Deconvoluting Topography and Spatial Physiological Activity of Live

Deconvoluting Topography and Spatial Physiological Activity of Live Macrophage Cells by Scanning Electrochemical Microscopy in Constant-Distance Mode...
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Anal. Chem. 2010, 82, 8371–8373

Letters to Analytical Chemistry Deconvoluting Topography and Spatial Physiological Activity of Live Macrophage Cells by Scanning Electrochemical Microscopy in Constant-Distance Mode Xiaocui Zhao, Piotr M. Diakowski, and Zhifeng Ding* Department of Chemistry, The University of Western Ontario, London, ON, Canada N6A 5B7 Detection of reactive oxygen species (ROS) released from live macrophage cells (RAW264.7) without any addition of external redox mediators using constant-height and constant-distance mode scanning electrochemical microscopy (SECM) was presented in this Letter. The successful separation of the ROS profile from the topography of cells in the physiological condition was demonstrated by recording the amperometric current and probing position in the z-direction along with lateral coordinates at each pixel where an alternating current (AC) was kept constant. It was discovered that the nucleus region of the cell releases more ROS than other organelle regions and the height of the cell is approximately 4.8 µm. To our best knowledge, this work reports the first spatially monitored ROS release without the influence of cell morphology using SECM. Scanning electrochemical microscopy (SECM) can be used to evaluate interfacial reactivities by scanning an ultramicroelectrode tip in the vicinity of substrates.1,2 Biological applications of SECM have been recently highlighted in several review articles.3-9 SECM was used to monitor cell topography,10-12 study cellular * To whom correspondence should be addressed. E-mail: [email protected]. Tel: (519)661-2111Ext. 86161. Fax: (519)661-3022. (1) Wittstock, G.; Burchardt, M.; Pust, S. E.; Shen, Y.; Zhao, C. Angew. Chem., Int. Ed. 2007, 46, 1584–1617. (2) Amemiya, S.; Bard, A. J.; Fan, F.-R. F.; Mirkin, M. V.; Unwin, P. R. Annu. Rev. Anal. Chem. 2008, 1, 95-131. (3) Yasukawa, T.; Kaya, T.; Matsue, T. Electroanalysis 2000, 12, 653–659. (4) Amemiya, S.; Guo, J.; Xiong, H.; Gross, D. A. Anal. Bioanal. Chem. 2006, 386, 458–471. (5) Bard, A. J.; Li, X.; Zhan, W. Biosens. Bioelectron. 2006, 22, 461–472. (6) Schulte, A.; Schuhmann, W. Angew. Chem., Int. Ed. 2007, 46, 8760–8777. (7) Roberts, W. S.; Lonsdale, D. J.; Griffiths, J.; Higson, S. P. J. Biosens. Bioelectron. 2007, 23, 301–318. (8) Honda, A.; Komatsu, H.; Kato, D.; Ueda, A.; Maruyama, K.; Iwasaki, Y.; Ito, T.; Niwa, O.; Suzuki, K. Anal. Sci. 2008, 24, 55–66. (9) Zhang, M.; Girault, H. H. Analyst 2009, 134, 25–30. (10) Liebetrau, J. M.; Miller, H. M.; Baur, J. E.; Takacs, S. A.; Anupunpisit, V.; Garris, P. A.; Wipf, D. O. Anal. Chem. 2003, 75, 563–571. (11) Kurulugama, R. T.; Wipf, D. O.; Takacs, S. A.; Pongmayteegul, S.; Garris, P. A.; Baur, J. E. Anal. Chem. 2005, 77, 1111–1117. (12) Wang, W.; Xiong, Y.; Du, F.-Y.; Huang, W.-H.; Wu, W.-Z.; Wang, Z.-L.; Cheng, J.-K.; Yang, Y.-F. Analyst 2007, 132, 515–518. 10.1021/ac101524v  2010 American Chemical Society Published on Web 09/28/2010

Figure 1. Illustration on SECM imaging of live cells in constant height mode (a) and constant-distance mode (b). Dotted line represents the probe scan path. Red color indicates localized cellular activity.

