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Combined System for the Simultaneous Optical and Electrochemical Monitoring of Intra- and Extracellular NO Produced by Glioblastoma Cells Nazare´ Pereira-Rodrigues,† Naomi Zurgil,‡ Seung-Cheol Chang,§ James R. Henderson,§ Fethi Bedioui,*,† Calum J. McNeil,§ and Mordechai Deutsch‡
Laboratoire de Pharmacologie Chimique et Ge´ ne´ tique, Ecole Nationale Supe´ rieure de Chimie de Paris, UMR CNRS 8151/U INSERM 640, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France, Physics Department, The Biophysical Interdisciplinary Schottenstein Center for the Research and the Technology of the Cellome, Bar Ilan University, IL-52900 Ramat Gan, Israel, and School of Clinical & Laboratory Sciences, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, U.K.
A combined, optospectroscopic and electrochemical assay system for the simultaneous monitoring of intra- and extracellular production of biologically important species has been developed and assessed. The present model system evaluates intra- and extracellular nitric oxide produced by stimulated glioblastoma multiform cell line (A172). The production of endogenous NO was induced by phorbol-12-myristate-13-acetate and inhibited by Nω-nitro-L-arginine methyl ester. Intracellular production of NO was monitored via fluorescence image analysis using a 4,5-diaminofluorescein probe, while extracellular NO release was monitored via a chemically modified electrode, which was incorporated into an optically transparent cell chip. The results indicated that there was no mutual interference between the optical and electrochemical measurement systems. The response time of the combined optical/electrochemical system was found to be in the range of a few tens of seconds. Nitric oxide has been shown to be a highly diffusible and reactive molecule that plays a major role in several physiological processes, such as neurotransmission, immune response, and vasodilatation.1-3 In addition, the balance between intra- and extracellular NO levels plays a role in the control of major cell functions, including reactivity, proliferation, and, subsequently, apoptosis.2,4 Furthermore, in cells, NO may have coexisting beneficial and detrimental effects,4 and its metabolic interaction with other intra- or extracellular reactive molecules, such as reduced oxygen species (superoxide, hydrogen peroxide, etc.) is quite intricate.4-6 Due to the numerous physiological functions * Corresponding author. Phone: 33 155 42 63 88. Fax: 33 144 27 67 50. E-mail:
[email protected]. † Ecole Nationale Supe´rieure de Chimie de Paris. ‡ Bar Ilan University. § University of Newcastle upon Tyne. (1) Bolly, R. J. Mol. Cell. Cardiol. 2001, 33, 1897-1918. (2) Contestabile, A.; Ciani, E. Neuro. Chem. 2004, 45, 903-914. (3) Parker, L., Ed. Nitric Oxide. Part B, Physiological and Pathological Processes; Methods in Enzymology Vol 269; Part B, Academic Press: San Diego, CA, 1996. (4) Wink, D. A.; Mitchell, J. B. Free Radical Biol. Med. 1998, 25, 434-456. 10.1021/ac0483163 CCC: $30.25 Published on Web 03/30/2005
© 2005 American Chemical Society
performed by NO and the complexity of its interactions in biological systems, the proper investigation of its endogenous production and kinetics, in response to cell modulation, would benefit greatly from an ability to carry out simultaneous online monitoring of its generation and spatial distribution. Ideally, both intracellular and extracellular monitoring would be performed in the same cell sample. To address this issue it is necesssray to employ a combination of analytical techniques in order to upgrade existing systems presently based solely on optical fluorescence measurements. Extracellular measurement of NO efflux is now well documented, and electrochemical methods are recognized as the only analytical way to determine the concentration of NO in solution, without disturbing NO metabolism and the associated regulatory pathways.8-10 On the other hand, fluorescent techniques have been proposed to determine intracellular levels of NO released by mammalian cells.11 Attempts to perform unrelated asynchronous measurements of intra- and extracellular NO, utilizing different cell samples, have been previously made using electrochemical and fluorescence measurements. Recently, a few tentative studies have been conducted on the electrochemical determination of NO in complement to fluorescence measurements,7 but not as complementary simultaneous measurements. At the present time, no combined direct sensing means are available for the simultaneous local monitoring of both intra- and extracellular NO. The present study is dedicated, through a specially designed transparent optoelectronic cell chip,12 to allow the performance of real-time simultaneous monitoring of intra- and extracellular (5) Ford, P. C.; Wink, D. A.; Stanbury, D. M. FEBS Lett. 1993, 326, 1-3. (6) Grisham, M. B.; Jourd’Heuil, D.; Wink, D. A. Am. J. Physiol. 1999, 276, G315-G321. (7) Ishida, H.; Hirota, Y.; Higashijima, N.; Ishiwata, K.; Chokoh, G.; Matsuyama, S.; Murakami, E.; Nakazawa, H. Pathophysiology 2004, 11, 77-80. (8) Malinski, T., Electrochemical measurement of nitric oxide in biological systems. In Encyclopedia of Electrochemistry; Bard, A. J., Stratmann, M., Eds.; Wiley-VCH: Weinheim, 2002’ Vol. 9, pp 229-256. (9) Bedioui, F.; Villeneuve, N. Electroanalysis 2003, 15, 5-18. (10) Taha, Z., Talanta 2003, 61, 3-10. (11) Brown, L. A.; Key, B. J.; Lovick, T. A. J. Neur. Methods 1999, 92, 101-110. (12) Chang, S-C.; Pereira Rodrigues, N.; Zurgil, N.; Henderson, J. R.; Bedioui, F.; McNeil, C. J.; Deutsch, M. Biochem. Biophys. Res. Commun. 2005, 327, 979-984.
