Simultaneous Detection of Reactive Oxygen and Nitrogen Species

Anal. Chem. 2010, 82, 1411–1419

Simultaneous Detection of Reactive Oxygen and Nitrogen Species Released by a Single Macrophage by Triple Potential-Step Chronoamperometry Christian Amatore,* Ste´phane Arbault,*,† and Alaric C. W. Koh UMR CNRS-ENS-UPMC 8640 “PASTEUR” and LIA CNRS XiamENS, Ećole Normale Supe´rieure, 24 rue Lhomond, 75231 PARIS Cedex 5, France Macrophages produce reactive oxygen and nitrogen species (ROS/RNS) in response to immunological challenges. We have previously reported the real-time detection and quantification of released ROS/RNS by immunostimulated macrophages using constant potential amperometry, at four different potentials, with platinized carbon microelectrodes. As a methodological extension to that work, we sought to develop an electroanalytical method that would allow for the simultaneous monitoring of several ROS/RNS. Triple potential-step chronoamperometry at platinized carbon microelectrodes was found to provide satisfactory sensitivity and signal/noise ratio for this purpose. The title method was applied to the detection of endogenously produced ROS/RNS by single IFN-γ/LPS/ PMA stimulated RAW 264.7 macrophages. Significantly higher fluxes of H2O2, ONOO-, and NO• responses were detected over stimulated macrophages as compared to unactivated macrophages, consistent with the endogenous production of primary NO• and O2•- by both the inducible isoform of nitric oxide synthase (iNOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymatic systems in stimulated cells. Crucially, significant temporal variations in the release of each of the aforementioned species was evidenced using this method, which would not have been achievable with the use of either constant potential amperometry or classical biochemical methods such as the Griess assay. A primary role of macrophages is the phagocytosis of pathogens and cellular debris. It is generally accepted that the antimicrobial activities of phagocytic macrophages involve the release of reactive oxygen species (ROS) and reactive nitrogen species (RNS).1-6 However, despite the key role played by macrophages in the immune system, the nature and identities of * To whom correspondence should be addressed. E-mail: Christian.Amatore@ ens.fr. (C.A.); [email protected]. (S.A.). Phone: +33-1-4432-3388 (C.A.); +33-5-4000-8939 (S.A.). Fax: +33-1-4432-3863 (C.A.); +33-5-4000-2717 (S.A.). † Present address: Universite´ de Bordeaux 1, Institut des Sciences Molećulaires UMR5255, ENSCPB, 16 avenue Pey Berland, 33607 PESSAC, France. (1) Ischiropoulos, H.; Zhu, L.; Beckman, J. S. Arch. Biochem. Biophys. 1992, 298, 446–451. (2) Vazquez-Torres, A.; JonesCarson, J.; Balish, E. Infect. Immun. 1996, 64, 3127–3133. 10.1021/ac902486x  2010 American Chemical Society Published on Web 01/26/2010

the actual reactive species involved in the phagocytosis process are still widely debated.7-9 As such, some of the recent studies in our group were focused on the electroanalytical detection of ROS/RNS that were endogenously produced and released by activated macrophages.10,11 Indeed, as we12,13 and others14-19 have described, electrochemistry offers the possibility of direct, rapid, and real-time measurements of various electroactive (bio)chemical species, such as neurotransmitters or ROS/RNS, released by living cells. Microelectrodes, with their micrometric sizes and unique physicochemical properties, can be positioned in close proximity to individual cells and directly detect trace amounts of the analytical target(s) with up to attomole, submicrometer, subsecond resolutions.10,14,20-23 Making use of the unique advantages mentioned above, we have recently reported the use of amperometry at platinized (3) Alvarez, M. N.; Trujillo, M.; Radi, R. Methods Enzymol. 2002, 359, 353– 366. (4) Lindgren, H.; Stenman, L.; Tarnvik, A.; Sjostedt, A. Microbes Infect. 2005, 7, 467–475. (5) MacMicking, J.; Xie, Q. W.; Nathan, C. Annu. Rev. Immunol. 1997, 15, 323–350. (6) Gantt, K. R.; Goldman, T. L.; McCormick, M. L.; Miller, M. A.; Jeronimo, S. M. B.; Nascimento, E. T.; Britigan, B. E.; Wilson, M. E. J. Immunol. 2001, 167, 893–901. (7) Fukuto, J. M.; Ignarro, L. J. Acc. Chem. Res. 1997, 30, 149–152. (8) Halliwell, B.; Zhao, K.; Whiteman, M. Free Radical Res. 1999, 31, 651– 669. (9) Fang, F. C. Nat. Rev. Microbiol. 2004, 2, 820–832. (10) Amatore, C.; Arbault, S.; Chen, Y.; Crozatier, C.; Tapsoba, I. Lab Chip 2007, 7, 233–238. (11) Amatore, C.; Arbault, S.; Bouton, C.; Coffi, K.; Drapier, J. C.; Ghandour, H.; Tong, Y. H. ChemBioChem 2006, 7, 653–661. (12) Amatore, C.; Arbault, S.; Bruce, D.; de Oliveira, P.; Erard, M.; Vuillaume, M. Faraday Discuss. 2000, 319–333. (13) Amatore, C.; Arbault, S.; Guille, M.; Lemaitˆre, F. Chem. Rev. 2008, 108, 2585–2621. (14) Travis, E. R.; Wightman, R. M. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 77–103. (15) Wightman, R. M. Science. 2006, 311, 1570–1574. (16) Schulte, A.; Schuhmann, W. Angew. Chem., Int. Ed. 2007, 46, 8760–8777. (17) Borgmann, S. Anal. Bioanal. Chem. 2009, 394, 95–105. (18) Wang, W.; Zhang, S.; Li, L.; Wang, Z.; Cheng, J.; Huang, W. Anal. Bioanal. Chem. 2009, 394, 17–32. (19) Privett, B. J.; Shin, J. H.; Schoenfisch, M. H. Anal. Chem. 2008, 80, 4499– 4517. (20) Amatore, C.; Arbault, S.; Bruce, D.; de Oliveira, P.; Erard, M.; Vuillaume, M. Chem.sEur. J. 2001, 7, 4171–4179. (21) Amatore, C.; Arbault, S.; Bouret, Y.; Guille, M.; Lemaitˆre, F.; Verchier, Y. Anal. Chem. 2009, 81, 3087–3093. (22) Amatore, C.; Arbault, S.; Erard, M. Anal. Chem. 2008, 80, 9635–9641. (23) Liao, W.; Ho, J. A. Anal. Chem. 2009, 81, 2470–2476.

