Small volume in vitro analyte sensor

• US 20030201194A1
• Filed: 06/09/2003
• Published: 10/30/2003
• Est. Priority Date: 02/06/1997
• Status: Abandoned Application

• Current Assignee: Therasense Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Therasense Incorporated (Abbott Laboratories Incorporated)
• Inventors: Heller, Adam, Say, James, Vreeke, Mark S., Feldman, Benjamin J.
• Application Number:
  US10/457,585
• Publication Number:
  US 20030201194A1
• Time in Patent Office: Days
• US Class Current: 205/777.5
• CPC Class Codes: A61B 2560/0223 of calibration, e.g. protocols for calibrating sensors
• CPC Class Codes: A61B 2562/0295 Strip shaped analyte sensors for apparatus classified in A61B5/145 or A61B5/157 disposable multilayered or laminated electrodes G01N27/307; details of handling chemical test elements G01N33/4875
• CPC Class Codes: A61B 5/1411 by percutaneous method, e.g. by lancet
• CPC Class Codes: A61B 5/14532 for measuring glucose, e.g. by tissue impedance measurement
• CPC Class Codes: A61B 5/14546 for measuring analytes not otherwise provided for, e.g. ions, cytochromes
• CPC Class Codes: A61B 5/1486 using enzyme electrodes, e.g. with immobilised oxidase
• CPC Class Codes: A61B 5/14865 invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors
• CPC Class Codes: A61B 5/1495 Calibrating or testing of in-vivo probes
• CPC Class Codes: A61B 5/150022 for capillary blood or interstitial fluid
• CPC Class Codes: A61B 5/150358 Strips for collecting blood, e.g. absorbent optical reagent test strips G01N21/8483; chemical reagent test strips G01N33/4875, G01N33/52
• CPC Class Codes: A61B 5/150412 Pointed piercing elements, e.g. needles, lancets for piercing the skin blade-like piercing elements A61B5/150442
• CPC Class Codes: A61B 5/150503 Single-ended needles hollow piercing element tip design A61B5/150396, needle sets for catheters A61M25/06, needles for bringing media into the body A61M5/32
• CPC Class Codes: A61B 5/15105 Purely manual piercing, i.e. the user pierces the skin without the assistance of any driving means or driving devices
• CPC Class Codes: A61B 5/15113 Manually triggered, i.e. the triggering requires a deliberate action by the user such as pressing a drive button
• CPC Class Codes: A61B 5/15144 comprising driving means, e.g. a spring, for retracting the piercing unit into the housing
• CPC Class Codes: A61B 5/157 Devices characterised by integrated means for measuring characteristics of blood
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: G01N 2333/904 acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: G01N 27/3272 : Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels optical biosensors G01N33/52
• CPC Class Codes: G01N 27/3273 : Devices therefor, e.g. test element readers, circuitry details not specific to biochemical electrodes G01N33/4875
• CPC Class Codes: G01N 33/5438 : Electrodes

Abstract

A sensor designed to determine the amount and concentration of analyte in a sample having a volume of less than about 1 μL. The sensor has a working electrode coated with a non-leachable redox mediator. The redox mediator acts as an electron transfer agent between the analyte and the electrode. In addition, a second electron transfer agent, such as an enzyme, can be added to facilitate the electrooxidation or electroreduction of the analyte. The redox mediator is typically a redox compound bound to a polymer. The preferred redox mediators are air-oxidizable.

The amount of analyte can be determined by coulometry. One particular coulometric technique includes the measurement of the current between the working electrode and a counter or reference electrode at two or more times. The charge passed by this current to or from the analyte is correlated with the amount of analyte in the sample. Other electrochemical detection methods, such as amperometric, voltammetric, and potentiometric techniques, can also be used.

The invention can be used to determine the concentration of a biomolecule, such as glucose or lactate, in a biological fluid, such as blood or serum. An enzyme capable of catalyzing the electrooxidation or electroreduction of the biomolecule is provided as a second electron transfer agent.

Claims

1. A method for determining a concentration of an analyte in a sample, comprising the steps of:

contacting a sample with an electrochemical sensor comprising;

a facing electrode pair comprising a working electrode and a counter electrode; and

a sample chamber for holding the sample in electrolytic contact with the working electrode, the sample chamber comprising a measurement zone positioned between the working and counter electrodes, wherein the measurement zone is sized to contain a volume of less than about 1 μ

L of sample; and

determining the concentration of the analyte in the sample by coulometry.

