filter paper with 6 mm long and 6 mm wide was put on the surface of the electrode region (Scheme 1d). The paper used here is to construct a thin-layer electrochemical cell based on its porous structure. It is notable that the paper could not only store the reagent solution but also electrically connect working, reference and counter electrodes for electrochemical detection. By coupling the three-electrode system in a cover made of elastomeric material, PDMS (polydimethylsiloxane), the paper-based analytical device was developed for voltammetric analysis (Scheme 1e). Cyclic voltammetry (CV) was measured with scan rate at 0.1 V/s. Differential pulse voltammetry (DPV) was performed with amplitude at 25 mV, pulse width at 0.2 s, pulse
Scheme 1. Schemes of stepwise fabrication of the analytical device. (a) A piece of punched adhesive tape with a circle hole (diameter: 4 mm) was attached on the ITO glass. (b) Electrodeposition of AuNPs on the electrode region. (c) NB4 cell suspension in pH 7.4 PBS with the volume of 10 mL was dropped on the electrode region. (d) A piece of filter paper with 6 mm long and 6 mm wide was covered on the hole. (e) An Ag/AgCl wire and a Pt wire were integrated with the ITO glass to form the electrochemical detection system. (f) Electrochemical analysis after addition of the stimuli.
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period at 0.5 s, quite time at 2 s. Amperometric measurements were performed under a constant potential of 0.4 V (vs. Ag/AgCl). A 0.1 M KNO3 solution containing 1 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture was used for electrochemical impedance spectroscopy (EIS) measurement. Prior to each experiment, a stream of highly pure nitrogen was blown gently inside the detection device to maintain the nitrogen atmosphere throughout the experiments. 2.4. Cell culture and collection The human NB4 leukemia cell line was obtained as a gift from the Affiliated Hospital of Nantong University (Nantong, China). The cells were cultured in RPMI medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL, Sigma) and streptomycin (100 units/mL, Sigma) at 37 C in a humidified 5% CO2 incubator. To study the cytotoxic effect of sodium selenite on cell viability, NB4 cells were treated with different dose of sodium selenite for 24 h. Cells without treatment were used as negative control. The cells were counted using a Petroff–Hausser counter (USA). 2.5. Electrochemical detection of H2O2 released from cells The cells were separated from the culture medium by performing centrifugation at 1000 rpm for 5 min and washed with sterile pH 7.4 PBS. The sediment was suspended in deoxygenated PBS to obtain a homogeneous cell suspension for electrochemical measurements. Firstly, the cell suspension of 10 uL was dropped on the electrode region. After a steady state background was attained, ascorbic acid as the stimuli was injected into the electrode region, and the response current corresponding to the electrocatalytic reduction of H2O2 released from the cells was recorded at 0.4 V (Scheme 1f). 2.6. Analysis of cell viability by bioassay 3–(4,5–dimethyl–2–thiazolyl)–2,5–diphenyl–2–H–tetrazolium bromide (MTT) method was used to determine the cell viability. NB4 cells were seeded in 96 well plates at a density of 2 105 cells per well and stabilized for 24 h. Then cells were treated with various concentrations of sodium selenite for 24 h. At the end of the treatment period, cells were incubated with new culture medium containing MTT solution (5 mg/mL) for 2 h at 37 C. The culture supernatant was all removed from the wells, and dimethyl sulfoxide (DMSO) was added to completely dissolve the formazan crystals. The absorbance of each well was measured at a wave length of 540 nm. The percent cell viability was calculated from the percent ratio of the absorbance obtained from each treatment and that of the control.
3. Results and discussion 3.1. Characterization of the AuNPs/ITO Energy-dispersive X-ray spectroscopy (EDS) was employed to characterize the element change of the electrodes during the modification process. The presence of In, Sn, O and Si atoms are characteristics of the glass substrate ITO (Fig. 1A). New signal of Au at 2.12 keV in the EDS spectra indicates that AuNPs have been incorporated under proposed conditions (Fig. 1B). The electroactive surface area of the disposable sensor was evaluated by cyclic voltammetry using ferricyanide as a redox probe under different scan rate. As shown in Fig. 2A, the peak potential exhibits slight shifts with increasing scan rate from 30 to 350 mV/s, indicating some kinetic limitations [25]. The peak current (Ip) increases linearly with the increasing of the square root of the potential scan rate (v1/2), which suggests that the reaction on the electrode was nearly reversible and mainly diffusion controlled (Fig. 2B). According to the Randles–Sevcik equation [26], the effective electroactive area of the AuNPs modified ITO was calculated as 0.671 cm2, which was about 5.3 times of its geometric area (0.126 cm2), indicating that the effective electroactive sites of electrode was increased. 3.2. Electrocatalytic reduction of H2O2 at the disposable electrode The electrocatalytic characteristics of the prepared disposable electrode were evaluated for the reduction of H2O2. Fig. 3A shows the typical cyclic voltammograms of H2O2 reduction by different electrodes in pH 7.4 PBS containing 10 mM H2O2. The AuNPs/ITO performs much better electrocatalytic activity, that the current response of the AuNPs/ITO (curve d) is much higher than that of bare ITO (curve b). As a comparison, there is no current response without H2O2 (curve a and c), confirming that the cathodic current is generated via catalytic reduction of H2O2 by the electrodes. The results presented in curve b show that the reduction current can only be observed at the potential more negative than 0.4 V, demonstrating that electrocatalytic activity of bare ITO electrode toward the reduction of H2O2 is low. Obviously, the AuNPs electrodeposited on the ITO surface largely enhanced the conductivity and increased the active surface area of the electrode, resulting in the best electrocatalytical performance toward the reduction of H2O2. Because differential pulse voltammetric (DPV) is highly sensitive with low detection limits, DPV technique was used for the determination of H2O2 with such a disposable electrode. Fig. 3B shows the DPV response of H2O2 at various concentrations. The corresponding catalytic currents increase linearly with the H2O2 concentration ranging from 0.1 to 15 mM with a correlation
Fig. 1. The EDS spectra of (A) ITO and (B) AuNPs/ITO.