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carbon microelectrodes for the direct, real-time analysis of ROS/ RNS released by macrophages stimulated with interferon-γ (IFNγ) and lipopolysaccharide (LPS) to mimic an in vivo inflammatory situation.24 This method allowed for the detection and quantification of several different reactive species, despite their low release fluxes (in the order of several amol · cell-1 · s-1). In particular, the presence of peroxynitrite (ONOO-/ONOOH) was demonstrated, thereby providing, to the best of our knowledge, the first direct evidence of the production of this highly elusive species by immunostimulated macrophages. While the method reported in our previous work24 has proven useful in obtaining key biological insights to endogenous ROS/ RNS production and release by single cells, there were, nonetheless, two main difficulties associated with it. First, cellular “bursts” of ROS/RNS could only be monitored at a single potential for each measurement, since the large capacitive currents associated with the simultaneous release of various species prevented any scanning of the electrode potential25,26 at scan rates comparable with the duration of a single event. Second, and more importantly perhaps, cellular variability, as well as possible slight sensitivity differences between the microelectrodes, meant that the response obtained over one cell cannot be directly compared with that obtained over another.12 To allow for statistically meaningful averaging of charges, a large number of measurements had to be made. Typically, for the various experimental conditions tested, 15-35 independent measurements were made at each of the four working potentials. As each individual measurement lasted slightly over an hour, the total time required was long and tedious. In other words, while we were able to determine the average rates of ROS/RNS release, any temporal evolution of the flux of each species could not be followed at a single cell. In this paper, the development of an electroanalytical method that circumvents the above difficulties is presented. Our strategy is to use a single microelectrode to make successive chronoamperometric measurements at multiple potentials. Indeed, the large time constants, τ ) RC, of the platinized microelectrodes required for monitoring ROS/RNS and the large changes of capacitances during burst events prevented the use of transient electroanalytical techniques.12,27 Nonetheless, amperometry allows a time-separation between Faradaic and capacitive components. Some relatively broad, weak peaks were previously observed in the amperometric responses of immunochallenged macrophages.24 We, thus, hypothesized that it could be feasible to analyze the “plateau”-like responses previously detected during induced phagocytosis24 through multiple potential-step chronoamperometry, provided that the duration of each successive potential-step was sufficiently long to minimize interferences arising from capacitance charging currents of the platinized microelectrodes. The charging currents were generally observed to decay by more than 95% of their initial values within tens of seconds (data not shown). This was still relatively short with respect to the response time scales (in the order of several minutes to several tens of (24) Amatore, C.; Arbault, S.; Bouton, C.; Drapier, J.; Ghandour, H.; Koh, A. C. W. ChemBioChem 2008, 9, 1472–1480. (25) Sˇtulı´k, K.; Amatore, C.; Holub, K.; Mareeèk, V.; Kutner, W. Pure Appl. Chem. 2000, 72, 1483–1492. (26) Montenegro, M. I.; Queiro´s, M. A.; Daschbach, J. L. Microelectrodes: Theory and applications; Kluwer Academic Publishers; New York, 1991. (27) Amatore, C.; Arbault, S.; Bruce, D.; de Oliveira, P.; Erard, M.; Sojic, N.; Vuillaume, M. Analusis 2000, 28, 506–517.