2. The method of claim 1, wherein the measurement zone is sized to contain a volume of less than about 0.5 μ

L of sample.

3. The method of claim 2, wherein the measurement zone is sized to contain a volume of less than about 0.2 μ

L of sample.

4. The method of claim 3, wherein the measurement zone is sized to contain a volume of less than about 0.1 μ

L of sample.

5. The method of claim 1, wherein the sample chamber is sized to contain a volume of less than about 1 μ

L of sample.

6. The method of claim 5, wherein the sample chamber is sized to contain a volume of less than about 0.5 μ

L of sample.

7. The method of claim 6, wherein the sample chamber is sized to contain a volume of less than about 0.2 μ

L of sample.

8. The method of claim 1, wherein the step of determining the concentration of the analyte comprises:

electrolyzing at least 90% of analyte present in the measurement zone by applying a potential across the working and counter electrodes;

determining an electrical charge used to electrolyze the analyte; and

correlating the electrical charge with the concentration of the analyte in the sample.

9. The method of claim 8, wherein at least 90% of the analyte is electrolyzed in less than about 5 minutes.

10. The method of claim 9, wherein at least 90% of the analyte is electrolyzed in less than about 1 minute.

11. The method of claim 8, wherein the step of determining an electrical charge comprises the steps of:

measuring a current generated at the working electrode at two or more times as the analyte is electrolyzed; and

integrating the measured currents over time to obtain the electrical charge used to electrolyze the analyte.

12. The method of claim 1, wherein the step of determining the concentration of the analyte by coulometry comprises the steps of:

electrolyzing a portion of the analyte by applying a potential across the working and the counter electrode;

measuring a current generated at the working electrode at two or more times during the electrolysis;

extrapolating a current curve based on the measured currents;

integrating the current curve over time to obtain an electrical charge necessary to electrolyze at least 90% of the analyte; and

correlating the electrical charge with the concentration of the analyte in the sample.

13. The method of claim 1, wherein the sensor further comprises a non-leachable enzyme on the working electrode.

14. The method of claim 13, wherein the enzyme is immobilized on the working electrode.

15. The method of claim 1, wherein the sensor further comprises a non-leachable redox mediator on the working electrode.

16. The method of claim 15, wherein the redox mediator is immobilized on the working electrode.

17. The method of claim 15, wherein the redox mediator comprises a polymer and a redox species ionically, covalently, or coordinatively bound to the polymer.

18. The method of claim 17, wherein the redox species is coordinatively bound to the polymer.

19. The method of claim 15, wherein the redox mediator comprises an air-oxidizable redox mediator.

20. The method of claim 19, wherein the air-oxidizable redox mediator comprises Os[4,4′

-dimethoxy-2,2′

-bipyridine]₂Cl^+/+2 or Os[4,7-dimethoxy-1,10-phenanthroline]₂Cl^+/+2 complexed with poly(1-vinyl imidazole) or poly(4-vinylpyridine).

21. The method of claim 15, wherein at least 90% of the redox mediator is in an oxidized state prior to introduction of the sample in the sensor.

22. The method of claim 15, wherein the sensor further comprises a non-leachable second electron transfer agent on the working electrode.

23. The method of claim 22, wherein the second electron transfer agent is immobilized on the working electrode.

24. The method of claim 22, wherein the second electron transfer agent comprises an enzyme.

25. The method of claim 24, wherein the analyte is glucose.

26. The method of claim 25, wherein the enzyme is a glucose oxidase.

27. The method of claim 1, wherein the sensor further comprises sorbent material disposed in the measurement zone.

28. The method of claim 27, wherein the step of contacting the sample with an electrochemical sensor further comprises contacting the sample with the sorbent material to wick the sample into the measurement zone.

29. The method of claim 1, wherein the working electrode and counter electrode have a separation distance of less than about 0.2 mm.

30. The method of claim 29, wherein the separation distance is less than about 0.1 mm.

31. The method of claim 30, wherein the separation distance is less than about 0.05 mm.

32. The method of claim 1, wherein the sensor comprises two or more facing electrode pairs.

33. A method of determining the concentration of an analyte in a sample, comprising the steps of:

contacting a sample with an electrochemical sensor, wherein the sensor comprises a working electrode, a non-leachable redox mediator on the working electrode, and a sample chamber sized to contain a volume of less than about 1 μ

L of sample in electrolytic contact with the working electrode; and

determining the concentration of the analyte in the sample by coulometry.

