CA2133994A1 - Nitric oxide sensor - Google Patents

Abstract

A nitric oxide (NO) microsensor (34) based on catalytic oxidation of NO comprises a thermally-sharpened carbon fiber with a tip diameter of about 0.5-0.7 µm coated with several layers of p-type semiconducting polymeric porphyrin and cationic exchanger deposited thereon. The microsensor (34) which can be operated in either the amperometric voltametric or coulometric mode utilizing a two or three electrode system, is characterized by a liner response up to about 300 µM, a response time better than 10 msec and a detection limit of about 10 nM. The sensor (34) of the present invention also discriminates against nitrite, the most problematic interferant in NO measurements. The amount of NO released from a single cell can thus be selectively measured in situ by a porphyrinic microsensor (34) of the invention. A larger scale sensor (12) utilizing porphyrin and cationic exchanger deposited on larger fibers or wires, platinum mesh or tin indium oxide layered on glass, can also be employed when measurement of NO
concentration in chemical media, tissue or cell culture is desired.

Description

WO 93/21518 prL~/US93/037 NITRIC OXIDE SENSOR
RELATED APPLICATION
This is a continuation of U.S. Application Serial No. 07/871,463, entitled "Nitric Oxide Sensor,U filed April 21, 1992, by Tadeusz Malinski, incorporated herein by5 reference.
FIELD OF THE INVENTION
The present invention generally relates to sensors and sensing techniques which can selectively and quantitatively deteet NO in solution in both biological and chemical media. More specifically, the present invention relates to NO sensors which 10 utilize conductive catalytic materials deposited on microfibers or other supports to monitor the presence or release of NO using amperometric, voltammetric or coulometric methods.
BACKGROUND OF THE INVENTION
Nitric oxide (NO) has recently been shown to be a key bioregulatory molecule 1~ in a number of physiological processes. For example, NO plays a major role in the biological activity of endothelium derived relaxing fac~or (EDRF), abnormalities in which are associated with acute hypertension, diabetes, ischaemia and atherosclerosis. NO
is also considered a retrograde messenger in the central nervous system, appears to be involved in the regulation of macrophage cytotoxic activity and platelet aggregation 20 inhibition, and has been implicated in endotoxic shock and genetic mutations. In ad~ition, a number of drugs and other xenobiotics are metabolized to produce NO as either the eflector molecule or as a harmful m~tabolite. See e.g. Furchgott, P(.F. et al., Natvre 288:37~376 (19803; Palmer, P~.M. et al., Nature 327:~24-526 (1987); Furchgott, R.F., "Mechanism of Vasodilation", IV;401414 (ed. Vanhoutte, P.M.~ (Raven, NY) 25 (1988); Ignaro, LJ. et al., PNAS (USA) 84:926~9269 (1987); Wei, E.P. et al., Clr. Res.
57:781-~87 (1985); Piper, G.M. et al., J. ArT1. J. Pt~ysiolO 24:48254833 (1988);Vanbethuysen, K.M. et al., J. Clin. Invest. 79:26~-274 (1987~; Freiman, P.C. et al., Circ.
Res. 58:783-789 (1986); and Schuman, E.M. et al., Science 254:1503 (1991).
Several different methods have been employed in the past to measure NO
30 concentration in aqueous solution. For example, analysis of the ultimate aerobic oxidation products of NO, i.e. nitrite/nitrate ~NO2-/NO3-), has been used as a measure of NO. Monitoring of UV-vis spectral changes resulting from the conversion of oxyhemoglobin to met-hemoglobin has also been used as an indicator of NO
concentration. These methods, however, provide only an indirect and thus less 35 accurate measurement of NO. NO has also been measured in biological systems WO 93/21518 213 3 9 9 ~ PCr/US93/03701~

