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A disposable indium-tin-oxide sensor modified by gold nanorod–chitosan nanocomposites for the detection of H2O2 in cancer cells†
Received 17th March 2014, Accepted 19th May 2014
Chunmei Yu,‡ Zhenkun Zhu,‡ Qiuhong Wang, Wei Gu, Ning Bao and Haiying Gu*
DOI: 10.1039/c4cc01972e www.rsc.org/chemcomm
Integrating a disposable ITO electrode modified by gold nanorod– chitosan nanocomposites, a paper-based electroanalytical device for the real-time detection of H2O2 released from cancer cells has been developed. It provided a portable platform for biological and biomedical studies dealing with living cells.
Developing portable and simple analysis tools is of vital importance for practical applications.1 These devices provide powerful platforms for analysis in many disciplines with the features of analyzing small sample volumes, minimizing reagent consumption and reducing processing time.2 One proposal to reduce the time and cost of these analyses is related to the development of miniaturized devices.3 An indium tin oxide (ITO)-based electrode has been widely used as an electrochemical sensing platform due to its wide potential window and stable electrochemical properties.4 Additionally, ITO was accustomed to be used as the substrate for fabricating disposable sensors because of its mass production and low cost.5 Compared with the conventional electrodes, the main advantages of the disposable sensor are that problems of carry-over and surface fouling are alleviated. Hydrogen peroxide (H2O2), formed by disproportionation of unstable ROS superoxide ions (O2 ), is one of the major contributors to the oxidative stress damage because of its long lifetime to diffuse to other cellular compartments.6 Mounting evidence suggests that an excessive amount of H2O2 would induce various kinds of biological damage, leading to aging, neurodegeneration as well as cancer.7 Thus real-time detection of intracellular H2O2 is of great value in elaborating its regulation of signal transduction pathways and searching for new therapeutic strategies for diseases. Recently, electrochemical methods based on the specific high activity of natural enzymes toward H2O2 reduction have been mostly reported.8 The main shortcoming is that the School of Public Health, Nantong University, Nantong 226019, P. R. China. E-mail: [email protected], [email protected]; Fax: +86 513 85012900; Tel: +86 513 85012913 † Electronic supplementary information (ESI) available: Experimental details and additional figures and tables. See DOI: 10.1039/c4cc01972e ‡ These authors have contributed equally to this work.
This journal is © The Royal Society of Chemistry 2014
enzymes would not maintain their activity under the detection conditions because of their intrinsic nature. Metal nanoparticles (NPs) have been explored as alternative electrochemical catalysts for H2O2 detection.9 Gold nanorods (GNRs), which are rod-shaped nanoparticles, possess several advantages over the spherical gold nanoparticles, such as high surface area and good electron mediation capability.10 These additional features have been exploited to improve electrochemical sensing performances.11 Herein, we reported nonenzymatic detection of H2O2 released from living cancer cells using a paper-based electroanalytical device. The fabrication process is schematically shown in Fig. 1. A sheet of ITO chip modified by the polystyrene sulfonate (PSS)
Fig. 1 Schemes of (A) fabrication of the PSS–GNRs–chitosan nanocomposite. (B) Stepwise fabrication of the disposable electrode. (a) A piece of punched adhesive tape with a hole (diameter: 4 mm) was attached on the ITO glass. (b) PSS–GNRs–chitosan nanocomposite with 6 mL was dropped on the electrode region and was allowed to dry at room temperature. (c) K562 cell suspension in pH 7.4 PBS with the volume of 8 mL was dropped on the hole. (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. (C) The threeelectrode detection device was integrated in a PDMS cover.