34. The method of claim 33, wherein the sample chamber is sized to contain a volume of less than about 0.5 μ

L of sample.

35. The method of claim 34, wherein the sample chamber is sized to contain a volume of less than about 0.2 μ

L of sample.

36. The method of claim 33, wherein the redox mediator is immobilized on the working electrode.

37. The method of claim 33, wherein the sensor further comprises a non-leachable second electron transfer agent on the working electrode.

38. The method of claim 37, wherein the second electron transfer agent is immobilized on the working electrode.

39. The method of claim 37, wherein the second electron transfer agent comprises an enzyme.

40. The method of claim 33, wherein the sensor further comprises a sorbent material within the sample chamber to reduce the volume of sample that the sample chamber is sized to hold.

41. The method of claim 33, wherein the redox mediator comprises a transition metal complex.

42. The method of claim 41, wherein the transition metal complex is an osmium, ruthenium, iron, or cobalt complex.

43. The method of claim 42, wherein the transition metal complex comprises an osmium complex.

44. The method of claim 43, wherein the osmium complex comprises osmium complexed with at least one ligand having a nitrogen-containing heterocycle.

45. The method of claim 44, wherein the ligand having a nitrogen-containing heterocycle comprises 2,2′

-bipyridine, 1,10-phenanthroline, or a derivative thereof.

46. The method of claim 45, wherein the ligand having a nitrogen-containing heterocycle comprises a mono-, di-, or polyalkoxy derivative of 2,2′

-bipyridine or 1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking.

47. The method of claim 45, wherein the nitrogen-containing heterocycle comprises 2,2′

-bipyridine, 4,4′

-dimethyl-2,2′

-bipyridine, 4,4′

-dialkoxy-2,2′

-bipyridine, 1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline, or 4,7-dialkoxy-1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking.

48. The method of claim 43, wherein the osmium complex comprises osmium complexed with a polymeric ligand.

49. The method of claim 48, wherein the polymeric ligand comprises a nitrogen-containing heterocycle.

50. The method of claim 49, wherein the polymer comprises poly(4-vinyl pyridine) or poly(1-vinyl imidazole).

51. An electrochemical sensor for determining the concentration of an analyte in a sample, the sensor comprising:

a facing electrode pair comprising a working electrode and a counter electrode;

a non-leachable redox mediator on the working electrode; and

a measurement zone positioned between the working electrode and counter electrode, wherein the measurement zone is sized to contain a volume of less than about 1 μ

L of sample.

52. The sensor of claim 51, wherein the measurement zone is sized to contain a volume of less than about 0.5 μ

L of sample.

53. The sensor of claim 52, wherein the measurement zone is sized to contain a volume of less than about 0.2 μ

L of sample.

54. The sensor of claim 53, wherein the measurement zone is sized to contain a volume of less than about 0.1 μ

L of sample.

55. The sensor of claim 51, wherein the redox mediator is immobilized on the working electrode.

56. The sensor of claim 51, wherein the redox mediator is an air-oxidizable redox mediator.

57. An electrochemical sensor comprising:

two or more facing electrode pairs, each electrode pair comprising a working electrode, a counter electrode and a measurement zone disposed approximately between the working and counter electrodes, wherein the measurement zone is sized to hold less than about 1 μ

L of sample; and

non-leachable redox mediator on the working electrode of at least one of the electrode pairs.

58. The sensor of claim 57, wherein the redox mediator is immobilized on the working electrode of at least one of the electrode pairs.

59. The sensor of claim 57, wherein the measurement zone of at least one of the electrode pairs is sized to hold less than about 0.5 μ

L of sample.

60. The sensor of claim 59, wherein the measurement zone of at least one of the electrode pairs is sized to hold less than about 0.2 μ

L of sample.

61. The sensor of claim 60, wherein the measurement zone of at least one of the electrode pairs is sized to hold less than about 0.1 μ

L of sample.

62. The sensor of claim 57, wherein at least one electrode pair comprises a working electrode including non-leachable enzyme and redox mediator, and wherein at least one electrode pair comprises a working electrode including non-leachable redox mediator in the absence of the enzyme.

63. The sensor of claim 62, further comprising a third electrode pair having no redox mediator or enzyme on the working electrode.

64. The sensor of claim 57, further comprising sorbent material disposed in the measurement zone of at least one of the electrode pairs.