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Fig. 2. Cyclic voltammograms of AuNPs/ITO electrode in 0.1 M KCl containing 1 mM K3Fe(CN)6 at different scan rate (a h: 50, 100, 150, 200, 250, 300, 350 and 400 mV s Plots of oxidation and reduction peak currents vs. square root of scan rate.
1
). (E)
Fig. 3. (A) Cyclic voltammograms of ITO (a, b) and AuNPs/ITO (c, d) in 0.1 M PBS containing 0 (a, c) and 10 mM H2O2 (b, d). Scan rate: 100 mV s 1. (B) DPV curves of H2O2 in pH 7.4 PBS with different concentrations (a–h): 0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5 and 15 mM. (C) Plot of the peak current against the concentration of H2O2.
coefficient of 0.9900 (Fig. 3C). Based on a signal-to-noise ratio (S/N) of 3, a detection limit of 0.08 mM can be obtained. Additionally, comparisons about H2O2 determination by other electrochemical methods were presented in Table 1. Table 1 included some enzyme-based and non-enzyme based sensors. It could be found that some of these sensors are comparable or even slightly better than this work. However, the enzymes bear poor long-term stability, poor tolerance in experimental conditions, and high cost. This work is non-enzyme sensing, which can avoid the instability originating from the intrinsic nature of the enzyme. In addition, the application of ITO glass allows us to fabricate disposable electrodes in bulk and could be extensively utilized. The main advantages of them over conventional electrodes are that problems of carry-over and surface fouling are alleviated. Generally, the concentration of H2O2 in biological samples is relatively low and the linear range in this work can meet the requirements for the sample analysis. Therefore, such a paper-based device may be a good choice for monitoring H2O2 release from living cells.
Further, the selectivity of AuNPs/ITO toward H2O2 detection was examined at the potential of 0.4 V, as shown in Fig. 4. H2O2 solution was firstly injected into the paper-based electrochemical cell, and followed by the addition of interferents. It can be observed that there was obvious current response to the injection of 5 mM H2O2. The addition of four folds of glucose (Glu), uric acid (UA) and dopamine (DA) yielded little current change under the applied negative potential. While ascorbic acid (AA) of 5 mM found to cause about 12.3% increase of the peak current. When a second 5 mM H2O2 was added, the current increased proportionally even with the existence of the interferents, demonstrating that the proposed sensor has a superior selectivity to H2O2 over these biological compounds. In addition, six electrodes were fabricated using the same method, the relative standard deviation of the electrodes at 5 mM H2O2 was 5.6% thereby showing good reproducibility. Also, working electrodes were pre-prepared before measurements and can be stored for a period of time. When the electrode was stored at
Table 1 Comparison of the proposed H2O2 sensor with other electrochemical sensors. Electrode
Ep (V)
Linear range (mM)
Detection limit (mM)
Reference
HRP–HAP/GCE PB/Au-Chits/MCNTs/GCE Cyt c/Gel/ITO Cu–MOF/CPE Pt@Au/EDA/GCE graphene/pectin–CuNPs/GCE Pt48Pd52–Fe3O4/carbon HRP–attapulgite/GCE AuNPs/ITO
0.4 +0.1 0.05 0.2 +0.08 0.24 0.25 0.4 0.4
5–820 4–19.6 0.3–800 1–900 1–450 1–1000 0.02–0.1 and > 2 0.2–150 0.1–15
0.1 3.36 0.05 1.0 0.18 0.35 0.005 0.05 0.08
[27] [28] [29] [30] [11] [31] [32] [33] This work
HAP: hydroxyapatite; PB: Prussian blue; Chits: Chitosan; Gel: molecular hydrogel; Cu MOF: Cu based metal organic framework; CPE: carbon paste electrode; EDA: ethylenediamine.
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Fig. 4. Amperometric i–t response of the disposable electrode in pH 7.4 PBS at (c) 20 mM DA and (d) 5 mM AA.
