Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
  • B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
  • B01J31/18—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes containing nitrogen, phosphorus, arsenic or antimony as complexing atoms, e.g. in pyridine ligands, or in resonance therewith, e.g. in isocyanide ligands C=N-R or as complexed central atoms
  • B01J31/1805—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes containing nitrogen, phosphorus, arsenic or antimony as complexing atoms, e.g. in pyridine ligands, or in resonance therewith, e.g. in isocyanide ligands C=N-R or as complexed central atoms the ligands containing nitrogen
  • B01J31/181—Cyclic ligands, including e.g. non-condensed polycyclic ligands, comprising at least one complexing nitrogen atom as ring member, e.g. pyridine
  • B01J31/1815—Cyclic ligands, including e.g. non-condensed polycyclic ligands, comprising at least one complexing nitrogen atom as ring member, e.g. pyridine with more than one complexing nitrogen atom, e.g. bipyridyl, 2-aminopyridine
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/403—Cells and electrode assemblies
  • G01N27/404—Cells with anode, cathode and cell electrolyte on the same side of a permeable membrane which separates them from the sample fluid, e.g. Clark-type oxygen sensors
  • G01N27/4045—Cells with anode, cathode and cell electrolyte on the same side of a permeable membrane which separates them from the sample fluid, e.g. Clark-type oxygen sensors for gases other than oxygen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/0004—Gaseous mixtures, e.g. polluted air
  • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
  • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
  • G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
  • G01N33/0037—NOx
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
  • B01J2531/02—Compositional aspects of complexes used, e.g. polynuclearity
  • B01J2531/0238—Complexes comprising multidentate ligands, i.e. more than 2 ionic or coordinative bonds from the central metal to the ligand, the latter having at least two donor atoms, e.g. N, O, S, P
  • B01J2531/0241—Rigid ligands, e.g. extended sp2-carbon frameworks or geminal di- or trisubstitution
  • B01J2531/025—Ligands with a porphyrin ring system or analogues thereof, e.g. phthalocyanines, corroles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
  • B01J2531/80—Complexes comprising metals of Group VIII as the central metal
  • B01J2531/84—Metals of the iron group
  • B01J2531/842—Iron
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
  • B01J2531/80—Complexes comprising metals of Group VIII as the central metal
  • B01J2531/84—Metals of the iron group
  • B01J2531/845—Cobalt
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
  • B01J2531/80—Complexes comprising metals of Group VIII as the central metal
  • B01J2531/84—Metals of the iron group
  • B01J2531/847—Nickel
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters

Definitions

• the present invention generally relates to sensors and sensing techniques which can selectively and quantitatively detect NO in solution in both biological and chemical media. More specifically, the present invention relates to NO sensors which utilize conductive catalytic materials deposited on microfibers or other supports to monitor the presence or release of NO using amperometric, voltammetric or coulometric methods.
• Nitric oxide has recently been shown to be a key bioregulatory molecule in a number of physiological processes.
• NO plays a major role in the biological activity of endothelium derived relaxing factor (EDRF), abnormalities in which are associated with acute hypertension, diabetes, ischaemia and atherosclerosis.
• EDRF endothelium derived relaxing factor
• 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 inhibition, and has been implicated in endotoxic shock and genetic mutations.
• EDRF endothelium derived relaxing factor
• EDRF endothelium derived relaxing factor
• An optimal sensor for monitoring NO release should be sturdy and capable of sufficient miniaturization for in situ measurement in a single cell.
• the sensor should also be sensitive enough to produce an adequate signal to be observable at the low levels of NO secreted in biological environments. Due to the variation in the amount of NO secreted by different types of cells (e.g. from nanomoles/10 6 cells in macrophages to picomoles in endothelial cells), the signal produced by the sensor should also change linearly over a wide range of concentrations. See Marietta, M.A., Trends Biochem. ScL 14:488-492 (1989).