respiratory activities,13-16 and detect reactive oxygen species (ROS)17-20 and reactive nitrogen species (RNS)21,22 released from live cells. Typical SECM of live cells are performed in constant-height imaging mode where the probe is scanned in a constant reference plane above the sample (Figure 1a). The electrochemical current at the biased electrode allows for detection of metabolites (indicated by the red color in Figure 1), which is affected by both the tip-to-cell distance and chemical nature of the cells. The interpretation becomes problematic when variations in reactivity and topography are simultaneous. (13) Shiku, H.; Shiraishi, T.; Ohya, H.; Matsue, T.; Abe, H.; Hoshi, H.; Kobayashi, M. Anal. Chem. 2001, 73, 3751–3758. (14) Takii, Y.; Takoh, K.; Nishizawa, M.; Matsue, T. Electrochim. Acta 2003, 48, 3381–3385. (15) Zhu, R.; Macfie, S. M.; Ding, Z. J. Exp. Bot. 2005, 56, 2831–2838. (16) Holt, K. B.; Bard, A. J. Biochemistry 2005, 44, 13214–13223. (17) Kasai, S.; Shiku, H.; Torisawa, Y.-s.; Noda, H.; Yoshitake, J.; Shiraishi, T.; Yasukawa, T.; Watanabe, T.; Matsue, T.; Yoshimura, T. Anal. Chim. Acta 2005, 549, 14–19. (18) Zhao, X.; Petersen, N. O.; Ding, Z. Can. J. Chem. 2007, 85, 175–183. (19) Zhao, X.; Lam, S.; Jass, J.; Ding, Z. Electrochem. Commun. 2010, 12, 773– 776.

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The above inherent shortcomings can be overcome by constantdistance SECM (Figure 1b), where the electrode-to-cell distance is maintained constant during the scan by utilizing the distancedependent feedback signal.23 Shear force based distance control has been successfully introduced in this SECM mode, where the probe was attached to a piezoelectric tuning fork24-27 or a piezo dither.28-33 SECM was also combined with atomic force microscope (AFM).34 Sophisticated instrumentation and probe fabrication are required. More recently, intermittent contact SECM was developed using oscillation amplitude as the feedback signal.35 Vivier’s group proposed the constant-distance SECM by means of high frequency impedance detection.36,37 Bard’s group first reported alternating current (AC) amplitude at the SECM probe as the constant-distance signal with the predetermined conductive nature of the substrate.38 This technique has also been used to study the topography of neuron cells,11 which could not function properly in the case of massive neurotransmitter release. We recently illustrated that under the optimized experimental conditions, the AC signal was not affected by the local conductive properties of the substrate.39 This improved AC SECM appreciably with relatively simple instrumentation. ROS, including hydrogen peroxide, superoxide, etc., have been shown to play key roles in various physiological processes.40 Amatore’s group21,41-43 and Matsue’s group17 detected the ROS through the oxidation reaction in the positive potential region. We found that oxygen was reduced at -0.455 V vs Ag/AgCl while hydrogen peroxide was reduced at -0.745 V using differential pulse voltammetry (DPV).20 ROS released from COS-7 cells in (20) Zhao, X.; Zhang, M.; Long, Y.; Ding, Z. Can. J. Chem. 2010, 88, 569–576. (21) Amatore, C.; Arbault, S.; Bouton, C.; Coffi, K.; Drapier, J.-C.; Ghandour, H.; Tong, Y. ChemBioChem 2006, 7, 653–661. (22) Isik, S.; Schuhmann, W. Angew. Chem., Int. Ed. 2006, 45, 7451–7454. (23) Eckhard, K.; Schuhmann, W. Analyst 2008, 133, 1486–1497. (24) Zu, Y. B.; Ding, Z. F.; Zhou, J. F.; Lee, Y. M.; Bard, A. J. Anal. Chem. 2001, 73, 2153–2156. (25) Lee, Y.; Ding, Z. F.; Bard, A. J. Anal. Chem. 2002, 74, 3634–3643. (26) Florencia Garay, M.; Ufheil, J.; Borgwarth, K.; Heinze, J. Phys. Chem. Chem. Phys. 2004, 6, 4028–4033. (27) Takahashi, Y.; Shiku, H.; Murata, T.; Yasukawa, T.; Matsue, T. Anal. Chem. 2009, 81, 9674–9681. (28) Hengstenberg, A.; Kranz, C.; Schuhmann, W. Chem.sEur. J. 2000, 6, 1547– 1554. (29) Pitta Bauermann, L.; Schuhmann, W.; Schulte, A. Phys. Chem. Chem. Phys. 2004, 6, 4003–4008. (30) Etienne, M.; Anderson, E. C.; Evans, S. R.; Schuhmann, W.; Fritsch, I. Anal. Chem. 2006, 78, 7317–7324. (31) Eckhard, K.; Etienne, M.; Schulte, A.; Schuhmann, W. Electrochem. Commun. 2007, 9, 1793–1797. (32) Eckhard, K.; Schuhmann, W.; Maciejewska, M. Electrochim. Acta 2009, 54, 2125–2130. (33) Cougnon, C.; Bauer-espindola, K.; Fabre, D. S.; Mauzeroll, J. Anal. Chem. 2009, 81, 3654–3659. (34) Salomo, M.; Pust, S. E.; Wittstock, G.; Oesterschulze, E. Microelectron. Eng. 2010, 87, 1537–1539. (35) Mckelvey, K.; Edwards, M.; Unwin, P. Anal. Chem. 2010, 82, 68–74. (36) Gabrielli, C.; Huet, F.; Keddam, M.; Rousseau, P.; Vivier, V. J. Phys. Chem. B 2004, 108, 11620–11626. (37) Keddam, M.; Portail, N.; Trinh, D.; Vivier, V. ChemPhysChem 2009, 10, 3175–3182. (38) Wipf, D. O.; Bard, A. J. Anal. Chem. 1992, 64, 1362–1367. (39) Diakowski, P. M.; Ding, Z. Electrochem. Commun. 2007, 9, 2617–2621. (40) Rhee, S. G. Science 2006, 312, 1882–1883. (41) Arbault, S.; Pantano, P.; Sojic, N.; Amatore, C.; Best-Belpomme, M.; Sarasin, A.; Vuillaume, M. Carcinogenesis 1997, 18, 569–574. (42) Arbault, S.; Pantano, P.; Jankowski, J. A.; Vuillaume, M.; Amatore, C. Anal. Chem. 1995, 67, 3382–3390. (43) Arbault, S.; Sojic, N.; Bruce, D.; Amatore, C.; Sarasin, A.; Vuillaume, M. Carcinogenesis 2004, 25, 509–515.

