Stationary-State Oxidized Platinum Microsensor for Selective and On

Anal. Chem. 2001, 73, 3965-3974

Stationary-State Oxidized Platinum Microsensor for Selective and On-Line Monitoring of Nitric Oxide in Biological Preparations Andrea Cserey† and Miklo´s Gratzl*,†,‡

Department of Biomedical Engineering and Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106

Despite the multifaceted biomedical significance of NO, little progress has been achieved so far in the quantitative understanding of the signal transduction mechanisms where NO is involved. To help progress in this area, we propose a simple electrochemical NO sensor here, consisting of a glass sealed platinum microdisk electrode coated with cellulose acetate to reduce both surface fouling by proteins and response to potential interferences. A differential amperometry protocol is optimized to improve selectivity and provide a stationary oxidation state of the platinum surface, which prevents loss in sensitivity during long-term use. We found the oxidation of NO by O2 second order in [NO] with a rate constant of (8.0 ( 0.4) × 106 M-2 s-1, in good agreement with literature data obtained by other than electrochemical methods. The release rates of NO detected in cultures of activated macrophages were on the order of 20 pmol/ (106cells s) and correlated well with the nitrite content determined by the spectrophotometric Griess assay. Nitric oxide (NO) has been recognized as a fundamental biological messenger in the central and peripheral nervous systems and in the vascular endothelium.1 NO is also involved in host defense mechanisms and plays a critical role in inflammatory and degenerative diseases. Certain metabolites of NO (peroxinitrite, ONOO- and nitrous anhydride, N2O3) can be cytotoxic or mutagenic when present at sufficiently high concentrations.2,3 The physiological and pathophysiogical effects of NO depend on its actual spatial and temporal distribution. Little is known quantitatively about the factors determining this distribution in the implicated tissues, and mathematical modeling of NO signal transduction pathways is hampered by the lack of information on production and degradation rates. Selective and in situ monitoring of NO providing reproducible and quantitative data is therefore essential to draw solid conclusions on its signaling functions and their regulation. †

Department of Physiology and Biophysics. Department of Biomedical Engineering. (1) Nathan, C. FASEB J. 1992, 6, 3051-64. (2) Lewis, R. S.; Tamir, S.; Tannenbaum, S. R.; Deen, W. M. J. Biol. Chem. 1995, 270, 29350-5. (3) Gow, A. J.; Thom, S. R.; Ischiropoulos, H. Am. J. Physiol. 1998, 274, L1128. ‡

10.1021/ac010123h CCC: $20.00 Published on Web 07/18/2001

© 2001 American Chemical Society

Electrochemical oxidation or reduction is the only presently available approach to measure directly and in situ (on-line) the transient concentrations of NO with sufficient spatial and temporal resolution. There are three types of sensors utilizing the electrooxidation of NO available commercially. One of them is a Clarktype platinum gas electrode.4 Despite its low detection limit (10 nM), practical applications are limited due to the fragility of the sensor. A further problem is signal saturation at concentrations higher than 1 µM or at longer measurement times, probably caused by accumulation of nitrite in the internal compartment. Carbon fiber electrodes modified with immobilized Niporphyrin and coated with Nafion have widely been used in vivo, with a detection limit similar to that of the gas electrode.5-7 The Nafion coating reduces interference from nitrite and ascorbate. Ni-porphyrin was postulated to enhance the electrochemical oxidation of NO relative to that of potential interferences and thereby to provide selectivity. Later studies revealed that Ni2+ is not required in the porphyrin,6 and the oxidative electrochemical pretreatment usually applied on carbon fibers8 can provide the same sensitivity for NO as the Ni-porphyrin coating.6,7 Specificity for NO on these oxidized (pretreated) fibers is usually increased by elaborate immobilization of several layers of polymer films6,9,10 to exclude interfering anions and cations. A third approach, a carbon fiber electrode, directly coated with a hydrophobic membrane, is available from World Precision Instruments (ISO-NOP200). However, the catalog11 does not provide information on its design and connection to a reference electrode. Gold is also a suitable material for NO electrooxidation. Nitric oxide oscillations in optic nerve cells have been measured in vivo by a recessed gold microdisk coated with Nafion.12 However, the applied single-potential amperometric detection could not possibly (4) Shibuki, K.; Okada, D. Nature 1991, 349, 326-8. (5) Malinski, T.; Taha, Z. Nature 1992, 358, 676-8. (6) Wink, D. A.; Christodoluou, D.; Ho, M.; Krishna, M. C.; Cook, J. A.; Haut, H.; Randolph, K.; Sullivan, M.; Coia, G.; Murray, R.; Meyer, T. Methods Enzymol. 1995, 7, 71-7. (7) Lantoine, F.; Trevin, S.; Bedioui, F.; Devynck, J. J. Electroanal. Chem. 1995, 392, 85-90. (8) Gonon, F.; Fombarlet, C.; Buda, M. J. Anal. Chem. 1981, 53, 1386-9. (9) Mitchell, K. M.; Michaelis, E. K. Electroanalysis 1997, 10, 81-8. (10) Park, J. K.; Tran, P. H.; Chao, J. K. T.; Ghodadra, R.; Rangarajan, R.; Thakor, N. V. Biosens. Bioelectron. 1998, 13, 1187-95. (11) World Precision Instruments, Catalogue 2000, Sarasota, FL. (12) Buerk, D.; Riva, C. E. Microvasc. Res. 1998, 55, 103-12.