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coated GNRs–chitosan nanocomposite was prepared as the disposable working electrode. A piece of filter paper was used to construct a thin-layer electrochemical cell based on its porous structure.12 It is worth noting 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 disposable electrode in a paper-based analytical device, the electrochemical sensing of H2O2 can reach as low as 60 nM due to the enhanced catalytic activity of the nanocomposite. Most importantly, 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 electrodes. The method presented here allows the mass production of simple, reproducible yet inexpensive electrodes, which made it possible for the miniaturization of the corresponding device along with their ease of handling and manipulation in a disposable manner. A two-step procedure was employed to prepare the PSS–GNRs– chitosan nanocomposite (ESI†). The average length and the aspect ratio of the as-prepared GNRs are B50 nm and B5.1, respectively (Fig. S1, ESI†). In general, GNRs prepared by this method would contain a large amount of cetyltrimethylammonium bromide (CTAB) tightly packed onto the nanorod surface and show high cytotoxicity. In order to remove the CTAB bilayer, PSS was used to coat GNRs to increase the biocompatibility.13 The PSS coating of nanorods did not result in significant shift of SPR bands (Fig. S2, ESI†), which was in agreement with the previous report.14 The electrostatic interaction between the PSS and GNRs was characterized by zeta-potential measurements. CTAB coated GNRs have high positive surface charge due to the presence of a cationic surfactant. After replacement of CTAB with negatively charged polymer PSS, the surface potential decreases from 40.6 mV to approximately 31 mV for PSS-coated GNRs (Fig. S3, ESI†). The corresponding changes in zeta potential suggest a high density confirmation of PSS molecules on the surface of nanorods. Then, when the PSS–GNRs further dispersed in chitosan, they can bind to each other by electrostatic interaction since the PSS–GNRs are negatively charged and chitosan is positively charged at pH 5.0. The preparation process is schematically shown in Fig. 1A. The electrocatalytic characteristics of the disposable electrode were evaluated for the reduction of H2O2 in 0.1 M phosphate buffer (PBS, pH 7.4). The PSS–GNRs–chitosan nanocomposite modified electrode (curve b in Fig. 2A) generates a much higher reduction current than PSS–GNRs (curve b in Fig. 2B), chitosan modified ITO (curve b in Fig. 2C) and bare ITO (curve b in Fig. 2D) in PBS containing 50 mM H2O2. As a comparison, there is no peak signal without addition of H2O2 (curve a), confirming that the cathodic current is indeed generated from the catalytic reduction of H2O2. It is reported that the porous property of the gold nanoparticles and the chitosan nanocomposite modified electrode made the redox probe more accessible to the electrode surface, thus facilitating the electron transfer.15 Here, due to the high conductivity and large surface area of GNRs as well as the macroporous spatial structure of chitosan, the nanocomposite could function as an active catalyst and greatly promote electrochemical reduction of H2O2. DPV was used to study the H2O2 concentration-dependent electrocatalytic current on such a disposable electrode (Fig. S5, ESI†). The reduction peak currents are proportional to the
7330 | Chem. Commun., 2014, 50, 7329--7331
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Fig. 2 Voltammetric responses of (A) PSS–GNRs–chitosan modified ITO, (B) PSS–GNRs modified ITO, (C) chitosan modified ITO and (D) ITO electrode with (curve b) and without (curve a) 50 mM H2O2. Scan rate: 100 mV s 1.
concentration of H2O2 in the range of 1.0 10 7 to 1.0 10 4 M with a detection limit of 0.06 mM (S/N = 3). The analytical performance of the sensor developed in this study was compared with other electrochemical sensors with and without enzymes as shown in Table S1 (ESI†). It can be observed that the presently fabricated electrode exhibited excellent detection performance with a relatively low detection limit and fast response time, which was competent in real-time monitoring of H2O2 released from cells. The selectivity of such a disposable electrode toward H2O2 was extensively examined by the amperometric curve at 0.3 V (Fig. 3). Dopamine (DA), glucose (Glu) and uric acid (UA) yielded little current change under the applied negative potential. Two folds of ascorbic acid (AA) found to cause about 10.3% increase of the peak current (Table S2, ESI†). The current increased proportionally even with the existence of the interferents when one more 7.5 mM H2O2 was added, indicating a superior selectivity to H2O2 over these biological compounds and the suitability of the sensor to practical applications. Moreover, we found that the relative standard deviation (RSD) for six measurements with 10 mM H2O2 using six electrodes is within 7.8%, so the reproducibility of the proposed sensor is acceptable. The proposed sensor was applied to perform real-time detection of H2O2 in cancer cells. As discussed in previous reports, AA can be chosen as the stimuli to induce the generation of H2O2 from cells.16 Here, we selected AA as the stimuli and four kinds of cancer cells (human neuroblastoma SH-SY5Y, human leukemia cells K562, human hepatoma cell HepG2 and rat adrenal medulla pheochromocytoma PC12 cell) as model cells.
