Nitric oxide sensor

• US 5,603,820 A
• Filed: 06/07/1995
• Issued: 02/18/1997
• Est. Priority Date: 04/21/1992
• Status: Expired due to Term

• Current Assignee: United States As Represented By The Secretary Department of Health and Human Services
• Original Assignee: United States Department Of Health And Human Services, University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Wink, David A., Murray, Royce W., Sullivan, Melani, Christodoulou, Danae D., Malinski, Tadeusz, Meyer, Thomas J., Younathan, Janet
• Primary Examiner(s): Bell, Bruce F.
• Application Number:
  US08/477,620
• Time in Patent Office: 622 Days
• Field of Search: 204/418, 204/431, 204/432, 204/424, 204/426, 204/282, 204/290 R, 204/415, 205/780.5, 205/781, 205/782, 205/782.5, 205/783, 429/43, 429/44, 502/167, 502/163
• US Class Current: 205/781
• CPC Class Codes: B01J 2531/025 Ligands with a porphyrin ring system or analogues thereof, e.g. phthalocyanines, corroles
• CPC Class Codes: B01J 2531/842 Iron
• CPC Class Codes: B01J 2531/845 Cobalt
• CPC Class Codes: B01J 2531/847 Nickel
• CPC Class Codes: B01J 31/1658 immobilised by covalent linkages, i.e. pendant complexes with optional linking groups, e.g. on Wang or Merrifield resins
• CPC Class Codes: B01J 31/1815 with more than one complexing nitrogen atom, e.g. bipyridyl, 2-aminopyridine
• CPC Class Codes: G01N 27/4045 for gases other than oxygen

Abstract

An electrode sensor which may be used to specifically and quantitatively measure nitric oxide is provided, as well as a method of preparing and using such an electrode sensor to measure nitric oxide concentration in solution. A nitric oxide (NO) microsensor 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, which can be operated in either the amperometric, voltammetric or coulometric mode utilizing a two or three electrode system, is characterized by a linear response up to about 300 μM, a response time better than 10 msec and a detection limit of about 10 nM. The sensor 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 of the invention. A larger scale sensor 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.

Claims

1. A nitric oxide-specific electrode sensor, which comprises:

an electrically conductive substrate whose amperometric response is substantially unaffected by the presence of nitric oxide; and

an adherent and substantially uniform electrochemically active polymeric coating formed on a surface of said electrically conductive substrate which interacts with NO to change the redox potential of NO and the electrode sensor.

2. The electrode sensor of claim 1, wherein said electrochemically active polymeric coating is formed on the surface of said electrically conductive substrate by electrolytic polymerization.

3. The electrode sensor of claim 2, which additionally comprises a gas-permeable membrane coating that is permeable to nitric oxide and is not permeable to nitrite.

4. The electrode sensor of claim 1, wherein said electrically conductive substrate is selected from the group consisting of electrically conductive carbon, indium tin oxide, iridium oxide, nickel, platinum, silver, and gold.

5. The electrode sensor of claim 4, wherein said electrically conductive substrate is carbon.

6. The electrode sensor of claim 5, wherein said electrochemically active polymeric coating is a metallized or doped polymer.

7. The electrode sensor of claim 6, wherein said electrochemically active polymer coating is selected from the group consisting of metallized polymeric porphyrins, metallized polyphthalocyanines, polyvinylmetallocenes, metallized polyacetylenes, metallized polypyrrolines, and polymeric substituted glyoximes.

8. The electrode sensor of claim 7, wherein said electrochemically active polymeric coating is selected from the group consisting of metallized polymeric porphyrins and metallized polyphthalocyanines.

9. The electrode sensor of claim 8, wherein said electrochemically active polymeric coating is a metallized polymeric porphyrin which does not form metal-oxo bridges with said substrate.

10. The electrode sensor of claim 9, wherein said electrochemically active polymeric coating is selected from the group consisting of metallized polymeric pyrroles, pyridines, and ether porphyrins.

11. The electrode sensor of claim 10, wherein said electrochemically active polymeric coating is selected from the group consisting of metallized polymeric tetramethyl pyridine pyrrole and dimethyl ester porphyrins.

