Oxygen sensitive probe

• US 20060002822A1
• Filed: 09/06/2005
• Published: 01/05/2006
• Est. Priority Date: 03/07/2003
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Luxcel Biosciences Ltd. (Ametek Incorporated)
• Inventors: Fernandes, Richard, Papkovsky, Dmitri Boris
• Granted Patent: US 7,674,626 B2
• Time in Patent Office: Days
• US Class Current: 422/82.80
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 31/225 for oxygen, e.g. including dissolved oxygen
• CPC Class Codes: Y10S 436/905 Photochemical activation of reactions
• CPC Class Codes: Y10T 436/20 Oxygen containing

Abstract

An oxygen sensitive probe comprises a monofunctional derivative of an oxygen-sensitive photoluminescent dye covalently attached to a water soluble and/or hydrophilic macromolecular carrier. The probe may be a chemical conjugate of a monofunctional phosphorescent porphyrin dye and a poly(ethyleneglycol), polypeptide or polysaccharide.

Claims

1-43. -43. (canceled)

44. An oxygen sensitive probe comprising a monofunctional derivative of an oxygen-sensitive photoluminescent dye covalently attached to a water soluble hydrophilic macromolecular carrier.

45. The probe as claimed in claim 44, wherein the dye has an emission lifetime in the order of 10 to 1000 microseconds in deoxygenated aqueous solution.

46. The probe as claimed in claim 44, wherein the oxygen-sensitive photoluminescent dye comprises a single reactive chemical group for chemical conjugation to the macromolecular carrier.

47. The probe as claimed in claim 44, wherein the oxygen-sensitive photoluminescent dye is selected from any one or more of Pt(II)-porphyrin, Pd(II)-porphyrin, or closely related chemical structures.

48. The probe as claimed in claim 46, wherein the oxygen-sensitive photoluminescent dye is selected from any one or more of Pt(II)-coproporphyrin-I;

Pt(II)-coproporphyrin-III;

Pt(II)-uroporphyrin-I;

Pt (II) -tetrakis (p-carboxyphenyl)porphine;

Pt(II)-tetrakis(p-sulfophenyl)porphine;

derivatives or analogs thereof;

49. The probe as claimed in claim 44, wherein the oxygen-sensitive photoluminescent dye is selected from any one or more of Pt-porphyrin-ketones;

Pt-benzoporphyrins;

Pt-chlorins;

Pd-porphyrin-ketons;

Pd-benzoporphyrins;

or Pd-chlorins.

50. The probe as claimed in claim 46, wherein the oxygen-sensitive photoluminescent dye is a monofunctional p-isothyocyanatophenyl derivative of Pt-coproporphyrin-I.

51. The probe as claimed in claim 46, wherein the oxygen-sensitive photoluminescent dye is a monofunctional amino-, maleimido- or N-succinimido-derivative of Pt-coproporphyrin-I.

52. The probe as claimed in claim 44, wherein the macromolecular carrier comprises a polypeptide, polysaccharide, or a poly(ethyleneglycol).

53. The probe as claimed in claim 52, comprising a monofunctional derivative of an oxygen-sensitive photoluminescent dye covalently attached to a monofunctional macromolecular carrier.

54. The probe as claimed in claim 52, wherein the macromolecular carrier comprises poly(ethyleneglycol) (PEG) having molecular mass exceeding 2,000 Da.

55. The probe as claimed in claim 54, wherein the macromolecular carrier comprises any one or more of PEG-5,000 or PEG-20,000.

56. The probe as claimed in claim 54, wherein the macromolecular carrier comprises PEG-20,000 with one terminal amino group.

57. The probe as claimed in claim 54, wherein the macromolecular carrier comprises activated PEG-20,000 containing one or two terminal N-succinimide group.

58. The probe as claimed in claim 54, 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.

59. The probe as claimed in claim 52, wherein the macromolecular carrier comprises a polypeptide having molecular weight above 5,000 dalton.

60. The probe as claimed in claim 52, wherein the macromolecular carrier comprises albumin or immunoglobulin.

61. The probe as claimed in claim 52, wherein the macromolecular carrier comprises dextran or amino-dextran having molecular mass exceeding 5,000 Da.

62. The probe as claimed in claim 44, wherein the macromolecular carrier and oxygen-sensitive dye are present in a ratio of close to 1:

1 to 1;

2.

63. The probe as claimed in claim 44, 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.

64. The oxygen sensitive probe as claimed in claim 44, in combination with another probe.

69. The 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 45.

70. The method as claimed in claim 69, wherein the biological sample contains cells, microorganisms, subcellular organells, animal tissue or aquatic animals.

71. The method as claimed in claim 69, wherein prior to or after addition of the oxygen probe the sample is treated with a drug, hormone, or other effector(s).

72. The method as claimed in claim 69, for determining the number of cells, cell viability, drug or effector action on cells in a sample.

73. The method as claimed in claim 69, wherein the biological sample contains an oxygen dependent enzyme or enzymatic system and corresponding substrate(s).

74. The method as claimed in claim 73, for determining the concentration of enzyme substrate, or the rate of metabolism of this substrate.

75. The method as claimed in claim 73, for determining enzyme activity or inhibition.

76. The method as claimed in claim 69, wherein the luminescent intensity of the oxygen probe is monitored.

77. The method as claimed in claim 69, wherein the luminescent signal of the oxygen probe is measured by time-resolved fluorescence with microsecond time resolution.

78. The method as claimed in claim 77, wherein the delay and gate times are comparable with the lifetime of the oxygen probe in an air-saturated sample.

79. The method as claimed in claim 77, wherein time-resolved measurements of the oxygen probe are carried out at least two different gate times after excitation.

80. The method as claimed in claim 77, wherein the probe response to oxygen is optimised by optimising the parameters of time-resolved fluorescence measurements, namely the delay and/or gate time.

81. The method as claimed in claim 76, wherein the luminescent signal of the oxygen probe is measured using phase modulation technique.

82. The method as claimed in claim 76, wherein excitation of the oxygen probe is performed by a laser compatible with the probe.

65. A process for preparing an oxygen probe comprising reacting functionalised derivatives of an oxygen-sensitive photoluminescent dye and/or water soluble macromolecular carrier to form a stable chemical linkage.

66. The process as claimed in claim 65, wherein the dye is conjugated to the macromolecular carrier at a specific site(s).

67. The process as claimed in claim 65, wherein the dye is reacted with the carrier in a single step.

68. 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.

83. The method as claimed in claim 68, wherein the sample and the emission of oxygen probe are analysed with a fluorescence imaging system.

84. The method as claimed in claim 68 including the step of sealing the sample.

85. The method as claimed in claim 84 wherein the sample is sealed by covering the sample with a liquid seal material.

86. The method as claimed in claim 85 wherein the liquid seal comprises mineral oil.