using a Thermal Energy Analyzer (TEA), in which NO reacts with ozone to produce a characteristic chemilumineseent response. Downes, M.J. et al., Analyst 101:742-748 (1978). This approach, however, requires a lengthy regeneration time, the isolation of NO from solution, and cannot be miniaturized for in situ monitoring of N0 release.
5 Mass spectrometry has also been attempted, but has problems similar to the TEA approach. ~
Recently, a modified oxygen electrode for thç detection of nitric oxide has alsobeen reported. Shibuki, K., Neurosa Res. 9:6~76 (1990). This electrode, however, has a relatively large diameter (0.25 mm), a slow response time and a narrow 10 concentration range (1-3 ~.M). Although this method is advantageous in that it discriminates against the N02- produced in the outer solution of the electrode, it is not selective for NO in the presence of any NO2- produced in the electrode inner solution.
There is also some question of the validity of this technique owing to the small current observed and the lack of standards done in less than ~lM concentration.
Although the above-described methods can be used to measure NC3 in biological or chemical media, they are not sufficiently sensitive nor specific to provide a direct and accu!ate quantitative measurement of NO, particularly at low NO
concentra~ions. Furthermore, none of the methods or sensors employed to date canrapidly and selectively measure NO release by the cell In SitU in the presence of 20 oxygen and/sr N02-. Development of this methodology is crucial in order to evaluate endogenous NO release, distribution and reactivity on molecular ievel in biological systems.
Thus, there exists a need for a sensitive and selective sensor for direct quantitive measurements of N0. An optimal sensor for monitoring NO release should 25 be sturdy and capable of sufficient miniaturiza~ion for in Situ measurement in a single cell. The senscr should also be sensitive enough to produce an adequate signal to be observable at the low levels of N0 secreted in biological environments. Due to the variation in the amount of NO secreted by different types of cells (e.g. from nanomoles/106 cells in maorophages to picomoles in endothelial cells), the signal 30 produced by the sensor should also change linearly over a wide range of concentrations. See Marletta, M.A., Trends B~ochem. Sci. 14:488492 (1989). The short half-life of NO in biological systems, on the order of about 3 - 50 seconds, also mandates a fast response time. See Moncada, S. et al., Pharmacol. Rev. 43:10~142(1991). The NO sensor and method of the present invention exhibit these desirable 35 characteristics.

' WO 93/21518 21 ~ 3 9 9 '¦ PCr/US93/03701 SUMMARY OF THE INVENTION
The NO sensor and method of the present invention provide a direct and accurate measurement of NO in biological and chemical media. A sensor of the present invention generally comprises an electrode having a catalytic material capable 5 of catalyzing oxidation of NO coated with a cationic exchanger. The sensor provides a direct measurernent of NO through the redox reaction of NO - NO+ ~ e~ and is selective for NO through the discrimination of the cationic exchanger against nitrite (NO2~. Although the sensor can be fabricated on any scale, it can be miniaturized to provide a microsensor which can accurately measure NO in situ at the cell level.
In one preferred embodiment of the invention, the amount of NO released from a single cell can be selectively measured in situ by a microsensor with a response time better than about 10 msec. The rnicrosensor comprises a thermally-sharpenedconductive carbon fiber with a tip diameter of about 0.5-0.7 ~m covered with several layers of polymeric porphyrin capable of catalyzing NO oxidaiton with a cationicexchanger deposited thereon. Using either a two ~r three electrode arrangement, the ~nicrosensor can be operated in either the amperometric, voltammetric or coulometric mode. The microsensor is characterized by a linear response up to abou~ 300 ~M
and a detection limit oS about 10 nM NO concentration, which allows detection of NO
rsl~ase at the levels present in a single biological cell. Th8 sensor also discrimina~es against N02-, the most problematic interferant with current NO sensing techniques.
In other embodiments of the present invention, larger scale NO sensors are used to measure NO concentrations in chemical media, cell culture, extracellular fluids and tissue, rather than in single cells. For example, a carbon electrods Witll a larger tip diameter, platinum mesh or a tin indium oxide layered plate is coated with aconductive catalytic polymeric pvrphyrin and a cationic exchanger. A linear response and low detection limits similar to tl e NO microsensor are observed.
Other features and advantages of the present invsntion will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA and B depict preferred monomeric porphyrin structures used in sensors of the present invention.
Figurc 2 is a dmerential pulse voltammogram of NO at various concentrations.
Figure 3 is a graph showing nitric oxide response (nA) of NO solutions 3~ measured by a sensor of the invention.

2 1 3 3 9 9 4 PCI/US93/03701 .