• a sensor of the present invention generally comprises an electrode having a catalytic material capable of catalyzing oxidation of NO coated with a cationic exchanger.
• the sensor provides a direct measurement of NO through the redox reaction of NO - NO + + e " and is selective for NO through the discrimination of the cationic exchanger against nitrite
• the senor 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.
• 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 microsensor comprises a thermally-sharpened conductive 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 cationic exchanger deposited thereon.
• the microsensor can be operated in either the amperometric, voltammetric or coulometric mode.
• the microsensor is characterized by a linear response up to about 300 ⁇ M and a detection limit of about 10 nM NO concentration, which allows detection of NO release at the levels present in a single biological cell.
• the sensor also discriminates against NO 2 " , the most problematic interferant with current NO sensing techniques.
• larger scale NO sensors are used to measure NO concentrations in chemical media, cell culture, extracellular fluids and tissue, rather than in single cells.
• a carbon electrode with a larger tip diameter, platinum mesh or a tin indium oxide layered plate is coated with a conductive catalytic polymeric porphyrin and a cationic exchanger. A linear response and low detection limits similar to the NO microsensor are observed.
• FIGS 1A and B depict preferred monomeric porphyrin structures used in sensors of the present invention.
• Figure 2 is a differential pulse voltammogram of NO at various concentrations.
• Figure 3 is a graph showing nitric oxide response (nA) of NO solutions measured by a sensor of the invention.
• Figure 4A is a microscopic photograph of a carbon fiber microsensor of the present invention.
• Figure 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 growth patterns for poly-TMHPPNi, deposited from 5x1 O ⁇ M 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 microsensor in the differential pulse 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 mesh macrosensor to NO release by a cell culture grown directly on the sensor surface.
• the basic strategy used in the design of a preferred embodiment of the NO sensor is based on catalytic oxidation of NO which uses a specific potential unique to NO - NO + + e " .
• the normal oxidation potential for NO is about 1.0 V vs SCE on a 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 to be used as an analytical signal in microsystem.
• 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 nonconduct ⁇ ve base material; or the conductive catalytic material can itself comprise the conductive support.
• the third approach can be accomplished by fashioning the electrode 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 embodiment of this invention preferably comprises a solid conductive support coated with one or more layers of a conductive material capable of catalyzing oxidation of NO, hereinafter referred to as catalytic material.
• catalytic materials can be used in a sensor of the present invention, as long as the catalytic material exhibits electronic, 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 materials can contain central metals, preferably transition or amphoteric metals.
• Polymers which can also be used but require doping include, for example, polyvinylmetallocenes (e.g. ferrocene), 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 conductive material.
• Polymeric metalloporphyrins have been shown to have high catalytic effect for the electrochemical oxidation of several small organic and inorganic molecules. Bennett, J.E. et al., Chem. Materials 3:490-495 (1991).
• TSHPP tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin
• PUP meso-5'-O-p-phenylene-2',3'-O-isopropylidene uridine-tri(n-methyl-4- pyridinium)porphyrin
• the porphyrinic catalysts used in the present invention are also preferably covered with a thin layer of a cationic exchanger to prevent anion diffusion to the catalytic surface.
• Suitable cationic exchangers include AQ55D available from Kodak and Nafion. National, which is used in the Specific Examples, is a negatively charged cationic exchange polymer which prevents diffusion of anions like NO 2 " to the electroactive surface of the polymeric porphyrin, but is highly permeable to NO.
• the thin layer of polymeric porphyrin film can be electrochemically deposited, as described in detail below, on any solid conductive support.
• a conductive support can comprise a material that in itself is conductive 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.
• the catalytic component of the invention is preferably layered on a conductive support, the conductive catalytic material can also comprise the conductive support.
• Conductive support materials particularly suitable for smaller scale sensors of the invention include carbon fibers, and gold or platinum wire. Due to their mechanical properties as well as the possibility for controlled miniaturization, carbon fibers are preferable support materials for microsensors in single cell applications. See e.g. Maiinski, T. et al. Anal. Chem. Ada. 249:35-41 (1991); Bailey, F. et al., Anal. Chem. 63:395-398 (1991).