0.4 V to successive addition of 5 mM H2O2 and four interferents: (a) 20 mM Glu, (b) 20 mM UA,
room temperature no significant change in the amperometric response was observed in 5 days. 3.3. Detection of H2O2 released from NB4 cells As demonstrated above, the present disposable sensor for H2O2 with excellent analytical performance provided a platform for monitoring H2O2 in the physiological environment. Additionally, it is known that artificial stimulation exerted on cells would disrupt intracellular redox homeostasis and lead to efflux of H2O2 from cells [34]. As reported previously, AA in pharmacologic concentrations showed cytotoxic effects on cells through generating reactive oxygen species and can be chosen as the stimuli [35]. Here, the developed sensor was applied to monitor the cellular flux of H2O2 from the acute promyelocytic leukemia NB4 cells on exposure to AA by amperometry. Here, amperometric method is more convenient for the sample analysis because stimuli need to be added during the analysis process. As shown in Fig. 5, without the stimulation of AA, NB4 cells did not generate any measurable signal at the disposable electrode under the applied potential (curve a). When 0.5 mM AA was added, the current increased sharply during a very short period (in 1 s) to a peak value and then decreased to ca. 12% of the maximum in 5 s (curve c). It suggested that the current response should be ascribed to the reduction of H2O2, which was generated from cells upon the stimulation of AA and could be effectively reduced by the AuNPs/ ITO. In the control experiment, the addition of 0.5 mM AA did not produce any apparent current change at the disposable electrode in cell-free PBS solution (curve b), further demonstrating that the generated enhancement of the cathodic current is attributed to the reduction of H2O2 under the stimulation of AA. These results postulate the potential of the proposed device in studying the H2O2 release from living cells. The effect of AA dose on the amount of H2O2 released from the cells was also studied using the developed method. Fig. 6A depicts the time course of H2O2 released from the cells induced by different doses of AA. With the improving of the concentration of AA, the current response increased, suggesting that the amount of H2O2 flux increased accordingly and showed an AA dose–
dependent manner. In addition, from Fig. 6B, it can be observed that the release rate of H2O2 is enhanced by increasing the AA dose in the rising periods, demonstrating that the exocytosis generates rapidly with stimulation of a high dose of AA. After that, when the concentration of AA increased to 1.5 mM, the generated H2O2 by the cells changed little (Fig. 6B). This may be because the H2O2 released from cells has tended to be saturated. Considering the sensitivity and the possible interference, 0.5 mM AA is applied in the subsequent treatment of living cells. 3.4. Application of the sensor on the apoptosis of NB4 cells Because the amount of in vitro H2O2 reflects the growth and survival of living cells, we investigated the relation between the
Fig. 5. Current response of AuNPs/ITO in (a) PBS contained NB4 cells (1 105 cells/ mL), (b) PBS upon addition of 0.5 mM AA and (c) PBS contained NB4 cells (1 105 cells/mL) upon addition of 0.5 mM AA at 0.4 V.
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Fig. 6. (A) Time course of H2O2 release from the NB4 cells (1 105 cells/mL) induced by 0.25, 0.5, 0.75,1.0 and 1.5 mM (a–e) AA. (B) Dependence of the peak current on the AA dose. Results are presented as the mean SD of triplicate experiments.
extracellular H2O2 release and the apoptosis of NB4 cells treated by anticancer drug using the above method described. It has earlier been reported that sodium selenite is an effective cytotoxic agent in primary human acute myeloid leukemia cells [36]. Therefore, in the present study, we measured the H2O2 release from NB4 cells treated with sodium selenite. To investigate the effect of doses of sodium selenite on the H2O2 release, NB4 cells were allowed to grow for 24 h, then fresh culture medium containing different doses of sodium selenite was supplied and the response signals were detected after 24 h. Fig. 7A shows the variance of time courses of NB4 cells treated with 0, 2, 4, 8, 12 and 20 mM sodium selenite for 24 h on the disposable electrode. It can be seen that the amperometric response of cells treated with 2 mM sodium selenite did not exhibited significant change compared to that of the control. It suggested that lower dose of sodium selenite could not promote a significant inhibition
effect on the viability of NB4 cells. However, when the dose of sodium selenite in the culture medium increased from 4 to 20 mM, the current signals decreased significantly and tented to a relatively constant value (Fig. 7B). Since the H2O2 efflux has a positive relationship with the number of living cells, this decrease in peak current indicated the decrease of the amount of living cells in the culture medium, suggesting that sodium selenite treatment could restrict the growth of NB4 cells and induce apoptosis, reflecting a change of the physiological activity of living cells. These results showed the effect of sodium selenite on the cell viability of NB4 cells is dose–dependent. MTT assay was performed as comparison to prove the credibility of the proposed electrochemical method. Fig. 7C shows the viability curves of NB4 cells for the 24 h exposure to sodium selenite within suitable concentration ranges. The proliferation and viability of the cells treated with 2 mM sodium selenite were
Fig. 7. (A) Time course of H2O2 release from the NB4 cells induced by 0.5 mM AA after treatment with 0, 2, 4, 8, 12 and 20 mM of sodium selenite for 24 h. (B) Dependence of the peak current on the concentration of sodium selenite. Results are presented as the mean SD of triplicate experiments. (C) Cell viability of NB4 cells after exposure to different final concentrations of sodium selenite for 24 h obtained with MTT assay. Results are presented as the mean SD of triplicate experiments.