Figure 4A is a microscopic photograph of a carbon fiber microsensor of th present invention.
Fi~ure 4B is an electron scanning micrograph of the portion of the microsensor covered with a coat of isolating wax-resin mixture.
Figure 4C is an electron scanning micrograph of the thermally-sharpened tip of the microsensor covered with conductive polymeric porphyrin.
Figure 5 is a scan showing the grou~h patterns~or poly-TMHPPNi, deposited from 5 x 1 o4M TMHPPNi, 0.1 M NaOH solution by continuous scan cyclic voltammetry on a carbon fiber microelectrode.
Figures 6A, B, C and D are voltammograms showing the response of the rnicrosensor in the differential puise voltametric mode.
Figures 7A, B, C and D are scans showing the response of the microsensor in the amperometric mode.
Figure 8A - C are schematic overviews of macrosensors of the present invention used in cell culture.
Figure 9 shows the response of a platinum nlesh macrosensor to NO release by a cell culture grown directly on the sensor surface.
DETAIL~D DESCRIPTION OF THE PREFERRED EME~ODIMENTS
The basic strategy used in the design of a pref~rred embodiment of the NO
20 sensor is based on catalytic oxidation of NO which uses a specHic potential unique to NO - NO+ ~ e~. The normal oxidation potential for NO is about 1.0 V vs SCE ona standard platinum electrode, which potential can be lowered with various materials capable of catalytically oxidizing NO. The current or charge generated thereby is high enough ts be used as an analytical signal in microsystem.
In accordance with the principles of the pr~sent inv~ntion, a working electrode of a sensor of the present comprise a conductive solid support with a catalytic surface for NO oxidation. A catalytic surface on a conductive support can be provided using several approaches. For example a conductive catalytic material capable of catalyzing NO oxidation can be layered on a conductive solid support; the conductive catalytic material can be layered on a conductive material coated on a conductive or nonconductive base material; or the conductive catalytic material can itself comprise the conductive support. The third approach can be accomplished by fashioning theelectrode directly from the conductive catalytic material or by incorporating or doping a catalyst into the support material. A working electrode of a sensor of the described - 35 embodiment of this invention preferably comprises a solid conductive support coated ~`1 WO 93/21518 21 339 9 4 PCI/US93/03701 with one or more layers of a conductive material capable of catalyzing oxidation of NO, hereinafter referred to as catalytic material.
It will be appreciated that several types of catalytic materials can be used in a sensor of the present invention, as long as tha catalytic material exhibits electronic, 5 ionic or redox conductivity or semiconductivity, collectively referred to herein as conductivity. Such materials include, but are not limited to, polymeric porphyrins and polypthalocyanines. The above-mentioned rnaterials can contain central metals, preferably transition or amphoteric metals. Polymers which can also be used but require doping include, tor example, polyvinylmetaliocenes (e.g. ferrocene), 10 polyacetylene doped with different metal redox centers and polypyrroline doped with different redox centers such as, e.g. methyl viologen.
Preferred catalytic conductive materials for a sensor of the present invention are polymeric metalloporphyrins, which are organic p-type semiconductors with relatively high conductivity and which can be successfully deposited on a supporting 15 conductive material. Polymeric metalloporphyrins have been shown to have highcatalytic effect tor the electrochemical oxidation of several small organic and inorganic molecules. Bennett, J.E. et al., Chem. Materials 3:49~495 (1991). Polymeric porphyrins polymerized and copolymerized from monomeric porphyrins N,N'~i(~p-phenylene-10,1 5,2~tri(3-methoxy-4-hydroxyphenylkorphyrin;1,1 0,-phenantroline4,7-~0 diamine, and 5-p-(pyrole-1 -yl) phenylene-10,1 5,20-tri-(3-methoxy-4-hydroxyphenyl)porphyrin with Fe, Mn, Co and Ni as central metals are more preferred given their high catalytic effect for selective electrochemical oxidation of NO. Even more preferred compounds include tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin (TMHPP) and meso-5'-O-p-phenylene-2',3'-O-isopropylidene uridine-~ri(n-methyl-4 25 pyridinium)po7phyrin (PUP), shown in Figures 1A and B.
In order to discrimina~e against NO2-, the porphyrinic catalysts used in the present invention are also preterably covered with a thin layer of a cationic exchanger to prevent anion diffusion to the catalytic surfac~. Suitable cationic exchangers include AQ55D available from Kodak and Nafion. Nafion, which is used in the Specific 30 Examples, is a negatively charged cationic exchange polymer which prevents diffusion of anions 5ike N02- to the electroactive surface of the polymeric porphyrin, but is highly permeable to NO.
The thin layer ot polymeric porphyrin film can be electrochemically deposited, as described in detail below, on any solid conductive support. As previously 35 discussed, a conductive support can comprise a material that in itself is conductive W O 93/21518 PC~r/US93/03701 '~
213399 ~ 6-or a conductive or nonconductive base material coated with a conductive material.