Fig. 3 Amperometric responses of the disposable electrode toward 7.5 mM H2O2 and containing: (A) 50 mM DA, (B) 50 mM UA, (C) 50 mM Glu and (D) 15 mM AA at 0.3 V.
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Fig. 4 (A) Amperometric responses of the disposable electrode for the reduction of H2O2 released from SH-SY5Y cells without (a) and with (b) the addition of 1.0 mM AA. (B) The time course of H2O2 released from the SH-SY5Y cells induced by (a) 0.25, (b) 0.5, (c) 1.0, (d) 2.5 and (e) 5.0 mM AA. (C) Dependence of the flux of H2O2 released from cells on the AA dose. (D) Amount of H2O2 released from four kinds of cancer cells. The values are expressed as mean SD of three independent measurements.
As shown in Fig. 4A, without the stimulation of AA, SH-SY5Y cells did not generate any measurable signal at the disposable electrode under the applied potential of 0.3 V (curve a). When 1.0 mM AA was added, the current increased sharply in 0.5 s to a peak value, and then decreased to ca. 20% of the maximum in 3 s (curve b). In addition, after injecting 300 U mL 1 catalase, a selective scavenger of H2O2, into the solution, the current shown in curve b is found to decay gradually to almost the background, suggesting that H2O2 is either consumed or diffused away from the electrode surface. In the control experiment, upon the addition of AA in the cell-free detection solution, no obvious signal was observed (Fig. S6, ESI†), further suggesting that H2O2 was generated from cells by the stimulation of AA. These results postulate the potential of the proposed disposable electrode in studying the intracellular H2O2 released from living cells. The maximum current of ca. 0.0159 mA upon injection of 1.0 mM AA corresponds to about 1.69 mM H2O2 as calculated from the calibration curve in Fig. S6 (ESI†). The number of cells used in the measurements is about 4.0 104, and the mean amount of H2O2 released from each cell is 216 amol cell 1. The effect of AA dose on the amount of H2O2 released from the cells was also studied (Fig. 4B). The efflux of H2O2 increased upon increasing the concentration of AA (Fig. 4C), showing an AA dose-dependent manner. Taking into account the sensitivity and the possible interference, 1.0 mM AA is applied in subsequent treatment of cancer cells. The efflux amount of H2O2 from K562, HepG2 and PC12 cell lines is analyzed with the proposed method (Fig. S7, ESI†). The highest and the lowest H2O2 flux were obtained on K562 cells (535 81 amol cell 1) and PC12 cells (56 9.2 amol cell 1), respectively, showing a cell type-dependent manner (Fig. 4D). These results showed that low concentration of H2O2 in living cells can indeed be successfully detected by the developed electrode, which represents a new sensing platform for a reliable collection of kinetic information of cellular H2O2 release. Summarizing, we have designed a paper-based electroanalytical device using a disposable ITO chip modified by PSS–GNRs–chitosan
This journal is © The Royal Society of Chemistry 2014
Communication
