Small volume in vitro sensor and methods of making

• US 8,083,929 B2
• Filed: 09/29/2009
• Issued: 12/27/2011
• Est. Priority Date: 10/08/1998
• Status: Expired due to Term

• Current Assignee: Abbott Diabetes Care Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Diabetes Care Incorporated (Abbott Laboratories Incorporated)
• Inventors: Feldman, Benjamin J., Heller, Adam, Heller, Ephraim, Mao, Fei, Vivolo, Joseph A., Funderburk, Jeffery V., Colman, Fredric C., Krishnan, Rajesh
• Primary Examiner(s): Noguerola, Alex
• Application Number:
  US12/569,310
• Publication Number:
  US 20100032317A1
• Time in Patent Office: 819 Days
• Field of Search: 20440301-40315, 205/777.5, 205/778, 205/792
• US Class Current: 205/792
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: G01N 27/3271 Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001
• 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 27/3274 Corrective measures, e.g. error detection, compensation for temperature or hematocrit, calibration coding of calibration information G01N33/48771
• CPC Class Codes: Y10T 156/1052 with cutting, punching, tearing or severing
• CPC Class Codes: Y10T 29/49002 Electrical device making
• CPC Class Codes: Y10T 29/49004 including measuring or testing of device or component part
• CPC Class Codes: Y10T 29/49007 Indicating transducer
• CPC Class Codes: Y10T 29/49126 Assembling bases
• CPC Class Codes: Y10T 29/49128 Assembling formed circuit to base
• CPC Class Codes: Y10T 29/4913 Assembling to base an electrical component, e.g., capacitor, etc.
• CPC Class Codes: Y10T 29/49147 Assembling terminal to base
• CPC Class Codes: Y10T 29/49155 Manufacturing circuit on or in base
• CPC Class Codes: Y10T 29/49204 Contact or terminal manufacturing
• CPC Class Codes: Y10T 29/49208 by assembling plural parts
• CPC Class Codes: Y10T 29/4921 with bonding
• CPC Class Codes: Y10T 29/49794 Dividing on common outline

Abstract

A sensor utilizing a non-leachable or diffusible redox mediator is described. The sensor includes a sample chamber to hold a sample in electrolytic contact with a working electrode, and in at least some instances, the sensor also contains a non-leachable or a diffusible second electron transfer agent. The sensor and/or the methods used produce a sensor signal in response to the analyte that can be distinguished from a background signal caused by the mediator. 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, using techniques such as coulometry, amperometry; and potentiometry. An enzyme capable of catalyzing the electrooxidation or electroreduction of the biomolecule is typically provided as a second electron transfer agent.

Claims

1. A sensor for determining the concentration of glucose in a sample fluid, the sensor comprising:

a working electrode disposed on a first substrate and a counter/reference electrode disposed on a second substrate, wherein the working electrode comprises palladium and the counter/reference electrode comprises gold, and wherein the working electrode and the counter/reference electrode are in a facing configuration and separated by an effective distance in a range of 25 to 1000 μ

m;

a spacer comprising a first part and a second part disposed between the first and second substrates, wherein the first part of the spacer, the second part of the spacer, the first substrate, and the second substrate define a first aperture along a first side edge of the sensor and a second aperture along a second side edge of the sensor, wherein either the first aperture or the second aperture is used for sample application and the sensor has no more than two apertures;

a sample chamber for holding the sample fluid and sized to contain a volume of no more than about 1 μ

L of sample, wherein the sample chamber has a length which extends from the first aperture to the second aperture and a substantially uniform width between the first part of the spacer and the second part of the spacer along the entire length of the sample chamber, and wherein the width of the sample chamber is not wider than either the first aperture or the second aperture; and

a glucose dehydrogenase and a diffusible redox mediator comprising ferricyanide disposed in the sample chamber;

wherein the first and second substrates define a sensor comprising a proximal end having a width and a distal end having a width, the distal end being configured for insertion into a sensor reader, wherein the width of the distal end is greater than the width of the proximal end.

2. The sensor of claim 1, wherein the sample chamber is sized to contain a volume of no more than about 0.5 μ

L of sample.

3. The sensor of claim 1, wherein the sample chamber is sized to contain a volume of no more than about 0.25 μ

L of sample.

4. The sensor of claim 1, wherein the working electrode and the counter/reference electrode are separated by an effective distance in a range of 50 to 500 μ

m.

5. The sensor of claim 1, wherein the sample chamber is bounded on at least two sides by the working electrode and the counter electrode.