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provide selectivity for NO in the presence of potential interferences (e.g., catecholamines, purins, and indoleamines). Another problem with oxidative amperometry on noble metal electrodes (gold and platinum as well) is surface oxidation: it can proceed over time to such an extent that NO electrooxidation becomes inhibited.13 Electrochemical reduction of NO can provide better specificity since most potential interferences cannot be reduced.14 On the other hand, electroreduction of oxygen may interfere with NO detection. Besides, the concurrently generated oxygen radicals may be toxic, as well as the coating electropolymerized from [Cr(v-tpy)2]3+ applied to catalyze NO reduction. Despite the great potential in electrochemical sensing and the large body of work on different technical approaches, little progress has been achieved so far in the quantitative understanding of the signal transduction mechanisms where NO is involved. One of the reasons is that the actual NO levels reflect its rate of biosynthesis as well as degradation, coupled with complex mass transport. The other difficulty is related to NO sensing itself: the sensors currently available all suffer from some technical drawbacks or insufficient understanding of their theoretical basis, as reviewed above. The approach proposed here may help solving these problems. We have developed a glass-sealed and cellulose acetate-coated platinum microdisk electrode utilizing NO oxidation with differential pulse amperometry. The polished flat microdisk surface surrounded by a relatively thick insulating layer is meant for flat preparations such as brain slices, endothelium, or a monolayer of cultured cells. The approach has several advantages. Compared to a gas electrode or sensors based on carbon fiber, this electrode is more durable and its tip cannot be damaged easily during the measurements. The fabrication procedure is quick and yields practically identical electrodes. The polished, glass-insulated microdisk design allows for an effective coating procedure as well. This is in contrast to carbon fibers, where nitrite exclusion and selectivity can be compromised due the difficulties in achieving a uniform Nafion coating.15 Selectivity for NO is achieved here by a combination of an appropriate differential amperometry protocol and size exclusion of the cellulose acetate coating, without the need for polymer immobilization or electrode pretreatment. The voltage protocol also ensures a stationary oxidized surface of the platinum and therefore prevents loss in sensitivity during longterm use. To test the approach, NO release from macrophages was monitored in situ. EXPERIMENTAL SECTION Apparatus. The electrochemical measurements were performed with BAS (BioAnalytical Systems) 100B/W and CH 660 (CH Instruments) electrochemical analyzers. All potentials are expressed versus a junctionless Ag|AgCl reference electrode in the given electrolyte (containing 0.15 or 0.12 M chloride). The spectrophotometric assays were run on a tunable microplate reader (Molecular Devices) and a Shimadzu UV 1601 spectrophotometer. Procedures. Data analyis and numerical procedures were carried out with SigmaPlot 3.0 (Jandel Scientific). (13) Dutta, D.; Landolt, D. J. Electrochem. Soc. 1972, 119, 1321-5. (14) Maskus, M.; Pariente, F.; Toffanin, A.; Shapleigh, J. P.; Abruna, H. D. Anal. Chem. 1996, 68, 3128-34. (15) Pihel, K.; Walker, Q. D.; Wightman, R. M. Anal. Chem. 1996, 68, 2084-9.

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Electrode Fabrication and Coating. A platinum wire (99.99%, Goodfellow) with a diameter of 25 or 75 µm was sealed into a glass capillary (i.d. 0.58 mm, o.d. 1 mm, A-M Systems) over the flame of an alcohol burner. The protruding part of the platinum was then cut, and the tip was polished with sandpaper (320 grit) and fine grade polish paper (600 and 800 grit, all from Buehler). This procedure yielded a flat glass insulation (∼400 µm thick) around the exposed microdisk. Silver epoxy conducting polymer (H-20E, Epo-Tek) was used to make the electrical connection. Bare electrodes were cathodically pretreated before each voltammetry and chronoamperometry (but not differential pulse amperometry) experiment by constant-current electrolysis at 0.3 mA/cm2 for 2 min. Prior to coating it with cellulose acetate (CA) or Nafion, the electrode surface area was determined by chronoamperometry in 5 mM K3[Fe(CN)6]/0.5 M KCl, applying the microdisk equation16 to the current. The values were within 8% of the nominal surface area (4.4 × 10-5 cm2), indicating that the fabrication procedure yields practically identical, disk-shaped electrodes. The sealed microdisk was then coated with cellulose acetate by immersing the sensor tip into a 2% (w/w) solution of CA in acetone and then drying it with a heat blower (∼60 °C) for 1 min. Nafion coatings were made similarly from a 5% (w/w) solution of Nafion (DuPont) in ethanol. The membrane thickness (d) was calculated also from chronoamperometry results in 5 mM K3[Fe(CN)6]/0.5 M KCl. The stationary current (Itf∝) gave a reasonable estimate for the thickness d ) FADmKc*/Itf∞, where c* is the bulk concentration, Dm is the diffusion coefficient in the membrane, and K is the partition coefficient between the polymer phase and the background electrolyte solution. This estimation is valid if DmK is at least 1 order of magnitude less than the diffusion coefficient in the background electrolyte. Under these conditions, the current is limited by transport through (and within) the membrane and thus it is not dependent on the radius of the electrode. The partition coefficients were determined by spectrophotometry from the absorbance of K3[Fe(CN)]6 at 420 nm17 and found to be 0.06 ( 0.01 for Nafion and 0.16 ( 0.02 for CA. The diffusion coefficients in the membranes were calculated from the transient chronoamperometry current at 60e t e 110 ms, when the flux is limited by planar diffusion inside the membrane and can be described by Cottrell’s equation: I ) nFA(Dm)0.5Kc/(πt)0.5. The coefficients were found to be (8.7 ( 0.9) × 10-7 cm2/s in CA and 1.0 ( 0.2 × 10-6 cm2/s in Nafion, satisfying the above criterion. The calculated thickness of the membranes varied between 11 and 15 µm for CA and between 8 and 12 µm for Nafion. Procedures with Nitric Oxide. Calibrations for NO are usually done by adding small volumes of a stock, saturated solution into the calibration buffers with a gastight syringe. The stock solution is made by bubbling pure NO gas directly from a cylinder.4-9,12 Since the reaction of NO with O2 is fast in gas phase and in solution at millimolar concentrations18 (the saturation level is 1.93 mM at 25 °C 19), preparing and handling the stock solution would require extreme care to exclude even traces of oxygen. Therefore, (16) Shoup, D.; Szabo, A. J. J. Electroanal. Chem. 1982, 140, 237-45. (17) Dewulf, D.; Bard A. J. J. Macromol. Sci. 1989, A26, 1205-9. (18) Caccia, S.; Denisov, I.; Perrella, M. Biophys. Chem. 1999, 76, 63-72. (19) Handbook of Physical Quantities; Grigoriev, J. S., Meilikov, E. Z., Eds.; CRC Press: Boca Raton, FL, 1997.

