Immobilized enzymes in biocathodes

• US 20050095466A1
• Filed: 08/31/2004
• Published: 05/05/2005
• Est. Priority Date: 11/05/2003
• Status: Active Grant

• Current Assignee: St. Louis University
• Original Assignee: St. Louis University
• Inventors: Topcagic, Sabina, Treu, Becky, Minteer, Shelley D.
• Granted Patent: US 8,859,151 B2
• Time in Patent Office: Days
• US Class Current: 429/401
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: H01M 4/8605 Porous electrodes
• CPC Class Codes: H01M 8/16 Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
• CPC Class Codes: Y02E 60/50 Fuel cells

Abstract

Disclosed is an improved biofuel cell having a cathode comprising a dual function membrane, which contains an oxygen oxidoreductase enzyme immobilized within a buffered compartment of the membrane and an electron transport mediator which transfers electrons from an electron conducting electrode to the redox reaction catalyzed by the oxygen oxidoreductase enzyme. The improved biofuel cell also has an anode that contains an oxidoreductase enzyme that uses an organic fuel, such as alcohol, as a substrate. An electric current can flow between the anode and the cathode.

Claims

1. A biocathode comprising:

(a) an electron conductor;

(b) at least one enzyme capable of reacting with a reduced form of an electron mediator and an oxidant to produce an oxidized form of the electron mediator and water; and

(c) an enzyme immobilization material comprising an electrocatalyst, the enzyme immobilization material being capable of immobilizing and stabilizing the enzyme, the material being permeable to the oxidant, an oxidized form of the electrocatalyst being capable of gaining electrons from the electron conductor to produce a reduced form of the electrocatalyst that is capable of reacting with an oxidized form of the electron mediator to produce a reduced form of the electron mediator and an oxidized form of the electrocatalyst;

wherein the electrocatalyst is present in a concentration sufficient to make the enzyme immobilization material conduct electrons.

5. A biofuel cell for generating electricity comprising:

a fuel fluid;

an electron mediator;

an anode capable of a reaction to oxidize the fuel fluid; and

a biocathode of claim 1.

9. The biocathode of claim 1 wherein the enzyme immobilization material comprises a micellar or inverted micellar structure.

10. The biocathode of claim 1 wherein the enzyme immobilization material comprises a modified perfluoro sulfonic acid-PTFE copolymer.

11. The biocathode of claim 1 wherein the electron conductor comprises a carbon-based material, a metallic conductor, a semiconductor, a metal oxide or a modified conductor.

12. The biocathode of claim 1 wherein the electron conductor comprises a carbon-based material.

13. The biocathode of claim 1 wherein the electron conductor comprises carbon cloth, carbon paper, carbon screen printed electrodes, carbon black, carbon powder, carbon fiber, single-walled carbon nanotubes, double-walled carbon nanotubes, multi-walled carbon nanotubes, carbon nanotube arrays, diamond-coated conductors, glass carbon, mesoporous carbon, graphite, uncompressed graphite worms, delaminated purified flake graphite, high performance graphite, highly ordered pyrolytic graphite, pyrolytic graphite or polycrystalline graphite.

14. The biocathode of claim 1 wherein the enzyme immobilization material is modified with a hydrophobic cation larger than NH₄⁺.

15. The biocathode of claim 14 wherein the hydrophobic cation comprises an ammonium-based cation, quaternary ammonium cation, alkyltrimethylammonium cation, organic cation, phosphonium cation, triphenylphosphonium, pyridinium cation, imidazolium cation, hexdecylpyridinium, ethidium, viologen, methyl viologen, benzyl viologen, bis(triphenylphosphine)iminium metal complex, bipyridyl metal complex, phenanthroline-based metal complex, [Ru(bipyridine)₃]²⁺ or [Fe(phenanthroline)₃]³⁺.

16. The biocathode of claim 14 wherein the hydrophobic cation comprises a quaternary ammonium cation represented by formula 1

17. The biocathode of claim 16 wherein R₁, R₂, R₃ and R₄ are independently hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl wherein at least one of R₁, R₂, R₃ and R₄ is other than hydrogen.

18. The biocathode of claim 16 wherein R₁, R₂, R₃ and R₄ are the same and are methyl, ethyl, propyl, butyl, pentyl or hexyl.

19. The biocathode of claim 10 wherein the modified perfluoro sulfonic acid-PTFE copolymer is modified with tetrabutylammonium halide, triethylhexylammonium halide or trimethyldodecylammonium halide.

20. The biocathode of claim 10 wherein the modified perfluoro sulfonic acid-PTFE copolymer is modified with tetrabutylammonium halide or triethylhexylammonium halide.