Conductive materials which do not need to be coated with additional conductive materials are preferred. It will also be appreciated that, although the catalytic component of the invention is preferably layered on a conductive support, the 5 conductive catalytic material can also compris~ the conductive s~pport. Conductive support materials particularly suitable for sm~r scale sensors of the invention include carbon fibers, and gold or platinum wire. ~ùe to their mechanical properties as well as the possibility for controlled miniaturization, carbon f~bers are preferable support materials for microsensors in single cell applications. See e.g. Malinski, T. et al. Anat.
10 Chem. Acta. 249:35-41 ~1991); Bailey, F. et al., Anlal~ Chem. 63:395-3g8 (1991).
It will be appreciated that the dimension of the sensor of the invention can be varied to produce virtually any size sensor, including microsensors with a tip diameter of about 1 ~m or less and macrosensors, including fibers with a larger tip diameter (e.g. about 1 - 10 mm) and metallic mesh and conductive layered plates. Thus, while 15 Specific Examples Il-V describe the production and use of a microsensor for use in small environments such as single cells ar syr:apses, the same techniques can beapplied to a larger support, such as described in Specific Examples I and IV, toproduce convenient macrosensors for tissue, cell culture or chemical media studies.
In measuring N0, a two or preferably thres electrode system can be employed.
20 The workin~ electrode, comprising the coated carbon fiber, with mesh or plate, is connected to a conductive lead wire (e.g. copper) with conductive (~.9. silver) epoxy, with the lead wir~ connecting to the voltammetric analyzer, potenffostat or coulometric measuring instrument. The auxiliary or counterelectrode generaliy comprises a chemically inere conductivs material such as a nobet metal (e.g. platinum wire), carbon 25 or tin indium oxide which is also connected to the measuring instrument with a lead wire. In a three electrode system, a reference electrode, such as a standard calomel electrode (SC~3, is also employed and connected to the measuring instrument witha third conductive lead wire.
In use, the working electrode, with the other electrode(s) in proximity, is placed 30 into the analytic solution. It will be appreciated that by "analytic solution" is meant any aqueous or nonaqueous solution in which N0 is to be detected or measured~ The term thus includes both chemical and biological media, including tissue fluids and extracellular and cellular fluids. It will also be appreciated that the sensor of the invention can be used quantitatively to detect the presence of N0 and also 35 quantitatively to measure the levels o~ N0 present in the analytic solution. To detect ~j WO 93/21518 2 1 3 3 ~ 9 ~ PCT/US93/03701 or measure NO release in a singie biological cell, a microsensor of appropriate dimension can be either inserted into or placed close to the cell membrane.
The cell membrane surface concentration of NO is influenced by the following factors: release of NO due to the action of bradykinin~ adsorption and chemisorption 5 o~ NO on the surface of the cell membrane, oxidation by 2 and organic molecules, and diffusion into other cells and to the bulk solution. The decay in NO response following the addition of standard amounts of oxygen was studied. Decreases of only 22% and 35% were observed a~ter 4 and 10 min respectiveiy, tollowing the addition of 100 ~M 2 to a 20 ~lM NO solution in the absence of biological material. These 10 measurements in the presence of 2 indicate that Hs role in the oxidation of NO may have been overestimated, and that NO oxidation is due mainly to the biological material as was previously suggested by Monocada. See Moncada, S. et ai., .Pharmacol. Rev. 43:109-142 (1991).
SPECIFIC E)CANIPLE I
A carbon macroelectrode covered with cont1uctive porphyrin polymer was prepared as ~ollows. A glassy carbon electrode ~GC~) (diameter about 2 mm) was coated wrth conductive polymeric porphyrins by cyclic voltammetry or controlled potential oxidation (4 min3 at 0.7 V vs SCE of the monomeric porphyrin in 0.1 M NaOH
solution (5 ml). The auxiliary electrod~ was a platinum (Pt) rod and the re~erence 20 electrode was a standard calomel electrode (SCE). The porphyrin-coated (about 0.8 -1.5 nmlcm2) electrode was removed from the solution and stored in 0.1 M base. The porphyrins used were Ni2+~ Co2~, or Fe3+ TMHPP or PUP as shown in Figures 1A
- and E3. The porphyrin-coated electrodes were then further coated with 4 ~l of 5%
- Nafion solution.
2~ A stock solution of saturated nitric oxide was prepared anaerobically in pH =
7.4 (0.1 M) phosphate buffer. This stock solution was then added in the correct volume to obtain the desired final concentration of NO (10, 20 and 40 ~M). The slectrochemical celi had a Pt rod counter electrode and SCE re~erence electrode and the working electrode was the glassy carbon electrode coated with polymeric 30 porphyrin film and Nafion, as described above. It will, also be appreciated that a two electrode arrangement, i.e. the working and auxiliary electrode, can be utilized.
Measurements were performed in 5 ml phosphate buffer (pH = 7.4, 0.1 M) which served as the supporting electrolyte. All solutions were degassed prior to use and kept under nHrogen. A base line scan was taken using linear sweep voltammetry 35 (range = 0 to +0.9 V vs SCE) or differential pulse voltammetry (range = +0.4 to 0.9 WO 93/21~18 213 ~ 9 9 ~ PCl`/US93/03701 ?e~