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NO, produced in and secreted from the same measured cell sample. The feasibility of the integrated concept and methodology in monitoring intracellular and leaked NO is tested here on 4,5diaminofluorescein (DAF-2)-stained A172 glioma cells, cultured on the cell chip platform with positioned modified electrodes, having organized layers of nickel tetrasulfonated phthalocyanine and Nafion, to measure the amperometric current related to the anodic oxidation of NO in solution at 0.75 V versus Ag/AgCl with the expected and requested performances in terms of sensitivity to NO and selectivity against interfering analytes, in particular nitrite, ascorbate, and L-arginine.13,14 As for the fluorescence measurements, the fluorophore DAF-2 has been used as an intracellular NO indicator. The membrane-permeable DAF-2 diacetate (DAF-2 DA) is first loaded into cells, where the ester bonds are hydrolyzed by intracellular esterases, generating DAF2, and remains accumulated in the cell. DAF-2 is nonfluorescent until oxidized by NO. In a wide range of dye concentrations, the evolution of the measured signal is linear, indicating that elevation in the fluorescence intensity truly reflects an increase in NO production.15,16 Although it has been recently shown that this diaminofluorescein derivate interacts not only with NO but also with other compounds released in the cell, it has been concluded that, following careful experimental design with NO inhibitors, DAF-2 is a good choice for intracellular NO probing.17-19 EXPERIMENTAL SECTION Chemicals. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, penicillin, streptomycin, and Hanks’ balanced salt solution (HBSS) were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). DAF-2 DA was purchased from Calbiochem (La Jolla, CA). DAF-2 DA stock solution (5 mM) in DMSO (storage at -20 °C) was diluted before measurement in HBSS to a final concentration of 10 µM and was kept in the dark at 4 °C. Phorbol 12-myristate 13-acetate (PMA), tetramethylrhodamine methyl ester (TMRM), and Nωnitro-L-arginine methyl ester (L-NAME) were obtained from SigmaAldrich (St. Louis, MO). PMA stock solution in DMSO (1 mg/ mL) was kept at -20 °C and was diluted before measurement in HBSS to a final concentration of 4 µg/mL. L-NAME was stored as a powder (-20 °C), and a stock solution (1 mM) was prepared de novo in water, kept on ice for 1 day, and diluted before measurement in HBSS to a final concentration (10 µM). Nickel tetrasulfonated phthalocyanine (NiTSPc) (Sigma-Aldrich) and Nafion (Sigma-Aldrich), were reagent grade and used as received. Cell Culture. A glioblastoma multiform cell line (A172) suspended in DMEM containing 10% FBS, 1 mM sodium pyruvate, 200 IU/mL penicillin, and 200 g/mL streptomycin was seeded onto the surface of sterile and circular glass slides (diameter 1 cm, thickness 250 µm), which were placed in the bottom of 24(13) Pontie´, M.; Gobin, C.; Pauporte´, T.; Bedioui, F.; Devynck, J. Anal. Chim. Acta 2000, 411, 175-185. (14) Brunet, A.; Pailleret, A.; Devynck, M. A.; Devynck, J.; Bedioui, F. Talanta 2003, 61, 53-59. (15) Kojima, H.; Sakurai, K.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Chem. Pharm. Bull. 1998, 46, 373-375. (16) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453. (17) Brown, L. A.; Key, B. J.; Lovick, T. A. Neurosci. Lett. 2000, 294, 9-12. (18) von Bohlen und Halbach O. Nitric Oxide 2003, 9 (4), 217-228. (19) Strijdom, H.; Muller, C.; Lochner, A. J. Mol. Cell. Cardiol. 2004, 37 (4), 897-902.
