Amperometric Ultramicrosensors for Peroxynitrite Detection and Its

Christian Amatore , Stéphane Arbault , Delphine Bruce , Pedro de Oliveira , Marie Erard , Monique Vuillaume. Chemistry - A European Journal 2001 7 (1...
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Anal. Chem. 2000, 72, 5313-5321

Amperometric Ultramicrosensors for Peroxynitrite Detection and Its Application toward Single Myocardial Cells Jian Xue,† Xiangyang Ying, Junshui Chen, Yuezhong Xian, and Litong Jin*

Department of Chemistry, School of Chemistry & Life Science, East China Normal University, Shanghai 200062, PR. of China Jiye Jin

Department of Chemistry, Faculty of Engineering, Gifu University, Gifu 501-11, Japan

The research studied the concentration variation of peroxynitrite anion (OdN-O-O-) released from cultured neonatal myocardial cells induced by ischemia/reperfusion and studied the protective effect of melatonin on the injury. For this purpose, amperometry peroxynitrite ultramicrosensors (UMS) were fabricated and constructed by electropolymerizing inorganic macromolecular film of tetraaminophthalocyanine manganese(II) and coating chemically with poly(4-vinylpyridine). Under optimum conditions, the UMS showed high selectivity and sensitivity to peroxynitrite determination with a calculated detection limit of 1.8 × 10-8 mol/L (S/N of 3). The detection of peroxynitrite was based on electrocatalytic reduction of peroxynitrite. The mechanism of catalysis was also discussed. The UMS should be promising for in vivo measurement of peroxynitrite without interference or fouling. Peroxynitrite released from myocardial cells both in the ischemic period and in the reperfusion period was measured directly. This approach may lead to important information for myocardial cells on the mechanism of injury and prospective treatments of medicine such as melatonin. Free radicals play a central role not only in many pathological conditions and chronic diseases but also in certain normal biological processes.1-3 Superoxide anion, generated from cells and tissues, was thought to be one of the strongest oxidants known.4 Nitric oxide, another important free-radical species, is a widespread intracellular and intercellular diffusible messenger that passes through most cells and tissues with little consumption or direct reaction.5 In the past several years, many papers considered * Corresponding author: (e-mail) [email protected]; (telephone) (86)2162232627; (fax) (86)21-62451876. † E-mail: [email protected]. (1) Beckman, J. S.; Koppenol, W. H. Am. J. Physiol. 1996, 271, C1424-1437. (2) Pryor, W. A.; Squadrito, G. L. Am. J. Physiol. 1995, 268, L699-L722. (3) Stamler, J. S.; Singel, D. J.; Loscalzo, J. Science 1992, 158, 1898-1902. (4) Akaike, T.; Noguchi, Y.; Ijiri, S.; Setoguchi, K.; Suga, M.; Zheng, Y. M.; Dietzshold, B.; Maeda, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 24482453. (5) Lancester, J. R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8137-8141. 10.1021/ac000701e CCC: $19.00 Published on Web 10/06/2000

© 2000 American Chemical Society

independently the damage roles of superoxide and nitric oxide. However, what is more important in vivo is the recombination of NO and the superoxide anion radical, to form potent oxidant peroxynitrite.6-7 There is plenty of evidence suggesting that O2itself is not particularly toxic8 and that the direct toxicity of NO is modest, but the major mechanism of injury associated with NO in vivo more likely results from peroxynitrite, which is much more reactive than NO and O2-. Peroxynitrite anion (OdN-O-O-, PON) is a potent biological oxidant8 generated, for example, from endothelial cells, Kupffer cells, neutrophils, and macrophages. The attack it makes on lipids,9 carbohydrates,10 proteins,11 nucleic acids,12 DNA, and R1proteinase inhibitor11 causes irreversible cellular damage. Peroxynitrite has been considered as a valid mediator of cellular injury in myocardial ischemia and reperfusion injury, shock, and inflammation in the past several years. The concept of a PON-mediated cardiotoxicity has important clinical implications in situations of myocardial ischemia followed by reperfusion.13-15 The endogenous formation of peroxynitrite contributes to myocardial ischemia/ reperfusion injury16 and has also been implicated in the pathogenesis of atherosclerosis,17 septic shock,18 and neurodegenrative (6) Beckman, J. S.; Ischiropoulos, H.; Zhu, L.; Van der Woerd,; Smith, C.; Chen, J.; Harrison, J.; Martin, J. C.; Tsai, M. Arch. Biochem. Biophys. 1992, 298, 438-445. (7) Ignarro, L. J.; Fukuto, J. M.; Griscavage, J. M.; Rogers, N. E.; Byrns, R. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8103-8107. (8) Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.; Freeman, B. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1620-1624. (9) Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A. Arch. Biochem. Biophys. 1991, 288, 481-487. (10) Moro, M. A.; Darley-Usmar, V. M.; Lizasoain, I.; Su, Y.; Knowles, R. G.; Radomski, M. W.; Moncada, S. J. Pharmacol. 1995, 116, 1999-2004. (11) Moreno, J. J.; Pryor, W. A. Chem. Res. Toxicol. 1992, 5, 425-431. (12) Salgo, M. G.; Squadrito, G. L.; Pryor, W. A. Biochem. Biophys. Res. Commun. 1995, 215, 1111-1118. (13) Schultz, R.; Dodge, K. L.; Lopaschuk, G. D.; Clanachan, A. S. Am. J. Physiol. 1997, 272, H1212-H1219. (14) Ischida, H.; Ichimori, K.; Hirota, Y.; Fukahori, M.; Nakazawa, H. Free Radical Biol. Med. 1996, 20, 343-350. (15) Thiemermann, C.; Bowes, J.; Myint, F. P.; Vane, J. R. Proc. Natl. Acad. Sci. U.S.A.1997, 94, 679-683. (16) Yasmin, W.; Strynadka, K. D.; Schulz, R. Cardiovasc. Res. 1997, 33, 4422432. (17) Markert, M.; Andrewss, P. C.; Babior, B. M. Methods Enzymol. 1984, 105, 358-365.