V vs SCE). Aliquots of the N0 stock solution were introduced to the cell via a gas-tight syringe. The final dilution was taken as the final N0 concentration. A ~ypical N0 response by differential pulse voltammetry is shown in Figure 2 (Epa = 0.7 V vs SCE), which shows the results using a GCE/Ni(ll)TMPP Nafion-coated (4 ~L) electrode. As 5 shown in Figure 3, the electrode gave a linear response (as did all three electrodes) in the range of lNO] - 1 = 100 llM (0.7 V vs SCE).
SPECIFIC EXAMPLE l!
Carbon rnicrofiber conductive supports for th:e microsensor were produced by threading an individual carbon fiber (7 ~m) through the pulled end of a capillary tube 10 with approximately 1 cm left protruding. Non-conductive epoxy was put at the glass/fiber interface. When the epoxy that was drawn into the tip of the capillary dried, the carbon fiber was sealed in place; The carbon flber was sharpened following standard procedure using a microburner. See Bailey, F. et al., Anal. Chem. 63:395-398 (1991). The sharpened fiber was immersed in melted wax-resin (5:1 ) at controlled 15 temperature 10r 5 - t5 sec. After cooling to ro~m temperature, the fiber was sharpened again. During burning, the flame temperc~ture and the distance of the flber from the centcr o~ the tlame need to be carefully con~rolled. While the diameter of the sharpened lip is smaller, the tip length is larger, with the overall effect ot the resulting electrode being a slim ~ylinder with a small diameter rather than a short taper. This 20 geometry aids in implantation and increases the active surface area. Scanningeléctron microscopy of the f~ber produced shows that the wax is burned approximately to the top of the sharpened tip. The area of the tip, controllably fabricated with appropriate dimensions, is the only part of the carbon fiber where electrochemical processes can occur. A typical length of the electrochemically active tip is between 25 4 6 ~m. For the sensor to be implanted into a cell, this length must be smaller than ths thickness of the cell. The unsharpened end of the carbon fiber was attached to a copper wire lead with silver epoxy.
Referring now to the Figures, Figure 4A is a microscopic photograph of a complete N0 microsensor of the present invention. Figure 4~ is an electron scanning 30 ~micrograph illustrating the part of the microsensor covered with the coat of isolating wax-resin mixture. Figure 4C is an electron scanning micrograph of the thermally-sharpened tip of the microsensor covered with conductive polymeric porphyrin andNafion as described below.