we have chosen to generate NO chemically from KNO2 by an excess amount of ascorbic acid,20 which readily reduces nitrite to NO at a pH lower than 3. A solution containing 30 mM KNO2, 30 mM HCl, and 90 mM NaCl was purged with nitrogen for 15 min in a rubber-sealed glass vial (1.5-mL gas chromatography sample vial, Alltech) to remove dissolved oxygen. Then crystalline ascorbic acid was added to make a final concentration of 30 mM, and the vial was sealed. When bubble formation stopped, the concentration of NO was determined by drawing samples from the solution with a gastight syringe (Hamilton) and analyzing it according to Griess.7 The concentration was found to be 2.4 ( 0.3 mM (at room temperature) and constant for at least 30 min, indicating that NO did not escape from the vial. After 30 min, the vial was again purged with nitrogen for 5 min to remove NO and the solution was tested for the presence of nitrite. The concentration of the remaining nitrite was always less than 2% of the initial NO concentration, indicating that ascorbic acid in excess sufficiently preserves NO under these conditions. The saturated solution of NO was used either directly (i.e., electrodes immersed into the reaction vial) or diluted up to 10 times with deoxygenized buffers. During the measurements, no further care was taken to exclude oxygen from the solution. Chronoamperometry experiments were carried out with a 75µm-diameter bare electrode in saturated solutions of NO generated by a stoichiometric amount of ascorbic acid in this case. The potential was stepped from 500 to 700 mV. The current was corrected for the background recorded in the same solution after NO was removed by purging with nitrogen. The number of electrons transferred in NO electrooxidation was calculated from both the intercepts and the slopes of the current versus t-1/2 plots based on the microdisk equation,16 using data obtained between 10 and 40 ms. The diffusion coefficient of NO was taken as D ) 1.54 × 10-5 cm2/s 19 (at infinite dilution). Electrode calibrations were done by adding small volumes of the saturated NO solution into the calibration buffers with a gastight syringe. The final nitrite concentration in the buffers was determined by the Griess assay. The measured values correlated within 5% to the calculated ones based on the concentration of the saturated solution. Cell Culture. Two murine macrophage cell lines, BAC-1 and RAW 264.7, were cultured in Dulbecco’s modified Eagle’s medium containing 10% iron supplemented calf serum and 1% penicillin/ streptomycin mixture (Sigma). Cell density was kept ∼106 cells/ mL. The medium on the BAC-1 cells contained also 25% L-cell conditioned medium to provide macrophage colony stimulating factor. Some BAC-1 macrophage cultures were activated by a 15-h incubation with the same medium also containing 500 ng/mL lipopolysaccharide (LPS, from Escherichia coli K12). RAW cultures needed only 5.5 h of incubation with LPS. Prior to the measurements, the medium was washed out and exchanged for a balanced buffer containing (in mM) NaCl (108), KCl (5), CaCl2 (1.5), MgCl2 (0.5), KH2PO4 (0.5), Na2HPO4 (0.5), HEPES (25), glucose (10), and in some cases, L-arginine (2). The pH was adjusted to 7.0 with NaOH. Immediately after the exchange, the buffer was tested for nitrite. (20) Beake, B. D.; Moodie, R. B.; Smith, D. J. Chem. Soc., Perkin Trans. 2 1995, (7), 1251-2.