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not much different from those of the control cells. However, treatment of the cells with higher doses of sodium selenite resulted in a greater decrease in proliferation and viability, as indicated in previous reports [37]. The results were in accordance with that of electrochemical method. The slight difference between two methods might be attributed to the different culture conditions and assay procedures used [38]. Therefore, monitoring of H2O2 release from cells using the proposed approach could be used as a credible means for evaluating the drug-induced apoptosis. 4. Conclusion In this work, a paper-based electroanalytical device using a disposable ITO glass modified by AuNPs as the working electrode has been designed. Due to the unique catalytic activity of AuNPs, the electrode exhibited low detection limit for the reduction of H2O2. The paper-based analytical device provides a simple platform for reliable collection of kinetic information on cellular H2O2 release and can be further applied to evaluate the druginduced apoptosis of living cells efficiently. It should be pointed out that owing to its low cost and miniature size, the working electrode can be fabricated as a one-time-use disposable sensor, and thus it can eliminate complicated and laborious cleaning steps needed while using common solid electrodes. In addition, the volume of sample required for each test decreased down to only 10 mL. This non-enzymatic sensor also can avoid the complex fabrication procedure and inherent instability of enzyme-based sensors. We envision that our approach for designing flexible analytical device with nanomaterial decorating can offer new insights on designing cost-effective disposable miniaturized sensors for further physiological and pathological investigations. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21375066 and 21475070), the Natural Science Foundation of Jiangsu Province (BK20151267 and BK2011047), the Qing Lan Project of Jiangsu Province, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Application Research Item of Nantong City (MS12015046) and the Nantong university postgraduate student science and technology innovation projects (YKC15061). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. electacta.2016.04.073. References [1] C.D. Fernando, P. Soysa, Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H2O2) scavenging activity of plant extracts, Methods X 2 (2015) 283–291. [2] V.B. Koman, C. Santschi, N.R. von Moos, V.I. Slaveykova, O.J. Martin, Portable oxidative stress sensor: Dynamic and noninvasive measurements of extracellular H2O2 released by algae, Biosens. Bioelectron. 68 (2015) 245–252. [3] J. Zhao, Y.L. Yan, L. Zhu, X.X. Li, G.X. Li, An amperometric biosensor for the detection of hydrogen peroxide released from human breast cancer cells, Biosens. Bioelectron. 41 (2013) 815–819. [4] C. Klomsiri, L.C. Rogers, L. Soito, A.K. McCauley, S.B. King, K.J. Nelson, L.B. Poole, L.W. Daniel, Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling, Free Radic. Biol. Med. 71 (2014) 49–60. [5] H. Patel, J. Chen, M. Kavdia, Induced peroxidase and cytoprotective enzyme expressions support adaptation of HUVECs to sustain subsequent H2O2 exposure, Microvasc. Res. 103 (2016) 1–10. [6] A. Sevanian, P. Hochstein, Mechanisms and consequences of lipid peroxidation in biological systems, Annu. Rev. Nutr. 5 (1985) 365–390.
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Contents lists available at ScienceDirect
Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Paper–based analytical device for detection of extracellular hydrogen peroxide and its application to evaluate drug–induced apoptosis Qiuhong Wang1, Weibo Li1, Dongping Qian, Yubin Li, Ning Bao, Haiying Gu* , Chunmei Yu* School of Public Health, Nantong University, Nantong 226019, PR China
A R T I C L E I N F O
Article history: Received 29 January 2016 Received in revised form 22 March 2016 Accepted 14 April 2016 Available online 19 April 2016 Keywords: paper-based device Au nanoparticles NB4 cells hydrogen peroxide cellular cytotoxicity
A B S T R A C T
Developing cost-effective and simple analysis tools is of vital importance for practical applications in bioanalysis. Here, a disposable paper-based analytical device based on Au nanoparticles modified indium tin oxide electrode has been designed, which was applied for the reliable and non-enzymatic detection of H2O2. Due to the excellent electrocatalytic activity of Au nanoparticles, the disposable electrode exhibited favorable performance toward H2O2 reduction in the linear concentration range from 0.1 to 15 mM. The detection limit has been estimated to be 0.08 mM, which was lower than certain enzymes and other metal nanomaterials-based sensors. Because of these analytical advantages, the constructed device was used to study the extracellular H2O2 release from NB4 cells and further applied to evaluate sodium selenite induced apoptosis. The results obtained by electrochemical method are correlated well with the results of MTT assays. The developed paper-based sensor is easy-to-fabricate and portable, providing an effective platform for cellular H2O2 sensing and can be used to study the dynamic biological process involving H2O2 in biological and biomedical applications. ã 2016 Elsevier Ltd. All rights reserved.
1. Introduction Developing a rapid and accurate detection assay of hydrogen peroxide (H2O2) in biological samples is of broad interest in the fields of biology and biomedicine [1–3]. As a major reactive oxygen species in living organisms, H2O2 is produced by almost all oxidases in mitochondria and its appropriate level is essential for intracellular signaling transduction and normal cell functions [4,5]. But the excessive H2O2 quantity can induce different kinds of biological damages connected with lipid peroxidation [6], DNA damage [7] and tumor promotion [8], etc. Therefore, quantitative detection of H2O2 in cellular environment and monitoring its dynamic release process are essential to fully understand its roles in cellular physiology, and can further provide reliable diagnosis of pathological conditions. Over the past years, considerable efforts have been paid on the development of electrochemical methods for determination of H2O2 in living cells [9,10]. Enzyme-based electrochemical biosensors have received considerable attention due to their good
* Corresponding authors. Tel.: +0086 513 85012913. E-mail addresses: [email protected] (H. Gu), [email protected] (C. Yu). These authors have contributed equally to this work.
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http://dx.doi.org/10.1016/j.electacta.2016.04.073 0013-4686/ ã 2016 Elsevier Ltd. All rights reserved.