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Figure 1. (a) Endogenous coproduction within a macrophage’s phagosome of NO• and O2•- by iNOS and NADPH oxidase enzymatic systems, leading to the formation and release of various reactive oxygen and nitrogen species. (b) Positioning of a platinized carbon fiber microelectrode directly above a single immunostimulated macrophage, in the artificial synapse configuration, to monitor in real time the release of several electroactive ROS/RNS.

minutes), suggesting that multiple potential-step chronoamperometric studies should indeed be possible. So, as a proof-ofprinciple, such a method was used to detect ROS/RNS released by single living macrophages during induced phagocytosis. The method was sufficiently sensitive to characterize the different ROS/RNS released by immunostimulated macrophages. In addition, this method also permitted us to directly evidence significant temporal variations in the fluxes of the various reactive species and compare them to the responses of nonactivated control cells. EXPERIMENTAL SECTION Chemicals. Murine interferon-γ (IFN-γ; specific activity 2 × 107 units · mg-1) was provided by R&D systems. Escherichia coli lipopolysaccharide (LPS) and phorbol 12-myristate 13acetate (PMA) were from Sigma. Unless otherwise stated, phosphate-buffered saline (PBS) was used in all experiments. PBS (pH 7.4) was prepared by dissolving commercial tablets (Sigma) in water. Purified water from a Milli-Q purification system (resistivity ) 18 MΩ · cm-1; Millipore) was used in the preparation of all solutions. Microelectrode Fabrication. The preparation of microelectrodes has previously been described in detail.11,28 Briefly, individual carbon fibers (10 µm diameter; Thornel P-55S, Cytec) were sealed into pulled-glass capillaries (O.D. 1.2 mm, I.D. 0.7 mm; GC120F-10, Clark Electrochemical Instruments), and the protruding carbon fibers were insulated by electrochemical (28) Arbault, S.; Pantano, P.; Jankowski, J. A.; Vuillaume, M.; Amatore, C. Anal. Chem. 1995, 67, 3382–3390.

deposition of poly(oxyphenylene) following a literature procedure.29 The tip of the insulated carbon fiber was then polished at an angle of 45° on a diamond particle whetstone microgrinder (EG-4, Narishige) to expose a clean, elliptical surface. Dendritic platinum black was deposited on the carbon surface by reducing hydrogen hexachloroplatinate (Sigma) in the presence of lead acetate (Sigma) at -60 mV versus SSCE. Alternatively, electrodeposition of a nanostructured, mesoporous platinum film (H1-Pt) was done, with slight modifications, according to the literature procedure.30,31 In both cases, the temporal evolution of the platinization process was followed on a computer and continued until the desired deposition charge (5 µC) was reached. Cell Culture and Activation. The murine macrophage RAW 264.7 (ATCC) cell line was cultured at 37 °C under a humidified 5% CO2 atmosphere in Dulbecco’s Modified Eagle’s medium (DMEM) containing 1.0 g · L-1 D-glucose and sodium pyruvate (Invitrogen). The growth medium, supplemented with 5% heatinactivated fetal bovine serum (E.U. origin; Gibco) and 20 µg.mL-1 gentamicin (Sigma), was renewed every 2 to 3 days. Prior to electrochemical studies (18-24 h), confluent monolayers of RAW 264.7 cells were harvested mechanically and resuspended in Petri dishes (35 mm diameter; Nunc), without (control) or with the addition of IFN-γ (20 units · mL-1 for constant potential studies; 100 units · mL-1 for triple potentialstep studies) and LPS (50 ng · mL-1 for constant potential studies; 1 µg · mL-1 for triple potential-step studies) to induce the expression of the inducible isoform of nitric oxide synthase (iNOS). Single-Cell Measurements. Experiments were performed at controlled room temperature (22 ± 1 °C) on the stage of an inverted microscope (Axiovert 135, Zeiss) placed in a Faraday cage. Immediately prior to measurements, the medium was emptied from a Petri dish containing adherent macrophages, which was then rinsed three times and filled with PBS. For singlecell measurements of stimulated macrophages, the added PBS contained PMA (0.6 µM) to additionally activate reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Note that the exchange of the culture medium by PBS is a common practice in amperometric detection of cellular release since components present in the culture medium would lead to fast inactivation of microelectrodes’ surfaces. It was also checked that PBS did not alter the NADPH oxidases activity when cell responses (data not shown)32 were induced solely by PMA (0.6 µM in PBS). Constant potential amperometric measurements and data treatment were carried out as described previously.24 The platinized microelectrode electroactive surface was held at ca. 1 µm above the tested macrophage to ensure that the collection efficiency remained close to unity even if the cell underwent some morphological changes over the course of the measurement. In some cases, it was noted that the macrophage either migrated away from the microelectrode or “attempted” to phagocytize the electrode; in such cases, the measurements were interrupted and discarded from the analyses. For multiple potential-step chrono(29) Kawagoe, K. T.; Jankowski, J. A.; Wightman, R. M. Anal. Chem. 1991, 63, 1589–1594. (30) Attard, G. S.; Bartlett, P. N.; Coleman, N. R.; Elliott, J. M.; Owen, J. R.; Wang, J. H. Science 1997, 278, 838–840. (31) Evans, S. A. G.; Elliott, J. M.; Andrews, L. M.; Bartlett, P. N.; Doyle, P. J.; Denuault, G. Anal. Chem. 2002, 74, 1322–1326. (32) Ghandour, H. PhD thesis, Universite´ Pierre et Marie Curie, 2006.

