Engineered proteins for analyte sensing

• US 6,197,534 B1
• Filed: 07/15/1999
• Issued: 03/06/2001
• Est. Priority Date: 07/17/1998
• Status: Expired due to Term

• Current Assignee: University of Maryland Baltimore County (University of Maryland)
• Original Assignee: University of Maryland Baltimore County (University of Maryland)
• Inventors: Rao, Govind, Tolosa, Leah, Eichhorn, Lisa, Lakowicz, Joseph R.
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/354,106
• Time in Patent Office: 600 Days
• Field of Search: 435/14, 435/23, 435/25, 435/968, 536/1.11, 536/123.13, 536/123.1
• US Class Current: 435/14
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

Disclosed are methods and sensors for detecting the presence or concentration of an analyte in a sample, preferably a sugar such as glucose, which preferably utilizes a labeled engineered protein, or fragment thereof, that is capable of binding the analyte to be detected.

Claims

1. A method for determining the presence or concentration of an analyte in a sample, comprising the steps of:

a) providing a protein sensing molecule that is capable of binding said analyte in said sample, said molecule having a detectable quality that changes in a concentration-dependent manner when said molecule is bound to said analyte;

b) exposing said sensing molecule to said sample; and

c) measuring any change in said detectable quality to thereby determine the presence or concentration of said analyte in said sample.

2. The method of claim 1, wherein the analyte is a sugar.

3. The method of claim 2, wherein the sugar is selected from the group consisting of glucose, lactose, galactose, sucrose and maltose.

4. The method of claim 3, wherein the sugar is glucose.

5. The method of claim 1, wherein the sensing molecule is selected from the group consisting of glucose/galactose binding protein, hexokinase and glucokinase.

6. The method of claim 4, wherein the sensing molecule is a glucose/galactose binding protein.

7. The method of claim 1, wherein the sensing molecule is a modified protein selected from the group consisting of modified glucose/galactose binding protein, modified hexokinase and modified glucokinase.

8. The method of claim 7, wherein the protein is modified by substituting at least one cysteine residue therein.

9. The method of claim 8, wherein the protein is modified by substituting two cysteine residues therein.

10. The method of claim 8, wherein the protein is a modified glucose/galactose binding protein that contains a cysteine residue at one or both of positions 26 and 182.

11. The method of claim 1, wherein the sensing molecule is a fusion protein which contains at least one detectable segment.

12. The method of claim 11, wherein the sensing molecule is a fusion protein which contains two detectable segments.

13. The method of claim 11, wherein the detectable segment comprises green fluorescent protein or fluorescent variants thereof.

14. The method of claim 12, wherein one detectable segment comprises green fluorescent protein or fluorescent variants thereof, and the other detectable segment comprises blue fluorescent protein or fluorescent variants thereof.

15. The method of claim 1, wherein the detectable quality results from at least one detectable label associated with the sensing molecule.

16. The method of claim 15, wherein the label is a fluorescent label.

17. The method of claim 15, wherein the label is a non-fluorescent energy transfer acceptor.

18. The method of claim 1, wherein the sensing molecule comprises an energy donor moiety and an energy acceptor moiety, each bound to the sensing molecule and spaced such that there is a detectable signal change when the sensing molecule is bound to the analyte.

19. The method of claim 13, wherein the sensing molecule further comprises an energy donor moiety or an energy acceptor moiety.

20. The method of claim 1, wherein the detectable quality is a detectable spectral change.

21. The method of claim 20, wherein the detectable spectral change is a change in fluorescent decay time.

22. The method of claim 20, wherein the detectable spectral change is a change in fluorescent intensity.

23. The method of claim 20, wherein the detectable spectral change is a change in fluorescent anisotropy or polarization.