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diseases.19 The relative importance of peroxynitrite production to irreversible injury of the animal and particularly of the human heart, however, is far from clear. Thus, understanding of the role of peroxynitrite and its accurate determination will provide insight into viral pathogenesis at a molecular level. Due to the great importance of the research mentioned above, point of interest was concentrated upon the measurement of peroxynitrite in biological systems. However, it is indeed extremely difficult because of the low concentration, high activity, and fleeting presence of the peroxynitrite. Currently, most of the techniques for peroxynitrite measurement are indirectly based on chemical detection of the decompostion products removed from biological systems. Peroxynitrite generation is usually measured by a immunohistochemistry,20 chemiluminescence,21,22 fluorescence,23 and UV-visible spectroscopy.24 In most cases, the contribution of peroxynitrite has been inferred from detection of 3-nitrotryrosine because the nitration of tyrosine residues to produce nitrotyrosine is a sensitive marker elicited by peroxynitrite. Recently, the immunohistochemical assay of nitrotyrosine, as a robust method and gold standard for detecting peroxynitrite, has been studied extensively.25,26 Althrough the above methods demonstrated wide detection range and low detection limits, the techniques could not be applied in real-time determination in vivo because of the amount of complex pretreatment and technical problems. For measurement of peroxynitrite and to obtain accurate information in vivo, it is necessary to set up a sensitive, selective, reliable, easy-to-obtain, fast-response method due to the varying concentrations of peroxynitrite in the biological systems. An electrochemical method has been developed for determination of some electroactive biologicals and more and more attention is being paid to it since it has more advantages, such as direct detection, high sensitivity, measurement in vivo, and so on. As yet, there are almost no reports to describe electrochemical methods for measurement of peroxynitrite except for Sodum et al.27 who determined 3-nitrotyrosine by high-pressure liquid chromatography with a dual-electrode electrochemical detector. Synthetic metal porphyrin, pathalocyanine, and Schiff base complexes have been promoted to be an extensive area of research to mimic the enzyme active site in enzymatic systems, especially for the monooxygenase of the cytochrone P-450 family.28 Recently we developed several microsensors for the direct determination of NO based on electropolymerized film of a metal (18) Reed, J. W.; Ho, H. H.; Jolly, W. L. J. Am. Chem. Soc. 1974, 96, 453-454. (19) Crow, J. P.; Spruell, C.; Chen, J.; et al. Free Radical Biol. Med. 1994, 16, 331-338. (20) Viera, L.; Ye, Y. Z.; Estevez, A. G.; Beckman, J. S. Methods Enzymol. 1999, 301, 373-381. (21) Morot, G. T. Y.; Moulian, N.; Meunier, F. A.; Blanchard, B.; Angaut-Petit, D.; Faille, L.; Ducrocq, C. Nitric Oxide: Biol. Chem. 1997, 1, 330-345. (22) Wang, P.; Zweier, J. L. J. Biol. Chem.1996, 271, 29223-29230. (23) Possel, H.; Noack, H.; Augustin, W.; Keilhoff, G.; Wolf, G. FEBS Lett. 1997, 416, 175-178. (24) Vander, V. A.; Eiserich, J. P.; O’Neill, C. A. Arch. Biochem. Biophys. 1995, 319, 341-349. (25) Digerness, S. B.; Harris, K. D.; Kirklin, J. W.; Urthaler, F.; Viera, L.; Beckman, J. S.; Darley-Usmar, V. Free Radical Biol. Med. 1999, 27, 1386-1392. (26) Bauersachs, J.; Bouloumie, A.; Fraccaro, D.; Hu, K.; Busse, R.; Ertl, G. Circulation 1999, 100, 292-298. (27) Sodum, R. S.; Akerkar, S. A.; Fiala, E. S. Anal. Biochem. 2000, 280, 278285. (28) Mansay, D.; Battioni, P. In Bioinorganic Catalysis; Reedijk, J., Ed.; Marcel Dekker: New York, 1993; p 395.