In Situ Monitoring. The measurements were carried out at room temperature in 35-mm dishes containing a total buffer volume of 2 mL above the cells attached to the bottom. The coated working electrode was soaked in the buffer overnight before the experiments. The reference electrode was also coated with CA. The buffer was continuously stirred above the cells by bubbling air through a thin Teflon tube (i.d. 0.5 mm). The flow rate of air was adjusted with a Cole Palmer N-032-15 flowmeter to such a rate that the buffer was well stirred without decreasing the solubility of NO at micromolar concentrations. We tested the loss of NO under these conditions by the Griess assay and found less than 10% decrease at concentrations below 8 µM. The electrodes were immersed to ∼1 mm above the cell layer. The time constant of the current response to a stepwise change in the concentration with this stirring rate was much smaller (3 s) than the time constant of biological release (minutes), and therefore, the effect of stirring on the concentration profiles can be neglected. At the end of each experiment, density and viability of the cells were determined by the Trypan blue staining method with a standard hemacytometer (Sigma) and the nitrite content in the balanced buffer was measured. Despite the time spent at room temperature, at least 85% of the cells always remained viable. Reagents. Millipore deionized water and Aldrich or Sigma analytical grade chemicals were used in all experiments, except for L-N-nitroarginine methyl ester (L-NAME), which was purchased from Calbiochem. Griess reagents were 2 g/L sulfanilamide in 3 M HCl and 1 g/L N-(1-naphthyl ethylenediamine hydrochloride in deionized water. RESULTS AND DISCUSSION Electrooxidation of NO and Nitrite on Platinum. We have chosen to develop a platinum microdisk electrode to detect NO selectively and on-line for three reasons. First, a disk-shaped electrode is more advantageous to study efflux from a flat layer of cell populations, such as endothelial cells or cultured macrophages, than a cylindrical fiber.21 Second, platinum does not require surface pretreatment for NO electrooxidation, unlike carbon. Third, a durable insulated microdisk can be fabricated from platinum easily without specialized equipment or the use of hazardous materials (see Experimental Section). The mechanism of NO electrooxidation on platinum is not fully understood. Systematic studies have been carried out mostly in strong acidic electrolytes.13,22 Under these conditions, NO oxidation is stepwise and irreversible.13 The reaction rates of both steps in the potential region of oxygen coverage were found to strongly decrease with increasing oxidation state of the platinum. We carried out further voltammetric studies (Osteryoung square wave voltammetry, OSWV) on both nitrite and NO electrooxidation in order to find the optimal conditions for selective and on-line monitoring of NO. We found the oxidation (peak) potential for nitrite is independent of pH in the range of 4.3-9.0 (i.e., well above the pKa ) 3.3 of HNO2). OSWV, which gives a peak response at the half-wave potential, was applied to find the range of fastest increase in the oxidation rates (Figure 1). Electrochemical cleaning (see Experimental Section) was necessary before each run to ensure a (21) Kashyap, R.; Gratzl, M. Anal. Chem. 1999, 71, 2814-20. (22) Encyclopedia of Electrochemistry of the Elements; Bard, A. J., Ed.; Marcel Dekker: New York, 1978; Vol. VIII.


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In contrast to nitrite oxidation, the peak potential for NO was independent of pH in the entire range studied (1.2-8.1) and different from that for nitrite (Figure 1). The shape of the forward and reverse currents of OSWV did not indicate adsorption. We calculated the number of electrons transferred in NO electroxidation from chronoamperometry data and found n ) 1.6 ( 0.1 at pH7.4 (data not shown, see Experimental Section for the conditions). These observations are consistent with the following overall reaction:

2NO + 2H2O f NO2- + NO3- + 6H+ + 4e-

Figure 1. OSWV on a 25-µm disk. Parameters step, 4 mV; frequency, 15 Hz; amplitude, 5 mV. Difference currents are shown. The pH was adjusted with 0.15 M Na2HPO4 and 0.15 M citric acid in 0.15 M NaCl. (A) Representative set of voltammograms in 25 mM KNO2. (B) Representative set of voltammograms of NO. Dashed line, saturated solution of NO. Solid lines, diluted NO solutions with concentrations of 319 and 262 µM with increasing pH, respectively. (C) Peak currents as a function of pH. Solid lines, regressions. Dashed line, theoretical dependence of the formal potential of ascorbic acid on pH, Ef ) E0 - (RT/2F) ln (1/([H+]2 + [H+]K1 + K1K2)) + EAg|AgCl, where K1 and K2 are the acid dissociation constants. Error bars indicate standard deviations of three to five experiments.

standard deviation less than 7% of the peak currents. For comparison, we determined the pH dependence of ascorbic acid oxidation the same way and found that the peak potential followed the formal potential as expected, in agreement with the literature data23 (Figure 1C). Cyclic voltammetry experiments with a cylindrical platinum electrode (l ) 3 mm, d ) 75 µm) revealed irreversible electrooxidation of nitrite, with peak currents depending linearly on the square root of the scan rate (25-750 mV/s, data not shown). Differential pulse amperometry results (see discussion later) were used to calculate the number of electrons transferred resulting in n ) 1.7 ( 0.1. The overall reaction of nitrite electrooxidation that is consistent with these observations is

NO2- + H2O f NO3- + 2H+ + 2e-

(1)

However, involvement of platinum oxide in the reaction mechanism cannot be excluded (such as NO2- + PtO f NO3- + Pt, coupled with Pt + H2O f PtO +2e-+ 2H+). At low pH, the peak potential is higher than the constant value observed above the pKa of HNO2 and dependent on pH (Figure 1A,C), indicating a different reaction mechanism. (23) Ruiz, J. J.; Aldaz, A.; Dominquez, M. Can. J. Chem. 1978, 55, 2799-805.

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(2)