12. The electrode sensor of claim 11, wherein said electrochemically active polymeric coating is selected from the group consisting of polymeric tetramethyl pyridine pyrroles and dimethyl ester porphyrins metallized with Ni²⁺, Co²⁺, or Fe³⁺.

13. The electrode sensor of claim 12, wherein said electrochemically active polymeric coating is Ni²⁺ or Fe³⁺ polymeric tetramethyl pyridine pyrrole.

14. The electrode sensor of claim 12, wherein said electrochemically active polymeric coating is Fe³⁺ polymeric dimethyl ester porphyrin.

15. The electrode sensor of claim 12, which additionally comprises a gas-permeable membrane coating that is permeable to nitric oxide and is not permeable to nitrite.

16. A method of detecting nitric oxide in solution, which method comprises connecting an electrode sensor of claim 1 to a potentiostat, calibrating the potentiostat and electrode sensor for known concentrations of nitric oxide in solution, contacting said electrode sensor with an unknown sample, and detecting the presence or absence of nitric oxide in said unknown sample by comparing the measured current to a current for a sample of known nitric oxide concentration at a particular potential.

17. The method of claim 16, wherein said sample of known nitric oxide concentration has no nitric oxide.

18. The method of claim 16, wherein said the concentration of nitric oxide in said unknown sample is determined by comparing the measured current to currents observed for samples of more than one known nitric oxide concentration.

19. The method of claim 18, wherein said electrochemically active polymer coating of said electrode sensor is selected from the group consisting of polymeric tetramethyl pyridine pyrroles and dimethyl ester porphyrins metallized with Ni²⁺, Co²⁺, Fe³⁺ and said electrically conductive substrate is carbon.

20. The method of claim 18, wherein said electrode sensor comprises a gas-permeable membrane coating that is permeable to nitric oxide and is not permeable to nitrite and said electrochemically active polymeric coating of said sensor is formed on the surface of said electrically conductive substrate by electrolytic polymerization.

21. The method of claim 18, wherein said electrode sensor comprises a gas-permeable membrane coating that is permeable to nitric oxide and is not permeable to nitrite and said electrochemically active polymeric coating of said sensor is formed on the surface of said electrically conductive substrate by electrolytic polymerization, and said electrochemically active polymer coating is selected from the group consisting of polymeric tetramethyl pyridine pyrroles and dimethyl ester porphyrins metallized with Ni²⁺, Co²⁺, or Fe³⁺.

22. An electrode sensor for detecting the presence of NO in an analytic solution, the electrode sensor detecting an electrical signal developed between the electrode sensor and a counterelectrode, the electrode sensor comprising:

a) a conductive support having a catalytic surface for catalyzing NO oxidation and thereby generating a 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.

23. The sensor of claim 22, wherein the conductive support having a catalytic surface comprises a conductive material with a layer of catalytic material disposed thereon.

24. The sensor of claim 23, wherein the cationic exchanger comprises a chemically stable perfluorosulfonic acid ion exchange resin.

25. The sensor of claim 23, wherein the catalytic material comprises a polymer selected from the group consisting of polymeric porphyrins, polypthalocyanines, polyvinylmethallocenes, polyacetylenes and polypyrrolines.

26. The sensor of claim 23, wherein the catalytic material comprises a carbon fiber.

27. The sensor of claim 23, wherein the catalytic material comprises a polymeric metalloporphyrin.

28. The sensor of claim 23, wherein the conductive material comprises tin indium oxide.

29. The sensor of claim 23, wherein the fiber is about 1 μ

m or less in diameter at one tip.

30. The sensor of claim 22, wherein the conductive material is a material selected from the group consisting of carbon, platinum and gold.

31. The sensor of claim 30, wherein the conductive material comprises platinum mesh.

32. A sensor system for measuring the level of NO in an analytic solution comprising:

a) an electrode sensor 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 electrode sensor and the counterelectrode in response to the oxidation of NO.

33. The sensor system of claim 32, wherein the conductive support comprises a conductive material and the catalytic material comprises a polymeric porphyrin.

34. The sensor system of claim 33, wherein the fiber of the electrode sensor comprises a carbon fiber, the counterelectrode comprises an inert conductive material and the reference electrode comprises a standard calomel electrode.