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Schiff base29-31 and for direct monitoring of the variation of superoxide anion released from myocardial cells stimulated by acetylcholine.32 The mechanism of electrocatalysis is that NO plays the role of a strong axial ligand of the central metallic cation of the polymerzied Schiff base and makes it possible to be oxidized in the appropriate potential range because the electroactive materials polymerzied on the electrode surface are unsaturated coordination compounds, and some smaller moleculars such as NO and O2 have lone-pair electrons. Metallophthalocyanines, as macrocyclic complexes, have been exploited extensively on electrocatalysis. In this paper, we prepared the ultramicrosensor modified with electropolymerized film of tetraaminophthalocyanine manganese(III) (MnIII(TAPc)), to catalyze the peroxynitrite reduction. Similar to NO and O2, the peroxynitrite has several lonepair electrons, one of which could fill the empty orbit of polymerized Mn(TAPc) and form axial coordination compouds. Therefore, poly-Mn(TAPc) was suggested to be a considerable electrochemical mediator for catalytical reduction of peroxynitrite. For real-time detection of peroxynitrite, we have fabricated, for the first time, a novel amperometric ultramicrosensor for peroxynitrite measurement in vivo. Poly-Mn(TAPc) was used as electroactive material to catalyze peroxynitrite electroreduction, and PVP was applied to form a selective film against macromolecules, such as protein. The peroxynitrite ultramicrosensor (UMS) was used as the detector and operated at 0.0 V vs Ag/AgCl so that the effect of interferences such as dopamine, ascorbic acid, and uric acid can be minimized effectively. The pharmacological effect of melatonin on ischemia/reperfusion injury of myocardial cells was also discussed. EXPERIMENTAL SECTION Synthesis. Synthesis of Peroxynitrite. Peroxynitrite was synthesized according to the procedures described elsewhere8 in a quenched-flow system by the reaction of nitrite with acidified H2O2. Peroxynitrite was also biomimetic synthesized from potassium superoxide and nitric oxide as described in the literature.33 Excess H2O2 was eliminated by treatment with MnO2 powder. The peroxynitrite stock solution was stored at -18 °C. Peroxynitrite concentration was verified by ultraviolet spectrophotometry at 302 nm ( ) 1670 mol-1 L cm-1) just before the electroanalytical experiments.24 Synthesis of Tetraaminophthalocyanine Manganese(II). Mn(TAPc) was synthesized and purified according to the modified procedure described by Achar et al.34 In a 500-mL three-necked flask containing 90 mL of nitrobenzene, 3.15 g of manganese chloride pentahydrate, 12.5 g of 3-nitrophthalic acid, 1.7 g of ammonium chloride, 0.17 g of ammonium molybdate, and excess urea (20 g) were finely ground and placed. The reaction mixture was strictly stirred at 185 °C for 4.5 h. The solid product was (29) Tu, H. P.; Xue, J.; Cao, X. N.; Zhang, W.; Jin, L. T. Analyst 1999, 125, 163167. (30) Mao, L. Q.; Tian, Y.; Shi, G. Y.; Liu, H. Y.; Jin, L. T.; Yamamoto, K.; Jin, J. Y. Anal. Lett. 1998, 31, 1991-2007. (31) Tu, H. P.; Mao, L. Q.; Cao, X. N.; Jin, L. T. Electroanalysis 1999, 11, 6974. (32) Xue, J.; Xian, Y. Z.; Ying, X. Y.; Chen, J. S.; Wang, L.; Jin, L. T. Anal. Chim. Acta 2000, 405, 77-85. (33) Koppenol, W. H.; Kissner, R.; Beckman, J. S. Methods Enzymol. 1996, 269, 296-302. (34) Achar, B. N.; Fohlen, G. M.; Parker, J. A.; Keshavayya, J. Polyhedron 1987, 6, 1463-1467.