nanocomposites as the working electrode, which provides a simple platform for the electrochemical detection of the intracellular H2O2 released from cancer cells. The proposed method offers several advantages. First of all, the method is sensitive, selective, and reliable compared with enzyme-based strategies. Second, preparation of such a low-cost disposable electrode avoided complicated operation, time consuming pretreatment and expensive electrode material. The single-use disposable electrode was prepared ‘‘as new’’ for each experiment and the sample volume needed in each experiment is only 8 mL. In this way, the proposed device is a promising tool for further physiological and pathological studies dealing with living cells. To be sure, despite these advantages, the fabrication of the sensing platform with more simplicity and higher specificity is worth of further optimization regarding the application of such devices in biological studies. All in all, the present work will promote the development of portable analytical tools and provide new opportunities for analytical and bioanalytical fields. We thank the National Natural Science Foundation of China (81001263, 21175075 and 21075070), the Natural Science Foundation of Jiangsu Province (BK2011047, BK2009152) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Notes and references ¨ller, A. Schlichtiger and H. Schlebusch, TrAC, 1 (a) P. B. Luppa, C. Mu Trends Anal. Chem., 2011, 30, 887; (b) L. Yan, Z. Zhu, Y. Zou, Y. S. Huang, D. W. Liu, S. S. Jia, D. M. Xu, M. Wu, Y. Zhou, S. Zhou and C. Y. J. Yang, J. Am. Chem. Soc., 2013, 135(10), 3748. 2 M. A. Burns, Science, 2002, 296, 1818. 3 G. M. Whitesides, Nature, 2006, 442, 368. 4 (a) S. K. Mishra and B. D. Gupta, Analyst, 2013, 138, 2640; (b) C. Liu, X. H. Wei and Y. F. Tu, Talanta, 2013, 111, 156. 5 (a) M. Z. Dai, Y. L. Lin, H. C. Lin, H. W. Zan, K. T. Chang, H. F. Meng, J. W. Liao, M. J. Tsai and H. Cheng, Anal. Chem., 2013, 85(6), 3110; (b) J. Yang, J. R. Strickler and S. Gunasekaran, Nanoscale, 2012, 4, 4594; (c) C. M. Yu, Z. K. Zhu, L. Wang, Q. H. Wang, N. Bao and H. Y. Gu, Biosens. Bioelectron., 2014, 53, 142. 6 (a) C. Amatore, S. Arbault, M. Guille and F. Lemaitre, Chem. Rev., 2008, 108, 2585; (b) D. Trachootham, J. Alexandre and P. Huang, Nat. Rev. Drug Discovery, 2009, 8, 579. 7 (a) W. Dorge and H. M. Schipper, Aging Cell, 2007, 6, 361; (b) B. Halliwell, J. Neurochem., 1992, 59, 1609; (c) H. Ohshima, M. Tatemichi and T. Sawa, Arch. Biochem. Biophys., 2003, 417, 3. 8 (a) C. X. Guo, X. T. Zheng, S. R. Ng, Y. C. Lai, Y. Lei and C. M. Li, Chem. Commun., 2011, 47, 2652; (b) X. M. Wu, Y. J. Hu, J. Jin, N. L. Zhou, P. Wu, H. Zhang and C. X. Cai, Anal. Chem., 2010, 82, 3588; (c) J. Zhao, Y. L. Yan, L. Zhu, X. X. Li and G. X. Li, Biosens. Bioelectron., 2013, 41, 815; (d) Z. Matharu, J. Enomoto and A. Revzin, Anal. Chem., 2013, 85(2), 932. 9 X. L. Sun, S. J. Guo, Y. Liu and S. H. Sun, Nano Lett., 2012, 12(9), 4859. 10 (a) S. E. Lohse and C. J. Murphy, J. Am. Chem. Soc., 2012, 134, 15607; (b) C. Wang, Z. Ma, T. Wang and Z. Su, Adv. Funct. Mater., 2006, 16, 1673. ´, L. Cornu, T. W. Napporn, J. Rousseau and B. K. Kokoh, 11 S. Hebie J. Phys. Chem. C, 2013, 117, 9872. 12 (a) L. J. Sun, Q. M. Feng, Y. F. Yan, Z. Q. Pan, X. H. Li, F. M. Song, H. Yang, J. J. Xu, N. Bao and H. Y. Gu, Biosens. Bioelectron., 2014, 60, 154; (b) M. Santhiago, J. B. Wydallis, L. T. Kubota and C. S. Henry, Anal. Chem., 2013, 85, 5233. 13 A. P. Leonov, J. Zheng, J. D. Clogston, S. T. Stern, A. K. Patri and A. Wei, ACS Nano, 2008, 2, 2481. 14 C. L. Chen, L. R. Kuo, C. L. Chang, Y. K. Hwu, C. K. Huang, S. Y. Lee, K. Chen, S. J. Lin, J. D. Huang and Y. Y. Chen, Biomaterials, 2010, 31, 4104. 15 L. Ding, C. Hao, Y. D. Xue and H. X. Ju, Biomacromolecules, 2007, 8, 1341. 16 H. C. Chang, X. M. Wang, K. K. Shiu, Y. L. Zhu, J. L. Wang, Q. W. Li, B. A. Chen and H. Jiang, Biosens. Bioelectron., 2013, 41, 789.