35. The sensor system of claim 33, wherein the conductive material comprises tin indium oxide.

36. The sensor system of claim 33, wherein the conductive material comprises platinum mesh.

37. The sensor system of claim 33, wherein the counterelectrode comprises a layer of tin indium oxide.

38. The sensor system of claim 32, further comprising:

d) a reference electrode conductively connected to the measuring instrument.

39. 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.

40. The sensor of claim 39, wherein the conductive support comprises a conductive fiber having a tip diameter about 1 μ

m or less in diameter.

41. The sensor of claim 39, wherein the metalloporphyrin comprises tetrakis (3-methoxy-4-hydroxy-phenyl)porphyrin or meso-5'"'"'-0-P-phenylene-2'"'"',3'"'"'-0-isopropylidine uridinetri(n-methyl-4-pyridinium)porphyrin.

42. The sensor of claim 39, wherein the conductive support comprises inert metallic material.

43. The sensor of claim 42, wherein the conductive support comprises tin indium oxide.

44. The sensor of claim 42, wherein the cationic exchanger is a chemically stable perfluorosulfonic acid ion exchange resin.

45. The sensor of claim 42, wherein the conductive support comprises platinum.

46. An electrode sensor for a sensor for measuring the level of NO in an analytic solution, the sensor measuring an electrical signal developed between the electrode sensor and a counterelectrode, the electrode sensor 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 at 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 interfering anions to the catalytic layer surface, which interfering anions would adversely affect the NO oxidation electrical signal.

47. A method of manufacturing a nitric oxide-specific electrode sensor, which comprises contacting an electrically conductive substrate whose amperometric response is substantially unaffected by the presence of nitric oxide with a compound which forms an electrochemically active polymeric coating which interacts with NO to change the redox potential of NO and the electrode sensor.

48. The method of claim 47 wherein said electrochemically active polymeric coating is formed on the surface of said electrically conductive substrate by electrolytic polymerization.

49. The method of claim 48, wherein said electrically conductive substrate is selected from the group consisting of electrically conductive carbon, indium tin oxide, iridium oxide, nickel, platinum, silver, and gold.

50. The method of claim 49, wherein said electrochemically active polymeric coating is a metallized or doped polymer.

51. The method of claim 50, wherein said electrochemically active polymeric coating is selected from the group consisting of metallized polymeric porphyrins and metallized polyphthalocyanines.

52. The method of claim 51, wherein said electrically conductive substrate is carbon and said electrochemically active polymeric coating is selected from the group consisting of polymeric tetramethyl pyridine pyrroles and dimethyl ester porphyrins metallized with Ni²⁺, Co²⁺, or Fe³⁺.

53. The method of claim 52, wherein said electrochemically active polymeric coating is Ni²⁺ or Fe³⁺ polymeric tetramethyl pyridine pyrrole.

54. The method of claim 52, wherein said electrochemically active polymeric coating is Fe³⁺ polymeric dimethyl ester porphyrin.

55. The method of claim 52, which additionally comprises applying a coating of a gas-permeable membrane that is permeable to nitric oxide and excludes nitrite.

56. A method of directly measuring NO in an analytic solution generally comprising the steps of:

a) providing an electrode sensor 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 electrode sensor and the counterelectrode;

d) placing the electrode sensor in the analytic solution;

e) placing the counterelectrode in the analytic solution; and

f) measuring the electrical signal developed between the electrode sensor and the counterelectrode.

57. The method of claim 56 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.

58. The method of directly measuring NO in an analytic solution as in claim 56, the provided electrode sensor being a microsensor in which the electrically conductive substrate is a glass carbon fiber having a tip approximately 1 μ

m or less in diameter, in which the electrochemically active polymeric coating is a metalloporphyrin selected from the group consisting of tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin and meso-5'"'"'-0-P-phenylene-2'"'"',3'"'"'-0-isopropylidine uridine-tri(n-methyl-4-pyridinium)porphyrin, the porphyrin surface coverage (Γ

) being in the approximate range of 0.7-1.2 nmol cm², and in which the cationic exchanger comprises a chemically stable perfluorosulfonic acid ion exchange resin disposed on the electrochemically active polymeric coating,

the method conducted by a technique selected from the group consisting of differential pulse voltammetry and amperometry.