washed with alcohol until free from nitrobenzene. The product was treated twice with 100 mL of 1.0 mol/L hydrochloric acid and 1.0 mol/L sodium hydroxide. The chloride-free manganese(II) 4,9,16,23-tetranitrophthalocyanine was dried at 125 °C. About 5 g of finely ground manganese(II) 4,9,16,23-tetranitrophthalocyanine was placed in 100 mL of water. To this slurry, 100 g of Na2S. 9H2O was added and the resultant mixture was stirred at 50 °C for 5 h. The separated product was treated with 750 mL of 1.0 mol/L hydrochloric acid and then treated with 500 mL of 1.0 mol/L sodium hydroxide, stirred for 1 h, and centrifuged to separate the dark claret solid complex. The product was repeatedly treated with water, stirred, and centrifuged until the material was free from sodium hydroxide and sodium chloride. The pure manganese complex was dried in a vacuum. The structure was characterized by FT-IR: FT-IR spectra data (KBr, cm-1) 3281, 3183 (γ-NH2); 1345, 1258, 1060, 1090 (γC-N); 826, 868 (δAr); 735, 752, 950, 1607(δN-H). Chemicals. Hanks balanced salt solution (HBSS) was prepared as follows: NaCl 8.0 g/L, Na2HPO4‚2H2O 0.06 g/L, KCl 0.4 g/L, KH2PO4 0.06 g/L, MgSO4‚7H2O 0.2 g/L, glucose 1.0 g/L, and CaCl2‚2H2O 0.19 g/L, pH7.4 adjusted with H3PO4 and NaHCO3. HBSS was used as buffer solution in all experiments except real sample analysis. Minimum essential medium (MEM) was used as cell culture medium and purchased from Gibco BRL Inc. For fabrication of ultramicrosensors, Tetra-n-butylammonium perchlorate (TBAP) was prepared by the reaction of tetra-nbutylammonium bromide with sodium perchlorate. It was recrystallized by acetyl acetate (mp 212.5∼213.5 °C). Tetraaminophthalocyanine manganese(II) (MnTAPc) and peroxynitrite was synthesized as described below. Poly(4-vinylpyridine) (25% cross-linked, Aldrich) and other reagents, such as H2O2, NaNO2, uric acid (Sigma), ascorbic acid (Sigma), epinephrine (Sigma), norepinephrine (Sigma), dopamine (Sigma), reduced and oxidized glutathione (Sigma), melatonin (Sigma), and Nω-nitro-L-arginine-methyl ester (L-NAME) (Sigma), were at least AR grade and used as received without any pretreatment. Apparatus. All electrochemical measurements were performed with an Electrochemical Workstation (model 660A, CH Instruments Inc.) in a 10-mL beaker. For sensor fabrication and peroxynitrite measurement, the three-electrode system included a Au wire (diameter 0.5 mm) as counter electrode, a modified Ag/AgCl wire (diameter 0.2 mm) as reference electrode, and a planar platinum electrode (diameter 200 µm), a planar carbon fiber microelectrode (diameter 15 µm), or a cylindrical carbon fiber ultramicroelectrode (diameter 600 nm-1 µm) as working electrode. A Varian Cary 50 Probe UV-visible spectrophotometer (Varian Instruments, San Fernando, CA) and S-250 scanning electron microanalyzer (Cambridge Instruments) were used to characterize poly-Mn(TAPc) film modified electrodes. For real sample analysis, oxygen partial pressure was controlled through a self-made gaseous oxygen electrochemical sensor. An XDS-1 model stereomicroscope (Chongqing Optical & Electrical Instrument Co.) equipped with video and a WK-2 model micromanipulator (Xi′an Northwest Optical & Electrical Co.) were used as instruments for guiding micromanipulation.

Procedures. Isolation of Myocardial Cell Culture and Injured Model. The myocardial cell culture was prepared from the separated cells of coarsely minced neonatal rat (about 1-3 days) ventrical by incubation with 0.10% trypsin at 37 °C according to the modified method of Harary35 and Keith.36 The resultant cell suspensions were centrifuged at 1500 rpm and suspended in a MEM supplemented with 20% fetal bovine serum (Huamei Inc.), 100 units/mL penicillin, and 100 units/mL streptomycin. The suspensions were diluted to 1 × 106cells/mL so that contact between cells in the cultures was minimized. Then the cells were grown in MEM statically at 37 °C in a atmosphere of 5% CO2 and 95% air. After 6-8 h, the most myocardial cells (MCs) settled to the bottom of the culture well, appeared to be typical myofibrils, and beat synchronously. To injure the myocardial cells, glycerine was used as a substitute for glucose at double concentration to mimic hypoglycemia, and the oxygen partial pressure of the culture medium was controlled critically in the range of 12.0 ( 0.1% to imitate hypoxia. Electrode Activation, Modification, and Characterization. Prior to its modification, the planar microelectrode surface was polished with 0.5-µm diamond paste on a polishing microcloth and subsequently ultrasonicated thoroughly with acetone, 1.0 mol/L NaOH, HNO3 (1:1), and deionized water, respectively. Electrochemical pretreatment was performed by scanning the potential ranging from -0.30 to +1.20 V for 10 cycles in a 0.50 mol/L sulfuric acid solution. Then the pretreated microelectrode was allowed to air-dry and placed into dimethyl sulfoxide (DMSO) solution containing 5.0 mmol/L monomer of Mn(TAPc) and 0.1 mol/L TBAP as supporting electrolyte. Mn(TAPc) was deposited onto the microelectrode surface by means of scanning potential between -0.5 and +1.0 V at a scan rate of 100 mV/s. After consecutively cycling for 30 cycles, the electrode was taken out from the electroploymerization solution, rinsed with acetone and distilled water, and allowed to air-dry. Surface coverages of the poly-Mn(TAPc)-modified electrodes were calculated by integration of the voltammetric wave and assuming n value of 1 with a result of 7.8 × 10-8mol/cm2. Prior to the determination of peroxynitrite, the poly-Mn(TAPc)modified microelectrode was further coated with PVP twice by depositing 1 µL of 1% (w/v) PVP/methanol solution on the surface of the electrode and then allowing the methanol to evaporate in air. Fabrication of Peroxynitrite Ultramicrosensors. The manufacture of the carbon fiber cylindrical ultramicroelectrode was reported.37-39 In the present work, we prepared the matrix ultramicroelectrode by a modified method. In brief, a 7-µm carbon fiber (Togai Inc.) connected to a copper wire with silver conductive paint was aspirated into a standard glass capillary (Esselte Leitz GmbH & Co. KG), which was pulled in advance into a pipet with a tip of ∼50 µm. The extremity of the pipet was fixed with an ethanol flame, and the other end was filled with epoxy resin in order to ensure tightness. The carbon fiber was treated in an etch solution (35) Harary, I.; Farley, B. Science 1960, 131, 1674-1675. (36) Keith, A. W.; Nanette, H. B. J. Mol. Cell Cardiol. 1992, 24, 741-749. (37) Meulemans, A.; Poulain, B.; Baux, G.; Tauc, L.; Henzel, D. Anal. Chem. 1986, 58, 2088-2091. (38) Armstrong-James, M.; Fox, K.; Millar, J. J. Neurosci. Methods 1980, 2, 431434. (39) Malinski, T.; Taha, Z. Nature 1992, 358, 676-677.