Again, involvement of PtO in the reaction mechanism cannot be excluded. A smaller, second oxidation peak in OSWV was observed in diluted NO solutions (Figure 1B, solid lines), indicating the presence of nitrite. Both peaks disappear after removal of NO by purging the solution with nitrogen, confirming that nitrite is present as an electrooxidation product. The second peak is absent in the case of the saturated, acidic solution of NO, when it contains excess amount of ascorbic acid (as shown in Figure 1B, dashed line). When NO is generated by the stoichiometric amount of ascorbic acid, the first peak becomes smaller and a second peak appears around the potential of nitrite oxidation (as shown in Figure 5), consistent with an expected catalytic effect of ascorbic acid in acidic solutions. The height of the second peak is approximately one-third the first one, further supporting the stoichiometry of reaction 2 (taking into account the ratio of the diffusion coefficients). From the above considerations, it follows that, for a sensitive and stable response, a platinum-based NO sensor must operate in the platinum oxidation region with a stationary surface. An unstable oxidation state of the platinum surface may also influence the selectivities. Differential pulse amperometry is the electroanalytical technique that can provide this condition and best rejects the background (platinum oxidation) current. Selectivity for NO by Differential Pulse Amperometry. Since most likely interferences occurring in biological preparations are oxidized at much lower potentials than NO (and nitrite), as measured by OSWV (Figure 2, Table 1), a proper differential amperometry protocol that selects only the NO electrooxidation wave should prevent most interfering agents from obscuring the signal. The relatively large difference current of dopamine oxidation (Figure 2) results from adsorption of the oxidation product, indicated by a sharp peak in the reverse current, while the serotonin response reflects adsorption of the reactant (not shown). Carbon monoxide is oxidized in an irreversible adsorptive way: both the forward and reverse currents were sharp peaks in the oxidative direction and conventional cyclic voltammetric peak currents depended linearly on the scan rate (data not shown). The physiologically important products of NO oxidation, nitrosothiols, N-nitroso compounds, and NO2, cannot be oxidized on platinum.22 A protocol that can provide the necessary selectivity for NO and a stationary oxidized Pt surface at the same time must contain pulses at three different potential levels (Figure 3). Two levels are used to bracket the NO oxidation potential (“detection

Figure 2. Representative set of background corrected voltammograms (OSWV) of various potential interferents on a 25-µm disk. Parameters step, 4 mV; frequency, 15 Hz; amplitude, 5 mV. Difference currents are shown, except in the case of CO, where the forward current is plotted. Electrolyte balanced buffer without glucose. The concentration was 2.5 mM of all interferences, except for CO, which was 200 µM.

window”). A third (reducing) level serves for surface cleaning. This latter pulse is optimized such that it is not in the oxygen reduction region, and it ensures that the active surface area of the electrode remains the same after several hours of continuous pulsing (tested by chronoamperometry in 5 mM [K3Fe(CN)6]). When the pulse length is appropriately adjusted (see below), the difference of the currents at the end of the two positive pulses (differential current) is sensitive to NO, while interferences that are oxidized at lower potentials than NO in a diffusion limited fashion have very little contribution to it. Kinetically controlled or adsorptive oxidation of other electroactive compounds contributes more, but this interference is much less than in the case of constant potential amperometry. To achieve good selectivity at a microdisk electrode when the positive pulses follow each other (triple pulse amperometry, TPA), the duration of these pulses has to be long enough to ensure that the steady-state current dominates at the end of the pulse, when the current is sampled (assuming diffusion-limited current for the interfering agents). This pulse length can be calculated from the microdisk chronoamperometry equation16

I)

nFAxDc + 4nFrDc xπt

(3)

where all symbols have their usual meaning. For instance, with pulse durations of 500 ms at a 25-µmdiameter disk and a typical D ) 5 × 10-6 cm2/s, the steady-state component is ∼3 times the transient term and provides sufficient selectivity (Figure 3A, solid line). To increase the temporal resolution and the sensitivity while ensuring selectivity at the same time, differential normal pulse

amperometry (DNPA) with shorter pulses can be applied (Figure 3B). In this case, the larger, transient current dominates at the end of the pulse. At a larger disk or cylindrical electrode, the transients are longer and DNPA is the only option to provide sufficient selectivity with high (>0.1 Hz) temporal resolution. When a short pulse TPA is applied to a carbon fiber,7 as on a larger disk electrode (Figure 3A, dashed line), selectivity is not ideal and an increase in the concentration of interferences results in a decrease in the NO-sensitive differential current. Short-pulse DNPA has a further advantage: it can prevent the electrode from time-dependent and surface poisoning polymerization of the aminochrome product of dopamine electrooxidation.24 A significant reduction in interference even by nitrite can be achieved with a proper DNPA protocol, despite the small difference between the potentials of nitrite and NO oxidation and the higher potential value for nitrite (Figure 1C, Table 1). Adjusting the detection window for NO in such a way that the higher positive potential falls just below the nitrite peak oxidation potential (700 mV) reduced the sensitivity for nitrite by ∼65% compared to the sensitivity obtained by a pulse of 780 mV (data not shown). From the latter sensitivity (55 ( 3 pA/µM, by the 780-mV pulse), the number of electrons transferred can be calculated from eq 3 (with D ) 7.9 × 10-6 cm2/s at infinite dilution19). This calculation is valid if the irreversible oxidation of nitrite is fast enough to result in diffusion-limited current at the end of the oxidizing pulse. If this condition does not hold, the calculation yields a smaller number for n. Therefore, from the results, n ) 1.7 ( 0.1, we could reasonably conclude that the number of electrons transferred is 2. The same considerations apply to the chronoamperometry experiments of NO (see Experimental Section). Selectivity for NO on a Cellulose Acetate-Coated Electrode. To further reduce interference from nitrite and homovanillic acid, and at the same time render the sensor less prone to protein adsorption in biological media, two polymer coatings, Nafion and CA,25 were tested for anion as well as protein exclusion. The efficiency of size exclusion by CA is well demonstrated by cyclic voltammetry in K3[Fe(CN)6] (Figure 4). The plateau current values indicate that permeability (defined as P ) DmK/d) for the negatively charged ferricyanide ion is reduced to the same extent in CA as in Nafion at around the same coating thickness. The reaction kinetics does not seem to be affected by either membrane. Besides ferricyanide both coatings exclude ascorbate, nitrite, DOPAC, and homovanillate well (Figure 5). However, due to its cation exchanger property, Nafion can accumulate cations such as dopamine or norepinephrine, providing a basis for their sensitive in vivo determination.15 We also observed a second oxidation peak of the highly permeable dopamine, shifted positive by ∼250 mV from the first one. In practice, such an effect on the oxidation of highly permeable cations would reduce the selectivity for NO. In contrast to Nafion, permeability of compounds in CA seems to be determined by a combination of effects of the molecular weight and negative charge density, which is best demonstrated by the large difference between the oxidation peak heights of (24) Lane, R. F.; Hubbard, A. T. Anal. Chem. 1976, 48, 1287-93. (25) Pariente, F.; Alonso, J. L.; Abruna, H. D. J. Electronal. Chem. 1994, 379, 191-7.