• 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.
• 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.
• Specific Examples Il-V describe the production and use of a microsensor for use in small environments such as single cells or synapses, the same techniques can be applied to a larger support, such as described in Specific Examples I and IV , to produce convenient macrosensors for tissue, cell culture or chemical media studies.
• a two or preferably three electrode system can be employed.
• the working electrode comprising the coated carbon fiber, with mesh or plate, is connected to a conductive lead wire (e.g. copper) with conductive (e.g. silver) epoxy, with the lead wire connecting to the voltammetric analyzer, potentiostat or coulometric measuring instrument.
• the auxiliary or counterelectrode generally comprises a chemically inert conductive material such as a nobel metal (e.g. platinum wire), carbon or tin indium oxide which is also connected to the measuring instrument with a lead wire.
• a reference electrode such as a standard calomel electrode (SCE) is also employed and connected to the measuring instrument with a third conductive lead wire.
• SCE standard calomel electrode
• analytic solution any aqueous or nonaqueous solution in which NO is to be detected or measured.
• the term thus includes both chemical and biological media, including tissue fluids and extracellular and cellular fluids.
• the sensor of the invention can be used quantitatively to detect the presence of NO and also quantitatively to measure the levels of NO present in the analytic solution.
• 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 of NO on the surface of the cell membrane, oxidation by O 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 after 4 and 10 min respectively, following the addition of 100 ⁇ M O 2 to a 20 ⁇ M NO solution in the absence of biological material.
• a carbon macroelectrode covered with conductive porphyrin polymer was prepared as follows.
• a glassy carbon electrode (GCE) (diameter about 2 mm) was coated with conductive polymeric porphyrins by cyclic voltammetry or controlled potential oxidation (4 min) at 0.7 V vs SCE of the monomeric porphyrin in 0.1 M NaOH solution (5 ml).
• the auxiliary electrode was a platinum (Pt) rod and the reference electrode was a standard calomel electrode (SCE).
• the porphyrin-coated (about 0.8 - 1.5 nm/cm 2 ) electrode was removed from the solution and stored in 0.1 M base.
• porphyrins used were Ni 2+ , Co 2+ , or Fe 3+ TMHPP or PUP as shown in Figures 1A and B.
• the porphyrin-coated electrodes were then further coated with 4 ⁇ l of 5% Nation solution.
• Aliquots of the NO stock solution were introduced to the cell via a gas- tight syringe. The final dilution was taken as the final NO concentration.
• Carbon microfiber conductive supports for the microsensor were produced by threading an individual carbon fiber (7 ⁇ m) through the pulled end of a capillary tube 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 seared in place. The carbon fiber 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 temperature for 5 - 15 sec. After cooling to room temperature, the fiber was sharpened again.
• the flame temperature and the distance of the fiber from the center of the flame need to be carefully controlled. While the diameter of the sharpened lip is smaller, the tip length is larger, with the overall effect of the resulting electrode being a slim cylinder with a small diameter rather than a short taper.
• This geometry aids in implantation and increases the active surface area. Scanning electron microscopy of the fiber 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 4-6 ⁇ m. For the sensor to be implanted into a cell, this length must be smaller than the thickness of the cell.
• the unsharpened end of the carbon fiber was attached to a copper wire lead with silver epoxy.
• Figure 4A is a microscopic photograph of a complete NO microsensor of the present invention.
• Figure 4B is an electron scanning 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 and National as described below.
• Poly-TMHPPNi was deposited from a solution of 0.1 M NaOH containing 5 x 10 "4 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 1.1 V, on a carbon fiber microelectrode (16 ⁇ m 2 surface area), as generally described in 16 .
• peaks la and lc correspond to the oxidation of Ni(ll) to Ni(lll) and reduction of Ni(lll) to Ni(ll), respectively, in the film.