65. The sensor of claim 57, wherein the redox mediator is an air-oxidizable redox mediator.

66. An electrochemical sensor for determining the concentration of an analyte in a sample, the sensor comprising:

a facing electrode pair comprising a working electrode and a counter electrode;

a measurement zone positioned between the working electrode and counter electrode; and

sorbent material disposed at least partially within the measurement zone to reduce the volume of sample needed to fill the measurement zone;

wherein the measurement zone is sized to contain a volume of less than about 1 μ

L of sample.

67. The sensor of claim 66, wherein the measurement zone is sized to contain a volume of less than about 0.5 μ

L of sample.

68. The sensor of claim 67, wherein the measurement zone is sized to contain a volume of less than about 0.2 μ

L of sample.

69. The sensor of claim 68, wherein the measurement zone is sized to contain a volume of less than about 0.1 μ

L of sample.

70. The sensor of claim 66, wherein the sensor further comprises non-leachable redox mediator on the working electrode.

71. The sensor of claim 70, wherein the redox mediator is immobilized on the working electrode.

72. The sensor of claim 66, wherein the sorbent material has a void volume of between about 5% and 50% of the total volume of the sorbent material.

73. The sensor of claim 72, wherein the void volume is about 10% to about 25% of the total volume of the sorbent material.

74. An electrochemical sensor for determining the concentration of an analyte in a sample, the sensor comprising:

a working electrode;

a non-leachable redox mediator on the working electrode;

a sample chamber for holding the sample in electrolytic contact with the working electrode; and

sorbent material disposed within the sample chamber;

wherein the sample chamber is sized to contain a volume of less than about 1 μ

L of sample.

75. The sensor of claim 74, wherein the sample chamber is sized to contain a volume of less than about 0.5 μ

L of sample.

76. The sensor of claim 75, wherein the sample chamber is sized to contain a volume of less than about 0.2 μ

L of sample.

77. A method for determining the concentration of an analyte in a sample, comprising the steps of:

contacting the sample with an electrochemical sensor;

the sensor comprising;

an electrode pair comprising a working electrode and a counter electrode;

a sample chamber for holding the sample in electrolytic contact with the working electrode; and

a sorbent disposed within the sample chamber;

wherein the sample chamber is sized to hold less than about 1 μ

L of sample; and

determining the concentration of the analyte by coulometry.

78. The method of claim 77, wherein the method further comprises wicking the sample into the sample chamber using the sorbent material.

79. The method of claim 77, wherein the electrode pair is a facing electrode pair.

80. A sensor for the determination of the concentration of an analyte in a sample having a volume of less than about 1 μ

L, comprising;

a support; and

an air-oxidizable redox mediator coated on the support to form a working surface for contacting sample;

wherein, at least 90% of the air-oxidizable redox mediator is in an oxidized state prior to introduction of sample.

81. The sensor of claim 80, wherein the air-oxidizable redox mediator is immobilized on the support.

82. A method for determining a concentration of an analyte in a sample, comprising the steps of:

contacting the sample with a sensor comprising a support; and

an air-oxidizable redox mediator coated on the support to form a working surface, wherein, at least 90% of the air-oxidizable redox mediator is in an oxidized state prior to introduction of a sample; and

correlating the concentration of the analyte in the sample to a change in oxidation state of the redox mediator in the presence of analyte.

83. The method of claim 82, wherein the sensor is an optical sensor.

84. The method of claim 83, wherein the step of correlating the concentration of the analyte comprises:

irradiating the redox mediator with light;

measuring the response of the redox mediator to irradiation by light; and

correlating the concentration of the analyte to the measured response of the redox mediator.

85. The method of claim 84, wherein the step of measuring the response of the redox mediator to irradiation by light comprises measuring the absorption of the irradiated light by the redox mediator.

86. The method of claim 84, wherein the step of measuring the response of the redox mediator to irradiation by light comprises measuring the transmittance of the irradiated light by the redox mediator.

87. The method of claim 84, wherein the step of measuring the response of the redox mediator to irradiation by light comprises measuring the fluorescence of the redox mediator after irradiation by light.

88. The method of claim 84, wherein the step of measuring the response of the redox mediator to irradiation by light comprises measuring the reflection of light by the redox mediator.

89. The method of claim 82, wherein the sensor is an electrochemical sensor.

90. The method of claim 89, wherein the step of correlating the concentration of the analyte comprises:

applying an electrical potential across the redox mediator;

measuring a current at one or more intervals, the current being generated in response to the electrolysis of the redox mediator in the presence of the analyte; and

correlating the concentration of the analyte to the measured current.