24. The method of claim 20, wherein the detectable spectral change is spectral shift of the emission spectrum.

25. The method of claim 21, wherein the change in fluorescent decay time is determined by time domain measurement.

26. The method of claim 21, wherein the change in fluorescent decay time is determined by frequency domain measurement.

27. The method of claim 20, wherein the detectable spectral change is a change in time-resolved anisotropy decay.

28. The method of claim 27, wherein the change in time-resolved anisotropy decay is determined by time domain measurement.

29. The method of claim 27, wherein the change in time-resolved anisotropy decay is determined by frequency domain measurement.

30. A sensor for determining the presence or concentration of an analyte in a sample, which comprises:

a) a protein sensing molecule that is capable of binding to the analyte in said sample, said molecule having a detectable quality that changes in a concentration-dependent manner when said molecule is bound to the analyte;

b) a radiation source which is capable of causing said sensing molecule to emit said detectable quality; and

c) means for detecting changes in said detectable quality in response to said analyte binding.

31. The sensor of claim 30, wherein the analyte is a sugar.

32. The sensor of claim 31, wherein the sugar is selected from the group consisting of glucose, lactose, galactose, sucrose and maltose.

33. The sensor of claim 32, wherein the sugar is glucose.

34. The sensor of claim 30, wherein the sensing molecule is selected from the group consisting of glucose/galactose binding protein, hexokinase and glucokinase.

35. The sensor of claim 33, wherein the sensing molecule is a glucose/galactose binding protein.

36. The sensor of claim 30, wherein the sensing molecule is a modified protein selected from the group consisting of modified glucose/galactose binding protein, modified hexokinase and modified glucokinase.

37. The sensor of claim 36, wherein the protein is modified by substituting at least one cysteine residue therein.

38. The sensor of claim 37, wherein the protein is modified by substituting two cysteine residues therein.

39. The sensor of claim 37, wherein the protein is a modified glucose/galactose binding protein that contains a cysteine residue at one or both of positions 26 and 182.

40. The sensor of claim 30, wherein the sensing molecule is a fusion protein which contains at least one detectable segment.

41. The sensor of claim 40, wherein the sensing molecule is a fusion protein which contains two detectable segments.

42. The sensor of claim 40, wherein the detectable segment comprises green fluorescent protein or fluorescent variants thereof.

43. The sensor of claim 41, wherein one detectable segment comprises green fluorescent protein or fluorescent variants thereof, and the other detectable segment comprises blue fluorescent protein or fluorescent variants thereof.

44. The sensor of claim 30, wherein the detectable quality results from at least one detectable label associated with the sensing molecule.

45. The sensor of claim 44, wherein the label is a fluorescent label.

46. The sensor of claim 44, wherein the label is a non-fluorescent energy transfer acceptor.

47. The sensor of claim 30, wherein the sensing molecule comprises an energy donor moiety and an energy acceptor moiety, each bound to the sensing molecule and spaced such that there is a detectable signal change when the sensing molecule is bound to the analyte.

48. The sensor of claim 42, wherein the sensing molecule further comprises an energy donor moiety or an energy acceptor moiety.

49. The sensor of claim 30, wherein the detectable quality is a detectable spectral change.

50. The sensor of claim 49, wherein the detectable spectral change is a change in fluorescent decay time.

51. The sensor of claim 49, wherein the detectable spectral change is a change in fluorescent intensity.

52. The sensor of claim 49, wherein the detectable spectral change is a change in fluorescent anisotropy or polarization.

53. The sensor of claim 49, wherein the detectable spectral change is spectral shift of the emission spectrum.

54. The sensor of claim 50, wherein the change in fluorescent decay time is determined by time domain measurement.

55. The sensor of claim 50, wherein the change in fluorescent decay time is determined by frequency domain measurement.

56. The sensor of claim 49, wherein the detectable spectral change is a change in time-resolved anisotropy decay.

57. The sensor of claim 56, wherein the change in time-resolved anisotropy decay is determined by time domain measurement.

58. The sensor of claim 56, wherein the change in time-resolved anisotropy decay is determined by frequency domain measurement.