6. The sensor of claim 1, wherein the half-wave potential of the redox mediator, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, is about the same as the potential of the counter/reference electrode.

7. The sensor of claim 1, wherein the half-wave potential of the redox mediator, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, is no more than about −

150 millivolts relative to the potential of the counter/reference electrode.

8. The sensor of claim 1, wherein the first substrate comprises a cutout portion at the distal end to expose a portion of the counter/reference electrode disposed on the second substrate.

9. The sensor of claim 1, wherein the second substrate comprises a cutout portion at the distal end to expose a portion of the working electrode disposed on the first substrate.

10. A method for determining a concentration of a glucose in a sample, comprising the steps of:

contacting a sample with an electrochemical sensor comprising;

a working electrode disposed on a first substrate and a counter/reference electrode disposed on a second substrate, wherein the working electrode comprises palladium and the counter/reference electrode comprises gold, and wherein the working electrode and the counter/reference electrode are in a facing configuration and separated by an effective distance in a range of 25 to 1000 μ

m;

a spacer comprising a first part and a second part disposed between the first and second substrates, wherein the first part of the spacer, the second part of the spacer, the first substrate, and the second substrate define a first aperture along a first side edge of the sensor and a second aperture along a second side edge of the sensor, wherein either the first aperture or the second aperture is used for sample application and the sensor has no more than two apertures;

a sample chamber for holding the sample fluid and sized to contain a volume of no more than about 1 μ

L of sample, wherein the sample chamber has a length which extends from the first aperture to the second aperture and a substantially uniform width between the first part of the spacer and the second part of the spacer along the entire length of the sample chamber, and wherein the width of the sample chamber is not wider than either the first aperture or the second aperture; and

a glucose dehydrogenase and a diffusible redox mediator comprising ferricyanide disposed in the sample chamber;

generating a sensor signal at the working electrode, and

determining the concentration of the glucose using the sensor signal.

11. The method of claim 10, wherein determining the concentration of the glucose comprises determining the concentration of the glucose by coulometry using the sensor signal.

12. The method of claim 10, wherein determining the concentration of the glucose comprises determining the concentration of the glucose by amperometry using the sensor signal.

13. The method of claim 10, further comprising:

providing calibration data on a batch of electrodes to the measurement instrument, said calibration data comprising information related to a magnitude of a background charge for the batch of electrodes;

wherein the step of determining the concentration of the glucose comprises determining the concentration of the glucose using the sensor signal and the calibration data.

14. The method of claim 10, wherein determining the concentration of the glucose comprises determining the concentration of the glucose by potentiometry using the sensor signal.

15. The method of claim 10, wherein generating a sensor signal comprises operating the sensor at an applied potential of no more than about +100 mV between the working electrode and the counter/reference electrode when the redox mediator is reduced at the counter/reference electrode.

16. The method of claim 10, wherein generating a sensor signal comprises operating the sensor at an applied potential of about 0 mV between the working electrode and the counter/reference electrode when the redox mediator is reduced at the counter/reference electrode.

17. The method of claim 10, wherein generating a sensor signal comprises operating the sensor at an applied potential of no more than about −

100 mV between the working electrode and the counter/reference electrode when the redox mediator is reduced at the counter/reference electrode.

18. The method of claim 10, wherein the first substrate comprises a cutout portion at the distal end to expose a portion of the counter/reference electrode disposed on the second substrate.

19. The method of claim 10, wherein the second substrate comprises a cutout portion at the distal end to expose a portion of the working electrode disposed on the first substrate.

20. The method of claim 10, further comprising obtaining the sample from a finger of a subject.

21. The method of claim 10, further comprising obtaining the sample from a region of a subject having a lower nerve end density as compared to a fingertip.

22. The method of claim 21, wherein the region of a subject having a lower nerve end density as compared to a fingertip is selected from the group consisting of:

a forearm region, and a thigh region.