91. The method of claim 82, wherein the redox mediator comprises a transition metal complex.

92. The method of claim 91, wherein the transition metal complex comprises an osmium complex.

93. The method of claim 92, wherein the osmium complex comprises osmium complexed with at least one ligand having a nitrogen-containing heterocycle.

94. The method of claim 93, wherein the ligand having a nitrogen-containing heterocycle comprises 2,2′

-bipyridine, 1,10-phenanthroline, or a derivative thereof.

95. The method of claim 94, wherein the ligand having a nitrogen-containing heterocycle comprises a mono-, di-, or polyalkoxy derivative of 2,2′

-bipyridine or 1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking.

96. The method of claim 95, wherein the nitrogen-containing heterocycle comprises 4,4′

-dialkoxy-2,2′

-bipyridine or 4,7-dialkoxy-1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking.

97. The method of claim 96, wherein the nitrogen-containing heterocycle comprises 4,4′

-dimethoxy-2,2′

-bipyridine or 4,7-dimethoxy-1,10-phenanthroline.

98. The method of claim 92, wherein the osmium complex comprises osmium complexed with a polymeric ligand.

99. The method of claim 98, wherein the polymeric ligand comprises a nitrogen-containing heterocycle.

100. The method of claim 99, wherein the polymer comprises poly(4-vinyl pyridine) or poly(1-vinyl imidazole).

101. The sensor of claim 100, wherein the redox mediator comprises Os[4,4′

-dimethoxy-2,2′

-bipyridine]₂Cl^+/+2 or Os[4,7-dimethoxy-1,10-phenanthroline]₂Cl^+/+2 complexed with poly(1-vinyl imidazole).

102. The sensor of claim 82, wherein the sensor further comprises a second electron transfer agent coated on the support and in contact with the redox mediator.

103. The sensor of claim 102, wherein the second electron transfer agent is an enzyme.

104. The sensor of claim 102, wherein the second electron transfer agent is immobilized on the support.

105. The sensor of claim 82, wherein the air-oxidizable redox mediator is immobilized on the support.

106. A method for measuring analyte in a patient sample, the method comprising:

contacting the patient with an analyte measuring device, the device comprising;

sample acquisition means for producing a patient sample; and

an electrochemical sensor for measuring analyte in the sample;

wherein the electrochemical sensor comprises;

a facing electrode pair, comprising a working electrode and a counter electrode; and

a measurement zone positioned between the working electrode and counter electrode, wherein the measurement zone is sized to contain a volume of less than about 1 μ

L of sample;

acquiring a sample using the sample acquisition means;

transporting a portion of the sample to the measurement zone of the electrochemical sensor; and

determining the concentration of the analyte in the sample by coulometry.

107. The method of claim 106, wherein the sample acquisition means comprises a skin-piercing member and the step of acquiring a sample comprises piercing the patient'"'"'s skin to produce a sample.

108. The method of claim 107, wherein the skin piercing member comprises a lancet.

109. The method of claim 106, wherein the electrochemical sensor further comprises a sorbent material disposed within the measurement zone of the sensor.

110. The method of claim 109, wherein said transporting comprises:

wicking the sample into the measurement zone using the sorbent material.

111. The method of claim 106, wherein the measurement zone is sized to contain a volume of less than about 0.5 μ

L of sample.

112. The method of claim 111, wherein the measurement zone is sized to contain a volume of less than about 0.2 μ

L of sample.

113. The sensor of claim 112, wherein the measurement zone is sized to contain a volume of less than about 0.1 μ

L of sample.

114. The method of claim 106, wherein the sensor further comprises a non-leachable redox mediator.

115. The method of claim 114, wherein the redox mediator is an air-oxidizable redox mediator.

116. A method for measuring analyte in a patient sample, the method comprising:

contacting the patient with an analyte measuring device, the device comprising;

sample acquisition means for producing a patient sample; and

an electrochemical sensor for measuring analyte in the sample;

wherein the electrochemical sensor comprises;

a working electrode;

non-leachable redox mediator on the working electrode; and

a sample chamber for holding the sample in electrolytic contact with the working electrode, wherein the sample chamber is sized to contain a volume of less than about 1 μ

L of sample;

acquiring a sample using the sample acquisition means;

transporting a portion of the sample to the sample chamber of the electrochemical sensor; and

determining the concentration of the analyte in the sample by coulometry.

