Long lifetime anisotropy (polarization) probes for clinical chemistry, immunoassays, affinity assays and biomedical research

• US 5,660,991 A
• Filed: 10/28/1994
• Issued: 08/26/1997
• Est. Priority Date: 10/28/1994
• Status: Expired due to Term

• Current Assignee: Joseph R. Lakowicz
• Original Assignee: Joseph R. Lakowicz
• Inventors: Terpetschnig, Ewald, Szmacinski, Henryk, Lakowicz, Joseph R.
• Primary Examiner(s): ACHUTAMURTHY, PONNATHAPURA N
• Application Number:
  US08/330,743
• Time in Patent Office: 1,033 Days
• Field of Search: 435/7.1, 435/7.93, 435/968, 435/7.5, 435/501, 435/536, 435/546, 435/164, 435/172
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

A method of conducting an immunoassay of a sample of interest is described, including the steps of (A) coupling a luminescent asymmetric metal-ligand complex to the sample of interest to form a coupled sample, (B) exciting the coupled sample with linearly polarized electromagnetic energy to cause the coupled sample to emit fluorescent light; and (C) measuring the polarization of the fluorescent emission as a measure of a biological characteristic of the sample of interest.

Claims

1. A method of conducting an assay of a sample of interest, comprising the steps of:

coupling a luminescent asymmetric transition metal-ligand complex to an analyte in the sample of interest to form a coupled analyte, wherein the asymmetric transition metal-ligand complex-coupled analyte is capable of emitting polarized fluorescent light after being excited with linearly polarized electromagnetic light energy;

exciting the coupled analyte with linearly polarized electromagnetic light energy to cause the coupled analyte to emit polarized fluorescent light; and

measuring the polarization of the fluorescent light emission as a measure of a biological characteristic of the analyte in the sample of interest.

2. A method of claim 1, wherein the metal in said metal-ligand complex is selected from the group consisting of ruthenium, osmium, rhenium, rhodium, iridium, tungsten and platinum.

3. A method of claim 1, wherein the analyte of interest is an antigenic substance or with a molecular weight over 2,000.

4. A method of claim 1, wherein the exciting electromagnetic energy is a linearly polarized light pulse, and the method further comprises the step of measuring the polarization of the fluorescent light only after background autoflourescence of the coupled sample has subsided.

5. A method of claim 4, wherein the exciting step is implemented by a light source selected from the group consisting of a flash lamp, a modulated lamp, an electroluminescent device, a light-emitting diode, a diode laser, and an amplitude modulated laser.

6. A method of claim 4, wherein the light pulse and the fluorescent light are transmitted through optical fibers.

7. A method of claim 1, wherein the measuring step is performed using an implanted patch containing the coupled sample.

8. A method of claim 1, wherein a steady state of polarization is dependent on a characteristic of the coupled sample or any uncoupled analyte which is present in said sample.

9. A method of claim 1, wherein intensity decay or polarization decay is dependent on the coupled analyte.

10. A method of claim 3, wherein the antigenic substance is a protein, nucleic acid, or polysaccharide.

11. A method of claim 3, wherein the antigenic substance is a cellular or cell surface antigen, glycopeptide, lipoprotein, or glycolipid.

12. A method of claim 1, wherein the amount of analyte is estimated from time-dependent anisotropy decay as measured following pulsed excitation.

13. A method of claim 1, wherein the amount of analyte is determined from emission anisotropy decay measured with amplitude-modulated excitation by phase-modulation fluorometry.

14. A method of claim 1, wherein the immunoassay is a competitive immunoassay.

15. A fluorescence polarization assay for quantifying the amount of an analyte in a sample, comprising the steps of:

(a) mixing (1) an asymmetric transition metal-ligand complex conjugated to a molecule which specifically binds said analyte with (2) said sample, wherein the asymmetric transition metal-ligand complex is capable of emitting polarized light after being excited with linearly polarized light;

(b) exciting the mixture of step (a) with linearly polarized light to cause the complex to emit polarized light;

(c) measuring the polarization of the light emitted by said complex;

(d) calculating the amount of analyte in the sample by correlating the polarization measured in step (c) with the polarization of light emitted from a control sample containing a known amount of analyte.