amperometric studies, a platinized carbon microelectrode was first positioned in the Petri dish, away from all cells (>30 µm). The recording was then started, and the microelectrode was poised in sequence at the chosen potential steps; in most cases, this corresponds to a triple potential-step measurement at +300, +450, and +650 mV versus SSCE, with step durations of 20 s each. The waveform was imposed on a potentiostat (either AMU130 or Tacussel PRG-DEL, Radiometer Analytical) by a signal generator (LW110, LeCroy), programmed using its dedicated software interface (ArbExplorer, LeCroy). The electrode was cycled for 20 min in PBS for precalibration and was then positioned just above the cell of interest. After a measurement of ca. 30 min, the electrode was again repositioned away from all cells, and the measurement was continued for 10 min postcalibration. The recorded data were treated using a self-written program33 to extract and calculate the relevant information. Using a commercial software (Origin version 7.0, OriginLab), the best fit for the baseline evolution over time was then obtained on the basis of data from the 20 min precalibration and 10 min postcalibration periods during which the microelectrode was positioned away from all cells. This was subtracted from the recorded response to yield the final amperograms. Data for which baseline fits poorly correlate with measured values (R2 < 0.8) were discarded. The histograms and Gaussian distribution curves presented in Figures 3 and 5 were constructed using commercial software (Origin version 7.0). RESULTS AND DISCUSSION Measurement Protocol: Potential-Step Duration. In our previous work,24 releases of ROS/RNS were detected in real-time by a sequence of amperometric measurements performed individually at four different constant potentials of +300, +450, +650, and +850 mV versus a sodium-saturated calomel reference electrode (SSCE); averaging the signals obtained at each potential for a large number of cells then permitted for statistically relevant analyses. These four potentials were selected on the basis of previous in vitro voltammetric studies of the oxidation of H2O2, ONOO-, NO•, and NO2- phosphate-buffered saline (PBS) solutions (see Figure 3 of ref 11). Hence, linear combinations of the measured currents obtained at each potential specifically characterized (see eqs 1-4 of ref 24) and quantified (see eqs 5-6 of ref 24) individually the statistical time dependence of the four aforementioned reactive species released by single macrophages. When the chronoamperometric step-sequence of Figure 2 was used, only the measured currents near the end of each step were further analyzed to minimize capacitive contributions. Specifically, the current data at time ts corresponded to the mean of the response from ts-0.2 s to ts-0.1 s, where ts is the time at which the potential is stepped. When a platinized microelectrode was cycled in PBS away from any cells at the four detection potentials (see Figure 2, top), the average of the current between ts-0.2 s and ts-0.1 s for each successive step at a given potential was observed to decrease exponentially with each cycle (see Figure 2, bottom). This prompted us to define the following measurement protocol: First, a fit of the current decay was determined for each potential based on the chronoamperometric response obtained (33) Koh, A. C. W. PhD thesis, Universite´ Pierre et Marie Curie, 2009.


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Figure 2. Measured current (bottom, solid line) resulting from a repetitive sequence of potential steps (top) applied to a platinized carbon microelectrode. The series of four dashed lines are the best fits taking into account the mean current near the end of each step (see text). From bottom to top: +300, +450, +650, and +850 mV versus SSCE, respectively.

when the microelectrode was positioned far from any cell. Different fits, which agreed with an exponential dependence with time, were assumed to correspond to the background current evolution (polarization) at each potential and were then arithmetically subtracted from the measured currents at the same potentials when the microelectrode was positioned just above a single cell; a series of background subtracted faradaic components measuring the cell response were, thus, obtained. The faradaic signals, based on our previous work,24 were expected to be of the order of a few picoamperes or less. This implies that, for our proposed method to be viable, the duration of each potential step had to be chosen such that the background amperometric current fluctuations were within the range of 1 pA or less during the period over which the currents were averaged (0.1 s). This corresponded to a maximal gradient of -10 pA · s-1. Considering a signal/noise factor of 5, each potential step should then be sufficiently long for the background current to be decaying at a rate of less than 2 pA · s-1 at the end of each step, while being as short as possible to obtain the best temporal resolution. The mean gradients of the decaying response at various times, after a potential step from +300 to +450 mV versus SSCE, are listed in Table 1. It is noted that though the gradient at 20 s was about twice that at 30 s, using a 20 s duration provided a 50% improvement in temporal resolution. Therefore, potential steps of 20 s durations were optimal for the purpose of this work. Note that Table 1 reports decay rates rather than time constants since the background current was not strictly exponential, as would be expected for a simple RC capacitive component. This certainly reflected the fact that the dendritic nature of the electrode surface (Pt black) corresponded to a composite equivalent circuit. Yet, the fact that the background current had a capacitive origin was evidenced by the fact that it 1414


