BIOSENSORS HAVING IMPROVED SAMPLE APPLICATION AND MEASURING PROPERTIES AND USES THEREOF

• US 20040031698A1
• Filed: 01/04/2001
• Published: 02/19/2004
• Est. Priority Date: 01/04/2001
• Status: Active Grant

• Current Assignee: Tyson Bioresearch Inc.
• Original Assignee: Tyson Bioresearch Inc.
• Inventors: Huang, Tong-Yuh, Lee, Jin Po
• Granted Patent: US 6,793,802 B2
• Time in Patent Office: Days
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/001 Enzyme electrodes

Abstract

The present invention relates to biosensors having improved sample application and measuring properties and their uses for detection, preferably, quantitative measurement, of analyte in a liquid sample. In particular, the invention provides for a biosensor having a sample application and reaction chamber facilitating the speed and uniformity of sample application, especially small volume sample application, via capillary flow. The invention also provides for a biosensor having multiple circuits that lead to improved assay consistency and accuracy. Methods for assaying analytes or enzymes using the biosensors are further provided.

Claims

1. A biosensor for electrochemical analysis of a liquid sample, which biosensor comprises:

a) an insulating base plate having a first end and a second end;

b) an electrode system on said insulating base plate, wherein said electrode system comprises a working electrode and a counter electrode, said working and counter electrodes have conductive leads for connecting said electrodes to a readout device for electrochemical measurement on said first end of said base plate, said working electrode is engulfed by said counter electrode on all sides except the side leading to said conductive leads, and there is a gap space between said working and counter electrodes;

c) a reaction area as part of said electrode system, said reaction area occupies at least a portion of said working electrode, said counter electrode and the gap space between said working electrode and said counter electrode in a direction perpendicular to said conductive leads, said reaction area is a complete cross-section of said electrode system in a direction perpendicular to said conductive leads, said reaction area is defined by covering the non-reaction-area with a layer comprising a dielectrical material, and said reaction area comprises an enzyme that catalyzes a reaction involving an analyte to be analyzed or a substrate that is involved in a reaction catalyzed by an enzyme to be analyzed; and

d) a sample application and reaction chamber, wherein the bottom of said chamber is said reaction area defined in c), the top of said chamber is a cover that covers at least said reaction area, said top has an opening above said reaction area for sample application, the two side walls of said chamber in a direction perpendicular to said conductive leads are formed by said layer comprising said dielectrical material defined in c), and the two sides of said chamber in a direction parallel to said conductive leads are left open as air vents.

2. The biosensor of claim 1, wherein the insulating base plate comprises vinyl polymer(s), polymide(s), polyester(s), nylon, nitrocellulose or a combination thereof.

3. The biosensor of claim 1, wherein the working and counter electrodes are made of substantially identical material(s) within the reaction area.

4. The biosensor of claim 1, wherein the gap space between the working electrode and the counter electrode is kept substantially constant within the reaction area.

5. The biosensor of claim 1, wherein the width of the working electrode is about twice of the width of the counter electrode within the reaction area.

6. The biosensor of claim 1, wherein the surface area of the working electrode is substantially identical to the surface area of the counter electrode within the reaction area.

7. The biosensor of claim 1, wherein the electrode system is screen-printed onto the insulating base plate.

8. The biosensor of claim 7, wherein the working and counter electrodes comprise carbon paste and the conductive leads comprise conductive silver paste.

9. The biosensor of claim 1, wherein the dielectrical material is vinylpolyester(s), polyimide(s) or a combination thereof.

10. The biosensor of claim 1, wherein the thickness of the dielectrical material proximal to the first end is substantially higher than the thickness of the dielectrical material proximal to the second end.

11. The biosensor of claim 10, wherein the thickness of the dielectrical material proximal to the first end is about three times of the thickness of the dielectrical material proximal to the second end.

12. The biosensor of claim 1, wherein the top of the sample application and reaction chamber is the corresponding part of the cover for the entire biosensor.

13. The biosensor of claim 12, wherein the cover for the entire biosensor is a lamina adhered to the non-reaction-area and the opening on the top is a punched hole formed on said lamina.

14. The biosensor of claim 13, wherein the punched hole of the lamina has an arcuate part of the sampling slot protruding into the reaction area to form a convex, said convex serves as the passage for the sample fluid to the reaction area and the arcuate part of the convex provide an auxiliary of propulsion for a quick draw of the sample fluid.