Chem. Commun., 2014, 50, 7329--7331 | 7331
COMMUNICATION
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Cite this: Chem. Commun., 2014, 50, 7329
A disposable indium-tin-oxide sensor modified by gold nanorod–chitosan nanocomposites for the detection of H2O2 in cancer cells†
Received 17th March 2014, Accepted 19th May 2014
Chunmei Yu,‡ Zhenkun Zhu,‡ Qiuhong Wang, Wei Gu, Ning Bao and Haiying Gu*
DOI: 10.1039/c4cc01972e www.rsc.org/chemcomm
Integrating a disposable ITO electrode modified by gold nanorod– chitosan nanocomposites, a paper-based electroanalytical device for the real-time detection of H2O2 released from cancer cells has been developed. It provided a portable platform for biological and biomedical studies dealing with living cells.
Developing portable and simple analysis tools is of vital importance for practical applications.1 These devices provide powerful platforms for analysis in many disciplines with the features of analyzing small sample volumes, minimizing reagent consumption and reducing processing time.2 One proposal to reduce the time and cost of these analyses is related to the development of miniaturized devices.3 An indium tin oxide (ITO)-based electrode has been widely used as an electrochemical sensing platform due to its wide potential window and stable electrochemical properties.4 Additionally, ITO was accustomed to be used as the substrate for fabricating disposable sensors because of its mass production and low cost.5 Compared with the conventional electrodes, the main advantages of the disposable sensor are that problems of carry-over and surface fouling are alleviated. Hydrogen peroxide (H2O2), formed by disproportionation of unstable ROS superoxide ions (O2 ), is one of the major contributors to the oxidative stress damage because of its long lifetime to diffuse to other cellular compartments.6 Mounting evidence suggests that an excessive amount of H2O2 would induce various kinds of biological damage, leading to aging, neurodegeneration as well as cancer.7 Thus real-time detection of intracellular H2O2 is of great value in elaborating its regulation of signal transduction pathways and searching for new therapeutic strategies for diseases. Recently, electrochemical methods based on the specific high activity of natural enzymes toward H2O2 reduction have been mostly reported.8 The main shortcoming is that the School of Public Health, Nantong University, Nantong 226019, P. R. China. E-mail: [email protected], [email protected]; Fax: +86 513 85012900; Tel: +86 513 85012913 † Electronic supplementary information (ESI) available: Experimental details and additional figures and tables. See DOI: 10.1039/c4cc01972e ‡ These authors have contributed equally to this work.
This journal is © The Royal Society of Chemistry 2014
enzymes would not maintain their activity under the detection conditions because of their intrinsic nature. Metal nanoparticles (NPs) have been explored as alternative electrochemical catalysts for H2O2 detection.9 Gold nanorods (GNRs), which are rod-shaped nanoparticles, possess several advantages over the spherical gold nanoparticles, such as high surface area and good electron mediation capability.10 These additional features have been exploited to improve electrochemical sensing performances.11 Herein, we reported nonenzymatic detection of H2O2 released from living cancer cells using a paper-based electroanalytical device. The fabrication process is schematically shown in Fig. 1. A sheet of ITO chip modified by the polystyrene sulfonate (PSS)
Fig. 1 Schemes of (A) fabrication of the PSS–GNRs–chitosan nanocomposite. (B) Stepwise fabrication of the disposable electrode. (a) A piece of punched adhesive tape with a hole (diameter: 4 mm) was attached on the ITO glass. (b) PSS–GNRs–chitosan nanocomposite with 6 mL was dropped on the electrode region and was allowed to dry at room temperature. (c) K562 cell suspension in pH 7.4 PBS with the volume of 8 mL was dropped on the hole. (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. (C) The threeelectrode detection device was integrated in a PDMS cover.
Chem. Commun., 2014, 50, 7329--7331 | 7329
View Article Online
Published on 19 May 2014. Downloaded by CAL POLY POMONA UNIV LIB/PER on 22/10/2014 20:45:40.