selectivity and sensitivity [11,12]. However, the enzyme-based biosensors are limited by some serious disadvantages, such as environmental instability, high cost, and a complicated immobilization procedure. Besides, the majority of other sensors for H2O2 detection usually use a glassy carbon electrode or Au electrode as working electrodes in a standard three-electrode system [13–15]. Unfortunately, these electrodes are limited by some serious disadvantages for analyzing biological samples. For example, the surface of electrodes could be easily contaminated during studies and the electrodes should be polished physically to obtain a clean and refreshed surface for the following detection. In addition, the treatment process requires complex operation and is timeconsuming, which further hinders the practical applications of the sensors. To overcome these obstacles, simplified and disposable paper-based analytical devices could be explored as alternative to construct sensors for H2O2 detection. Paper is an attractive substrate for the development of cheap, point-of-care sensors because it is easily available, portable, disposable and biocompatible [16–19]. The first paper-based sensor was reported in 1883 [20], and since that time the field has evolved from lateral flow assays to three-dimensional origami devices [21,22]. Researchers have put significant efforts into developing highly sensitive paper-based sensors for biological analysis. Crooks’s group reported a self-powered aptamer-based
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origami paper analytical device for electrochemical detection of adenosine [23]. Noiphung et al. developed electrochemical paperbased analytical devices with integrated plasma isolation for determination of glucose from whole blood samples [24]. So far, it is believed that the paper-based electrochemical sensors could open more opportunities for real time analysis of biological analytes that requires low sample volumes. In the present work, we describe a paper-based electrochemical sensor for detection of extracellular H2O2 and its application to evaluate drug-induced apoptosis in NB4 cells. Indium tin oxide (ITO) was selected as the substrate electrode, which was further modified by Au nanoparticles (AuNPs) through electrochemical deposition. By coupling the AuNPs/ITO electrode in a paper-based analytical device, the electrochemical sensing of H2O2 can reach as low as 0.08 mM. Subsequently, the fabricated device was used to monitor H2O2 release from NB4 cells and further applied to evaluate sodium selenite induced apoptosis. The cellulose paper used here provided an effective strategy for cell-based electrochemical sensors. Living cells could be well trapped by the fiber matrix of paper and thus cells could be maintained in good conditions conveniently. In addition, the porous structure of paper allows external materials, such as stimuli, to reach cells without disturbance so that responses from cells could be well investigated. In this way, the developed paper-based sensor provided a useful platform for in vitro electrochemical sensing of H2O2 in living cells with high sensitivity, highlighting the potential application of such devices in cell biology study and drug screening. 2. Experimental 2.1. Materials and reagents Hydrogen tetrachloroaurate trihydrate (HAuCl4, 99%), ascorbic acid (AA, 99%) and sodium selenite (purity 98%) were purchased from Sigma (St. Louis, MO, USA) and used without further purification. H2O2 (30%), glucose, dopamine and urine was purchased from Shanghai Chemical Reagent Company and was freshly prepared before being used. Indium tin oxide (ITO) conductive glass (355.6 406.4 1.1 mm STN, 10 V/cm2) was purchased from Nanbo Display Technology Co. LTD (Shenzhen, China). The qualitative filter papers (Whatman No.1) were from Whatman International Ltd. (Maidstone, United Kingdom). Phosphate buffer saline (PBS, pH 7.4) containing 87.2 mM Na2HPO4, 14.1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl was used as the electrolyte. A fresh solution of H2O2 was prepared daily. Other
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chemicals were of analytical grade. All the solutions were prepared with doubly distilled water. 2.2. Design and fabrication of the disposable electrode ITO plate was cut into pieces of 20 mm 10 mm to fabricate disposable electrodes. The ITO glasses were ultrasonically cleaned sequentially with acetone, absolute ethanol, and deionized water for 5 min, respectively, and dried with nitrogen gas. Subsequently, a section of plastic adhesive tape was punched with a circle hole (4 mm diameter) and attached on the ITO surface (Scheme 1a) to provide an identical detection area. Subsequently, the Au nanoparticles (AuNPs) were electrochemically deposited on the prepared ITO glass by cyclic voltammetry scanning from 0.2 V to 1.0 V in 0.1 M KCl solution containing 0.5 mM HAuCl4 at a scan rate of 50 mV s 1 for 10 cycles (Scheme 1b). After rinsed clearly with twice-distilled deionized water, AuNPs/ITO electrode was developed and allowed to dry at room temperature. The UV–vis spectra were recorded on an UV-2450 spectrophotometer (Shimadzu, Japan). The scanning electron microscopy (SEM) images were obtained with a JSM-6510 scanning electron microscope (JEOL Ltd., Japan). Electronic dispersive spectroscopy (EDS) analysis was obtained by using a Hitachi S3400 SEM (Hitachi, Japan). 2.3. Electrochemical measurements The electrochemical experiments were performed with a CHI1230B workstation (CH Instrumentation, Shanghai, China). A three-electrode system comprised the AuNPs/ITO as the working electrode, a platinum wire as the auxiliary electrode, and Ag/AgCl as the reference electrode. Sample solutions in pH 7.4 PBS with the volume of 10 mL was dropped on the electrode region of the AuNPs/ ITO (Scheme 1c). Then a piece of Whatman filter paper with 6 mm long and 6 mm wide was put on the surface of the electrode region (Scheme 1d). The paper used here is to construct a thin-layer electrochemical cell based on its porous structure. It is notable that the paper could not only store the reagent solution but also electrically connect working, reference and counter electrodes for electrochemical detection. By coupling the three-electrode system in a cover made of elastomeric material, PDMS (polydimethylsiloxane), the paper-based analytical device was developed for voltammetric analysis (Scheme 1e). Cyclic voltammetry (CV) was measured with scan rate at 0.1 V/s. Differential pulse voltammetry (DPV) was performed with amplitude at 25 mV, pulse width at 0.2 s, pulse
Scheme 1. Schemes of stepwise fabrication of the analytical device. (a) A piece of punched adhesive tape with a circle hole (diameter: 4 mm) was attached on the ITO glass. (b) Electrodeposition of AuNPs on the electrode region. (c) NB4 cell suspension in pH 7.4 PBS with the volume of 10 mL was dropped on the electrode region. (d) A piece of filter paper with 6 mm long and 6 mm wide was covered on the hole. (e) An Ag/AgCl wire and a Pt wire were integrated with the ITO glass to form the electrochemical detection system. (f) Electrochemical analysis after addition of the stimuli.