15. The biosensor of claim 1, wherein the insulating base plate is transparent, whereby the liquid sample is introduced on the opposite side of the transparent insulating base plate and the liquid sample movement can be monitored through the transparent insulating base plate.

16. The biosensor of claim 1, further comprising an electron transfer mediator in the reaction area.

17. The biosensor of claim 16, wherein the electron transfer mediator is selected from the group consisting of ferrocene and its derivatives, osmium complex, tetrathiofulvalene, phenazine ethosulfate, benzoquinone and hexacyanoferrate.

18. The biosensor of claim 1, wherein the non-reaction area proximal to the second end is divided into two corner portions and one middle portion, and the middle portion is made part of the reaction area by either not covering the middle portion with a layer comprising a dielectrical material or by making the thickness of the dielectrical material of the two corner portions substantially higher than the thickness of the dielectrical material of the middle portion, whereby the reaction area occupies a T-shaped area comprising the complete cross-section in a direction perpendicular to said conductive leads and a strip area in a direction parallel to said conductive leads from the cross-section to the edge of the second end.

19. The biosensor of claim 18, wherein the thickness of the dielectrical material proximal to the first end and the thickness of the two corner portions proximal to the second end is about three times of a unit thickness and the thickness of the dielectrical material of the middle portion proximal to the second end is about one time of the unit thickness.

20. A method for assaying an analyte or an enzyme in a liquid sample, which method comprises:

a) contacting a liquid sample containing or suspected of containing an analyte or an enzyme with the biosensor of claim 1 in the presence of a suitable electron transfer mediator under suitable conditions whereby the analyte in the sample liquid, if there is any, is involved in a reaction catalyzed by the enzyme comprised in the reaction area of the biosensor, or the enzyme in the sample liquid, if there is any, catalyzes a reaction involving the substrate comprised in the reaction area of the biosensor, said reaction involving the analyte or substrate, in conjunction with the electron transfer mediator, leads to the generation of a current that is capable of being detected by the biosensor; and

b) detecting the current generated in step a), whereby the presence or amount of the analyte or enzyme in the sample liquid is assessed.

21. The method of claim 20, wherein the volume of the liquid sample to be contacted with the biosensor is from about 1.5 microliters to about 3.0 microliters.

22. The method of claim 20, wherein the analyte to be detected is glucose.

23. The method of claim 22, wherein the enzyme comprised in the reaction area of the biosensor is glucose oxidase and the electron transfer mediator used in the assay is potassium ferricyanide.

24. A method for assaying an analyte or an enzyme in a liquid sample, which method comprises:

a) contacting a liquid sample containing or suspected of containing an analyte or an enzyme with the biosensor of claim 16 under suitable conditions whereby the analyte in the sample liquid, if there is any, is involved in a reaction catalyzed by the enzyme comprised in the reaction area of the biosensor, or the enzyme in the sample liquid, if there is any, catalyzes a reaction involving the substrate comprised in the reaction area of the biosensor, said reaction involving the analyte or substrate, in conjunction with the electron transfer mediator comprised in the reaction area of the biosensor, leads to the generation of a current that is capable of being detected by the biosensor; and

b) detecting the current generated in step a), whereby the presence or amount of the analyte or enzyme in the sample liquid is assessed.

25. A method for assaying an analyte or an enzyme in a liquid sample, which method comprises:

a) contacting a liquid sample containing or suspected of containing an analyte or an enzyme with the biosensor of claim 18 in the presence of a suitable electron transfer mediator under suitable conditions whereby the analyte in the sample liquid, if there is any, is involved in a reaction catalyzed by the enzyme comprised in the reaction area of the biosensor, or the enzyme in the sample liquid, if there is any, catalyzes a reaction involving the substrate comprised in the reaction area of the biosensor, said reaction involving the analyte or substrate, in conjunction with the electron transfer mediator, leads to the generation of a current that is capable of being detected by the biosensor; and

b) detecting the current generated in step a), whereby the presence or amount of the analyte or enzyme in the sample liquid is assessed.