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Figure 1. Diagram of the experiment arrangement toward single myocardial cell: (a) Au auxiliary electrode, (b) cylindrical carbon fiber ultramicrosensor, (c) modified Ag/AgCl reference electrode, (d) a living and beating myocardial cell, (e) Hanks balanced saline solution, and (f) stereomicroscope.

(containing K2Cr2O7/H2SO4/H2O, 1:50:50 w/w) with a constant potential of 4.0 V. In this process, the carbon fiber had an obvious change in length, but only an indistinctive change in diameter. Every 10 s the fiber was viewed under the stereomicroscope until the length was only ∼20 µm. Then the carbon fiber was etched with flowing etch solution; i.e., the solution flowed from the fiber tip straight to the glass capillary. The process was continued until a tip of ∼1 µm was obtained. Modification of the poly-Mn(TAPc) and PVP on the cylindrical carbon fiber ultramicroelectrode was described as above. Electrochemical Determination of Peroxynitrite. Prior to peroxynitrite determination, the ultramicrosensors were placed in 5.0 mL of buffer solution, and the potential was cycled between -0.2 and +1.0 V(vs SCE) at a scan rate of 100 mV/s for ∼5 min. Peroxynitrite determination was performed by subsequently adding aliquots of peroxynitrite stock solutions with a gastight syringe. The current of the electrocatalytic reduction of peroxynitrite was recorded after each injection. Real Sample Analysis. After being washed and filled with HBSS, MCs in growth medium kept in an incubator at 37.0 ( 0.5 °C attaching to the floor of Petri dishes were counted under the stereomicroscope. In terms of the culture medium, the experimental samples were divided into 15 groups, including 3 cell-free groups, 3 normal groups, 3 ischemia groups and 6 ischemia/ reperfusion groups. With the guidance of the stereomicroscope, the three-electrode system (including cylindrical carbon fiber peroxynitrite UMS, Au wire, and Ag/AgCl wire) was bound together so that distances between each electrode were unchanged and then the peroxynitrite UMS was punctured into living, beating MCs (the arrangement diagram was shown in Figure 1). Peroxynitrite generation was monitored with calibrated peroxynitrite carbon fiber cylindrical ultramicrosensors by the DPA technique in all real sample analysis. All numerical data are expressed as means ( SEM. Data were analyzed by analysis of variance with Scheffe’s F-test. Statistical significance was considered at the P < 0.05 level and indicated in the figures and text. 5316 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

Figure 2. (A) Consecutive cyclic voltammograms of 5 mmol/L Mn(TAPc) in DMSO on the 15-µm platinum microelectrode at a potential range of -0.50 to +1.0 V (vs Ag/AgCl) with a scan rate of 100 mV/s. (B) UV-visible absorption spectrum of 1.0 × 10-5mol/L Mn(TAPc) in DMSO (a); UV-visible absorption spectrum of poly-Mn(TAPc) on ITO electrodes at 0.0 V vs Ag/AgCl in HBSS without peroxynitrite (b); same as (a) but at 0.80 V vs Ag/AgCl (c); poly-Mn(TAPc) on ITO electrodes at 0.0 V in HBSS containing 1.0 × 10-5mol/L peroxynitrite (d).

RESULTS AND DISCUSSION Preparation and Characterization of UMS. Cyclic voltammetry of 5.0 mmol/L Mn(TAPc) in DMSO was carried out over the potential range of -0.5 to +1.0 V. Figure 2A shows a series of consecutive cyclic voltammograms for a 200-µm platinum planar electrode at a constant scan rate of 100 mV/s with a 5.0 mmol/L Mn(TAPc) solution in DMSO (0.1 M TBAP). On the first voltammetric scan at the pretreated electrode, a cathodic peak was observed at a peak potential value of -0.35 V. Upon scan reversal to +1.0 V, three significant anodic peaks were observed at -0.23, 0.10, and 0.75 V, respectively. At the beginning of scan period, the redox wave at the potentials of 0.60 and 0.75 V disappeared gradually, and the anodic current (related to the oxidation of TAPc cycles, which was consist with the electrochemical cyclic scan process of TAPc in DMSO) around 0.80 V increased gently. On further potential scanning through the reported potential range, two additional anodic peaks shifted to