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Table 1. Voltammetric and Differential Pulse Amperometric Characteristics of Compounds Potentially Interfering with NO Detection voltammetric (OSWV) characteristicsa interference dopamine COd serotonin ascorbic acid Dopac uric acid H2O2 homovanillic acid nitrite glucose arginine L-NAME albumin (bovine serum)

peak potential (mV)

half-peak width (mV)

peak ht (nA)

76 ( 13 223 ( 5 264 ( 5 300 ( 5 400 ( 18 438 ( 3 503 ( 15 626 ( 6 695 ( 4

270 ( 8 67 ( 2 147 ( 9 333 ( 4 307 ( 4 173 ( 4 232 ( 9 305 ( 11 183 ( 2 n/ag n/a n/a n/a

5.6 ( 0.3 6.9 ( 0.6 2.9 ( 0.4 3.2 ( 0.1 2.6 ( 0.2 1.5 ( 0.1 2.1 ( 0.2 2.7 ( 0.4 3.9 ( 0.05

sensitivityb by DNPA (pA/µM) 0.1 -4.5e 0.3 0.5 0.2 4.2 1.6 4.1 ,0.1 -12e,f -11.5e,f -2.7e (200 pA mL/mg)

selectivityc 6910 -154 2300 1380 3460 165 432 168 .6000 -58 -60 -256

a A representative set of OSWV is shown in Figure 2. b Sensitivity, the mean slope of 3-5 calibrations for the given interfering agent with a CA-coated 75-µm disk in the balanced buffer not containing glucose. The values refer to the differential current of DNPA. The potential sequence is the same as in Figure 6. The membrane permeability for Fe[(CN)6]3- was 7.0 × 10-5 cm/s. The interference was added sequentially in 100 µM increments into a continuously stirred solution up to a final concentration of 400 µM. c Selectivity, defined as the ratio of sensitivity for NO to the sensitivity for the given interference under the same conditions. See text under Kinetics of NO Oxidation by Dissolved Oxygen for NO sensitivity. d OSWV data refer to the forward current. Sensitivity and selectivity values will be published later. e See text for explanation for negative values. f When the concentration is above 200 µM. g n/a, not applicable.

Figure 4. Cyclic voltammetry in 5 mM K3[Fe(CN)6] /0.5 M KCl on 75-µm disks. Initial potential, 400 mV; sweep rate, 20 mV/s. The inset shows the currents on the coated electrodes enlarged. The permeability of the CA and Nafion coatings for [Fe(CN)6]3- was 7.0 × 10-5 and 5.6 × 10-5 cm/s, respectively. Figure 3. Typical TPA and DNPA recordings. Electrolyte balanced buffer without glucose. At each step, a mixture of dopamine, DOPAC, uric acid, and ascorbic acid was added to yield a concentration of 0.5, 60, 20, and 200 µM, respectively, and the solution was stirred for 10 s. Spikes are resulted from the onset of stirring. (A) Solid line, differential current of TPA on a 25-µm disk. The pulse sequence is shown in the inset. The current was sampled and averaged during the last 17 ms of each pulse. Dashed line, differential current of TPA on a 75-µm disk at a pulse sequence of -150 mV, 1000 ms; 580 mV, 100 ms; and 780 mV, 100 ms. (B) DPNA on a 75-µm disk with the pulse sequence shown in the inset. The current was sampled and averaged during the last 50 ms of each pulse.

nitrite and HNO2 and the small current of dopamine oxidation (Figure 5). Therefore, large cations do not permeate so well as they do in Nafion and a possible positive shift in the oxidation 3970 Analytical Chemistry, Vol. 73, No. 16, August 15, 2001

potential would interfere less with NO detection. Small, neutral molecules are highly permeable in CA: the voltammetric peaks of both hydrogen peroxide and NO are higher than at a bare electrode. On the basis of all discussions above, DNPA with three potential levels applied to a larger, CA-coated platinum disk is a well-suited analytical method to monitor the average NO efflux from a monolayer of cells selectively and on-line. A larger disk provides greater sensitivity and lower detection limit for NO than a smaller diameter one, due to its larger surface area and the improved signal-to-noise ratio, but single-cell applications would certainly require a microdisk with a diameter smaller than 10 µm. DNPA is preferable to TPA to improve selectivity on a membranecoated electrode, for any size electrode. The diffusion coefficient

Figure 5. Representative set of background-corrected voltammograms (OSWV) on 75-µm disks. Parameters step, 4 mV; frequency, 15 Hz; amplitude, 5 mV. Electrolyte balanced buffer without glucose. Concentration of all interferences was 2.5 mM, except for H2O2, which was 1.2 mM. Solid lines, bare electrode. Dashed lines, CA-coated electrode, with a permeability for [Fe(CN)6]3- of 7.0 × 10-5 cm/s. Dotted lines, Nafioncoated electrode, with a permeability for [Fe(CN)6]3- of 5.6 × 10-5 cm/s.