21. The biocathode of claim 10 wherein the modified perfluoro sulfonic acid-PTFE copolymer is modified with triethylhexylammonium halide.

22. The biocathode of claim 1 wherein the enzyme comprises an oxidoreductase.

23. The biocathode of claim 1 wherein the enzyme comprises a glucose oxidase, alcohol-based oxidase or cholesterol-based oxidase.

24. The biocathode of claim 1 wherein the enzyme comprises laccase, cytochrome C oxidase, bilirubin oxidase or peroxidase.

25. The biocathode of claim 1 wherein the enzyme comprises an oxygen oxidoreductase having an optimum activity at a pH between about 6.5 and about 7.5.

26. The biocathode of claim 1 wherein the enzyme comprises bilirubin oxidase.

27. The biocathode of claim 1 wherein the electron mediator comprises a metalloprotein, a conjugated organic compound, a sugar, a sterol, a fatty acid or a coenzyme or substrate of an oxidase.

28. The biocathode of claim 1 wherein the oxidized form of the electron mediator comprises stellacyanin, bilirubin, glucose or cholesterol.

29. The biocathode of claim 1 wherein the oxidized form of the electron mediator comprises bilirubin.

30. The biocathode of claim 1 wherein the electrocatalyst for the electron mediator comprises organometallic cations with standard reduction potentials greater than +0.4 volts.

31. The biocathode of claim 1 wherein the electrocatalyst for the electron mediator comprises osmium, ruthenium, iron, nickel, rhodium, rhenium, or cobalt complexes.

32. The biocathode of claim 1 wherein the reduced form of the electrocatalyst for the electron mediator comprises Ru(phen)₃⁺², Fe(phen)₃⁺², Ru(bpy)₃⁺², Os(bpy)₃⁺² or Os(terpy)₃⁺².

33. The biocathode of claim 1 wherein the reduced form of the electrocatalyst for the electron mediator comprises Ru(bpy)₃⁺².

34. The biocathode of claim 1 wherein the concentration of the electrocatalyst is from about 100 mM to about 3M.

35. The biocathode of claim 1 wherein the concentration of the electrocatalyst is from about 250 mM to about 2.25M.

36. The biocathode of claim 1 wherein the concentration of the electrocatalyst is from about 500 mM to about 2M.

37. The biocathode of claim 1 wherein the concentration of the electrocatalyst is from about 1 M to about 1.5M.

38. The biofuel cell of claim 5 wherein the oxidant comprises oxygen or peroxide.

39. The biofuel cell of claim 5 wherein the oxidant comprises oxygen.

40. The biofuel cell of claim 5 wherein the fuel fluid comprises ammonia, methanol, ethanol, propanol, isobutanol, butanol and isopropanol, allyl alcohols, aryl alcohols, glycerol, propanediol, mannitol, glucuronate, aldehyde, carbohydrates, glucose, glucose-1, D-glucose, L-glucose, glucose-6-phosphate, lactate, lactate-6-phosphate, D-lactate, L-lactate, fructose, galactose-1, galactose, aldose, sorbose, mannose, glycerate, coenzyme A, acetyl Co-A, malate, isocitrate, formaldehyde, acetaldehyde, acetate, citrate, L-gluconate, beta-hydroxysteroid, alpha-hydroxysteroid, lactaldehyde, testosterone, gluconate, fafty acids, lipids, phosphoglycerate, retinal, estradiol, cyclopentanol, hexadecanol, long-chain alcohols, coniferyl-alcohol, cinnamyl-alcohol, formate, long-chain aldehydes, pyruvate, butanal, acyl-CoA, steroids, amino acids, flavin, NADH, NADH₂, NADPH, NADPH₂ or hydrogen.

41. The biofuel cell of claim 5 wherein the fuel fluid comprises methanol, ethanol or propanol.

42. The biofuel cell of claim 5 wherein the fuel fluid comprises ethanol.

43. The biofuel cell of claim 5 wherein the anode is a bioanode.

44. The biofuel cell of claim 43 wherein the bioanode comprises a modified perfluoro sulfonic acid-PTFE copolymer.

45. The biofuel cell of claim 44 wherein the modified perfluoro sulfonic acid-PTFE copolymer is modified with tetrabutylammonium halide, triethylhexylammonium halide or trimethyldodecylammonium halide.

46. The biofuel cell of claim 44 wherein the modified perfluoro sulfonic acid-PTFE copolymer is modified with triethylhexylammonium halide.

47. The biofuel cell of claim 43 wherein the bioanode comprises a PQQ-dependent alcohol dehydrogenase which has a PQQ molecule electrostatically associated with it.