the potential values of -0.16 and +0.02 V, respectively, and cathodic peak at ∼-0.35 V shifted a little negatively. Another broad cathodic wave around the potential value of 0.0 V was observed. The current of both redox waves, which could be due to the redox processes of MnII/III and MnIII/IV, respectively, increased with continued potential cycling. The shape of the voltammetric feature is that of a surface-immobilized redox couple, suggesting the formation of a redox-active polymer film on the electrode surface. Further proof could be obtained from the data of the UV-visible spectrum and the scanning electron micrograph (SEM). Subsequently, the poly-Mn(TAPc)-modified electrodes were removed from the Mn(TAPc) solution, rinsed with DMSO and water, and coated with 1% PVP twice. The poly-Mn(TAPc)/PVPmodified electrodes were placed in HBSS and cycled in the potential range of -0.2 to +1.0 V; two anodic peaks were observed with formal potential values of 0.52 and 0.78 V at the first scanning. Upon further cycling, a cathodic wave was observed at the potential of 0.0 V and the current increased continuously until after ∼15 cycles. The UV-visible spectrum of the tetraaminophthalocyanine manganese complexes after polymerization is shown as Figure 2B. The absorption spectrum of Mn(TAPc) in DMSO solution (curve a) is compared with that of the polymer film on a optically transparent indium-tin oxide electrode (curve b). This clearly indicates that a poly-Mn(TAPc) film was present on the electrode surface. Experiments identical to these described above for platinum electrodes were performed on electrochemical pretreated carbon fiber electrodes. Unlike on the platinum electrodes, there was not obvious redox wave in the potential range used above. However, both anodic current and cathodic current were increasing with consequent cycling in DMSO containing Mn(TAPc). And such a modified electrode also demonstrated electrocatalytical activity toward peroxynitrite reduction as described below similar to the modified electrode with platinum as the matrix electrode. Electrocatalysis of Mn(TAPc) toward Peroxynitrite Reduction. According to the data of quantum chemistry, the cloud density of every atom were given as follows:

The O* atom has maximum cloud density; therefore, we suggested that it provide the lone-pair electron to the center manganese atom (cloud density of -0.252) of Mn(TAPc) when they form an axial coordination compound. At the following chemical reaction, peroxynitrite was oxidized to nitric dioxygen and nitrite.40-41 The following equations show the detailed catalysis process:

poly-(TAPc)Mn(III)(H2O) + OdN-O-O- f [poly-(TAPc)Mn(III)-OONO]- + H2O (1) 2[poly-(TAPc)Mn(III)sOONO]- f 2poly-(TAPc)Mn(IV) ) O + NO2- + NO2 (2)

poly-(TAPc)Mn(IV)dO + 2e +2H+ f poly-(TAPc)Mn(III)(H2O) (3)

The UV-visible absorption spectrum (see Figure 2B) of the poly-Mn(TAPc) film on a ITO-transparent electrode in HBSS was compared with that of similar polymer after addition of peroxynitrite and that of the electrooxidized form of the poly-Mn(TAPc) film (constant potential at 0.8 V vs Ag/AgCl in HBSS) in the absence of peroxynitrite. In terms of the spectrum data, the polyMn(TAPc) film electrooxidized at 0.8 V and the film oxidized by peroxynitrite had a similar spectrum. This result suggests that the oxidation by peroxynitrite changes the poly-Mn(TAPc) film spectrum and yields new compounds. Response of Peroxynitrite at UMS. The responses of peroxynitrite at UMS were assayed in HBSS solution incubated at 37.0 °C. Cyclic voltammetric tests in the absence and presence of a certain concentration peroxynitrite were performed to assess the activity of the UMS. Figure 3a shows the cyclic voltammetric response at a 15-µm planar carbon fiber-modified microelectrode in the absence of peroxynitrite. After 5.0 × 10-7 mol/L peroxynitrite was introduced, an enhancement of the cathodic peak is clearly noted (as shown in Figure 3b). Curve c in Figure 3 corresponded to the electroreduction of 1.0 × 10-6 mol/L peroxynitrite on poly-Mn(TAPc)/PVP-modified microelectrode. When the same concentration of peroxynitrite used for curve c was injected onto a PVP-modified microelectrode, only an anodic current increase at the potential value of 0.80 V took place (Figure 3d), which may be due to the electrooxidation of NO2- at the PVPmodified microelectrode. Ten seconds after addition of peroxynitrite, the anodic current decreased down to ∼5% of the original current. It may be that the reaction results in an extremely unstable substance and the peroxynitrite is oxidized rapidly in the neutral solution. One of the purposes of the present investigations was developing a rapid, real-time, and in vivo detection technique. As mentioned above, electrochemical techniques exhibited excellent advantages as compared to other techniques such as chemiluminescence. Therefore, it could be used to measure bioactive substance concentrations, which often vary in biological systems. To monitor the variation of the peroxynitrite concentration, we employed differential pulse amperometry (DPA), a sensitive technique. Differential pulse amperometry was performed according to the following procedure: the initial potential of the ultramicrosensor was set at 0.20 V for 100 ms to clean the work electrode and then the applied potential as first pulse potential was kept for 100 ms sequentially. After this potential delay, the potential subsequently was jumped from 0.20 to 0.0 V. At 0.0 V, the potential was maintained for 100 ms. The current measured was the difference between the values at 0.20 and 0.0 V. Figure 4B illustrates a typical DPA response of peroxynitrite at the carbon fiber UMS (600 nm in diameter and 18 µm in length) in HBSS solution incubated at 37.0 °C. Corresponding to each current increase, 1.0 × 10-7mol/L PON was delivered with a microinjector. (40) Marla, S. S.; Lee, J.; Groves, J. T. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14243-14248. (41) Balavoine, G. G. A.; Geletii, Y. V.; Bejan, D. Nitric Oxide: Biol. Chem. 1997, 1, 507-521.