for most interfering agents in the membrane phase is much smaller than in water, elongating the transient currents and thereby decreasing the selectivity for NO by TPA, as discussed previously. The values of sensitivities and selectivities achieved with a CAcoated 75-µm microdisk for a range of interferences are summarized in Table 1. Sensitivities are highest and selectivities are lowest for nitrite, homovanillic acid, and hydrogen peroxide, since these compounds have the smallest molecular weights, oxidize close to NO, or both. (Although exclusion of interferences by CA is determined by size and charge density, the overall selectivity in differential pulse amperometry is also influenced by the oxidation potential of the given interference.) The negative sensitivity for serotonin represents a decrease in the differential current while both oxidizing currents are increasing, and it very likely results from the adsorptive nature of oxidation. Upon addition of arginine, L-NAME, and serum albumin (none of which is electroactive), currents at all potentials decrease, as well as the differential current. This observation is also consistent with (potential-dependent) adsorption. Interference by these compounds is decreased by ∼10-fold compared to a bare electrode. Sensitivity and Detection Limit. Calibrations with a CAcoated electrode were first carried out in a deaerated acidic solution containing ascorbic acid in order to prevent fast oxidation

of NO by dissolved oxygen.18 Even under these conditions, the rate of chemical oxidation becomes significant at longer times, as indicated by the gradually increasing decays in the differential current upon sequential addition of NO (Figure 6). Therefore, we determined the sensitivity from one-step additions of NO monitored for only ∼90 s at different concentrations (between 1.5 and 5 µM) and found 175 ( 21 pA/µM. This value is higher than expected at a bare electrode from eq 3, indicating higher permeability for NO in the membrane phase, consistent with OSWV results (Figure 5). The membrane is thin enough for NO to diffuse in and accumulate during the three pulses preceding the pulse of 700 mV (300 ms). Since ascorbic acid is excluded from the vicinity of the electrode surface by the CA coating, a catalytic effect cannot account for the increased sensitivity. We observed higher sensitivity for NO at neutral pH26 as well (see calibration method under Kinetics of NO Oxidation by Dissolved Oxygen). Another advantage of the membrane coating is that it smoothes convective noise when the diffusion layer thickness due to convective transport would become smaller than the membrane thickness. The noise amplitude is higher on the currents measured with a bare electrode when the solution is continuously stirred26 (not shown). (26) Cserey, A. Ph.D. Dissertation, Case Western Reserve University, Cleveland, OH, 2001.


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Figure 6. Calibrations on 75-µm disks by a DNPA pulse sequence of -150 mV, 100 ms; 500 mV, 100 ms; -150 mV, 100 ms; and 700 mV, 100 ms. The current was sampled and averaged during the last 50 ms of each pulse. The change in the differential current, relative to the baseline current, is shown. At each step, a small volume of saturated NO solution was added while stirring for 10 s. Solid line, typical calibration with a CA-coated electrode in a solution containing 90 mM NaCl, 30 mM HCl, and 50 µM ascorbic acid. Each addition increased the concentration by 1.5 µM. The coating permeability for [Fe(CN)6]3- was 9.5 × 10-5 cm/s. Dashed line, typical calibration with a bare electrode in the balanced buffer without glucose. Each addition increased the concentration by 2.0 µM. The initial current spikes resulting from the onset of stirring were deleted from the data set.

The detection limit, as calculated from the sensitivity above and the standard deviation of the background current (5 pA), is around 50 nM, which is sufficient for a range of biological applications. A lower detection limit can be achieved by an arraylike arrangement of the platinum disks or with an even larger diameter disk, if necessary. Kinetics of NO Oxidation by Dissolved Oxygen. To evaluate data for NO concentrations obtained in the presence of oxygen, which is always the case in biological preparations, the chemical oxidation of NO has to be considered. The reaction rate follows an overall third-order kinetics with the rate-limiting step of reaction 4b18

d[NO]/dt ) -k[NO]2 [O2]

(4a)

2NO + O2 ) 2NO2

(4b)

NO2 + NO ) N2O3

(4c)

N2O3 + H2O ) 2NO2- + 2H+

(4d)

2NO2 + H2O ) NO2- + NO3-

(4e)

If the fast hydrolysis of NO222 (4e) is taken into account, in contrast to the physiology literature,18 the overall stoichiometry becomes

6NO + 2O2 + 3H2O ) 5NO2- +NO3- + 6H+ 3972


(4f)

Both the oxidation rate constant and the electrode sensitivity can be determined by adding NO in a relatively high concentration (∼10 µM) in one step and fitting the theoretical kinetics to the measured oxidation current.26 We found NO oxidation second order in [NO] with a rate constant of (8.0 ( 0.4) × 106 M-2 s-1 by fitting the current obtained with a bare electrode at pH 7.0 (under continuous stirring, in contrast to the conditions in Figure 6). The value is in good agreement with literature data obtained by other than electrochemical methods18 ((7.6 ( 1.2) × 106 M-2 s-1), in contrast to an earlier report claiming zero-order kinetics of NO oxidation measured with a porphyrin-coated carbon fiber sensor.5,18 When measured with CA-coated electrodes, the apparent rate constant for NO oxidation was found to be ∼8 times higher than in the case of the bare electrode26 (kapp) k[O2] ) (19 ( 4) × 103 M-1 s-1) consistent with a sensitivity for NO 2.8 times higher than that of a bare electrode. Selectivity values in Table 1 are based on the mean of this sensitivity (540 ( 60 pA/µM). Release Rate of NO from Cultured Macrophages. NO released from macrophages was monitored in situ for ∼20 min after incubation with LPS (Figure 7A,B). After each recording, the electrode was calibrated under the same conditions and sensitivity was obtained by curve fitting.26 Although the concentration of NO is an important indicator of NOS activity, the biologically more relevant information is the rate of release that can be calculated from our in situ data, due to the high temporal resolution of the recordings. We can reasonably assume that the initial concentrations of both NO and nitrite are zero, the only reactant consuming NO is O2, and the concentration is homogeneous in the dish (see Experimental Section). Therefore, the release rate of NO can be written as