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period at 0.5 s, quite time at 2 s. Amperometric measurements were performed under a constant potential of 0.4 V (vs. Ag/AgCl). A 0.1 M KNO3 solution containing 1 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture was used for electrochemical impedance spectroscopy (EIS) measurement. Prior to each experiment, a stream of highly pure nitrogen was blown gently inside the detection device to maintain the nitrogen atmosphere throughout the experiments. 2.4. Cell culture and collection The human NB4 leukemia cell line was obtained as a gift from the Affiliated Hospital of Nantong University (Nantong, China). The cells were cultured in RPMI medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL, Sigma) and streptomycin (100 units/mL, Sigma) at 37 C in a humidified 5% CO2 incubator. To study the cytotoxic effect of sodium selenite on cell viability, NB4 cells were treated with different dose of sodium selenite for 24 h. Cells without treatment were used as negative control. The cells were counted using a Petroff–Hausser counter (USA). 2.5. Electrochemical detection of H2O2 released from cells The cells were separated from the culture medium by performing centrifugation at 1000 rpm for 5 min and washed with sterile pH 7.4 PBS. The sediment was suspended in deoxygenated PBS to obtain a homogeneous cell suspension for electrochemical measurements. Firstly, the cell suspension of 10 uL was dropped on the electrode region. After a steady state background was attained, ascorbic acid as the stimuli was injected into the electrode region, and the response current corresponding to the electrocatalytic reduction of H2O2 released from the cells was recorded at 0.4 V (Scheme 1f). 2.6. Analysis of cell viability by bioassay 3–(4,5–dimethyl–2–thiazolyl)–2,5–diphenyl–2–H–tetrazolium bromide (MTT) method was used to determine the cell viability. NB4 cells were seeded in 96 well plates at a density of 2 105 cells per well and stabilized for 24 h. Then cells were treated with various concentrations of sodium selenite for 24 h. At the end of the treatment period, cells were incubated with new culture medium containing MTT solution (5 mg/mL) for 2 h at 37 C. The culture supernatant was all removed from the wells, and dimethyl sulfoxide (DMSO) was added to completely dissolve the formazan crystals. The absorbance of each well was measured at a wave length of 540 nm. The percent cell viability was calculated from the percent ratio of the absorbance obtained from each treatment and that of the control.
3. Results and discussion 3.1. Characterization of the AuNPs/ITO Energy-dispersive X-ray spectroscopy (EDS) was employed to characterize the element change of the electrodes during the modification process. The presence of In, Sn, O and Si atoms are characteristics of the glass substrate ITO (Fig. 1A). New signal of Au at 2.12 keV in the EDS spectra indicates that AuNPs have been incorporated under proposed conditions (Fig. 1B). The electroactive surface area of the disposable sensor was evaluated by cyclic voltammetry using ferricyanide as a redox probe under different scan rate. As shown in Fig. 2A, the peak potential exhibits slight shifts with increasing scan rate from 30 to 350 mV/s, indicating some kinetic limitations [25]. The peak current (Ip) increases linearly with the increasing of the square root of the potential scan rate (v1/2), which suggests that the reaction on the electrode was nearly reversible and mainly diffusion controlled (Fig. 2B). According to the Randles–Sevcik equation [26], the effective electroactive area of the AuNPs modified ITO was calculated as 0.671 cm2, which was about 5.3 times of its geometric area (0.126 cm2), indicating that the effective electroactive sites of electrode was increased. 3.2. Electrocatalytic reduction of H2O2 at the disposable electrode The electrocatalytic characteristics of the prepared disposable electrode were evaluated for the reduction of H2O2. Fig. 3A shows the typical cyclic voltammograms of H2O2 reduction by different electrodes in pH 7.4 PBS containing 10 mM H2O2. The AuNPs/ITO performs much better electrocatalytic activity, that the current response of the AuNPs/ITO (curve d) is much higher than that of bare ITO (curve b). As a comparison, there is no current response without H2O2 (curve a and c), confirming that the cathodic current is generated via catalytic reduction of H2O2 by the electrodes. The results presented in curve b show that the reduction current can only be observed at the potential more negative than 0.4 V, demonstrating that electrocatalytic activity of bare ITO electrode toward the reduction of H2O2 is low. Obviously, the AuNPs electrodeposited on the ITO surface largely enhanced the conductivity and increased the active surface area of the electrode, resulting in the best electrocatalytical performance toward the reduction of H2O2. Because differential pulse voltammetric (DPV) is highly sensitive with low detection limits, DPV technique was used for the determination of H2O2 with such a disposable electrode. Fig. 3B shows the DPV response of H2O2 at various concentrations. The corresponding catalytic currents increase linearly with the H2O2 concentration ranging from 0.1 to 15 mM with a correlation
Fig. 1. The EDS spectra of (A) ITO and (B) AuNPs/ITO.
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Fig. 2. Cyclic voltammograms of AuNPs/ITO electrode in 0.1 M KCl containing 1 mM K3Fe(CN)6 at different scan rate (a h: 50, 100, 150, 200, 250, 300, 350 and 400 mV s Plots of oxidation and reduction peak currents vs. square root of scan rate.
1
). (E)
Fig. 3. (A) Cyclic voltammograms of ITO (a, b) and AuNPs/ITO (c, d) in 0.1 M PBS containing 0 (a, c) and 10 mM H2O2 (b, d). Scan rate: 100 mV s 1. (B) DPV curves of H2O2 in pH 7.4 PBS with different concentrations (a–h): 0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5 and 15 mM. (C) Plot of the peak current against the concentration of H2O2.
coefficient of 0.9900 (Fig. 3C). Based on a signal-to-noise ratio (S/N) of 3, a detection limit of 0.08 mM can be obtained. Additionally, comparisons about H2O2 determination by other electrochemical methods were presented in Table 1. Table 1 included some enzyme-based and non-enzyme based sensors. It could be found that some of these sensors are comparable or even slightly better than this work. However, the enzymes bear poor long-term stability, poor tolerance in experimental conditions, and high cost. This work is non-enzyme sensing, which can avoid the instability originating from the intrinsic nature of the enzyme. In addition, the application of ITO glass allows us to fabricate disposable electrodes in bulk and could be extensively utilized. The main advantages of them over conventional electrodes are that problems of carry-over and surface fouling are alleviated. Generally, the concentration of H2O2 in biological samples is relatively low and the linear range in this work can meet the requirements for the sample analysis. Therefore, such a paper-based device may be a good choice for monitoring H2O2 release from living cells.