48. The biofuel cell of claim 47 wherein the bioanode and biocathode are not separated by a salt bridge or a polymer electrolyte membrane.

49. A method of generating electricity using the biofuel cell of claim 5 comprising

(a) oxidizing the fuel fluid at the anode and reducing the oxidant at the biocathode;

(b) oxidizing the reduced form of the electron mediator during the reduction of the oxidant at the biocathode;

(c) oxidizing the electrocatalyst; and

(d) reducing the electrocatalyst at the electron conductor.

51. The method of claim 49 wherein the oxidant is oxygen or a peroxide.

52. The method of claim 49 wherein the oxidant is oxygen.

53. The method of claim 49 wherein the fuel fluid comprises ammonia, methanol, ethanol, propanol, isobutanol, butanol and isopropanol, allyl alcohols, aryl alcohols, glycerol, propanediol, mannitol, glucuronate, aldehyde, carbohydrates, glucose, glucose-1, D-glucose, L-glucose, glucose-6-phosphate, lactate, lactate-6-phosphate, D-lactate, L-lactate, fructose, galactose-1, galactose, aldose, sorbose, mannose, glycerate, coenzyme A, acetyl Co-A, malate, isocitrate, formaldehyde, acetaldehyde, acetate, citrate, L-gluconate, beta-hydroxysteroid, alpha-hydroxysteroid, lactaldehyde, testosterone, gluconate, fafty acids, lipids, phosphoglycerate, retinal, estradiol, cyclopentanol, hexadecanol, long-chain alcohols, coniferyl-alcohol, cinnamyl-alcohol, formate, long-chain aldehydes, pyruvate, butanal, acyl-CoA, steroids, amino acids, flavin, NADH, NADH₂, NADPH, NADPH₂ or hydrogen.

54. The method of claim 49 wherein the fuel fluid comprises methanol, ethanol or propanol.

55. The method of claim 49 wherein the fuel fluid comprises ethanol.

56. The method of claim 49 wherein the enzyme comprises an oxidoreductase.

57. The method of claim 49 wherein the enzyme comprises a glucose oxidase, alcohol-based oxidase or cholesterol-based oxidase.

58. The method of claim 49 wherein the enzyme comprises laccase, cytochrome c oxidase, bilirubin oxidase or peroxidase.

59. The method of claim 49 wherein the enzyme comprises bilirubin oxidase.

60. The method of claim 49 wherein the electron mediator comprises a metalloprotein, a conjugated organic compound, a sugar, a sterol, a fatty acid or a coenzyme or substrate of an oxidase.

61. The method of claim 49 wherein the electron mediator comprises stellacyanin, bilirubin, glucose or cholesterol.

62. The method of claim 49 wherein the oxidized form of the electron mediator comprises bilirubin.

63. The method of claim 49 wherein the electrocatalyst for the electron mediator comprises organometallic cations with standard reduction potentials greater than +0.4 volts.

64. The method of claim 49 wherein the electrocatalyst for the electron mediator comprises osmium, ruthenium, iron, nickel, rhodium, rhenium, or cobalt complexes.

65. The method of claim 49 wherein the reduced form of the electrocatalyst for the electron mediator comprises Ru(phen)₃⁺², Fe(phen)₃⁺², Ru(bpy)₃⁺², Os(bpy)₃⁺² or Os(terpy)₃⁺².

66. The method of claim 49 wherein the reduced form of the electrocatalyst for the electron mediator comprises Ru(bpy)₃⁺².

2. A biocathode comprising:

(a) an electron conductor;

(b) at least one enzyme capable of reacting with a reduced form of an electron mediator and an oxidant to produce an oxidized form of the electron mediator and water; and

(c) an enzyme immobilization material comprising the electron mediator, the enzyme immobilization material being capable of immobilizing and stabilizing the enzyme, the material being permeable to the oxidant, an oxidized form of the electron mediator being capable of gaining electrons from the electron conductor to produce a reduced form of the electron mediator;

wherein the electron mediator is present in a concentration sufficient to make the enzyme immobilization material conduct electrons.

6. A biofuel cell for generating electricity comprising:

a fuel fluid;

an anode capable of a reaction to oxidize the fuel fluid; and

a biocathode of claim 2.

50. A method of generating electricity using the biofuel cell of claim 6 comprising

(a) oxidizing the fuel fluid at the anode and reducing the oxidant at the biocathode;

(b) oxidizing the reduced form of the electron mediator during the reduction of the oxidant at the biocathode; and

(c) reducing the electron mediator at the electron conductor.