• 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. Sensor fabrication was completed by dipping in the National solution (5%) for 15 - 20 sec and left to dry (5 min) and stored in pH 7.4 buffer. Since the Ni(ll)/Ni(lll) reaction requires diffusion of OH " to neutralize a charge generated in the poly-TMHPPNi and OH " cannot diffuse through National, the later absence of the Ni(ll)/Ni(lll) voltammetric peaks in 0.1 M NaOH demonstrated the integrity of the Nation film coverage.
• NO monitoring was done by differential 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 auxiliary electrode.
• the pulse amplitude was 40 mV and the phosphate buffer solution was pH 7.4.
• Differential pulse voltammograms were obtained for oxidation of NO on poly-TMHPPNi without National (depicted as A in Figure 6) and with Nation (depicted as C in Figure 6) and for 1 ⁇ M NO in the presence of 20 ⁇ M NO 2 " on poly-TMHPPNi without Congress (depicted as B in Figure 6) and with Congress (depicted as D in Figure 6).
• DPV of NO on poly-TMHPPNi without Congress showed a peak at 0.63 V in buffer pH 7.4 (see Figure 6A).
• DPV of a solution of 1 ⁇ M NO and 20 ⁇ M NO 2 " 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 NO 2 " and NO occur at a similar potential, but that the current increase is not proportional to the concentration of NO 2 " .
• the NO peak current with the Nafion-coated sensor was observed at 0.64 V (see Figure 6C). Although the observed current is lower, National coverage provides high selectivity against NO 2 " .
• the detection limit of the sensor is 2-4 orders of magnitude lower than the estimated amount of NO released per single cell (1-200 attomol/cel!) 1 ,13 .
• SPECIFIC 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 endotheiial cell culture were prepared according to previously described procedures. Using a computer-controlled micropositioner (0.2 mm X-Y-Z resolution), the microsensor could be implanted into a single cell, or placed on the surface of the cell membrane, or kept at a controlled distance from the cell membrane. Alternating current was measured in three electrode systems, as described above, at constant potential of 0.75 V modulated with 40 mV pulse in time intervals of 0.5 sec.
• the background shown in Figure 7A, was measured in cell culture medium at 37°C (DMEM-Dulbecco's Modified Eagle Medium, 100 mg/L D-glucose, 2 mM glutamine, 110 mg/L sodium pyruvate, 15% controlled process serum replacement TYPE I). No change of the background was observed after the addition of 50 nM of bradykinin to 5 ml of cell culture medium.
• 2 nm of NO were injected by microsyringe into the cell culture medium, a 5 mm distance from the microsensor.
• one microsensor was placed on the surface of the single endotheiial cell in the aortic ring, and another was implanted into the smooth muscle cell.
• FIG. 8A illustrates the use of a layer of indium oxide (14) as a counterelectrode (10), (10), whereas Figure 8B illustrates its use as a conductive layer of the working electrode of a macrosensor (12).
• BCH1 myocytes (16) were grown under standard culture conditions at 2 x 10 7 cell/cm 2 on a glass plate (18) ( Figures 8A and C) or on a plate layered with catalytic polymeric iron porphyrin with National coated thereon (20) ( Figure 8B).
• Figures 8A and B the tin indium oxide semiconductor layer in both cases was attached to the measuring instrument by a copper wire lead (22) with silver epoxy (24).
• FIG. 8C depicts the set up for NO measurements of the cell culture in Figure 8A.
• Cells were grown on a tin indium oxide (14) layered glass plate (18) placed in a Petri dish (20) with standard culture media (26).
• a microsensor working electrode (34) constructed as described in previous Examples was then used to measure NO release in situ.
• the culture was microscopically monitored (30 - inverted microscope) and the working electrode positioned with a micromanipulator (32).
• microsensor was attached to a measuring instrument such as a voltammetric analyzer (36) with the results fed to a computer (38) connected to a plotter (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.

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