Biosensor

• US 6,977,180 B2
• Filed: 08/28/2002
• Issued: 12/20/2005
• Est. Priority Date: 08/28/2001
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Benson, David E., Hellinga, Homme W., Conrad, David W.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US10/229,286
• Publication Number:
  US 20030129622A1
• Time in Patent Office: 1,210 Days
• Field of Search: 204/400, 204/403, 422/82.01, 422/82.02, 422/82.03, 435/5, 435/6, 435/7.31, 435/7.2, 435/14, 435/287.1, 435/287.2, 436/518, 436/524, 436/525, 436/149, 436/806, 436/815
• US Class Current: 436/518
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds

Abstract

The present invention relates, in general, to biosensors and, in particular, to bioelectronic sensors comprising a macromolecule immobilized on an electrode surface so that a redox cofactor that is site-specifically attached to the surface of the macromolecule is between the macromolecule and electrode surface ligand-mediated conformational changes alter the geometry of interaction between the redox cofactor and the electrode surface resulting in a change in electronic coupling between the cofactor and electrode.

Claims

1. A biosensor comprising:

(i) an electrode;

(ii) at least one molecule of a protein that undergoes a conformational change upon binding to a ligand;

(iii) a redox reporter bound to said molecule of said protein; and

(iv) a means for measuring a voltage or current,

wherein said redox reporter is bound to a surface of said molecule of said protein that faces said electrode so that said redox reporter is positioned between said molecule of said protein and said electrode, and wherein the conformational change that occurs upon binding of said ligand to said molecule of said protein results in a change in the interaction between said redox reporter and said electrode that is detectable potentiometrically or amperometrically.

2. The biosensor according to claim 1 wherein said electrode comprises gold, silver, copper, aluminum, platinum, iridium, palladium, rhodium, mercury, silicon, osmium, ruthenium, gallium arsenide, indium phosphide, mercury, cadmium telluride or carbon.

3. The biosensor according to claim 1 wherein said electrode is in the form of a foil, wire, wafer, chip or micro- or nano-particle.

4. The biosensor according to claim 1 further comprising a self-assembled monolayer (SAM) bound to said electrode.

5. The biosensor according to claim 4 wherein said SAM comprises molecules comprising a functional group that adheres to said electrode.

6. The biosensor according to claim 4 wherein said SAM comprises molecules comprising a pendant moiety that interacts with said molecule of said protein.

7. The biosensor according to claim 1 wherein said protein is a genetically engineered or chemically modified protein.

8. The biosensor according to claim 7 wherein said protein is engineered to bind to a ligand other than its wild type cognate ligand.

9. The biosensor according to claim 1 wherein said redox reporter comprises a redox-active metal or a redox-active organic molecule.

10. The biosensor according to claim 9 wherein said redox reporter comprises a transition metal.

11. The biosensor according to claim 10 wherein said transition metal is selected from the group consisting of ruthenium, osmium, iron, platinum, palladium, nickel, cobalt, copper and manganese.

12. The biosensor according to claim 9 wherein the redox reporter comprises a redox-active organic molecule.

13. The biosensor according to claim 1 wherein said redox reporter comprises a functional group suitable for covalent coupling to said molecule of said protein.

14. The biosensor according to claim 1 wherein said protein is a member of the periplasmic binding protein superfamily.

15. The biosensor according to claim 14 wherein said protein is selected from the group consisting of a glucose-binding protein, maltose binding protein, ribose binding protein, arabinose-binding protein, histidine-binding protein and glutamine-binding protein.

16. The biosensor according to claim 1 wherein said molecule of said protein is bound to said electrode, or to a SAM bound to said electrode, via a tether.

17. The biosensor according to claim 16 wherein said tether comprises an organic or organo-metallic moiety.

18. The biosensor according to claim 17 wherein said tether comprises a peptide, nucleic acid, carbohydrate or lipid moiety.

19. A method for detecting the presence of, or quantifying the amount of, a ligand in a sample or system, comprising:

contacting said biosensor according to claim 1 with said sample or system, wherein said molecule of said protein undergoes a conformational change upon binding to said ligand, and

measuring the voltage or current generated by interaction between said redox reporter and said electrode, wherein a change in said voltage or current is indicative of the presence of, or the amount of, said ligand in said sample or system.

20. The method according to claim 19 wherein said ligand is selected from the group consisting of an environmental pollutant, a therapeutic molecule, an endogenous biomolecule, a nutrient, a cell, a virus and a spore.

21. The method according to claim 19 wherein said ligand is a biological or chemical warfare agent or an explosive.

22. The method according to claim 19 wherein said ligand is glucose.

23. The method according to claim 19 wherein said sample or system is a physiological fluid of a human or non-human patient.

24. The method according to claim 23 wherein said physiological fluid is blood, urine, sweat or cerebrospinal fluid.

25. The method according to claim 19 wherein said sample or system is a water sample or system.

26. The method according to claim 25 wherein said water sample or system is drinking water, or sea, lake, or river water, sewage, ground water or surface water.

27. The method according to claim 19 wherein said system is a bioreactor or chemical reactor.