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Figure 3. Sampled cyclic voltammetric response to peroxynitrite anion in HBSS containing peroxynitrite at 0 (a), 5.0 × 10-7(b), and 1.0 × 10-6 mol/L (c) at a planar carbon fiber-modified microelectrode (diameter 15 µm); 1.0 × 10-6 mol/L peroxynitrite at a PVP-modified microelectrode (d); and 1.0 × 10-6 mol/L peroxynitrite at a bare electrode (e). Scan rate was 500 mV/s.

Every DPA signal had a rise time of less than 0.5 s. However, at only less than 10 s, a sharp decay of signals was observed, which contributed to the fast degradation of peroxynitrite. Such phenomena also came into being in the process of differential pulse voltammetric (DPV) scanning (figure is not shown). In addition, as shown in Figure 4B, the peroxynitrite UMS was inert to H2O2 and NO2- primarily due to its low operation potential. Sensitivity, Calibration (Detection Limits and Life Time), and Selectivity. We report here that our results obtained under optimum conditions. Figure 4A depicts the linear variations of the measured currents by DPA techniques at 0.0 mV vs Ag/AgCl with a cylindrical carbon fiber ultramicroelectrode modified with polyMn(TAPc)/PVP. It appears from these data that the sensitivity of the peroxynitrite UMS decreases from 2.4 × 10-3 to 8.2 × 10-4A mol-1 L when peroxynitrite concentration varies from 2.0 × 10-8 to 8.0 × 10-5mol/L. These data ensured that the calibration of the peroxynitrite electrodes is effective in HBSS and show that 5318 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

the analytical procedure presented here can be used to quantify peroxynitrite for peroxynitrite assay in vivo at submicromolar and micromolar concentrations. Interference by electroactive materials is the biggest drawback of the electrochemical analysis method. However, we could mitigate the interference insofar as possible and even diminish it. In this paper, on one hand the ultramicroelectrodes modified with poly-Mn(TAPc) were further coated chemically with PVP, a polymer film with positive charge, which could not only prevent the diffusion of positive electrical (e.g., dopamine) to the electrode surface but also diminish the electrode fouling, to enhance the selectivity of sensors. On the other hand, the peroxynitrite UMS was operated at 0.0 V vs Ag/AgCl; as a result, the majority of interference from electroactive materials could be diminished, because these materials were easily oxidized on the anode at potentials over 0.2 V vs Ag/AgCl. For evaluating the sensors’ selectivity, which is relative to a variety of potential interferences present in biological samples, we performed the interference tests in vitro with DPA techniques. The interfering agents include the neurotransmitters, their metabolites, and other species such as AA, UA, O2-, and NO2-. The results are summarized in Table 1. From Table 1, it is clear that the above substances have no apparent effects on the responses of the sensors and also demonstrate that the poly-Mn(TAPc)/ PVP-modified electrodes should have interference-free signals for peroxynitrite measurements in vivo. Some biological substances such as uric acid (UA) and glutathione (GSH) still shift DPA signals of peroxynitrite at UMS, but they did not interfere with the determination of peroxynitrite because they themselves did not bring out DPA responses at the peroxynitrite UMS. We suggest that the peroxynitrite be reduced chemically by these substances, which have been initially verified to be scavengers against PON.42-43 The detection limits of the sensors were defined as the analyte (peroxynitrite) concentration yielding a signal equal to 3 times the standard deviation of the background current. In the present paper, we performed experiments 8 times and obtained an average result with a calculated detection limit of ∼1.8 × 10-8mol/L. To investigate the lifetime, we prepared some modified electrodes, divided into four groups and stored in (1) humid air (4 °C) with saturated aqueous vapor tension, (2) HBSS (4 °C), (3) xeric air (4 °C), and (4) humid air (room temperature) with saturated aqueous vapor tension, respectively. The contrast of variations (shown in Figure 5) of the measured current by DPA expressed that condition 1 was more propitious than the others for storing of the UMS. Measurement of Peroxynitrite Released from Myocardial Cells. The present studies showed that peroxynitrite production by injured single myocardial cells was induced by ischemia/ reperfusion. To quantify the amount of peroxynitrite release from single myocardial cells, the above-described cylindrical carbon fiber peroxynitrite UMS were used to determine the dynamical variation of peroxynitrite concentration through monitoring the current with the DPA technique. When the peroxynitrite UMS was removed from the HBSS without cells and placed into the HBSS containing normal (42) Cheung, P. Y.; Schulz, R. Am. J. Physiol. 1997, 273, H1231-1238. (43) Xie, Y. W.; Kaminski, P. M.; Wolin, M. S. Circ. Res. 1998, 82, 891-897.

