Biosensor

• US 20080166747A1
• Filed: 04/18/2007
• Published: 07/10/2008
• Est. Priority Date: 10/16/2002
• Status: Abandoned Application

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Hellinga, Homme W., Looger, Loren L.
• Application Number:
  US11/785,591
• Publication Number:
  US 20080166747A1
• Time in Patent Office: Days
• US Class Current: 435/14
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: G01N 2333/195 from bacteria
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

Biosensors are made by attaching covalently or non-covalently at least one reporter group to one or more specific positions of a bacterial periplasmic binding protein (bPBP). Upon binding of ligand to the biosensor, there is a change in the signal transduced by the reporter group.

Claims

1. A biosensor for ligand, which comprises a glucose binding protein (GBP) and at least one reporter group attached at position 183 of said GBP, wherein binding of glucose in a glucose-binding pocket of said biosensor causes a change in signaling by said reporter group.

2. The biosensor according to claim 1, wherein said GBP is a W183C mutant.

7. The biosensor according to claim 1, wherein said reporter group is covalently attached.

8. The biosensor according to claim 1, wherein said reporter group is noncovalently attached.

9. The biosensor according to claim 1, wherein said reporter group is a redox cofactor.

10. The biosensor according to claim 1, wherein said reporter group is a fluorophore.

11. The biosensor according to claim 1, wherein said biosensor'"'"'s standard intensity change (Δ

I_std) upon binding of ligand is greater than 0.25.

12. The biosensor according to claim 11, wherein said Δ

I_std is greater than 0.9.

13. The biosensor according to claim 1, wherein said biosensor'"'"'s maximum value of standard ratiometric change (Δ

R_max) upon binding of ligand is greater than 1.25.

14. The biosensor according to claim 13, wherein said Δ

R_max is greater than 2.5.

16. A method of detecting presence or absence of glucose in a sample, which comprises:

contacting a biosensor according to claim 1 with said sample under conditions such that said biosensor is able to bind to glucose present in said sample;

comparing the signal transduced by said reporter group when said biosensor is contacted with said sample with the signal(s) transduced by said reporter group when said biosensor is contacted with at least one control sample containing a known quantity of glucose; and

determining the presence or absence of glucose in said sample from said comparison.

17. A method of quantitating amount or concentration of glucose in a sample, which comprises:

contacting a biosensor according to claim 1 with said sample under conditions such that said biosensor is able to bind to glucose present in said sample;

comparing the signal transduced by said reporter group when said biosensor is contacted with said sample against signals transduced by a series of control samples containing known quantities of glucose; and

calculating the quantity of glucose in said sample from said comparison.

18. A method of assaying for glucose in a sample, which comprises:

(a) contacting a biosensor according to claim 1;

(b) measuring a ratiometric change (Δ

R) for the signal transduced by said reporter group; and

(c) at least detecting or quantitating ligand present in said sample.

19. The method of claim 18, wherein said sample is comprised of a physiological fluid.

20. The method of claim 19, wherein said physiological fluid is selected from the group consisting of blood, interstitial fluid, lavage, perspiration, plasma, saliva, serum, and urine.

23. The method of claim 16, wherein said sample is comprised of a physiological fluid.

24. The method of claim 23, wherein said physiological fluid is selected from the group consisting of blood, interstitial fluid, lavage, perspiration, plasma, saliva, serum, and urine.

25. The method of claim 17, wherein said sample is comprised of a physiological fluid.

26. The method of claim 25, wherein said physiological fluid is selected from the group consisting of blood, interstitial fluid, lavage, perspiration, plasma, saliva, serum, and urine.

3-6. -6. (canceled)

15. (canceled)

21-22. -22. (canceled)