ELECTROCHEMICAL BIOSENSOR

• US 20100285514A1
• Filed: 01/27/2010
• Published: 11/11/2010
• Est. Priority Date: 01/27/2009
• Status: Active Grant

• Current Assignee: Purdue Research Foundation (Purdue University)
• Original Assignee: Purdue Research Foundation (Purdue University)
• Inventors: Franklin, Aaron D., Claussen, Jonathan Clay, Porterfield, D. Marshall, Fisher, Timothy S.
• Granted Patent: US 8,715,981 B2
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C23C 28/322 only coatings of metal elements only
• CPC Class Codes: C23C 28/345 with at least one oxide layer
• 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

Abstract

Networks of single-walled carbon nanotubes (SWCNTs) decorated with Au-coated Pd (Au/Pd) nanocubes are employed as electrochemical biosensors that exhibit excellent sensitivity (2.6 mA mM⁻¹ cm⁻²) and a low estimated detection limit (2.3 nM) at a signal-to-noise ratio of 3 (S/N=3) in the amperometric sensing of hydrogen peroxide. Biofunctionalization of the Au/Pd nanocube-SWCNT biosensor is demonstrated with the selective immobilization of fluorescently labeled streptavidin on the nanocube surfaces via thiol linking. Similarly, glucose oxidase (GOx) is linked to the surface of the nanocubes for amperometric glucose sensing. The exhibited glucose detection limit of 1.3_M (S/N=3) and linear range spanning from 10 μM to 50 mM substantially surpass other CNT-based biosensors. These results, combined with the structure'"'"'s compatibility with a wide range of biofunctionalization procedures, would make the nanocube-SWCNT biosensor exceptionally useful for glucose detection in diabetic patients and well suited for a wide range of amperometric detection schemes for biomarkers.

Claims

1. An apparatus for sensing a substance, comprising:

a substrate including an electrically conductive layer and an electrically insulating layer, the insulating layer including a plurality of pores each having a first end and second open end and a passage therebetween, the first end of each pore being open to the conductive layer;

a plurality of carbon nanotubes each having first and second ends, each said nanotube being grown within a different one of said pores, the first end of each said nanotube being in electrical contact with said conductive layer, the second end of each said nanotube extending out of the second open end of the respective pore; and

a plurality of nanoparticles, each said nanoparticle being electrodeposited to the extension of a different one of said nanotubes, each said nanoparticle having bonded to it an enzyme for converting the substance into products.

2. The apparatus of claim 1 wherein said nanoparticles are electrodeposited on the respective nanotube.

3. The apparatus of claim 1 wherein said nanoparticle is one of a nanosphere or nanocube.

4. The apparatus of claim 1 wherein said nanoparticle comprises platinum or palladium.

5. The apparatus of claim 1 which further comprises a layer of gold electrodeposited on each said nanoparticle.

6. The apparatus of claim 1 which further comprises a plurality of electrically conductive nanowires, each said nanowire being located within a different one of said pores and providing electrical continuity from the respective nanotube of the pore to said conductive layer.

7. The apparatus of claim 1 wherein said enzyme is covalently bonded to said nanoparticle.

8. The apparatus of claim 1 wherein said enzyme is bonded to said nanoparticle by a thiol linker.

9. The apparatus of claim 1 wherein the substance is glucose and the enzyme is glucose oxidase.

10. The apparatus of claim 1 wherein the substance is glutamate and the enzyme is glutamate oxidase.

11. The apparatus of claim 1 wherein the substance is glutamate and the enzyme is glutamate dehydrogenase.

12. The apparatus of claim 1 wherein at least one of the products is hydrogen peroxide.

13. The apparatus of claim 1 wherein said insulating layer comprises porous anodic alumina.

14. The apparatus of claim 1 wherein a characteristic overall average length of said nanoparticles is more than about 100 nanometers and less than about 300 nanometers.

15. The apparatus of claim 1 wherein said conductive layer comprises titanium.

16. The apparatus of claim 1 wherein said substrate includes a catalytic layer placed in between said conductive layer and said insulating layer, and said nanotubes are grown from said catalytic layer.