Further, the selectivity of AuNPs/ITO toward H2O2 detection was examined at the potential of 0.4 V, as shown in Fig. 4. H2O2 solution was firstly injected into the paper-based electrochemical cell, and followed by the addition of interferents. It can be observed that there was obvious current response to the injection of 5 mM H2O2. The addition of four folds of glucose (Glu), uric acid (UA) and dopamine (DA) yielded little current change under the applied negative potential. While ascorbic acid (AA) of 5 mM found to cause about 12.3% increase of the peak current. When a second 5 mM H2O2 was added, the current increased proportionally even with the existence of the interferents, demonstrating that the proposed sensor has a superior selectivity to H2O2 over these biological compounds. In addition, six electrodes were fabricated using the same method, the relative standard deviation of the electrodes at 5 mM H2O2 was 5.6% thereby showing good reproducibility. Also, working electrodes were pre-prepared before measurements and can be stored for a period of time. When the electrode was stored at
Table 1 Comparison of the proposed H2O2 sensor with other electrochemical sensors. Electrode
Ep (V)
Linear range (mM)
Detection limit (mM)
Reference
HRP–HAP/GCE PB/Au-Chits/MCNTs/GCE Cyt c/Gel/ITO Cu–MOF/CPE Pt@Au/EDA/GCE graphene/pectin–CuNPs/GCE Pt48Pd52–Fe3O4/carbon HRP–attapulgite/GCE AuNPs/ITO
0.4 +0.1 0.05 0.2 +0.08 0.24 0.25 0.4 0.4
5–820 4–19.6 0.3–800 1–900 1–450 1–1000 0.02–0.1 and > 2 0.2–150 0.1–15
0.1 3.36 0.05 1.0 0.18 0.35 0.005 0.05 0.08
[27] [28] [29] [30] [11] [31] [32] [33] This work
HAP: hydroxyapatite; PB: Prussian blue; Chits: Chitosan; Gel: molecular hydrogel; Cu MOF: Cu based metal organic framework; CPE: carbon paste electrode; EDA: ethylenediamine.
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Fig. 4. Amperometric i–t response of the disposable electrode in pH 7.4 PBS at (c) 20 mM DA and (d) 5 mM AA.
0.4 V to successive addition of 5 mM H2O2 and four interferents: (a) 20 mM Glu, (b) 20 mM UA,
room temperature no significant change in the amperometric response was observed in 5 days. 3.3. Detection of H2O2 released from NB4 cells As demonstrated above, the present disposable sensor for H2O2 with excellent analytical performance provided a platform for monitoring H2O2 in the physiological environment. Additionally, it is known that artificial stimulation exerted on cells would disrupt intracellular redox homeostasis and lead to efflux of H2O2 from cells [34]. As reported previously, AA in pharmacologic concentrations showed cytotoxic effects on cells through generating reactive oxygen species and can be chosen as the stimuli [35]. Here, the developed sensor was applied to monitor the cellular flux of H2O2 from the acute promyelocytic leukemia NB4 cells on exposure to AA by amperometry. Here, amperometric method is more convenient for the sample analysis because stimuli need to be added during the analysis process. As shown in Fig. 5, without the stimulation of AA, NB4 cells did not generate any measurable signal at the disposable electrode under the applied potential (curve a). When 0.5 mM AA was added, the current increased sharply during a very short period (in 1 s) to a peak value and then decreased to ca. 12% of the maximum in 5 s (curve c). It suggested that the current response should be ascribed to the reduction of H2O2, which was generated from cells upon the stimulation of AA and could be effectively reduced by the AuNPs/ ITO. In the control experiment, the addition of 0.5 mM AA did not produce any apparent current change at the disposable electrode in cell-free PBS solution (curve b), further demonstrating that the generated enhancement of the cathodic current is attributed to the reduction of H2O2 under the stimulation of AA. These results postulate the potential of the proposed device in studying the H2O2 release from living cells. The effect of AA dose on the amount of H2O2 released from the cells was also studied using the developed method. Fig. 6A depicts the time course of H2O2 released from the cells induced by different doses of AA. With the improving of the concentration of AA, the current response increased, suggesting that the amount of H2O2 flux increased accordingly and showed an AA dose–
dependent manner. In addition, from Fig. 6B, it can be observed that the release rate of H2O2 is enhanced by increasing the AA dose in the rising periods, demonstrating that the exocytosis generates rapidly with stimulation of a high dose of AA. After that, when the concentration of AA increased to 1.5 mM, the generated H2O2 by the cells changed little (Fig. 6B). This may be because the H2O2 released from cells has tended to be saturated. Considering the sensitivity and the possible interference, 0.5 mM AA is applied in the subsequent treatment of living cells. 3.4. Application of the sensor on the apoptosis of NB4 cells Because the amount of in vitro H2O2 reflects the growth and survival of living cells, we investigated the relation between the
Fig. 5. Current response of AuNPs/ITO in (a) PBS contained NB4 cells (1 105 cells/ mL), (b) PBS upon addition of 0.5 mM AA and (c) PBS contained NB4 cells (1 105 cells/mL) upon addition of 0.5 mM AA at 0.4 V.