3 WO 93/21518 21 3 3 9 9 ~ Pcr/us93~o37ol SPECIFIC E)CAMPLE lll The growth patterns for poly-TMHPPNi were examined. Poly-TMHPPNi was deposited from a solution of 0.1 M NaOH containing 5 x 104 M monomeric tetrakis(3-methoxy-4-hydroxy-phenyl)porphyrin (TMHPP), with Ni as a central metal (TMHPPNi)by continuous scan cyclic voltammetry from 0.0 to 101 V, on a carbon fiber microelectrode (16 ~m2 surface area), as generally described in 16. As shown in Figure 5, peaks la and Ic correspond to the oxidation of Ni(ll) to Ni(lll) and teduction of Ni(lll) to Ni(ll), respectively, in the film. The Ni(ll)/Ni(lll) redox couple observed at 0.5 V allows porphyrin surface coverage, (r), to be monitored (optimal r = 0.7-1.2 nmol cm2). Surface coverage is calculated ~rom the charge transferred under process la (r = 0.8 nM/cm2). The surface coverage depends upon the initiall concentration of TMHPPNi, electrolysis time and potential.
Following deposit on the fiber, the porphyrin film was conditioned by 5 - 10 scans from 0.4 to 0.9 V. At this stage, the electrode should be stored in 0.1 M NaOH.
1~ Sensor fabrication was completed by dipping in the Nafion solution ~5h) for 15 - 20 sec and lefl to dry (5 min) and stored in pH 7.4 buffer. Since the Ni(ll)/Ni(lll) reacffon requires dfflusion of OH- to neutralize a charge generated in the poly-TMHPPNi and OH- cannot diffuse through Nafion, the later absence of the Ni(ll)/l~li(lll) voltammetric peaks in 0.1 M NaOH demonstrated the integrity of the Na~lon film coverage.
SPECIFIC EXAMPLE IV
NO monitoring was done by dfflerential pulse voltammetry using a classic three electrode system: the sensor as the working electrode, a saturated calomel electrode (SCE) reference electrode and a platinum (PE) wire auxiiiary electrode. The pulse amplitude was 40 mV and the phospha~e buffer solution was pH 7.4. Differerltial pulse 25 voltammograms were obtained for oxidation of NO on poly-TMHPPNi without Na~lon (depicted as A in Figure 6) and with Nafion (depieted as C in Figure 6~ and for 1 I~M
NO in the presence o~ 20 ~M N02- on poly-TMHPPNi without Nafion (depicted as B
in Figure 6) and with Nafion (depicted as D in Figure 6).
DPV of NO on poly-TMHPPNi without Nafion showed a peak at 0.63 V in buffer 30 pH 7.4 (see ~igure 6A). DPV of a solution of 1 ~ M NO and 20 ~.M N02- showed a single peak at 0.80 V (see Figure 6B). The peak current was thus three times higher than that observed at 0.63 V for NO alone. This indicated that the oxidation of NO2-and NO occur at a similar potential, but that the current increase is not proportional to the concentration of NO2-. The NO peak current with the Nafion-coated sensor was 3~ observed at 0.64 V (see Figure 6C). Although the observed current is lower, Nafion W O 93/21518 Pc~r/uss3/o37ol ~ ~
213399~