R(t) )

d[NO(t)] + kapp[NO(t)]2 dt

(5)

where [NO(t)] is the measured concentration of NO. Figure 7C shows the calculated release rates for NO (both BAC and RAW cultures). The rates are in the order of 10 nM s-1/106 cells or 20 pmol s-1/106 cells and comparable to literature data.2 The increase in the rates is very likely the consequence of medium exchange: after incubation the cells need to adjust to the new environment. The increase was also observed in separate experiments with the Griess assay: the output of nitrite was less in the first half hour following medium exchange than in the second one (data not shown). The total amount of NO released during the measurements (integrated R(t)) correlates linearly to the concentration of nitrite found in the buffer by the Griess assay (Figure 7C). The intercept of the linear regression is negative, but zero is contained within the 99% confidence interval. The slope, in the case of BAC cultures, is 1.32, close to the value expected from eq 4e (6/5), supporting our assumptions in eq 5. This finding also indicates that the reaction of NO with superoxide anion (O2-) is negligible under our experimental conditions in BAC cultures. However, in the case of RAW cells, we found more nitrite with the Griess assay than we can expect from eq 4e. This can be attributed to the reaction of NO with superoxide (which degrades to nitrite and nitrate) and is in agreement with earlier findings2 based on indirect, chemiluminescent detection of NO in RAW cultures.

Figure 7. In situ monitoring of NO release from macrophages with a 75-µm CA coated-electrode with a DNPA pulse sequence of -150 mV, 100 ms; 500 mV, 100 ms; -150 mV, 100 ms; and 700 mV, 100 ms. The coating permeability for [Fe(CN)6]3- was 1.0 × 10-5 cm/s. Solid lines, LPS-activated cultures in a buffer containing 2 mM L-arginine; dotted lines, LPS-activated cultures in a buffer containing 2 mM L-NAME; dashed lines, control (inactivated) cells. (A) Measured concentration of NO normalized to 1 million cells in BAC-1 cultures. (B) Measured concentration of NO normalized to 1 million cells in RAW cultures. (C) Calculated rate of NO release normalized to 1 million cells. (D) Correlation between the concentration of nitrite at the end of the recording and the calculated amount of NO released. Closed circles, BAC-1; open circles, RAW cells.

The measured NO was released as a result of NOS activity and not by other mechanisms, as revealed by the decrease in concentration and release rate when the recordings were made in the presence of L-NAME, a NOS inhibitor, instead of L-arginine (Figure 7B,C). CONCLUSIONS A stationary oxidized state of the platinum electrode surface is necessary for sustained nitric oxide recordings with stable sensitivity. This cannot be achieved with regular oxidative amperometry that would lead to an increasing degree of surface inhibition. Therefore, an appropriate pulsing protocol is needed for NO sensing, alternating between oxidizing and reducing voltage levels. This inevitably leads to a relatively high background current due to surface oxide formation in each oxidizing step. It is possible to establish a differential normal pulse amperometry protocol that ensures both a stationary surface and a maximized useful signal due to NO. The appropriate potential steps provide excellent selectivity for NO over a large number of potential interferences, as well as

sufficiently high temporal resolution. A cellulose acetate coating is sufficient to protect the sensor surface from fouling and to reduce permeability of interferences whose signals differential amperometry cannot eliminate so effectively as it can others. At the same time, the CA membrane increases the sensitivity and lowers the detection limit for NO. The established pH independence of NO electrooxidation on platinum is a further advantageous feature of the sensor applied in biological research. Platinum might also be used for detection of carbon monoxide by an appropriately modified amperometry protocol. Since differential techniques can be easily tuned to detect either NO or CO at the same electrode, alternating this tuning between the oxidation peaks of the two species allows detection of both, virtually simultaneously with the same sensor. In this scenario, selectivity for CO would be provided by the exclusion properties of the cellulose acetate coating on the electrode. Carbon monoxide being a recently discovered putative messenger in NO signaling pathways, its simultaneous detection with NO from the same site is an attractive added benefit to this approach. Analytical Chemistry, Vol. 73, No. 16, August 15, 2001

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Chemical generation of NO by ascorbic acid not only does ensure accurate means of calibration but also can provide a convenient way for in vivo stimulations. ACKNOWLEDGMENT The authors gratefully acknowledge the valuable comments of Drs. Barry Miller, Ulrich Hopfer, and George Dubyak on the manuscript. Preliminary studies on this technique done in our laboratories by Huijun Xie and Vinil Bhardwaj are acknowledged. We thank Dr. Ravi Bellamkonda for providing the cell culture and

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laboratory facilities for the experiments. Macrophages were the generous gift of Dr. George Dubyak. Sylvia Kertesy’s help with the cell culture is greatly appreciated. The work was funded by the NSF, the Whitaker Foundation (Cost-Effective Health Care Technologies grant), and the Cystic Fibrosis Foundation.

Received for review January 25, 2001. Accepted May 7, 2001. AC010123H

Stationary-State Oxidized Platinum Microsensor for Selective and On

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