increased upon immersing the electrode deeper within the solution due to the capacitance between the electrode shaft and the solution. For this reason, all measurements were performed with an approximately constant PBS height in the Petri dish. Measurement Protocol: Choice of Microelectrode and Number of Measured Potentials. Before conducting serial measurements on single cells, different types of platinized carbon microfiber electrodes, having different carbon fiber insulations (glass-sealed, polymer, or epoxy) and different protocols of platinization, were tested in vitro with regard to their background current magnitude as well as their sensitivity toward faradaic information. In addition, certain microelectrodes were coated with a layer of wax along the body of the microelectrode in an attempt to reduce stray surface capacitance. Several microelectrodes of each type (n > 3) were tested using the above measurement protocol, over a period of 1.5 h in PBS. In the absence of any cells (i.e., in the absence of significant faradaic currents), the difference (Idiff) between the measured current (I response) and the background fit (Ifit) should be close to zero, provided the electrodes exhibited good stability over time. Hence, the dispersion of Idiff around the line Idiff ) 0 pA was used as an indicator of the microelectrode’s suitability for our purpose. It was noted that, for all types of electrodes, responses at +850 mV versus SSCE yielded the greatest dispersion. As described previously,24 the measurements made at +850 mV, in conjunction with those at +650 mV, provide information about the release of NO2-. Since nitrite ions are one of the most stable end products of RNS decomposition, its accumulation can in fact be measured, albeit not in real-time, by classical techniques such as chemiluminescence or the Griess method.34,35 Furthermore, NO2- is also of lower biological reactivity than NO• or other reactive intermediates like ONOO-. Consequently, in view of the lower analytical interest of NO2- and the greater dispersion at +850 mV, as well as the significant gain in temporal resolution (one data point per 60 s instead of 80 s), it was decided that subsequent measurements would only be made at three potentials, viz, +300, +450 and +650 mV versus SSCE, which would still allow for the analyses of H2O2, ONOO-, and NO•. Preliminary results (data not shown33) showed that glasssealed, epoxy-sealed, or wax coated microelectrodes provided higher dispersions of Idiff than polymer-insulated ones. As such, only this latter category of microelectrodes was kept for further experiments. Subsequently, two types of platinized surfaces were compared (i) electrodeposited platinum black, as used in our previous works;13,24 (ii) electrodeposited nanostructured platimum film consisting of a hexagonal array of mesopores.30,31 This second type of platinum, denoted hereafter as H1-Pt, has a specific surface area of 17-23 m2 · g-1, i.e., comparable to that of platinum black (20-26 m2 · g-1).30,36 Denuault and co-workers reported that microelectrodes with electrodeposited H1-Pt were excellent amperometric sensors for the detection of H2O231 and were, thus, of interest to this work. (34) Braman, R. S.; Hendrix, S. A. Anal. Chem. 1989, 61, 2715–2718. (35) Tarpey, M. M.; Wink, D. A.; Grisham, M. B. Am. J. Physiol., Regul. Integr. Comp. Physiol. 2004, 286, R431-R444. (36) Elliott, J. M.; Attard, G. S.; Bartlett, P. N.; Coleman, N. R.; Merckel, D. A.; Owen, J. R. Chem. Mater. 1999, 11, 3602–3609.

Table 1. Mean Decay Rates (n ) 10) of the Background Current at Various Times after a Potential-Step from +300 to +450 mV Versus SSCE in PBS for Platinized Carbon Microelectrodes time/s decay rate/pA · s-1

5 -20.9 ± 5.2

10 -6.9 ± 1.3

The results of triple potential-step (+300, +450, and +650 mV versus SSCE) chronoamperometric measurements in PBS in the absence of cells, using polymer-insulated carbon microelectrodes platinized with either type of Pt, are compared in Figure 3. These two types of microelectrodes are both relatively stable under the experimental conditions, as evidenced by the small dispersions; 99.9% and 96.7% of the data for Pt black electrodes and 98.8% and 92.6% of the data for H1-Pt electrodes have Idiff values that lie within ±2 pA and ±1 pA, respectively, of Idiff ) 0 pA (n > 1300). Hence, the current measurement uncertainty associated with the reproducibility and stability of these electrodes was 1 pA at most. This made them compatible for triple potential-step chronoamperometric studies of cellular ROS/RNS release. Nonetheless, microelectrodes with electrodeposited platinum black appeared to slightly outperform those with electrodeposited H1-Pt mesoporous film at +650 mV versus SSCE (Figure 3c), while the converse trend was observed at +450 mV (Figure 3b). Since electrode stability was of relatively greater concern at the higher potentials, polymer-insulated carbon microelectrodes with electrodeposited platinum black were selected for studies of responses from single phagocytotic macrophages. Prior to our single cell studies, the triple potential-step chronoamperometric analytical protocol was tested in vitro with a separate series of constant potential studies.24 Chronoamperometric responses of a platinized carbon microelectrode in PBS were recorded, during which mixtures of NO2-, NO• (generated by diethylamine-NONOate or DEA-NONOate), and/or ONOOsolutions were successively added with agitation. Note that DEA-NONOate was used as a NO• source instead of solutions of dissolved NO• gas since, as shown previously,37 this offers a reproducible and more convenient way of preparing NO• solutions. A baseline fit was calculated using data prior to additions, and this fit was subsequently subtracted from the recorded current after mixing. Using the same microelectrode, three separate constant potential amperometric studies were then carried out at +300, +450, and +650 mV versus SSCE in a second series of experiments. The same protocol was performed with continuous sampling, as in our previous work, so that the outcome of the present method could be compared to previous results. These tests33 showed that the present triplestep chronoamperometric method led to “constant potential” amperograms which were remarkably similar to those obtained directly from the three separate constant potential studies, albeit with a poorer temporal resolution due to the discontinuous sampling in the present method (every 20 s). These series of tests established that faradaic current variations in the order of 1 pA from the electrooxidation of released ROS/RNS may be detected with sufficient sensitivity and accuracy using the defined electroanalytical protocol; the method was, thus, applied to the real-time monitoring ROS/RNS production by single immunostimulated macrophages. (37) Amatore, C.; Arbault, S.; Bouret, Y.; Cauli, B.; Guille, M.; Rancillac, A.; Rossier, J. ChemPhysChem 2006, 7, 181–187.