117. An analyte measurement device comprising:

sample acquisition means for producing a patient sample;

an electrochemical sensor comprising;

a facing electrode pair comprising a working electrode and a counter electrode;

a non-leachable redox mediator on the working electrode; and

a measurement zone positioned between the working electrode and counter electrode;

wherein the measurement zone is sized to contain a volume of less than about 1 μ

L of sample; and

transport means for transporting the sample produced by the sample acquisition means to the measurement zone of the sensor.

118. The device of claim 117, wherein the sample acquisition means comprises a skin-piercing member.

119. The device of claim 118, wherein the skin-piercing member comprises a lancet.

120. The device of claim 117, wherein the redox mediator is immobilized on the working electrode.

121. The device of claim 117, wherein the redox mediator is an air-oxidizable redox mediator.

122. The device of claim 117, wherein the transport means comprises a wicking material, a capillary chamber, or a pump.

123. An analyte measurement device comprising:

sample acquisition means for producing a patient sample; and

an electrochemical sensor coupled to the sample acquisition means, comprising;

a facing electrode pair comprising a working electrode and a counter electrode;

a measurement zone positioned between the working electrode and counter electrode; and

sorbent material disposed within the measurement zone;

wherein the measurement zone is sized to contain a volume of less than about 1 μ

l of sample; and

transport means for transporting the sample produced by the sample acquisition means to the measurement zone of the sensor.

124. The device of claim 123, wherein the transport means is the sorbent material.

125. A method for determination of a concentration of an analyte in a sample, the method comprising the steps of:

contacting a sample with an electrochemical sensor comprising;

first and second electrode pairs, each pair comprising a working electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode, the sample chamber sized to contain a volume of less about 1 μ

L of sample;

wherein said first electrode pair includes a non-leachable redox mediator and a non-leachable enzyme on the working electrode;

and wherein said second electrode pair includes non-leachable redox mediator on the working electrode in the absence of enzyme;

measuring substantially simultaneously, and at two or more times, a first current generated at the first electrode pair and a second current generated at the second electrode pair; and

integrating the measured first currents over time to obtain a first charge;

integrating the measured second currents over time to obtain a second charge;

subtracting the second charge from the first charge to obtain a noise-reduced charge; and

correlating the concentration of the analyte to the noise-reduced charge.

126. The method of claim 125, wherein the first and second electrode pairs are facing electrode pairs and further comprise a counter electrode and a measurement zone between the working electrode and counter electrode, the measurement zone sized to contain a volume of less than about 1 μ

L of sample.

127. A method for determining a concentration of an analyte in a sample, the method comprising the steps of:

providing an electrochemical sensor, the sensor having one or more facing electrode pairs, each electrode pair comprising a working electrode, a counter electrode, and a measurement zone between the working electrode and the counter electrode, the measurement zone having a volume of less than about 1 μ

L, wherein the measurement zones of the one or more electrode pairs have approximately equal volumes, and wherein at least one of the electrode pairs further comprises redox mediator on its working electrode;

measuring a capacitance of at least one of the electrode pairs;

calculating the volume of the measurement zone from the capacitance measurement;

contacting the sample with the sensor; and

determining the concentration of the analyte in the sample by coulometry.

128. The method of claim 127, wherein the redox mediator is a non-leachable redox mediator.

129. A method of storing and packaging an analytical sensor comprising the steps of:

packaging an analytical sensor in an atmosphere containing molecular oxygen, the sensor comprising an air-oxidizable redox mediator.

130. The method of claim 129, wherein greater than 90% of the redox mediator is in an oxidized state after being stored for more than one month.

131. A method for determining a concentration of an analyte in a sample comprising the steps of:

contacting the sample with an electrochemical sensor, the sensor comprising a working electrode and a non-leachable redox mediator on the working electrode, wherein the molar amount of redox mediator in a reduced form prior to introduction of the sample is less than, on a stoichiometric basis, 5% of the expected molar amount of the analyte to be electrolyzed;

electrolyzing less than about 1 μ

L of sample; and

determining the concentration of the analyte in the sample by coulometry.

132. A method of determining a concentration of an analyte in a sample comprising the steps of:

contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a measurement zone bounded on at least two sides by the working and the counter electrodes, wherein the measurement zone is sized to contain less than about 1 μ

L of sample; and

determining the concentration of the analyte in the sample by coulometry.