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Fig. 6. (A) Time course of H2O2 release from the NB4 cells (1 105 cells/mL) induced by 0.25, 0.5, 0.75,1.0 and 1.5 mM (a–e) AA. (B) Dependence of the peak current on the AA dose. Results are presented as the mean SD of triplicate experiments.
extracellular H2O2 release and the apoptosis of NB4 cells treated by anticancer drug using the above method described. It has earlier been reported that sodium selenite is an effective cytotoxic agent in primary human acute myeloid leukemia cells [36]. Therefore, in the present study, we measured the H2O2 release from NB4 cells treated with sodium selenite. To investigate the effect of doses of sodium selenite on the H2O2 release, NB4 cells were allowed to grow for 24 h, then fresh culture medium containing different doses of sodium selenite was supplied and the response signals were detected after 24 h. Fig. 7A shows the variance of time courses of NB4 cells treated with 0, 2, 4, 8, 12 and 20 mM sodium selenite for 24 h on the disposable electrode. It can be seen that the amperometric response of cells treated with 2 mM sodium selenite did not exhibited significant change compared to that of the control. It suggested that lower dose of sodium selenite could not promote a significant inhibition
effect on the viability of NB4 cells. However, when the dose of sodium selenite in the culture medium increased from 4 to 20 mM, the current signals decreased significantly and tented to a relatively constant value (Fig. 7B). Since the H2O2 efflux has a positive relationship with the number of living cells, this decrease in peak current indicated the decrease of the amount of living cells in the culture medium, suggesting that sodium selenite treatment could restrict the growth of NB4 cells and induce apoptosis, reflecting a change of the physiological activity of living cells. These results showed the effect of sodium selenite on the cell viability of NB4 cells is dose–dependent. MTT assay was performed as comparison to prove the credibility of the proposed electrochemical method. Fig. 7C shows the viability curves of NB4 cells for the 24 h exposure to sodium selenite within suitable concentration ranges. The proliferation and viability of the cells treated with 2 mM sodium selenite were
Fig. 7. (A) Time course of H2O2 release from the NB4 cells induced by 0.5 mM AA after treatment with 0, 2, 4, 8, 12 and 20 mM of sodium selenite for 24 h. (B) Dependence of the peak current on the concentration of sodium selenite. Results are presented as the mean SD of triplicate experiments. (C) Cell viability of NB4 cells after exposure to different final concentrations of sodium selenite for 24 h obtained with MTT assay. Results are presented as the mean SD of triplicate experiments.
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not much different from those of the control cells. However, treatment of the cells with higher doses of sodium selenite resulted in a greater decrease in proliferation and viability, as indicated in previous reports [37]. The results were in accordance with that of electrochemical method. The slight difference between two methods might be attributed to the different culture conditions and assay procedures used [38]. Therefore, monitoring of H2O2 release from cells using the proposed approach could be used as a credible means for evaluating the drug-induced apoptosis. 4. Conclusion In this work, a paper-based electroanalytical device using a disposable ITO glass modified by AuNPs as the working electrode has been designed. Due to the unique catalytic activity of AuNPs, the electrode exhibited low detection limit for the reduction of H2O2. The paper-based analytical device provides a simple platform for reliable collection of kinetic information on cellular H2O2 release and can be further applied to evaluate the druginduced apoptosis of living cells efficiently. It should be pointed out that owing to its low cost and miniature size, the working electrode can be fabricated as a one-time-use disposable sensor, and thus it can eliminate complicated and laborious cleaning steps needed while using common solid electrodes. In addition, the volume of sample required for each test decreased down to only 10 mL. This non-enzymatic sensor also can avoid the complex fabrication procedure and inherent instability of enzyme-based sensors. We envision that our approach for designing flexible analytical device with nanomaterial decorating can offer new insights on designing cost-effective disposable miniaturized sensors for further physiological and pathological investigations. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21375066 and 21475070), the Natural Science Foundation of Jiangsu Province (BK20151267 and BK2011047), the Qing Lan Project of Jiangsu Province, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Application Research Item of Nantong City (MS12015046) and the Nantong university postgraduate student science and technology innovation projects (YKC15061). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. electacta.2016.04.073. References [1] C.D. Fernando, P. Soysa, Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H2O2) scavenging activity of plant extracts, Methods X 2 (2015) 283–291. [2] V.B. Koman, C. Santschi, N.R. von Moos, V.I. Slaveykova, O.J. Martin, Portable oxidative stress sensor: Dynamic and noninvasive measurements of extracellular H2O2 released by algae, Biosens. Bioelectron. 68 (2015) 245–252. [3] J. Zhao, Y.L. Yan, L. Zhu, X.X. Li, G.X. Li, An amperometric biosensor for the detection of hydrogen peroxide released from human breast cancer cells, Biosens. Bioelectron. 41 (2013) 815–819. [4] C. Klomsiri, L.C. Rogers, L. Soito, A.K. McCauley, S.B. King, K.J. Nelson, L.B. Poole, L.W. Daniel, Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling, Free Radic. Biol. Med. 71 (2014) 49–60. [5] H. Patel, J. Chen, M. Kavdia, Induced peroxidase and cytoprotective enzyme expressions support adaptation of HUVECs to sustain subsequent H2O2 exposure, Microvasc. Res. 103 (2016) 1–10. [6] A. Sevanian, P. Hochstein, Mechanisms and consequences of lipid peroxidation in biological systems, Annu. Rev. Nutr. 5 (1985) 365–390.
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