26. A biosensor for electrochemical analysis of a liquid sample, which biosensor comprises:

a) an insulating base plate having a first end and a second end; and

b) an electrode system on said insulating base plate, wherein said electrode system comprises a working electrode, a counter electrode, and two reference electrodes, said working, counter and reference electrodes have conductive leads for connecting said electrodes to a readout device for electrochemical measurement on said first end of said base plate, each of said reference electrode is diagonally positioned from said working or counter electrode and there is a gap space between said working/counter, working/reference and reference/reference electrodes, said working electrode and a first reference electrode diagonally positioned from said working electrode forms a first closed circuit and said counter electrode and a second reference electrode diagonally positioned from said counter electrode forms a second closed circuit, said first and second closed circuits are connected to form a third circuit, whereby said third circuit is closed only when both said first and second circuits are closed at the same time.

27. The biosensor of claim 26, wherein the insulating base plate comprises vinyl polymer(s) or polymide(s), polyester(s), nylon, nitrocellulose or a combination thereof.

28. The biosensor of claim 26, wherein the working, counter and reference electrodes are made of substantially identical material(s).

29. The biosensor of claim 26, wherein the gap space between the reference electrodes and the working or counter electrode is kept substantially constant.

30. The biosensor of claim 26, wherein the surface area of the working electrode is substantially identical to the surface area of the counter electrode.

31. The biosensor of claim 26, wherein the electrode system is screen-printed onto the insulating base plate.

32. The biosensor of claim 31, wherein the working, counter and reference electrodes comprise carbon paste and the conductive leads of the electrodes comprise conductive silver paste.

33. The biosensor of claim 26, wherein the reference electrodes are engulfed by the working and counter electrodes on all sides except the side leading to said conductive leads.

34. The biosensor of claim 26, wherein the reference electrodes are separated to the farthest distance without contacting either working or counter electrode.

35. The biosensor of claim 26, wherein at least a portion of the working, counter and reference electrodes and the gap space among the electrodes form a reaction area, said reaction area comprises an enzyme that catalyzes a reaction involving an analyte to be analyzed or a substrate that is involved in a reaction catalyzed by an enzyme to be analyzed.

36. The biosensor of claim 35, further comprising an electron transfer mediator in the reaction area.

37. The biosensor of claim 35, wherein the reaction area is a complete cross-section of the electrode system in a direction perpendicular to the conductive leads, the reaction area is defined by covering the non-reaction-area with a layer comprising a dielectrical material, and the biosensor further comprises a sample application and reaction chamber, wherein the bottom of said chamber is the reaction area, the top of said chamber is a cover that covers at least the reaction area, said top has an opening above the reaction area for sample application, the two side walls of said chamber in a direction perpendicular to said conductive leads are formed by the layer comprising the dielectrical material, and the two sides of said chamber in a direction parallel to said conductive leads are left open as air vents.

38. The biosensor of claim 37, wherein the dielectrical material is vinylpolyester(s), polyimide(s) or a combination thereof.

39. The biosensor of claim 37, wherein the thickness of the dielectrical material proximal to the first end is substantially higher than the thickness of the dielectrical material proximal to the second end.

40. The biosensor of claim 39, wherein the thickness of the dielectrical material proximal to the first end is about three times of the thickness of the dielectrical material proximal to the second end.

41. The biosensor of claim 37, wherein the top of the sample application and reaction chamber is the corresponding part of the cover for the entire biosensor.

42. The biosensor of claim 41, wherein the cover for the entire biosensor is a lamina adhered to the non-reaction-area and the opening on the top is a punched hole formed on said lamina.

43. The biosensor of claim 42, wherein the punched hole of the lamina has an arcuate part of the sampling slot protruding into the reaction area to form a convex, said convex serves as the passage for the sample fluid to the reaction area and the arcuate part of the convex provide an auxiliary of propulsion for a quick draw of the sample fluid.

44. The biosensor of claim 37, wherein the insulating base plate is transparent, whereby the liquid sample is introduced on the opposite side of the transparent insulating base plate and the liquid sample movement can be monitored through the transparent insulating base plate.

45. The biosensor of claim 37, further comprising an electron transfer mediator in the reaction area.

46. The biosensor of claim 45, wherein the electron transfer mediator is selected from the group consisting of ferrocene and its derivatives, osmium complex, tetrathiofulvalene, phenazine ethosulfate, benzoquinone and hexacyanoferrate.