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well cell culture plates and incubated at 37 °C with 5% CO2 until attaining confluence. Morphometric image analysis confirmed that the A172 cell line was phenotypically astrocytic. Staining the glioma cells with TMRM revealed high fluorescence intensity (FI), which was indicative of a high mitochondrial membrane potential and high energy level of the cells. Cell Preparation and Staining. A172 cells attached to the glass slide at the bottom of a 24-wells microplate were washed with 500 µL of HBSS modified to contain 10% glucose at pH 7.4. Cells were loaded with DAF-2 (500 µL, final concentration 10 µM in modified HBSS) for 15 min at 37 °C with 5% CO2. After staining incubation, the stained cells on the glass were washed twice with HBSS (500 µL) and immediately transferred to the opticalelectrochemical cell chip for optical-electrochemical measurements. Optical System and Operating Software. The optical measurements were performed using an inverted microscope, an XY stage, and a digital camera. The entire system was controlled by a specific homemade software package. In brief, a fully motorized inverted microscope IX-81 was equipped with a 100-W Hg lamp light source (Holder U-LH100HG), one internal and three external six-position filter wheels, condenser lens, and an adjustable/ controllable white light source for a transmitted light bright-field observation. The main microscope controller and PC interface was 1 × 2-UCB. The filters used in this study included a broad-band excitation filter (450 ( 35 nm), a long-pass dichroic mirror with a reflection/transmission cutoff wavelength of 495-500 nm, and an emission long-pass filter, having a cutoff wavelength of 520 nm. The camera is a cooled, highly sensitive digital camera, ORCA II, C4742-97, manufactured by Hamamatsu. The controlling software provides microscope automatization, enables experiment sequencing, image acquisition, and management, image acquisition, management and processing, image and experiment analysis. The software was designed and compiled by our Center’s computer experts, utilizing the IPP (Image Pro Plus) as a system environment. The optical data acquisition and central steps of the analysis procedures were as follows: Each set of image acquisition was initiated by first acquiring the bright-field image of a chosen view field, followed by the acquisition of a few fluorescent images, each taken at a different preset time point. The objects of interest in the fluorescent wide-field image were then determined by mapping their outlines, which were defined by their bright-field images, on the interrogated fluorescent field image. Next, the fluorescent background, which was determined by averaging over the FI detected by camera pixels spaced between the outlined regions, was subtracted from the fluorescent image. It should be emphasized that background signal determination and subtraction were performed separately for each of the acquired fluorescent field images. Data are presented as FI of individual cells, or mean FI of a cell population ( SD. Electrochemical Amperometric Measuring System and Procedure. Amperometric measurements were performed using structured millimetric disk electrodes, consisting of three working electrodes (2 made of gold and 1 of carbon, d ) 1 mm) (CRL). Recent studies by us and our collaborators have reported the use of such a planar electrode array configuration for the electrochemical measurement of extracellular NO, superoxide, and
Figure 1. Integrated optical-electrochemical experimental device for simultaneous detection of intra- and extracellular NO in vitro.