17. The apparatus of claim 1 wherein the carbon nanotube is a single walled carbon nanotube.

18. The apparatus of claim 1 wherein the carbon nanotube is a multiwalled walled carbon nanotube.

19. A method of making a sensor for detecting a substance, comprising:

providing a substrate having a plurality of pores aligned parallel to one another;

growing a carbon nanotube within each of the pores that extends out of a surface of the substrate;

establishing parallel electrical communication among the carbon nanotubes;

electrodepositing a metal nanoparticle on the portion of the nanotubes extending out of the surface; and

immobilizing an enzyme on the nanoparticles that converts the substance to products.

20. The method of claim 19 which further comprises coating the nanoparticle with a metal selected from the group including gold, platinum, palladium, copper, silver and nickel prior to said immobilizing.

21. The method of claim 19 wherein said electrodepositing includes limiting the diameter of the nanoparticles to an average of less than about 200 nanometers.

22. The method of claim 19 wherein said establishing electrical communication includes electrodepositing a metal in the respective pore.

23. The method of claim 22 wherein the metal is chosen from the group including platinum and palladium

24. The method of claim 19 which further comprises adding defect sites to the nanoparticles before said immobilizing an enzyme.

25. The method of claim 24 wherein said adding defects is by electrochemically treating the deposited nanoparticles in an acid bath with a time-varying voltage.

26. The method of claim 24 wherein said adding defects is by electrochemically treating the deposited nanoparticles in an alkali bath with a time-varying voltage.

27. The method of claim 19 which further comprise attaching linker molecules to the nanoparticles before said immobilizing an enzyme.

28. The method of claim 27 wherein said immobilizing an enzyme is with a thiol linker.

29. The method of claim 19 wherein said immobilizing an enzyme is by covalently bonding the enzyme proximate to a defect site on the nanoparticle.

30. The method of claim 19 wherein said immobilizing an enzyme includes cross-linking the enzyme with a linker and adhering the linker to the nanoparticle.

31. The method of claim 30 wherein the linker includes gluteraldehyde.

32. The method of claim 19 wherein said growing includes separating adjacent nanotubes by an average of more than about 100 nanometers.

33. A method for detecting a substance, comprising:

providing a substrate comprising a conductive layer having two surfaces and a layer of porous anodic alumina on one surface, a plurality of the pores each including a different carbon nanotube therein;

a plurality of the nanotubes each having a portion extending out of their respective pores;

attaching a different metal nanoparticle on each of the extending portions of the nanotubes;

providing common electrical communication by the conductive layer with each of the other ends of the nanotubes;

bonding an enzyme that assists in the reduction-oxidation reaction of the substance to each of the nanoparticles;

placing the substrate in a solution with the substance; and

amperometrically sensing current released from the reaction with the plurality of nanotubes.

34. The method of claim 33 wherein said bonding is by covalently bonding the enzyme to the nanoparticle.

35. The method of claim 33 wherein said attaching a nanoparticle is by electrodepositing the nanoparticle.

36. The method of claim 33 wherein said sensing includes applying a pulsed current in the solution.

37. The method of claim 33 which further comprises coating the nanoparticle with a metal selected from the group including gold, platinum, palladium, copper, silver and nickel prior to said immobilizing.

38. The method of claim 33 wherein said providing electrical communication includes electrodepositing a metal in the respective pore.

39. The method of claim 38 wherein the metal is chosen from the group including platinum and palladium

40. The method of claim 33 which further comprises adding defect sites to the nanoparticles before said bonding an enzyme.

41. The method of claim 33 which further comprise attaching linker molecules to the nanoparticles before said bonding an enzyme.

42. The method of claim 41 wherein said bonding an enzyme is with a thiol linker.

43. The method of claim 33 wherein said bonding an enzyme is by covalently bonding the enzyme proximate to a defect site on the nanoparticle.

44. The method of claim 33 wherein said bonding an enzyme includes cross-linking the enzyme with a linker and adhering the linker to the nanoparticle.

45. The method of claim 44 wherein the linker includes gluteraldehyde.