20 -1.9 ± 0.4

30 -0.9 ± 0.2

40 -0.5 ± 0.3

50 -0.4 ± 0.2

ROS/RNS Released by Single IFN-γ/LPS/PMA-Stimulated Macrophages. IFN-γ/LPS24 incubation is known to induce expression of the inducible isoform of nitric oxide synthase (iNOS, NOS2) in macrophages, though it is not expected to lead to NADPH oxidase activation.3 NADPH oxidases (NOX2), which are found in various professional phagocytes (macrophages, neutrophils, monocytes, and eosinophils), catalyze the production of superoxide (O2•-) by the one-electron reduction of oxygen, using NADPH as the electron donor.38,39 Since superoxidederived ROS are also damaging for the host, the enzymatic production of O2•- is tightly regulated. In the resting state, the subunits of NADPH oxidase are separated; the enzymes are, thus, inactive and must be assembled to become active. Thus, macrophages were preincubated with IFN-γ/LPS to induce the expression of iNOS, following which measurements were carried out in the presence of phorbol 12-myristate 13-acetate (PMA), a known NADPH oxidase activator.40,41 The simultaneous use of both activators significantly increased ROS/RNS production, as evidenced by the stronger amperometric responses observed compared to the case where only iNOS was activated (see Figure 4 for examples recorded at +650 mV versus SSCE). The effective quantities of each species released were determined as described previously.24 Overall, a IFN-γ/LPS/PMAstimulated macrophage (n > 14 at each measured potential) was found to release, on average, 4.2 ± 0.9 fmol of H2O2, 9.1 ± 1.9 fmol of ONOO-, 6.0 ± 1.2 fmol of free NO•, and 5.6 ± 0.4 fmol of NO2- over an hour. Owing to the stoichiometries of the above species production, the average primary fluxes of NO• and O2•- released by IFN-γ/LPS/PMA-stimulated macrophages were, thus, estimated to be 5.8 ± 0.2 amol · cell-1 · s-1 and 6.4 ± 0.2 amol · cell-1 · s-1, respectively. In comparison, the corresponding rates were 5.7 ± 0.7 amol · cell-1 · s-1 of NO• and 3.2 ± 0.5 amol · cell-1 · s-1 of O2•- for macrophages stimulated with IFN-γ/LPS only. This showed that the endogenous NO• production rate by immunostimulated macrophages remained largely unchanged following PMA addition, while that of O2•was doubled. This is consistent with the added activation of NADPH oxidases by PMA, which then function independently from iNOS to synthesize additional O2•-. In other words, the ROS/RNS detected were “additions” of fluxes from the concomitant activities of each enzymatic system. The main purpose of this paper is to demonstrate the applicability of multiple potential-step chronoamperometry for realtime simultaneous monitoring of several ROS/RNSs released by a single cell. The macrophages studied herein were, therefore, stimulated as described above by IFN-γ, LPS, and PMA. Note that higher concentrations of these activators were used than for the constant potential studies presented in Figure 4, enabling us to take advantage of the fact that the production of reactive species (38) (39) (40) (41)

Lambeth, J. D. Nat. Rev. Immunol. 2004, 4, 181–189. Bedard, K.; Krause, K. Physiol. Rev. 2007, 87, 245–313. Rossi, F. Biochim. Biophys. Acta 1986, 853, 65–89. Bellavite, P. Free Radical Biol. Med. 1988, 4, 225–261.


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Figure 3. Histograms of the difference (I diff) between the experimentally measured current (I response) and the calculated fit (I fit) of the background current decay for polymer-insulated carbon microelectrodes with either electrodeposited platinum black (black bars) or H1-Pt (white bars). Data were obtained at (a) +300 mV, (b) +450 mV, and (c) +650 mV versus SSCE.

Figure 4. Typical constant potential amperometric responses from two single RAW 264.7 macrophages stimulated with mixtures of either IFN-γ/LPS/PMA (black) or IFN-γ/LPS only (gray). These responses were obtained at +650 mV versus SSCE, allowing for H2O2, ONOO-, and NO• to be detected. Concentrations used 20 units · mL-1 IFN-γ, 50 ng · mL-1 LPS, 0.6 µM PMA. See Experimental Section for further details.

by macrophages increases with activators’ concentrations.42 The triple potential-step chronoamperometric measurements were carried as detailed in the Experimental Section. Briefly, at the start of each experiment, a platinized carbon microelectrode was first positioned away from any cells and cycled for about 20 min. The microelectrode was then positioned just above the membrane of a single cell, in a so-called “artificial synapse”13 configuration. Recording of the current response was continued for 30 min and, after which, for a further 10-20 min after the microelectrode was again repositioned at large distances from all cells. The recorded data from the initial and final parts of the experiment were then used to fit the background current time variations, which was subsequently subtracted from the overall response to obtain the faradaic contribution, Idiff. Typical reconstructed amperograms recorded through this procedure from immunostimulated and unactivated control cells (42) Kamei, K.; Haruyama, T.; Mie, M.; Yanagida, Y.; Aizawa, M.; Kobatake, E. Anal. Biochem. 2003, 320, 75–81.