3. A biocathode comprising:

(a) an electron conductor;

(b) at least one enzyme capable of reacting with a reduced form of an electron mediator and an oxidant to produce an oxidized form of the electron mediator and water; and

(c) an enzyme immobilization material comprising the electron mediator and an electrocatalyst, the enzyme immobilization material being capable of immobilizing and stabilizing the enzyme, the material being permeable to the oxidant, an oxidized form of the electrocatalyst being capable of gaining electrons from the electron conductor to produce a reduced form of the electrocatalyst that is capable of reacting with an oxidized form of the electron mediator to produce a reduced form of the electron mediator and an oxidized form of the electrocatalyst;

wherein the electrocatalyst is present in a concentration sufficient to make the enzyme immobilization material conduct electrons.

7. A biofuel cell for generating electricity comprising:

a fuel fluid;

an anode capable of a reaction to oxidize the fuel fluid; and

a biocathode of claim 3.

4. A biocathode comprising:

(a) an electron conductor;

(b) at least one enzyme capable of reacting with a reduced form of an electron mediator and an oxidant to produce an oxidized form of the electron mediator and water; and

(c) an enzyme immobilization material comprising the electron mediator, the enzyme immobilization material being capable of immobilizing and stabilizing the enzyme, the material being permeable to the oxidant, an oxidized form of an electrocatalyst being capable of gaining electrons from the electron conductor to produce a reduced form of the electrocatalyst that is capable of reacting with an oxidized form of the electron mediator to produce a reduced form of the electron mediator and an oxidized form of the electrocatalyst;

wherein the electron mediator is present in a concentration sufficient to make the enzyme immobilization material conduct electrons.

8. A biofuel cell for generating electricity comprising:

a fuel fluid;

an anode capable of a reaction to oxidize the fuel fluid; and

a biocathode of claim 4.

67. A fuel cell comprising a cathode and an anode, wherein the cathode comprises an electron conductor, a cathode enzyme, an electron transport mediator, and a membrane, wherein the cathode enzyme is immobilized within a buffered internal compartment of the membrane.

68. The fuel cell of claim 67 wherein the cathode enzyme is an oxygen oxidoreductase.

69. The fuel cell of claim 68 wherein the cathode enzyme is a bilirubin oxidase.

70. The fuel cell of claim 68 wherein the cathode enzyme is a laccase.

71. The fuel cell of claim 67 wherein the electron transport mediator is immobilized within the buffered internal compartment of the membrane.

72. The fuel cell of claim 71 wherein the electron transport mediator is a transitional metal compound.

73. The fuel cell of claim 71 wherein the electron transport mediator is a compound comprising ruthenium.

74. The fuel cell of claim 71 wherein the electron transport mediator is a Ru(bpy)₃⁺².

75. The fuel cell of claim 67 wherein the membrane is a modified ion exchange polymer membrane.

76. The fuel cell of claim 75 wherein the membrane is a quaternary ammonium salt treated perfluoro sulfonic acid-PTFE copolymer.

77. The fuel cell of claim 75 wherein the membrane is a tetrabutylammonium bromide treated Nafion®

membrane.

78. The fuel cell of claim 67 wherein the electron conductor is a carbon-based material selected from the group consisting of carbon cloth, carbon felt, carbon paper, carbon screen printed electrodes, carbon paper, carbon black, carbon powder, carbon fiber, single-walled carbon nanotubes, double-walled carbon nanotubes, multi-walled carbon nanotubes, carbon nanotubes arrays, diamond-coated conductors, glass carbon, mesoporous carbon, graphite, uncompressed graphite worms, delaminated purified flake graphite, high performance graphite, high performance carbon powders, highly ordered pyrolytic graphite, pyrolytic graphite and polycrystalline graphite.

79. The fuel cell of claim 78 wherein the electron conductor is carbon felt.

80. The fuel cell of claim 67 wherein the anode is a bioanode.

81. The fuel cell of claim 80 wherein the bioanode comprises an electron conductor, a redox polymer film, a modified ion exchange polymer membrane and an anode enzyme that is immobilized within a buffered internal compartment of the modified ion exchange polymer membrane.

82. The fuel cell of claim 81 wherein the redox polymer film is a polymethylene green.

83. The fuel cell of claim 81 wherein the modified ion exchange membrane is a quaternary ammonium salt treated perfluoro sulfonic acid-PTFE copolymer.

84. The fuel cell of claim 83 wherein the membrane is a tetrabutylammonium bromide treated Nafion®

membrane.