Figure 4. (A) Linear variations curve obtained from DPA responses with standard peroxynitrite solution. The solid line is the average of n ) 4 measurements with the same peroxynitrite UMS as sensors for monitoring in vivo. (B) DPA responses to injection of 1.0 × 10-7 mol/L peroxynitrite (a), 1.0 × 10-6 mol/L H2O2 (b), and 1.0 × 10-6 mol/L NO2- (c). Technical parameters: cleaning potential 0.20 V for 100 ms, first pulse potential 0.20 V for 100 ms, and second pulse potential 0.0 V for 100 ms. The indicated currents were the differences of currents at the first and second pulse potentials. (C) Typical differential pulse amperograms illustrating the variation of peroxynitrite concentration with time during the ischemia/ reperfusion period in a single myocardial cell (a), a myocardial cell treated with 100 µmol/L melatonin (b), a normal myocardial cell treated with 100 µmol/L melatonin (c) and cell-free HBSS treated with 100 µmol/L melatonin (d). The tehcnical parameters were the same as in Figure 4. Ischemia and reperfusion points are indicated by arrows. Melatonin was administrated at the beginning of the ischemia period. (D) Typical differential pulse amperograms illustrating the alteration of peroxynitrite production from a single myocardial cell subject to 0.2 mmol/L L-arginine (a), 4.0 mmol/L L-NAME (b), 500 units/mL SOD (c), 3.0 mmol/L L-NAME (d), etc.

myocardial cells, a basal level of peroxynitrite was undetectable with the DPV technique. After continuous ischemia, however, the peroxynitrite released from a single myocardial cell tardily was electroreduced on the UMS punctured into the cell. As shown in Figure 4C(a), the emission of peroxynitrite ∼7 ( 2 min after the beginning of ischemia was indicated on the DPA response by a significant increase in the current. A plateau segment was observed from the 15 ( 3 to 33 ( 5 min, and maximum peroxynitrite concentration (68.4 ( 7.9 nmol/L) released from intracellular myocardial was perceived at about 24 ( 6 min. These data demonstrated that ischemia injury induced cells to secrete peroxynitrite. In succession, for each group, reperfusion progressed at 30 min and 1 h soon after ischemia, respectively. Along with the reperfusion, the peroxynitrite concentration increased massively over again. Figure 4C (curve a) illustrates typically that the variation of peroxynitrite concentration was dependent upon time. For IRG (30 min) and IRG (1 h), the amount

of peroxynitrite released from the MCs increased rapidly such that at the period of reperfusion the amount of peroxynitrite was significantly higher as compared to the amount at the ischemia period. To ensure that the observed signals were due to cellular evolution of peroxynitrite from NO and O2- generation, various related experiments were carried out. Superoxide dismutase (SOD), a specific enzyme for dismutation of superoxide anion, was introduced into the culture medium at the period of reperfusion. The administration of SOD at the doses of 200 and 500 units/mL caused a decrease in peroxynitrite production of ∼17% for the dose of 200 units/mL and ∼29% for the dose of 500 units/ mL (P < 0.05 for both doses). NO generation could be inhibited in a dose-dependent manner by L-NAME, a NO synthase inhibitor. When several concentrations of L-NAME were injected into culture medium during reperfusion, DPA signal alteration was observed; 1.0 mmol/L L-NAME caused a 22% average reduction of DPA Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

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Table 1. In Vitro DPA Responses of the Cylindrical Carbon Fiber Ultramicrosensors for a Variety of Potential Interfering Agents Expressed as a Percentage of the Electrocatalytical Reduction Current of Peroxynitrite (1.0 µmol/L) in HBSSa interference (peroxynitrite) O2 O2H2O2 NO NO2NO3ascorbic acid (AA) uric acid (UA) dopamine (DA) 3,4-dihydroxyphenyl acetic acid (DOPAC) norepinephrine (NE) epinephrine (E) 5-hydrotryptamine (5-HT) 5-hydroxyindole-3-acetic acid (5-HIAA) reduced glutathione (GSH) oxidized glutathione (GSSG) NAD+ NADP+ NADH NADPH L-arginine

concn (µmol/L)

peroxynitrite (%)

satd 50 500 100 1000 1000 5000 5000 500 500

100 (0.204 ( 0.07 nA) 1.39 ( 0.10 1.02 ( 0.09 2.48 ( 0.66