Oxygen sensitive probe

1. An oxygen sensitive probe comprising a monofunctional derivative of an oxygen sensitive photoluminescent dye covalently attached to a water soluble hydrophilic macromolecular carrier selected from the group comprising a polypeptide, a polysaccharide, and a poly(ethyleneglycol), the oxygen-sensitive photoluminescent dye including a single reactive chemical group for chemical conjugation to the macromolecular carrier and the oxygen sensitive photoluminescent dye being a monofunctional p-isothiocyantophenyl derivative of Pt-coproporphyrin-I.

2. The probe as claimed in claim 1 , wherein the dye has an emission lifetime in the order of 10 to 100 microseconds in deoxygenated aqueous solution.

3. The probe as claimed in claim 1 , wherein the macromolecular carrier and oxygen sensitive dye are present in a ratio of close to 1:1 to 1:2.

4. The oxygen sensitive probe as claimed in claim 1 , in combination with another probe.

5. A method for monitoring oxygen uptake by chemical or biological samples comprising the step of adding an oxygen sensitive probe to a test sample and measuring changes in the probe emission which reflect changes in the dissolved oxygen concentration, wherein the probe comprises a monofunctional derivative of an oxygen-sensitive photoluminescent dye covalently attached to a water-soluble hydrophilic macromolecular carrier wherein the probe is a probe as claimed in claim 2 .

6. The probe as claimed in claim 1 , wherein the macromolecular carrier comprises a reactive chemical group for chemical conjugation to the monofunctional derivative of an oxygen sensitive photoluminescent dye.

7. The probe as claimed in claim 6 , wherein the reactive chemical group is selected from: an amino group, a thiol group, and an N-succinimide group.

8. The probe as claimed in claim 1 , wherein the macromolecular carrier is a monofunctional macromolecular carrier.

9. The probe as claimed in claim 1 , wherein the macromolecular carrier comprises poly(ethyleneglycol) (PEG) having a molecular mass exceeding 2,000 Da.

10. The probe as claimed in claim 9 , wherein the macromolecular carrier comprises any one or more of PEG-5,000 or PEG-20,000.

11. The probe as claimed in claim 9 , wherein the macromolecular carrier comprises PEG with one terminal amino group.

12. The probe as claimed in claim 9 , wherein the macromolecular carrier comprises PEG with one terminal thiol group.

13. The probe as claimed in claim 9 , wherein the macromolecular carrier comprises activated PEG containing one or two terminal N-succinimide group.

14. The probe as claimed in claim 1 , wherein the macromolecular carrier comprises a polypeptide having molecular weight above 5,000 dalton.

15. The probe as claimed in claim 14 , wherein the macromolecular carrier comprises albumin or immunoglobulin.

16. The probe as claimed in claim 1 , wherein the macromolecular carrier comprises dextran or amino-dextran having molecular mass exceeding 5,000 Da.

17. The probe as claimed in claim 1 , wherein the macromolecular carrier is capable of recognising and specifically binding to the surface of a particular cell or group of cells in a sample.

18. An oxygen sensitive probe comprising a monofunctional derivative of an oxygen sensitive photoluminescent dye covalently attached to a water soluble hydrophilic macromolecular carrier selected from the group comprising a polypeptide, a polysaccharide, and a poly(ethyleneglycol), the oxygen sensitive photoluminescent dye including a single reactive chemical group for chemical conjugation to the macromolecular carrier and the oxygen sensitive photoluminescent dye being a monofunctional amino, maleimido-, or N-succinimido-derivative of Pt-coproporphyrin-I.

19. The probe as claimed in claim 18 , wherein the dye has an emission lifetime in the order of 10 to 100 microseconds in deoxygenated aqueous solution.

20. The probe as claimed in claim 18 , wherein the macromolecular carrier and oxygen sensitive dye are present in a ratio of close to 1:1 to 1:2.

21. The oxygen sensitive probe as claimed in claim 18 , in combination with another probe.

22. The probe as claimed in claim 18 , wherein the macromolecular carrier comprises a reactive chemical group for chemical conjugation to the monofunctional derivative of an oxygen sensitive photoluminescent dye.

23. The probe as claimed in claim 22 , wherein the reactive chemical group is selected from: an amino group, a thiol group, and an N-succinimide group.

24. The probe as claimed in claim 18 , wherein the macromolecular carrier is a monofunctional macromolecular carrier.

25. The probe as claimed in claim 18 , wherein the macromolecular carrier comprises a polypeptide having molecular weight above 5,000 dalton.

26. The probe as claimed in claim 25 , wherein the macromolecular carrier comprises albumin or immunoglobulin.

27. The probe as claimed in claim 18 , wherein the macromolecular carrier comprises dextran or amino-dextran having molecular mass exceeding 5,000 Da.

28. The probe as claimed in claim 18 , wherein the macromolecular carrier is capable of recognising and specifically binding to the surface of a particular cell or group of cells in a sample.