85. The fuel cell of claim 81 wherein the anode enzyme is an oxidoreductase enzyme having an organic compound substrate.

86. The fuel cell of claim 85 wherein the anode enzyme is an alcohol dehydrogenase.

87. The fuel cell of claim 86 wherein the bioanode comprises a second bioanode enzyme.

88. The fuel cell of claim 87 wherein the second bioanode enzyme is an aldehyde dehydrogenase.

89. The fuel cell of claim 81 wherein the electron conductor is a carbon-based material selected from the group consisting of carbon cloth, carbon felt, carbon paper, carbon screen printed electrodes, carbon paper, carbon black, carbon powder, carbon fiber, single-walled carbon nanotubes, double-walled carbon nanotubes, multi-walled carbon nanotubes, carbon nanotubes arrays, diamond-coated conductors, glassy carbon, mesoporous carbon, graphite, uncompressed graphite worms, delaminated purified flake graphite, high performance graphite, high performance carbon powders, highly ordered pyrolytic graphite, pyrolytic graphite and polycrystalline graphite.

90. The fuel cell of claim 89 wherein the electron conductor is carbon felt.

91. The fuel cell of claim 80 wherein the fuel cell comprises a polymer electrolyte membrane to separate an anode compartment from a cathode compartment.

92. The fuel cell of claim 80 wherein the fuel cell does not comprise a salt bridge nor a polymer electrolyte membrane to separate an anode compartment from a cathode compartment.

93. A method of generating electrical power, comprising (a) oxidizing an organic fuel at an anode in the presence of at least one anode oxidoreductase enzyme, which is incorporated in the anode, (b) transferring electrons from an oxidized organic fuel to an anode electron conducting material by way of a redox polymer, (c) reducing an oxygen molecule at a cathode in the presence of an oxygen oxidoreductase enzyme, which is immobilized in a buffered compartment of a modified cathode ion exchange polymer membrane, and (d) transferring electrons from an electron conducting material to a substrate of the oxygen oxidoreductase by way of an electron transport mediator, which is immobilized in a buffered compartment of a modified ion exchange polymer membrane, such that an electric current is produced.

94. The method of claim 93 wherein the oxygen oxidoreductase is a bilirubin oxidase and the substrate is oxygen.

95. The method of claim 93 wherein the anode oxidoreductase enzyme is an alcohol reductase and the organic fuel is an alcohol.

96. The method of claim 93 wherein the redox polymer is a polymethylene green.

97. The method of claim 93 wherein the modified cathode ion exchange polymer is a quaternary ammonium bromide treated Nafion®

polymer.

98. The method of claim 93 wherein the electron transport mediator comprises a transition metal and aromatic ligand complex.

99. The method of claim 96 wherein the electron transport mediator is Ru(bpy)₃⁺².

100. The method of claim 93 wherein the anode is a bioanode, which comprises a modified anode ion exchange membrane and at least one anode oxidoreductase enzyme, which is immobilized within a buffered compartment of the modified anode ion exchange membrane.

101. The method of claim 100 wherein the modified anode ion exchange membrane is a quaternary ammonium bromide treated Nafion®

polymer.

102. The method of claim 100 wherein the anode oxidoreductase enzyme is an alcohol dehydrogenase.

103. The method of claim 100 wherein the bioanode comprises a first anode oxidoreductase enzyme and a second anode oxidoreductase enzyme, which are immobilized within the buffered compartment of the modified anode ion exchange membrane, wherein the first anode oxidoreductase enzyme is an alcohol dehydrogenase and the second anode oxidoreductase enzyme is an aldehyde dehydrogenase.

104. A biocathode useful in the acceptance of electrons from an electrical circuit, which comprises an electron conducting material juxtaposed to a dual use membrane, and a dual use membrane, wherein the dual use membrane comprises a modified ion exchange membrane, an immobilized cathode enzyme, and an electron transport mediator.

105. The biocathode of claim 104 wherein the modified ion exchange membrane is a quaternary ammonium salt-treated perfluoro sulfonic acid-PTFE copolymer.

106. The biocathode of claim 105 wherein the modified ion exchange membrane is a tetrabutylammonium bromide (TBAB) treated Nafion®

membrane.

107. The biocathode of claim 104 wherein the cathode enzyme is (a) immobilized within a buffered micelle of the modified ion exchange membrane and (b) an oxygen oxidoreductases enzyme.

108. The biocathode of claim 107 wherein the cathode enzyme is a bilirubin oxidase.

109. The biocathode of claim 104 wherein the electron transport mediator comprises a transition metal and an aromatic ligand together as a complex.

110. The biocathode of claim 109 wherein the electron transport mediator is Ru(bpy)₃⁺².