Communication
coated GNRs–chitosan nanocomposite was prepared as the disposable working electrode. A piece of filter paper was used to construct a thin-layer electrochemical cell based on its porous structure.12 It is worth noting 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 disposable electrode in a paper-based analytical device, the electrochemical sensing of H2O2 can reach as low as 60 nM due to the enhanced catalytic activity of the nanocomposite. Most importantly, 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 electrodes. The method presented here allows the mass production of simple, reproducible yet inexpensive electrodes, which made it possible for the miniaturization of the corresponding device along with their ease of handling and manipulation in a disposable manner. A two-step procedure was employed to prepare the PSS–GNRs– chitosan nanocomposite (ESI†). The average length and the aspect ratio of the as-prepared GNRs are B50 nm and B5.1, respectively (Fig. S1, ESI†). In general, GNRs prepared by this method would contain a large amount of cetyltrimethylammonium bromide (CTAB) tightly packed onto the nanorod surface and show high cytotoxicity. In order to remove the CTAB bilayer, PSS was used to coat GNRs to increase the biocompatibility.13 The PSS coating of nanorods did not result in significant shift of SPR bands (Fig. S2, ESI†), which was in agreement with the previous report.14 The electrostatic interaction between the PSS and GNRs was characterized by zeta-potential measurements. CTAB coated GNRs have high positive surface charge due to the presence of a cationic surfactant. After replacement of CTAB with negatively charged polymer PSS, the surface potential decreases from 40.6 mV to approximately 31 mV for PSS-coated GNRs (Fig. S3, ESI†). The corresponding changes in zeta potential suggest a high density confirmation of PSS molecules on the surface of nanorods. Then, when the PSS–GNRs further dispersed in chitosan, they can bind to each other by electrostatic interaction since the PSS–GNRs are negatively charged and chitosan is positively charged at pH 5.0. The preparation process is schematically shown in Fig. 1A. The electrocatalytic characteristics of the disposable electrode were evaluated for the reduction of H2O2 in 0.1 M phosphate buffer (PBS, pH 7.4). The PSS–GNRs–chitosan nanocomposite modified electrode (curve b in Fig. 2A) generates a much higher reduction current than PSS–GNRs (curve b in Fig. 2B), chitosan modified ITO (curve b in Fig. 2C) and bare ITO (curve b in Fig. 2D) in PBS containing 50 mM H2O2. As a comparison, there is no peak signal without addition of H2O2 (curve a), confirming that the cathodic current is indeed generated from the catalytic reduction of H2O2. It is reported that the porous property of the gold nanoparticles and the chitosan nanocomposite modified electrode made the redox probe more accessible to the electrode surface, thus facilitating the electron transfer.15 Here, due to the high conductivity and large surface area of GNRs as well as the macroporous spatial structure of chitosan, the nanocomposite could function as an active catalyst and greatly promote electrochemical reduction of H2O2. DPV was used to study the H2O2 concentration-dependent electrocatalytic current on such a disposable electrode (Fig. S5, ESI†). The reduction peak currents are proportional to the
7330 | Chem. Commun., 2014, 50, 7329--7331
ChemComm
Fig. 2 Voltammetric responses of (A) PSS–GNRs–chitosan modified ITO, (B) PSS–GNRs modified ITO, (C) chitosan modified ITO and (D) ITO electrode with (curve b) and without (curve a) 50 mM H2O2. Scan rate: 100 mV s 1.
concentration of H2O2 in the range of 1.0 10 7 to 1.0 10 4 M with a detection limit of 0.06 mM (S/N = 3). The analytical performance of the sensor developed in this study was compared with other electrochemical sensors with and without enzymes as shown in Table S1 (ESI†). It can be observed that the presently fabricated electrode exhibited excellent detection performance with a relatively low detection limit and fast response time, which was competent in real-time monitoring of H2O2 released from cells. The selectivity of such a disposable electrode toward H2O2 was extensively examined by the amperometric curve at 0.3 V (Fig. 3). Dopamine (DA), glucose (Glu) and uric acid (UA) yielded little current change under the applied negative potential. Two folds of ascorbic acid (AA) found to cause about 10.3% increase of the peak current (Table S2, ESI†). The current increased proportionally even with the existence of the interferents when one more 7.5 mM H2O2 was added, indicating a superior selectivity to H2O2 over these biological compounds and the suitability of the sensor to practical applications. Moreover, we found that the relative standard deviation (RSD) for six measurements with 10 mM H2O2 using six electrodes is within 7.8%, so the reproducibility of the proposed sensor is acceptable. The proposed sensor was applied to perform real-time detection of H2O2 in cancer cells. As discussed in previous reports, AA can be chosen as the stimuli to induce the generation of H2O2 from cells.16 Here, we selected AA as the stimuli and four kinds of cancer cells (human neuroblastoma SH-SY5Y, human leukemia cells K562, human hepatoma cell HepG2 and rat adrenal medulla pheochromocytoma PC12 cell) as model cells.