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are presented in Figure 5a-c. Significantly larger amounts of ROS/RNS were detected over immunostimulated cells than the controls at potentials of +450 and +650 mV versus SSCE. Conversely, the two responses at +300 mV versus SSCE appeared relatively close to each other (Figure 5a). The data gathered at each potential from several independent single cell measurements are presented as histograms in Figure 5d-f to study their significancy. For control cells at each potential, a single Gaussian curve centered at around Idiff ) 0 provided a good description of the detected currents distributions (R2 > 0.98; red curves in Figure 5d-f). This basal distribution was attributed to noise contamination and associated experimental setup stability. The half-widths were found to be in the range of 0.3-0.7, evidencing an acceptably low background noise inherent to this methodology. Conversely, for activated macrophages, single Gaussians were insufficient to describe satisfactorily the distributions recorded at each of the three potentials. Better correlations (R2 > 0.96; black curves in Figure 5d-f) were obtained when a weighted sum of two or three Gaussians (blue dashed curves in Figure 5d-f) was used for each distribution. It is noteworthy that, at each potential, the Gaussian component centered about Idiff ) 0 was similar to that recorded for control cells, and was, therefore, again attributed to the background signal noise. At all three potentials, the histograms clearly showed a higher occurrence of larger Idiff values for activated cells. The average values of Idiff for immunostimulated cells (µactivated) were also greater than those of the unactivated cells (µcontrol). Note that these results are consistent with those made using the aforementioned constant potential amperometric method (see Figure 4). Finally, the signal/noise ratio, calculated as [total area of other peak(s)]/ [area of peak centered around Idiff ) 0], was observed to increase from 1.2 at +300 mV to 4.3 at +450 mV and finally to 23.7 at +650 mV; this statistical trend perfectly agrees with visual inspection of Figure 5a-c, which were recorded on one cell. Taken together, these results demonstrate that the release of several ROS/ RNSs by a single cell can be effectively followed in real time by triple potential-step chronoamperometry at platinized microelectrodes. Evidencing Temporal Variations in ROS/RNS Production. As mentioned in the introduction, it is desirable that temporal evolutions, if any, in the cell production of the various reactive species can be followed. When monitoring the release at constant potentials by amperometry, time-dependent responses obtained at different potentials on different cells cannot be directly compared due to cellular variability. Conversely, the method

Figure 5. (a)-(c) Extracted amperograms recorded at (a) +300, (b) +450, and (c) +650 mV versus SSCE from an IFN-γ/LPS/PMA-stimulated (black squares) or an unactivated (red crosses) single macrophage. (d)-(f) Histograms of background subtracted currents, I diff, at (d) +300, (e) +450, and (f) +650 mV versus SSCE for responses of activated (solid bars; n > 10) and control (striped bars; n ) 6) macrophages. The red curves are the fitted single Gaussian distributions for control cells. The black curves represent multipeak Gaussian distributions, describing the responses monitored for activated cells (see blue dashed curves for each Gaussian component).

reported herein allowed three separate amperograms to be recorded simultaneously at the selected potentials for each single cell. This would then allow for a direct comparison of the responses, without concern of any cellular variability or changes to the microelectrode’s responsivity. Examples of such measurements are presented in Figure 6a-c. The figures clearly show the variability of ROS/RNS release from one cell to another; this establishes the interest of such single-cell analyses.43 We reported previously24 that two main features could be observed in the constant potential amperometric responses: (i) broad, relatively weak peaks and (ii) sharp amperometric spikes superimposed onto the broad features. In this work, the broad peaks were again observed in most responses but only amperometric spikes with sufficient half-widths (several tens of seconds) could be monitored due to the discontinuous monitoring. Indeed, one such example can be observed in Figure 6b. Interestingly, some of these spikes were detected at each of the three potentials (+300, +450, and +650 mV versus SSCE), though with different magnitudes, thus suggesting that they corresponded to sudden “bursts” of cocktails of ONOO-, NO•, and possibly H2O2. (See below; note that the currents due to each species are additive, and consequently, were “piled up” when the electrode potential was increased.) More interestingly, the broad features appeared to be mainly related to a release of NO•. Indeed, the amperograms at +650 mV were observed to be significantly larger than at +450 mV in most measurements. These results suggest that significant amounts of free NO• were continuously detected at the microelectrodes. This agrees with the fact that the iNOSs, once (43) Carlo, D. D.; Lee, L. P. Anal. Chem. 2006, 78, 7918–7925.