59. The method of claim 58, conducted by differential pulse voltammetry, with a pulse amplitude of approximately 40 mV, by obtaining differential pulse voltammograms for oxidation of NO at approximately 0.64 V;

wherein a linear relationship is observed between current and NO concentration for a range of NO concentration from approximately zero up to approximately 300 μ

M NO concentration, with a detection limit calculated at a signal/noise ratio equal to 3 of approximately 20 nM, and with a detection in a volume of approximately 10^-12 L of approximately 10^-2 attomoles.

60. A nitric oxide-specific electrode sensor for detecting the quantity of NO in an analytic solution, the electrode sensor detecting an electrical signal developed between the electrode sensor and a counterelectrode, in strength in proportion to the quantity of NO in the analytic solution, the electrode sensor comprising:

(a) an electrically conductive substrate whose amperometric response is substantially unaffected by the presence of nitric oxide;

(b) an adherent and substantially uniform electrochemically active polymeric coating formed on a surface of said electrically conductive substrate, said electrochemically active polymeric coating interacting with NO in said analytical solution to change the redox potential of NO and the nitric oxide-specific electrode sensor, the change being in strength related to the quantity of NO in the analytic solution, and to generate an NO detection electrical signal in strength related to the quantity of NO in the analytic solution; and

(c) a cationic exchanger disposed on a surface of said electrochemically active polymeric coating, the cationic exchanger being in contact with the analytic solution, the cationic exchanger allowing diffusion of NO therethrough but preventing the diffusion of anions to the surface of the electrochemically active polymeric coating that would mask the NO detection electrical signal;

wherein the sensor may be used quantitatively to measure the levels of NO present in the analytic solution.

61. A nitric oxide-specific electrode sensor as in claim 60, the nitric oxide-specific electrode sensor being a microelectrode in which the electrically conductive substrate is a glass carbon fiber having a tip approximately 1 μ

m or less in diameter, in which the electrochemically active polymeric coating is a metalloporphyrin selected from the group consisting of tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin and meso-5'"'"'-0-P-phenylene-2'"'"',3'"'"'-0-isopropylidine uridine-tri(n-methyl-4-pyridinium)porphyrin, the porphyrin surface coverage (Γ

) being in the approximate range of 0.7-1.2 nmol cm², and in which the cationic exchanger comprises a chemically stable perfluorosulfonic acid ion exchange resin disposed on the electrochemically active polymeric coating.

62. The nitric oxide-specific electrode sensor of claim 61, the glass carbon fiber having a length in the range of approximately 4 μ

m to approximately 6 μ

m.

63. The nitric oxide-specific electrode sensor of claim 61, the electrode providing an NO detection electrical signal in aqueous analytic solutions having a pH of approximately 7.4.

64. The nitric oxide-specific electrode sensor of claim 60, the electrode providing an NO detection electrical signal in strength proportional to the quantity of NO in the analytic solution for NO concentrations in the approximate range of 20 to 60 μ

M.

65. A method of manufacturing a nitric oxidespecific electrode sensor, which comprises depositing a metalloporphyrin selected from among the group consisting of tetrakis (3-methoxy-4-hydroxyphenyl)porphyrin and meso-5'"'"'0-P-phenylene-2'"'"',3'"'"'-0-isopropylidine uridine-tri (n-methyl-4-pyridinium)porphyrin on an electrically conductive glass carbon fiber microelectrode having a tip approximately 1 μ

m or less in diameter, by electrolytic polymerization, to an average surface coverage (Γ

) being in the approximate range of 0.7-1.2 nmol cm², to form an electrochemically active polymeric coating on the fiber which interacts with NO to change the redox potential of NO and the electrode sensor, and applying a coating on the metalloporphyrin of a cationic exchanger which comprises a chemically stable perfluorosulfonic acid ion exchange resin that is permeable to nitric oxide and exludes nitrite.

66. A method as in claim 65 further comprising depositing the metalloporphyrin from a solution of approximately 0.1M NaOH containing approximately 5×

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 approximately 0.0 to approximately 1.1 V, on the carbon fiber microelectrode, conditioning the resulting porphyrin film by multiple scans, storing the microelectrode in approximately 0.1M NaOH, and thereafter dipping the microelectrode in the cationic exchanger, drying the microelectrode, and storing the microelectrode in pH 7.4 buffer.