29. An oxygen sensitive probe comprising a monofunctional derivative of an oxygen sensitive photoluminescent dye covalently attached to a water soluble hydrophilic macromolecular carrier, the macromolecular carrier including poly (ethyleneglycol) (PEG) having a molecular mass exceeding 2,000 Da.

30. The probe as claimed in claim 29 , wherein the macromolecular carrier comprises any one or more of PEG-5,000 or PEG-20,000.

31. The probe as claimed in claim 29 , wherein the macromolecular carrier comprises PEG-20,000 with one terminal amino group.

32. The probe as claimed in claim 29 , wherein the macromolecular carrier comprises activated PEG-20,000 containing one or two terminal N-succinimide group.

33. The probe as claimed in claim 29 , wherein the oxygen sensitive probe is conjugated to the terminal group(s) of a high molecular weight PEG to give predominantly a 1:1 or 2:1 conjugate.

34. The probe as claimed in claim 29 , wherein the dye has an emission lifetime in the order of 10 to 100 microseconds in deoxygenated aqueous solution.

35. The probe as claimed in claim 29 , wherein the macromolecular carrier and oxygen sensitive dye are present in a ratio of close to 1:1 to 1:2.

36. The oxygen sensitive probe as claimed in claim 29 , in combination with another probe.

37. The probe as claimed in claim 29 , wherein the macromolecular carrier comprises poly(ethyleneglycol) (PEG) having a molecular mass exceeding 2,000 Da.

38. The probe as claimed in claim 37 , wherein the macromolecular carrier comprises any one or more of PEG-5,000 or PEG-20,000.

39. The probe as claimed in claim 37 , wherein the macromolecular carrier comprises PEG with one terminal amino group.

40. The probe as claimed in claim 37 , wherein the macromolecular carrier comprises PEG with one terminal thiol group.

41. The probe as claimed in claim 37 , wherein the macromolecular carrier comprises activated PEG containing one or two terminal N-succinimide group.

42. The probe as claimed in claim 29 , wherein the oxygen sensitive photoluminescent dye is a monofunctional derivative of Pt-coproporphyrin-I.

43. The probe as claimed in claim 29 , wherein the oxygen sensitive photoluminescent dye comprises a single reactive chemical group for chemical conjugation to the macromolecular carrier.

44. The probe as claimed in claim 29 , wherein the oxygen sensitive photoluminescent dye is selected from: a monofunctional p-isothiocyanatophenyl derivative of Pt-coproporphyrin-I, a monofunctional amino derivative of Pt-coproporphyrin-I, a monofunctional maleimido derivative of Pt-coproporphyrin-I, a monofunctional N-succinimido derivative of Pt-coproporphyrin-I, and a monofunctional carboxylic derivative of Pt-coproporphyrin-I.

45. The probe as claimed in claim 29 , wherein the macromolecular carrier comprises a reactive chemical group for chemical conjugation to the monofunctional derivative of an oxygen sensitive photoluminescent dye.

46. The probe as claimed in claim 45 , wherein the reactive chemical group is selected from: an amino group, a thiol group, and an N-succinimide group.

47. The probe as claimed in claim 29 , wherein the macromolecular carrier is a monofunctional macromolecular carrier.

48. The probe as claimed in claim 29 , wherein the macromolecular carrier comprises PEG with one terminal thiol group.

49. The probe as claimed in claim 29 , wherein the macromolecular carrier comprises PEG with one terminal amino group.

50. The probe as claimed in claim 29 , wherein the macromolecular carrier comprises activated PEG containing one or two terminal N-succinimide group.

51. The method as claimed in claim 5 , wherein the biological sample contains cells, microorganisms, subcellular organells, animal tissue or aquatic animals.

52. The method as claimed in claim 5 , wherein prior to or after addition of the oxygen probe the sample is treated with a drug, hormone, or other effector(s).

53. The method as claimed in claim 5 , for determining the number of cells, cell viability, drug or effector action on cells in a sample.

54. The method as claimed in claim 5 , wherein the biological sample contains an oxygen dependent enzyme or enzymatic system and corresponding substrate(s).

55. The method as claimed in claim 54 , for determining the concentration of enzyme substrate, or the rate of metabolism of this substrate.

56. The method as claimed in claim 54 , for determining enzyme activity or inhibition.

57. The method as claimed in claim 5 , wherein the luminescent intensity of the oxygen probe is monitored.

58. The method as claimed in claim 5 , wherein the luminescent signal of the oxygen probe is measured by time-resolved fluorescence with microsecond time resolution.

59. The method as claimed in claim 58 , wherein the delay and gate times are comparable with the lifetime of the oxygen probe in an air-saturated sample.

60. The method as claimed in claim 58 , wherein time-resolved measurements of the oxygen probe are carried out at least two different gate times after excitation.

61. The method as claimed in claim 58 , wherein the probe response to oxygen is optimised by optimising the parameters of time-resolved fluorescence measurements, namely the delay and/or gate time.

62. The method as claimed in claim 57 , wherein the luminescent signal of the oxygen probe is measured using phase modulation technique.

63. The method as claimed in claim 57 , wherein excitation of the oxygen probe is performed by a laser compatible with the probe.