coverage provides high selectivity against NO2-. Only a 1% increase in current and no change of potential was observed for oxidation of 1 ~.M NO in the presence o~ 20 ~M N02- (see Figure 6D). Thus the porphyrinic microsensor was selective for NO and insensitive for NO2- up to a ratio of at least 1:20.
A linear relationship was observed between current and NO concentration up to 300 ~M (r = 0.994; slope = 2.05 nA/IlM; n = 21). The response time (time tor the signal increase ~rom 10% to 75%) in the amperometric mode was less than 10 milliseconds. The detection limit calculated at a signal/noise ration = 3 was 20 nM
~or DPV and 10 nM for the amperometric method. Since, in a volume equivalent to that of an average singie cell (1 o~l2 L)~ about 1 o~2 attomoles (1 o~20 moles~ of NO can be detected, the detection limit of the sensor is 24 orders of magnitude iower than the estimated amount of NO released per single cell (1-200 attomol/cell)12~13.
SPECIFlt: EXAMPLE V
Amperometric detection of NO by the microsensor under various biological conditions was also studied. Ring segments from porcine aorta (about 2-3 mm wide) and porcine aorta endothelial cell culture were prepared according to previouslydescribed procedures.17 Using a computer-controlled micropositioner (0.2 mm X-Y-Z
resolution), ~he microsensor could be implanted into a single cell, or placed on the surface o~the cell membrane, or kep~ at a controlled distance~rom the cell membrane.
Alternating cuJrent was measured in three electrode systems, as described abovs, at constant potential of 0.75 V modulated with 4û mV pulse in time intervals of 0.5 sec. The background, shown in Figure 7A, was measured in cell cu~ure mediurn at 37C (DMEM-Dulbecco's Modrfled Eagie Medium, 100 mg/L D-glucose, 2 mM glutamine, 110 mg/Lsodium pyruvate, ~ 5% controlled process serum replacement25 TYPE 1). No change of the background was observed aiter the addition ~ 50 nM of bradykinin to 5 ml of cell culture medium. As shown in Figure 7B, 2 nm of NO were injected by microsyrlnge into the cell culture medium, a S mm distance from the microsensor. As shown in Figure 7C, one microsensor was placed on the surface of~he single endothelial cell in the aortic ring, and another was implanted into the 30 smooth muscle cell. 2 nm of bradykinin was injected into the medium near the endothelial cell. After 3 ~ 0.5 sec (n = 7), NO release was detected and a steady increase of surface concentration to a plateau at 450 + 40 nM was observed after 200 sec (see Figure 7C). After 16 min, the surface concentration of NO decreased to zero.
No signfficant difference in NO surface concentration was found for the endothelial cell 35 trom cell culture (430 ~ 40 nM, n = 7). NO was detected in a single smooth muscle d` 1 WO 93/21518 213 3 9 9 4 PCr/US93/û3701 cell within 6.0 ~ 0.5 s0c (n = 7) after injection of bradykinin and a maximum concentration (130 ~ 10 nM, n = 7) was observed after 90 sec (see Figure 7D). The observed current indicates that the initial concentration around the sensor is 230 nM
and decreases to 40 nM after 17 sec due mainly to depletion of NO by dfflusion and 5 also reaction with 2 SPECIFIC E)(ANIPLE Vl Sensors utilizing a layer of tin indium oxide on a glass plate base used as either a counterelectrode or as the conductive layer of a working electrode were also - constructed. Figure 8A illustrates the use of a layer of indium oxide (14) as a 10 counterelectrode (10), (10), whereas Figure 8B illustrates its use as a conductive l~yer of the working electrode of a macrosensor (12). BCH1 n-yocytes (16) were grown under standard culture conditions at 2 x 107 celllcm2 on a glass plate (18) (Figures 8A and C) or on a plate layered with catalytic polymeric iron porphyrin with Nafion coated thereon (20) (Figure 8B). As shown in Figures 8A and B the tin indium oxide 15 semiconductor layer in both cases was attaehed to the measuring instrument by a copper wire lead (22) with silver epoxy (24).
The schematic of Figure 8C depicts the set up for NO measurements of the cell culture in Figure 8A. Celis were grown on a tin indium oxide (14) layered glass plate ~183 placed in a Petri dish (20) with standard culture media (26). A microsensor 20 working electrode (34) constructed as described in previous Examples was then used to measure NO release in sit~. The culture was micr3scopically monitored (30 -inverted microscope) and the working electrode positioned with a micromanipulator (32). As shown in Figure 8C, microsensor was atta~hed to a measuring instrument such as a voltammetric analyzer (36) with the results fed ~o a computer (38) 25 connected to a plot~er (40) and printer (42) for result readout. NO response results observed were on the order of those in the previously described Specific Examples.
Similar results were also obtained in cell cultures grown on fine platinum mesh and are shown in Figure 9.
The foregoing discussion discloses and describes merely exemplary 30 embodiments of the present invention. One skiJled in the art will readily recognize from such discussion, and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as described herein and defined in the following claims.
All publications clted herein are incorporated by reference.