47. The biosensor of claim 37, wherein the non-reaction area proximal to the second end is divided into two corner portions and one middle portion, and the middle portion is made part of the reaction area by either not covering the middle portion with a layer comprising a dielectrical material or by making the thickness of the dielectrical material of the two corner portions substantially higher than the thickness of the dielectrical material of the middle portion, whereby the reaction area occupies a T-shaped area comprising the complete cross-section in a direction perpendicular to said conductive leads and a strip area in a direction parallel to said conductive leads from the cross-section to the edge of the second end.

48. The biosensor of claim 47, wherein the thickness of the dielectrical material proximal to the first end and the thickness of the two corner portions proximal to the second end is about three times of a unit thickness and the thickness of the dielectrical material of the middle portion proximal to the second end is about one time of the unit thickness.

49. A method for assaying an analyte or an enzyme in a liquid sample, which method comprises:

a) contacting a liquid sample containing or suspected of containing an analyte or an enzyme with the biosensor of claim 35 in the presence of a suitable electron transfer mediator under suitable conditions whereby the analyte in the sample liquid, if there is any, is involved in a reaction catalyzed by the enzyme comprised in the reaction area of the biosensor, or the enzyme in the sample liquid, if there is any, catalyzes a reaction involving the substrate comprised in the reaction area of the biosensor, said reaction involving the analyte or substrate, in conjunction with the electron transfer mediator, leads to the generation of a current that is capable of being detected by the biosensor; and

b) detecting the current generated in step a), whereby the presence or amount of the analyte or enzyme in the sample liquid is assessed.

50. The method of claim 49, wherein the volume of the liquid sample to be contacted with the biosensor is from about 1.5 microliters to about 3.0 microliters.

51. The method of claim 49, wherein the analyte to be detected is glucose.

52. The method of claim 51, wherein the enzyme comprised in the reaction area of the biosensor is glucose oxidase and the electron transfer mediator used in the assay is potassium ferricyanide.

53. A method for assaying an analyte or an enzyme in a liquid sample, which method comprises:

a) contacting a liquid sample containing or suspected of containing an analyte or an enzyme with the biosensor of claim 36 under suitable conditions whereby the analyte in the sample liquid, if there is any, is involved in a reaction catalyzed by the enzyme comprised in the reaction area of the biosensor, or the enzyme in the sample liquid, if there is any, catalyzes a reaction involving the substrate comprised in the reaction area of the biosensor, said reaction involving the analyte or substrate, in conjunction with the electron transfer mediator comprised in the reaction area of the biosensor, leads to the generation of a current that is capable of being detected by the biosensor; and

b) detecting the current generated in step a), whereby the presence or amount of the analyte or enzyme in the sample liquid is assessed.

54. A method for assaying an analyte or an enzyme in a liquid sample, which method comprises:

a) contacting a liquid sample containing or suspected of containing an analyte or an enzyme with the biosensor of claim 37 in the presence of a suitable electron transfer mediator under suitable conditions whereby the analyte in the sample liquid, if there is any, is involved in a reaction catalyzed by the enzyme comprised in the reaction area of the biosensor, or the enzyme in the sample liquid, if there is any, catalyzes a reaction involving the substrate comprised in the reaction area of the biosensor, said reaction involving the analyte or substrate, in conjunction with the electron transfer mediator, leads to the generation of a current that is capable of being detected by the biosensor; and

b) detecting the current generated in step a), whereby the presence or amount of the analyte or enzyme in the sample liquid is assessed.

55. A method for assaying an analyte or an enzyme in a liquid sample, which method comprises:

a) contacting a liquid sample containing or suspected of containing an analyte or an enzyme with the biosensor of claim 47 in the presence of a suitable electron transfer mediator under suitable conditions whereby the analyte in the sample liquid, if there is any, is involved in a reaction catalyzed by the enzyme comprised in the reaction area of the biosensor, or the enzyme in the sample liquid, if there is any, catalyzes a reaction involving the substrate comprised in the reaction area of the biosensor, said reaction involving the analyte or substrate, in conjunction with the electron transfer mediator, leads to the generation of a current that is capable of being detected by the biosensor; and

b) detecting the current generated in step a), whereby the presence or amount of the analyte or enzyme in the sample liquid is assessed.