16. A fluorescence polarization assay of claim 15, wherein the metal in said asymmetric metal-ligand complex is selected from the group consisting of ruthenium, osmium, rhenium, rhodium, iridium, tungsten and platinum.

17. A fluorescence polarization assay of claim 15, wherein the ligand in said metal-ligand complex comprises polypyridine or bipyridine and optionally further comprises CO, Cl, phosphine, nitrile or isonitrile groups.

18. A fluorescence polarization assay of claim 17, wherein said ligand contains a reactive group selected from the group consisting of a N-hydroxysuccinimide ester of a carboxylic acid, haloacetyl groups, sulfonyl chlorides, maleimides, and isothiocyanates.

19. A fluorescence polarization assay of claim 17, wherein said molecule is a receptor, antibody or lectin.

20. A fluorescence polarization assay of claim 15, wherein the ligand in said metal-ligand complex comprises a bipyrazyl group, a phenanthroline group.

21. A competitive fluorescence polarization assay for quantifying the amount of an analyte in a sample, comprising the steps of:

(a) mixing (1) a control containing a known amount of analyte conjugated to an asymmetric transmission metal-ligand complex with (2) a molecule which specifically binds the analyte, wherein the asymmetric transition metal-ligand complex is capable of emitting polarized light after being excited with linearly polarized light;

(b) exciting the mixture of step (a) with linearly polarized light to cause the complex to emit polarized light;

(c) measuring the polarization of the light emitted by the complex;

(d) adding the sample to the mixture to form a new mixture including analyte not conjugated which competes with the analyte conjugated to the asymmetric transition metal-ligand complex in binding to the molecule which specifically binds the analyte, thereby causing a change in polarization;

(e) measuring the change in polarization;

(f) calculating the amount of analyte in the sample by correlating the change in polarization with the control containing a known amount of analyte.

22. A competitive fluorescence polarization immunoassay of claim 21, wherein the metal in said asymmetric metal-ligand complex is selected from the group consisting of ruthenium, osmium, rhenium, rhodium, iridium, tungsten and platinum.

23. A competitive fluorescence polarization immunoassay of claim 21, wherein the ligand in said metal-ligand complex comprises polypyridine or bipyridine.

24. A competitive fluorescence polarization immunoassay of claim 23, wherein said ligand contains a reactive group selected from the group consisting of a N-hydroxysuccinimide ester of a carboxylic acid, haloacetyl groups, sulfonyl chlorides, maleimides, and isothiocyanates.

25. A competitive fluorescence polarization immunoassay of claim 23, wherein said molecule is a receptor, antibody or lectin.

26. A competitive fluorescence polarization immunoassay of claim 21, wherein the ligand in said metal-ligand complex comprises a bipyrazyl, a phenanthroline, or a related compound and optionally further comprises CO, Cl, phosphine, nitrile or isonitrile groups.

27. A method of conducting an affinity polarization assay of a sample of interest to quantify the amount of an analyte in the sample, comprising the steps of:

(a) mixing (1) a control containing a known amount of analyte conjugated to an asymmetric transition metal-ligand complex with (2) a molecule which has affinity for the analyte, wherein the asymmetric transition metal-ligand complex is capable of emitting polarized light after being excited with linearly polarized light;

(b) exciting the mixture of step (a) with linearly polarized light to cause the complex to emit polarized light;

(c) measuring the polarization of the light emitted by the complex;

(d) adding the sample to the mixture to form a new mixture including analyte not conjugated which competes with the analyte conjugated to the asymmetric transition metal-ligand complex in associating with the molecule which has affinity for the analyte, thereby causing a change in polarization;

(e) measuring the change in polarized emission;

(f) calculating the amount of analyte in the sample by correlating the change in polarization with the control containing a known amount of analyte.

28. A method of claim 27, wherein the molecule which has affinity for the analyte is selected from the group consisting of strepavidin, avidin, biotin and lectins.

29. A method of claim 27, wherein the molecule which has affinity for the analyte is a protein.

30. A method of claim 27, wherein the metal in said metal-ligand complex is selected from the group consisting of ruthenium, osmium, rhenium, rhodium, iridium, tungsten and platinum.