Fig. 3 Amperometric responses of the disposable electrode toward 7.5 mM H2O2 and containing: (A) 50 mM DA, (B) 50 mM UA, (C) 50 mM Glu and (D) 15 mM AA at 0.3 V.
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Fig. 4 (A) Amperometric responses of the disposable electrode for the reduction of H2O2 released from SH-SY5Y cells without (a) and with (b) the addition of 1.0 mM AA. (B) The time course of H2O2 released from the SH-SY5Y cells induced by (a) 0.25, (b) 0.5, (c) 1.0, (d) 2.5 and (e) 5.0 mM AA. (C) Dependence of the flux of H2O2 released from cells on the AA dose. (D) Amount of H2O2 released from four kinds of cancer cells. The values are expressed as mean SD of three independent measurements.
As shown in Fig. 4A, without the stimulation of AA, SH-SY5Y cells did not generate any measurable signal at the disposable electrode under the applied potential of 0.3 V (curve a). When 1.0 mM AA was added, the current increased sharply in 0.5 s to a peak value, and then decreased to ca. 20% of the maximum in 3 s (curve b). In addition, after injecting 300 U mL 1 catalase, a selective scavenger of H2O2, into the solution, the current shown in curve b is found to decay gradually to almost the background, suggesting that H2O2 is either consumed or diffused away from the electrode surface. In the control experiment, upon the addition of AA in the cell-free detection solution, no obvious signal was observed (Fig. S6, ESI†), further suggesting that H2O2 was generated from cells by the stimulation of AA. These results postulate the potential of the proposed disposable electrode in studying the intracellular H2O2 released from living cells. The maximum current of ca. 0.0159 mA upon injection of 1.0 mM AA corresponds to about 1.69 mM H2O2 as calculated from the calibration curve in Fig. S6 (ESI†). The number of cells used in the measurements is about 4.0 104, and the mean amount of H2O2 released from each cell is 216 amol cell 1. The effect of AA dose on the amount of H2O2 released from the cells was also studied (Fig. 4B). The efflux of H2O2 increased upon increasing the concentration of AA (Fig. 4C), showing an AA dose-dependent manner. Taking into account the sensitivity and the possible interference, 1.0 mM AA is applied in subsequent treatment of cancer cells. The efflux amount of H2O2 from K562, HepG2 and PC12 cell lines is analyzed with the proposed method (Fig. S7, ESI†). The highest and the lowest H2O2 flux were obtained on K562 cells (535 81 amol cell 1) and PC12 cells (56 9.2 amol cell 1), respectively, showing a cell type-dependent manner (Fig. 4D). These results showed that low concentration of H2O2 in living cells can indeed be successfully detected by the developed electrode, which represents a new sensing platform for a reliable collection of kinetic information of cellular H2O2 release. Summarizing, we have designed a paper-based electroanalytical device using a disposable ITO chip modified by PSS–GNRs–chitosan
This journal is © The Royal Society of Chemistry 2014
Communication
nanocomposites as the working electrode, which provides a simple platform for the electrochemical detection of the intracellular H2O2 released from cancer cells. The proposed method offers several advantages. First of all, the method is sensitive, selective, and reliable compared with enzyme-based strategies. Second, preparation of such a low-cost disposable electrode avoided complicated operation, time consuming pretreatment and expensive electrode material. The single-use disposable electrode was prepared ‘‘as new’’ for each experiment and the sample volume needed in each experiment is only 8 mL. In this way, the proposed device is a promising tool for further physiological and pathological studies dealing with living cells. To be sure, despite these advantages, the fabrication of the sensing platform with more simplicity and higher specificity is worth of further optimization regarding the application of such devices in biological studies. All in all, the present work will promote the development of portable analytical tools and provide new opportunities for analytical and bioanalytical fields. We thank the National Natural Science Foundation of China (81001263, 21175075 and 21075070), the Natural Science Foundation of Jiangsu Province (BK2011047, BK2009152) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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