glutamate production.20-22 However, these reports did not attempt simultaneous intracellular optical measurement using the specific format described for the first time for NO detection in the present study. In the present study, only the carbon working electrode of the array was specifically modified by electrodepositing NiTSPc and Nafion for NO detection, as described previously.9,13,14,22 An external Ag/AgCl electrode was used as a reference and counter electrode. Electrochemical detection of NO was performed by using a Petit Ampe`re potentiostat (BAS). The potential applied was 0.75 V versus Ag/AgCl which corresponded to NO oxidation. Integrated Optoelectrochemical Cell Chip. The combined system developed in this study is similar to that reported by us for the determination of intra- and extracellular superoxide anion.12 In brief, it comprises a specially designed 24-well microplate, where on each of the bottom surfaces of the transparent well, the structured 1-mm disk electrodes (described above) are positioned. The diameter of a well is designed to accommodate a circular glass coverslip, on which cells are grown. The latter is placed upside down, so that the attached cells face the electrodes. To avoid direct contact between cells and electrodes and to ensure a constant recurrent gap between the two in all wells, narrow (1 mm) slices of microscope cover glass (1 mm width, 160 µm height) were positioned as spacers between the glass coverslips containing the cells and the well bottom electrodes. The transparency of the well bottom, as well as the proximity of the attached cells to the bottom of the wells, permitted the performance of optospectroscopic measurement, by using an inverted microscope. Thus, while performing the amperometric measurements of extracellular NO, the FI signals of cells observed between electrodes were obtained. A schematic representation of a cell chip well is shown in Figure 1. All measurements were carried out in 500 µL of HBSS with 10% glucose at pH 7.4, which were found to represent the optimal (20) Oni, J.; Pailleret, A.; Isik, S.; Diab, N.; Radtke, I.; Blo¨chl, A.; Jackson, M.; Bedioui, F.; Schuhmann, W. Anal. Bioanal. Chem. 2004, 378, 1594-1600. (21) Castillo, J.; Isik, S.; Blo ¨chl, A.; Pereira Rodrigues, N.; Bedioui, F.; Cso ¨regi, E.; Schuhmann, W.; Oni, J. Biosens. Bioelectron. 2005, 20, 1559-1565. (22) Chang, S-C.; Pereira Rodrigues, N.; Henderson, J. R.; Cole, A.; Bedioui, F.; McNeil, C. J. Biosens. Bioelectron. In press.
conditions for simultaneous measurement (data not shown). Indeed, cells could remain active for over 1 h at room temperature. All manipulations were conducted on the cells within the wells by injecting the relevant biochemical into the well. For each such manipulation, the stimulation conditions for yielding both detectable optical and electrochemical signals were determined via a set of titration experiments (data not shown). For example, after achieving a stable baseline with cells suspended in 500 µL of HBSS, the production of NO was induced by adding a volume of 500 µL of PMA solution into a well, yielding a final working PMA concentration of up to 2 µg/mL. As a negative control, inhibition of NO production was carried out by incubating the cells with L-NAME for 15 min prior to PMA administration. The simultaneous monitoring of intra- and extracellular NO produced by glioblastoma A172 cell line, as well as the experiments related to activation and inhibition of NO production, were all carried out in real time, utilizing the same cell sample. While the amperometric measurements were carried out continuously, the optical measurements were performed sequentially, to minimize fluorophor bleaching. RESULTS AND DISCUSSION To negate a possible cross talk, mutual interference between the electrochemical and optical techniques, or both, each of the subsystems was separately examined under its complementary system environment. Regarding the electrochemical subsystem, in particular, three main issues were examined: the possible direct effects of DAF-2 on the amperometric measurements due to extracellular dye remaining after washing; the indirect effect of intracellular DAF-2-NO interactions, which might interfere with NO efflux; and finally, the possible photoeffects (ionization, photochemical processes, heating) induced in the illuminated electrode by the excitation light. Three different amperometric measurements of extracellular NO, each on a different batch (slide) of PMA-activated A172 glioblastoma cells, were performed. In the first measurement, cells were neither stained with DAF-2 nor illuminated (control). In the second, cells were stained with DAF-2 and non-illuminated, while in the third, they were unstained and illuminated. A typical set of Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
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Figure 2. Amperograms of extracellular NO response following the addition of PMA to unstained nonilluminated A172 cells (a), DAF-2stained non-illuminated cells (b), and unstained and illuminated cells (c).