Claims (27)

WE CLAIM:

1. A working electrode for a sensor of the type for detecting the presence of NO in an analytic solution, the working electrode detecting an electrical signal developed between the working electrode and a counterelectrode, the working electrode comprising:
a) a conductive support having a catalytic surface for catalyzing NO oxidation and thereby generating an NO detection electrical signal; and b) a cationic exchanger disposed on the catalytic surface in contact with the analytic solution, the cationic exchanger allowing diffusion of NO therethrough but preventing the diffusion of anions to the catalytic surface that would mask the NO detection electrical signal.

2. The sensor of Claim 1, wherein the conductive support having a catalytic surface comprises a conductive material with a layer of catalytic material disposed thereon.

3. The sensor of Claim 1, wherein the conductive material is a material selected from the group consisting essentially of carbon, platinum and gold.

4. The sensor of Claim 2, wherein the cationic exchanger comprises a chemically stable perfluorosulfonic acid ion exchange resin.

5. The sensor of Claim 2, wherein the catalytic material comprises a polymer selected from the group consisting essentially of polymeric porphyrins, polypthalocyanines, polyvinylmethallocenes, polyacetylenes and polypyrrolines.

6. The sensor of Claim 2, wherein the conductive material comprises a carbon fiber.

7. The sensor of Claim 2, wherein the catalytic material comprises a polymeric metalloporphyrin.

8. The sensor of Claim 2, wherein the conductive material comprises tin indium oxide.

9. The sensor of Claim 3, wherein the conductive material comprises platinum mesh.

10. The sensor of Claim 6, wherein the fiber is about 1 µm or less in diameter at one tip.

11. A sensor system for measuring the level of NO in an analytic solution comprising:
a) a working electrode comprising a conductive support coated with a conductive layer of catalytic material that will catalyze NO oxidation and generate a detection signal, and a layer of cationic exchanger disposed on the catalytic material and making the detection signal selective to NO;
b) a counterelectrode; and c) an instrument for detecting an electrical signal developed between the working electrode and the counterelectrode in response to the oxidation of NO.

12. The sensor system of Claim 11, wherein the conductive support comprises a conductive material and the catalytic material comprises a polymericporphyrin.

13. The sensor system of Claim 11, further comprising:
d) a reference electrode conductively connected to the measuring instrument.

14. The sensor system of Claim 12, wherein the fiber of the working electrode comprises a carbon fiber, the counterelectrode comprises an inert conductive material and the reference electrode comprises a standard calomel electrode.

15. The sensor system of Claim 12, wherein the conductive material comprises tin indium oxide.

16. The sensor system of Claim 12, wherein the conductive material comprises platinum mesh.

17. The sensor system of Claim 12, wherein the counterelectrode comprises a layer of tin indium oxide.

18. A method of directly measuring NO in an analytic solution generally comprising the steps of:
a) providing a working electrode comprising a conductive support having a layer of catalytic material disposed thereon with a layer of a cationic exchanger disposed on the catalytic material;
b) providing a counterelectrode;
c) providing an instrument for measuring an electrical signal developed between the working electrode and the counter electrode;
d) placing the working electrode in the analytic solution;
e) placing the counterelectrode in the analytic solution; and f) measuring the electrical signal developed between the working electrode and the counterelectrode.

19. The method of Claim 18 further comprising the step of:
g) providing a reference electrode; and h) placing the reference electrode in the analytic solution prior to measuring the electrical signal.

20. A sensor for measuring the level of NO in an analytic solution comprising a conductive support coated with a plurality of layers of a polymeric metalloporphyrin for detecting oxides of nitrogen, and a coating of a cationic exchanger on the plurality of layers of metalloporphyrin, which cationic exchanger allows passage of NO but not anions of other nitrogen oxides and makes the sensor selective for NO.

21. The sensor of Claim 20, wherein the conductive support comprises a conductive fiber having a tip diameter about 1 µm or less in diameter.

22. The sensor of Claim 20, wherein the metalloporphyrin comprises tetrakis (3-methoxy-4-hydroxy-phenyl) porphyrin or meso-5'-O-P-phenylene-2',3'-O-isopropylidine uridine-tri(n-methyl-4-pyridinium)porphyrin.

23. The sensor of Claim 20, wherein the conductive support comprises inert metallic material.

24. The method of Claim 20, wherein the cationic exchanger comprises Nafion.

25. The sensor of Claim 23, wherein the conductive support comprises tin indium oxide.

26. The sensor of Claim 23, wherein the conductive support comprises platinum.

27. A working electrode for a sensor of the type for measuring the level of NO in an analytic solution, the sensor measuring an electrical signal developed between the working electrode and a counterelectrode, the working electrode comprising:
a) a conductive support having a catalytic coating thereon comprising a polymeric metalloporphyrin layer disposed thereon for catalyzing NO oxidation, the oxidation developing an electrical signal as said electrode; and b) a cationic exchanger layer comprising a chemically stable perfluorosulfonic acid ion exchange resin disposed on the layer of catalytic material and in contact with the analytic solution which cationic exchanger layer prevents the diffusion of objectionable anions to the catalytic layer surface, which objectional anions would adversely affect the NO oxidation electrical signal.