23. A sensor for determining the concentration of glucose in a sample fluid, the sensor comprising:

a working electrode disposed on a first substrate and a counter/reference electrode disposed on a second substrate, wherein the working electrode comprises palladium and the counter/reference electrode comprises gold, and wherein the working electrode and the counter/reference electrode are in a facing configuration and separated by an effective distance in a range of 25 to 1000 μ

m;

a spacer comprising a first part and a second part disposed between the first and second substrates, wherein the first part of the spacer, the second part of the spacer, the first substrate, and the second substrate define a first aperture along a first side edge of the sensor and a second aperture along a second side edge of the sensor, wherein either the first aperture or the second aperture is used for sample application and the sensor has no more than two apertures;

a sample chamber for holding the sample fluid and sized to contain a volume of no more than about 1 μ

L of sample, wherein the sample chamber has a length which extends from the first aperture to the second aperture and a substantially uniform width between the first part of the spacer and the second part of the spacer along the entire length of the sample chamber, and wherein the width of the sample chamber is not wider than either the first aperture or the second aperture; and

a glucose-responsive enzyme and a diffusible redox mediator disposed in the sample chamber;

wherein the first and second substrates define a sensor comprising a proximal end having a width and a distal end having a width, the distal end being configured for insertion into a sensor reader, wherein the width of the distal end is greater than the width of the proximal end.

24. The sensor of claim 23, wherein the redox mediator comprises an effective diffusion coefficient, within the sensor, of no more than about 1×

10<sup>−

6</sup> cm/sec in the sample fluid.

25. The sensor of claim 23, wherein the effective diffusion coefficient of the redox mediator through the sample fluid is less than the effective diffusion coefficient of the glucose through the sample fluid.

26. The sensor of claim 23, wherein the effective diffusion coefficient of the redox mediator through the sample fluid is at least ten times less than the effective diffusion coefficient of the glucose through the sample fluid.

27. The sensor of claim 23, wherein the diffusible mediator comprises a molecular weight of at least 5,000 daltons.

28. The sensor of claim 23, wherein the sensor is configured and arranged so that the redox mediator is more readily electrolyzed on the working electrode than the counter electrode.

29. The sensor of claim 23, wherein the sensor comprises a molar amount of the redox mediator that is, on a stoichiometric basis, no more than an average normal physiological amount of the glucose.

30. The sensor of claim 23, wherein the sensor comprises a molar amount of the redox mediator that is, on a stoichiometric basis, no more than 20% of an average normal physiological amount of the glucose.

31. The sensor of claim 23, wherein the working electrode comprises a surface area of no more than about 0.01 cm² exposed in the sample chamber.

32. The sensor of claim 23, wherein the working electrode comprises a surface area of no more than about 0.001 cm² exposed in the sample chamber.

33. The sensor of claim 23, wherein the enzyme comprises an activity of no more than 1 unit/cm³.

34. The sensor of claim 23, wherein the sensor is configured and arranged so that the diffusible redox mediator precipitates when reacted at the counter electrode.

35. The sensor of claim 23, wherein the sensor is configured and arranged so that a mathematical product of the effective diffusion coefficient of the redox mediator and the concentration of the redox mediator is no more than 1×

10<sup>−

12</sup> moles cm<sup>−

1</sup> sec<sup>−

1</sup> when sample fluid fills the sample chamber.

36. The sensor of claim 23, wherein the sensor is configured and arranged so that the redox mediator oxidizes the glucose and the half-wave potential of the redox mediator, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, is no more than about +100 millivolts relative to the potential of the counter/reference electrode.

37. The sensor of claim 36, wherein the half-wave potential of the redox mediator, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, is about the same as the potential of the counter/reference electrode.

38. The sensor of claim 36, wherein the half-wave potential of the redox mediator, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, is no more than about −

150 millivolts relative to the potential of the counter/reference electrode.

39. The sensor of claim 23, wherein the sensor is configured and arranged so that the redox mediator reduces the glucose and the half-wave potential of the redox mediator, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, is no less than about −

100 millivolts relative to the potential of the reference electrode.

40. The sensor of claim 39, wherein the half-wave potential of the redox mediator, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, is about the same as the potential of the counter/reference electrode.

41. The sensor of claim 39, wherein the half-wave potential of the redox mediator, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, is no less than about +200 millivolts relative to the potential of the counter/reference electrode.

42. The sensor of claim 23, wherein a background signal generated by the diffusible redox mediator at no more than 25% of the signal generated by oxidation or reduction of the expected amount of glucose.

43. The sensor of claim 23, wherein the measurement zone is sized to contain a volume of no more than about 0.5 μ

L of sample.

44. The sensor of claim 23, wherein the working electrode and the counter/reference electrode are separated by an effective distance in a range of 50 to 500 μ

m.

45. The sensor of claim 23, wherein the first substrate comprises a cutout portion at the distal end to expose a portion of the counter/reference electrode disposed on the second substrate.

46. The sensor of claim 23, wherein the second substrate comprises a cutout portion at the distal end to expose a portion of the working electrode disposed on the first substrate.