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Figure 2. Simultaneously recorded (a) topography and (b) H2O2 and O2 release profile images of RAW 264.7 cells by keeping the electrode-to-cell distance of 8.5 µm. Their corresponding 3-D images are shown in (d) and (e). Excitation frequency was 20 kHz and amplitude was 0.050 V. A DC potential of -0.800 V vs Ag/AgCl was applied to the electrode with a diameter of 25 µm and RG of 3. Crosssection lines in (a) and (b) are plotted in (c), where the height and current are in the dotted and solid lines, respectively.

physiological conditions was also monitored at -0.800 V versus Ag/AgCl,18 and ROS were used as a probe for the inflammatory responses to bacteria induced infection of T24 human bladder cells.19 Herein, we explored if our AC- and direct current (DC)-SECM can be used to simultaneously obtain topographical and spatial ROS information in the case of live cells. It was hypothesized that the above two should be deconvoluted. The RAW264.7 macrophage cells were chosen owing to their massive ROS release (up to 24 fmol of superoxide21 or equivalent other ROS in about 50 s). EXPERIMENTAL SECTION The electrode preparation and instrumentation of the combined AC- and DC-SECM, while requiring only minor modifications of atypicalSECMsetup,canbefoundinSupportingInformation.15,39,44,45 The constant-distance regime was reached by keeping a constant AC, based on the fact that only negative AC feedback would be observed at a frequency of 20 kHz optimized for the cell culture medium (see also Supporting Information). RAW 264.7 cells and the culture protocol (detailed in Supporting Information) were from American Type Culture Collection, Virginia. RESULTS AND DISCUSSION Figure 2a and d shows cell topography image and the corresponding 3-D contour image, respectively. Simultaneously (44) Zhu, R.; Ding, Z. Can. J. Chem. 2005, 83, 1779–1791. (45) Zhu, R.; Nowierski, C.; Ding, Z.; Noeel, J. J.; Shoesmith, D. W. Chem. Mater. 2007, 19, 2533–2543.