results are depicted in the amperograms reported in Figure 2. The variance in the absolute maximum current amplitudes observed between the three conditions is related to the fact that A172 cell populations are not homogeneous, and cells vary in their degree of confluence and absolute cell number between slides, as well as in their physiological status. The highest current obtained under microscope illumination may be due in part to light-induced elevation of temperature in the surrounding solution, which could accelerate the NO diffusion process within the electrolyte. Nevertheless, the three time-dependent curves in Figure 2 display similar behavior and are similar in appearance to typical amperometric responses related to extracellular NO production obtained under standard conditions.8-10 Calibration of the NO-sensing electrode was performed in phosphate buffer solution (0.1 M, pH 7.4) by successive additions of NO stock solution prepared from NO gas, in a separate experiment before in vitro application (see Supporting Information, Figure 1s), as previously reported.13,14,22,23 The calibration curve 2736
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obtained showed good linearity up to 3.6 µM NO with a measured sensitivity of 93.6 nA/µM‚cm2. Given the well reported systemto-system variability and the difficulties in absolute measurement of NO concentration due to its high reactivity, the electrochemical measurements reported can only provide information on relative rates and dynamics. In the present study, therefore, on the basis of the independent calibration data, it can be estimated that the overall concentration of NO detected by the electrode under the experimental conditions employed lies in the order of 0.68-2.44 µM. The degree of similarity between the three curves reported in Figure 2 was further analyzed by comparing their current ratios, which were calculated for each of the curves at the same two time points, t1 ) 15 s and t2 > t1, after the PMA introduction Table and by defining this amperometric ratio parameter as AMR ) I(t2)/I(t1). The AMR values, calculated for three sets of time points for each of the three curves, are listed in Table 1. It can be seen clearly that the variations between corresponding AMR values are minimal, showing no significant evidence for interference by the fluorescence measurement environment (intracellular DAF-2 and illumination) during the electrochemical measurements. In addition, the fact that the amperograms of stained and unstained PMA-activated cells are similar negates the possibility of an interfering influence of extracellular DAF-2 remaining after washing. Concerning the fluorescence measurements, Figure 3 shows transmitted light and fluorescent images of PMA-activated, DAF-2 stained A172 glial cells. The cells are attached to the slide and face the NO-specific electrode (not seen in the figure). As it can be seen, the unoccupied area between the cells (Figure 3b) does not fluoresce, thus indicating zero or undetectable levels of autofluorescence of the glass or plastic materials comprising the cell chip. The electrode material is also not fluorescent when illuminated (data not shown). Positive DAF-2 staining was observed in most of the cells, and image analysis of the overall field of measurement indicates that the cells were heterogeneous with respect to their total FI. Moreover, the subcellular distribution of DAF-2 dye in single cells was also found to be heterogeneous and was chiefly localized in the cytoplasm. In the present optical measurement assay, the generation of intracellular NO was detected as early as 15 s after PMA introduction. The reason for this was purely technical: the physical introduction of PMA into the cell chip, as in many other applications, defocused the image. It could only be refocused manually, a step that lasted about 15 s. Consequently, the time dependence in all of the curves of FI(t) (see Figures 4 and 5) during the first 20 s after PMA administration seems to lag in comparison to the sharp rise of the related electrochemical signals shown in the previous figures. The FI(t) curves of three individual PMA-activated, DAF-2stained A172 glial cells, all simultaneously measured, are shown in Figure 4A. For each of the three cells, the FI data were acquired sequentially every 20 s, during the total of 90 s starting from PMA application (zero time point). As it can be seen, the three curves differ in the maximum values and appearance, indicating a high variability in the rate and level of NO generation in individual cells. (23) Lantoine, F.; Tre´vin, S.; Bedioui, F.; Devynck, J. J. Electroanal. Chem. 1995, 392, 85-89.
Figure 3. Transmitted light (a) and fluorescence (b) micrographs of individual A172 glioblastoma cells grown on a testing glass platform and stained with DAF-2. Table 1. Amperometric Ratios AMRs Calculated for Three Different Times Following Application of PMA to Unstained and Stained Cells, without and with Illumination (data taken from Figure 2) AMR experimental conditions
I(t2 ) 45 s)/ I(t1 ) 15 s)
I(t2 ) 60 s)/ I(t1 ) 15 s)
I(t2 ) 75 s)/ I(t1 ) 15 s)
unstained and non-illuminated cells DAF-2 stained and non-illuminated cells unstained and illuminated cells
0.73 0.88 0.82
0.66 0.80 0.76
0.60 0.71 0.70
Finally, as an additional control, the same volume of DMSO dissolved in HBSS was added in the absence of PMA. As can be seen (Figure 4A, dashed line), treatment of DAF-2-stained cells with DMSO only did not induce any alterations in the populationaveraged intracellular FI signal. The decay in FI seen in Figure 4A is most probably a result of fading. This supposition was verified by performing three experiments, similar to those described above, each on a different sample (a different slide containing A172 cells). The only differences between the three experiments were the starting point of measurements (after PMA application) and the total number of measurements. Since the illuminating dose per measurement was constant, the sample measured more times received the highest excitation dose. As it can be seen in Figure 4B (where each mark represents the averaged FI over all cells in the interrogation field), the maximum total FI in each experiment was similar and