activated, are capable of producing significant amounts of NO• over an extended period. Since NO• is assumed to diffuse rapidly across cell membranes, its release does not require a specific exocytotic process. Nevertheless, NO• spiked events could also be observed (see Figure 6d-f). Since these were in phase with significant current spikes at +300 and +450 mV versus SSCE, it is likely that they featured sudden vesicular releases, through individual exocytotic events of phagosomes.44 Such dichotomic release behavior would not have been observed if analyses of cell populations had been performed. Indeed, the observed linearity of nitrite accumulation with time in previous fluorimetric studies suggested a constant NO• production over time.24 We have, however, demonstrated herein that while that might be true when considering an entire population of cells (presumably due to averaging effects), at the single cell level, the rate of enzymatic NO• production varies significantly with time and proceeds through two different independent processes. Furthermore, for certain cells, the general aspect of the broad signals at +300 and +450 mV versus SSCE appeared to change with respect to one another over time. Unfortunately, this method, at present, lacks the precision required for the corresponding fluxes of ONOO- and H2O2 to be determined unambiguously when the faradaic components are too small, typically below 0.2 pA. This is principally due to the poor signal/noise ratio of the response at +300 mV, as we have discussed above. Nonetheless, since we are concerned here with the broad responses, an alternative approach would then be to study the (44) Di, A.; Krupa, B.; Bindokas, V. P.; Chen, Y. M.; Brown, M. E.; Palfrey, H. C.; Naren, A. P.; Kirk, K. L.; Nelson, D. J. Nat. Cell Biol. 2002, 4, 279–285.


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Figure 6. (a)-(c) Each figure shows amperograms at +300 (crosses), +450 (squares), and +650 mV (triangles) versus SSCE obtained for the same single immunostimulated macrophage. Measurements of three different single cells are reported, each performed with a freshly prepared microelectrode. The arrows indicate the time at which the microelectrode was positioned near and then removed from the surface of the cell. (d)-(f) NO• fluxes calculated from the corresponding responses shown immediately above in (a)-(c), through subtracting the responses at +450 mV (H2O2 and ONOO-) from those at +650 mV versus SSCE (H2O2, ONOO-, and NO•). Note that (a) is shown to illustrate the very small amplitudes of the currents detected at +300 and +450 mV versus SSCE for this particular cell; however, these data were not further analyzed in Table 2 since they could not afford any meaningful evaluations of the corresponding fluxes of H2O2 and ONOO- productions. Table 2. Time Dependence of ROS/RNS Production by Two Individual Stimulated Macrophagesa first 10 min H2O2/fmol ONOO-/fmol NO•/fmol

middle 10 min

last 10 min

cell 1

cell 2

cell 1

cell 2

cell 1

cell 2

1.7 4.6 20.3

1.2 2.3 13.6

0.4 3.2 9.0

1.0 ∼0 12.1

3.9 ∼0 ∼0

1.2 ∼0 8.1

a Note: Cells 1 and 2 are the single cells presented in Figure 6b,c, respectively.

temporal evolutions in the detected charges. The half-hour period of on-cell measurement was, thus, divided into three 10 min periods, and the total charge involved in oxidative processes at each of the three potentials was then obtained by time integration; the total amounts of H2O2, ONOO-, and NO•, released over each 10 min period, were then determined as described above. The amounts of the ROS/RNS produced by two different cells, corresponding to cells (b) and (c) of Figure 6, are listed in Table 2 for comparison. Present data evidence that the productions of H2O2, ONOO-, and NO• by immunostimulated macrophages are not constant but evolve over time. For example, in the initial 10 min, cell 1 produced H2O2, ONOO-, and NO• in relative proportions of ca. 1:3:12; in the following 10 min, the ratio changed to ca. 1:8: 22. Furthermore, as we report above, it is again seen that the production of ROS/RNS varies rather significantly from one cell to another. Indeed, we would like to stress once again that such temporal variations of ROS/RNS release at the single cell 1418


level would not have been detectable if a large population of cells was studied. It follows that many commonly used biochemical methods, like the Griess method or chemiluminescence studies, which are either bulk methods or detect decomposition products (i.e., NO2- and/or NO3-), would not have allowed these temporal features to be monitored. Furthermore, as mentioned above, single cell studies using constant potential amperometry do not permit direct comparisons of individual responses at different potentials, and hence, the release composition could not be analyzed at the single cell level.

CONCLUSION We have demonstrated herein the biological interest and analytical feasibility of applying triple potential-step chronoamperometry at platinized carbon microelectrodes to monitor time dependence and composition of ROS/RNS release by single cells. Using constant potential amperometry, IFN-γ/LPS/PMA-stimulated macrophages were shown to release greater amounts of ROS/RNS than macrophages activated with IFN-γ and LPS only; this can be explained by the additional activation of NADPH oxidase, a superoxide-producing enzyme. The triple potential-step chronoamperometric method was then successfully applied to the simultaneous detection of NO•, ONOO-, and H2O2 fluxes released by IFN-γ/LPS/PMA-stimulated macrophages. Finally, direct comparison of amperograms obtained over a single cell evidenced significant temporal variations in the production of

the reactive species. Optimization of the triple potential-step method is, however, necessary for better reliability and precision. ACKNOWLEDGMENT This work was financially supported by CNRS (research units: UMR 8640, LIA XiamENS), Ećole Normale Supe´rieure, Universite´ Pierre et Marie Curie Paris VI, and the French Ministry of Research (MESR). Jean-Claude Drapier and Cećile Bouton from

ICSN, UPR 2301 CNRS (Gif sur Yvette, France) are thanked for help in cell culturing and discussions. A.C.W.K. would also like to thank Ećole Polytechnique and MESR for the award of an AMX PhD fellowship.

Received for review November 1, 2009. Accepted January 3, 2010. AC902486X


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Simultaneous Detection of Reactive Oxygen and Nitrogen Species

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