recorded amperometric image is illustrated in Figure 2b and e (3-D image of Figure 2b) with constant electrode-to-cell distance of 8.5 µm. The cells have a diameter of 40 µm, approximately (Figure S1 in Supporting Information). The constant-distance regime was reached by maintaining a constant AC Figure S2 in Supporting Information). At EDC ) -0.800 V, the DC current depends on the extracellular concentrations of oxygen and hydrogen peroxide in the vicinity of RAW 264.7 cells.20 The evolution of the two species are summarized in Scheme S1 in Supporting Information, and their redox reactions have been investigated in a publication from our group20 and Amator’s group.21 Several cells and their thin membranes can be seen in the topographical image: the highest cell height was around 4.8 µm as read from the scale bars in Figure 2a,d. The lowest current was obtained for the substrate at the bottom of the glass Petri dish. To demonstrate the changes in topography and the ROS release profile over the cells, cross-section lines were drawn in Figure 2a (dotted line) and b (solid line). Their corresponding profiles were plotted in Figure 2c. The probe current is a consequence of nominal and extracellular ROS concentrations in the close environment of the cells. Relatively high current was recorded in the membrane regions of the cells. The primary ROS, superoxide, can be generated during the cell respiratory process in the mitochondria, by reduced nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome p450 reductase in the endoplasmic reticulum, and produced in the extracellular environment from the reduction of oxygen catalyzed by the activated NADPH oxidase.46 The higher current above nucleus regions demonstrated higher concentrations of hydrogen peroxide and oxygen as shown in Figure 2b and e. This spatial distribution may indicate that the detected hydrogen peroxide and oxygen, as the decomposition products of superoxide, draw their source mainly from the endoplasmic reticulum. Hydrogen peroxide and oxygen can diffuse locally inside the cells and travel freely through the cell membrane. A similar phenomenon was observed in the case of T24 cells in constant-height SECM mode.19,20 The difference in the height of two cells (two peaks in the dotted line in Figure 2c) is more obvious than the difference in the current (two peaks in the solid line in Figure 2c) obtained above the cells, which means the release of hydrogen peroxide and oxygen do not only follow the topography. In addition, the current peak above the cells was broader than the height peak of the cell, which shows the heterogeneous release of hydrogen peroxide and oxygen and their diffusion properties from the generation sites. The ROS release profiles were successfully separated from the topography using this simple and efficient combination. This is also applicable to other cells of interest.11,14,27,47 In contrast, while AC-SECM images in the constant-height mode showed a negative of cells (Figures S3a and c in Supporting Information), DC-SECM images did depend on the probe-to-cell distance: SECM image was switched from negative to positive when the probe was moved (46) Bedard, K.; Krause, K.-H. Physiol. Rev. 2007, 87, 245–313. (47) Zhu, L.; Gao, N.; Zhang, X.; Jin, W. Talanta 2008, 77, 804–808.

from 22.5 µm (Figure S3b in Supporting Information) to 17.1 µm (Figure S3d in Supporting Information). Other types of feedback signals, such as DC current for constant-distance control, were employed in the constant-distance SECM study of cells. A redox mediator normally was added to the solution, which inevitably caused, to some degree, a physiological environmental change to the cells. Oxygen in the bulk solution may be considered as an alternative to the mediator. However, it is difficult to maintain a constant current because the concentration of oxygen varies at the surface of the cell due to its physiological activity. The obvious disadvantage of this technique was that the topography was recorded at the expense of important chemical information. Matsue and co-workers27 has reported the shear force regulated constant-distance SECM study of single cells in a solution with a pH of 9.5, which was significantly derived from the actual physiological pH. It was costive to keep the real constant-distance regime due to the tip design and instrumentation demand. Wipf and Baur’s groups reported a similar experimental technique with PC12 cells to investigate vesicular release events by means of depolarizing solution, 105 mM K+.11 Constantdistance conditions, however, could not be maintained due to the significant impedance change caused by the depolarizing solution. In contrast, the method reported here is convenient and efficient in simultaneously recording the topography and biochemistry information of the cells without the introduction of any external chemicals, i.e., under physiological conditions. The most important benefit is that it can deconvolute the cellular topography from cellular activity and provide the spatial profile of the released hydrogen peroxide and oxygen. This experimental strategy is by no means limited to the RAW264.7 cells. CONCLUSIONS ROS release from RAW 264.7 macrophage cells, including the direct release of hydrogen peroxide and indirect generation of oxygen from the decomposition reactions of hydrogen peroxide and superoxide, was successfully monitored using DC-SECM in a constant-distance mode realized simply by keeping the AC constant. This experimental approach is an asset for studying many other cell lines. Insights into cell physiology and pathology are anticipated. ACKNOWLEDGMENT We appreciate the financial support for this research provided by NSERC, CIPI, OPC, CFI, OIT, PREA, and UWO. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 8, 2010. Accepted September 19, 2010. AC101524V

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