independent of the measurement starting point. This indicates no leakage of intracellular dye during the measurement. Further, in each of the three curves, as the time elapses and the total illumination dose increases, the FI signal decays. The fact that the less the illumination the slower is the decay clearly indicates the involvement of fading effects. Similar results were obtained when A172 cells were activated on a standard tissue culture plate (data not shown), indicating that the presence of NO electrodes did not interfere with the optical measurements. In addition to the above experiments, the simultaneous monitoring of intra- and extracellular NO produced by glioma A172 cell line, as well as the activation and inhibition of NO production, were all carried out in real time, using the same cell samples. Representative plots of simultaneous optical-electrochemical timedependent measurements of intra- and extracellular NO are shown in Figure 5. While both the amperometric and fluorescence signals
increase following the application of PMA, preincubation of cells with L-NAME for 15 min abolished the effect. Since L-NAME is a specific inhibitor of NO production, this result clearly demonstrates the specificity of the measured signal. Nevertheless, the increase of the amperometric signal is sharper and earlier than that of the fluorescence signal. This can be wrongly interpreted as if the oxidation of DAF-2 by intracellular NO lags after the electrochemical response. However, the fact that the total FI maximums in different preparations are similar and independent of the measurement starting point (Figure 4B) suggests that, if data acquisition could be started immediately after PMA administration, a sharp rise in the FI signal would be observed and would probably precede the amperometric signal. (Note that the amperometric responses shown here are produced by subtracting the signal recorded upon injection of the same volume of solvent (DMSO) with and without PMA.) Finally, the experimental conditions under which the results shown in Figures 2c and 5 were obtained are identical, except that in the latter case cells were stained. Consequently, the high degree of similarity observed between these two amperograms (Figure 2c and upper panel of Figure 5) clearly negates the possibility that, under the experimental conditions employed in this study, intracellular DAF-2 influences NO efflux. This opens up the way to simultaneous user-friendly optical and electrochemical measurements. In addition, although this aspect has not been fully assessed, it can be clearly stated that both the amperometric and the fluorescence signals demonstrate similar trends when monitoring either the rate of NO generation or its level following inhibition. It is recognized, however, that this finding may not hold true for other cell types or staining conditions, and these should be examined on a caseby-case basis. Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
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Figure 5. Simultaneous optical-electrochemical time-dependent fluoroamperograms of intra- and extracellular NO levels, produced by the same DAF-2-stained A172 glioblastoma cell population measured in the integrated optical-electrochemical subsystems. While both the amperometric and fluorescence signals increase following application of PMA (graphs a and c), the cells’ preincubation with L-NAME for 15 min abolished the effect (graphs b and d). Dashed and solid lines in the lower panel denote fitting curves.
Figure 4. Monitoring of intracellular NO generation in PMA stimulated individual DAF-2-stained A172 cells via sequential FI measurement. (A) Representative FI(t) curves of three individual DAF2-stained A172 glioblastoma cells (full lines and symbols). The control measurement is represented by the curve of averaged FI(t) signal over a DMSO-treated DAF-2-stained A172 cell population (dashed line and open circles). (B) Representative population-averaged FI(t) response curves of NO generation in three different samples (cellcontaining slides) of DAF-2-stained A172 glioblastoma cells. The illumination and measurements of each of the three samples, shown in the lower panel, were started at a different time after PMA introduction: 15 (2), 30 ([), and 60 (9) s. In both panels A and B, the bars represent the corresponding SD for each population measurement and the arrows mark the time of PMA administration. Solid and dashed lines denote fitting curves.
CONCLUSION The present study demonstrates, for the first time, the possibility of performing simultaneous detection of intracellular production and diffusion of NO into the extracellular space. The added value of the integrated optoelectrochemical cell chip methodology is undoubtedly linked to the fact that the amperometric measurements that occur on a cell-average basis may significantly complement optical measurements of individual cells and subpopulations. The amperometric signal can thus be normal-
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ized and better interpreted thanks to optical image analysis, which permits the assessment of the number and density of cells present in the measured sample, identifying individual cells or subsets of cells according to their NO production intensity or rate and also according to their physiological status. In addition, the proximity between the cell and the electrode can be easily controlled and taken into account in evaluating the amperometric signal, since the optical system can provide additional cytometric parameters for complex multiparametric experiments. The present study strongly suggests the possibility of incorporating extracellular biomolecule monitoring, via electrochemical measurements, into existing optical-based high-content screening protocols, thus adding a new dimension to these systems. ACKNOWLEDGMENT We acknowledge the financial support from the European Commission and the Horowitz foundation for the CELLSENS project (QLK-CT 2001-00244). Dr. M. Jackson and Dr. S. Dennison (CRL, U.K.) are acknowledged for providing the millimetric disk electrode structures. 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 November 15, 2004. Accepted February 11, 2005. AC0483163
