Method of conducting an assay of a sample containing an analyte of interest

• US 6,214,628 B1
• Filed: 01/14/1998
• Issued: 04/10/2001
• Est. Priority Date: 01/14/1998
• Status: Expired due to Fees

• Current Assignee: Joseph R. Lakowicz
• Original Assignee: Joseph R. Lakowicz
• Inventors: Murtaza, Zakir, Castellano, Felix, Lakowicz, Joseph R.
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): COOK, LISA V
• Application Number:
  US09/007,167
• Time in Patent Office: 1,182 Days
• Field of Search: 435/4, 435/968, 435/7.1, 435/7.5, 435/501, 435/536, 435/7.21, 435/546, 435/164, 435/172, 435/94, 435/95, 435/63, 422/82.07, 422/82.08, 422/82.09, 436/172, 436/518, 436/138, 436/546, 436/800, 436/536, 436/537, 436/68, 436/133, 436/163, 436/166, 436/74, 436/8, 382/6, 382/58, 382/191, 382/128, 382/133, 382/312, 382/129, 128/633, 128/634, 128/635, 250/461.2, 250/458.1, 250/459.1, 250/200, 250/338.1, 315/169.3, 356/73, 356/317, 356/318, 356/417, 356/346, 356/39
• US Class Current: 436/518
• CPC Class Codes: G01N 2333/765 Serum albumin, e.g. HSA
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/204998 Inorganic carbon compounds
• CPC Class Codes: Y10T 436/209163 Dissolved or trace oxygen or oxygen content of a sealed environment

Abstract

In accordance with the present invention, a method of conducting an assay of a sample containing an analyte of interest includes the step of forming a mixture so as to bring a metal-ligand complex into interactive proximity with the sample containing the analyte of interest. The mixture is irradiated with electromagnetic light energy so as to cause emission of light indicative of the analyte of interest. The emitted light is measured, and the measurement of the emitted light is utilized to measure the analyte of interest. The metal-ligand complex can be [Re(bcp)(CO)₃(4-COOHPy)]⁺, [Os(phen)₂(aphen)]²⁺, [Os(tpy)(triphos)]²⁺, [Os(tppz)₂]²⁺, and [Os(ttpy)₂]²⁺, or the like. Also, the present invention is directed to a metal-ligand complex of the formula [Re(bcp)(CO)₃(4-COOHPy)]⁺.

Claims

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

forming a mixture so as to bring a metal-ligand complex into interactive proximity with said sample containing said analyte of interest;

irradiating said mixture with electromagnetic light energy so as to cause emission of light indicative of said analyte of interest; and

measuring the emitted light and utilizing the measurement of the emitted light so as to measure the analyte of interest, wherein said metal-ligand complex is selected from the group consisting of [Re(bcp)(CO)₃(4-COOHPy)]⁺, [Os(phen)₂(aphen)]²⁺, [Os(tpy)(triphos)]²⁺, and [Os(ttpy)₂]²⁺.

2. A method as defined by claim 1, wherein said assay is used to characterize a high molecular weight antigen.

3. A method as defined by claim 2, wherein said analyte of interest is human serum albumin.

4. A method as defined by claim 1, wherein said assay is used to characterize protein hydrodynamics.

5. A method as defined by claim 4, wherein said analyte of interest is human serum albumin and bovine immunoglobin G.

6. A method as defined by claim 1,

wherein said assay is used to characterize a sample lipid; and

wherein said metal-ligand complex is coupled to a lipid so as to form a lipid-metal-ligand complex.

7. A method as defined by claim 1, wherein said analyte is oxygen.

8. A method as defined by claim 1,

wherein said assay is used to quantify said analyte of interest;

wherein prior to said forming step, a first binding partner and a second binding partner are added to said sample, wherein said first binding partner competes with the analyte for binding to said second binding partner, wherein one of said first and second binding partner is labeled with a metal-ligand complex and the other is labeled with a photoluminescent energy transfer acceptor, wherein the metal-ligand complex and photoluminescent energy transfer acceptor are chosen such that when the first binding partner binds to the second binding partner, the metal-ligand complex and the photoluminescent energy transfer acceptor are brought into interactive proximity, producing a detectable change in luminescence.

9. A method as defined by claim 8,

wherein subsequent to said measuring step, binding of the first binding partner to the second binder partner is quantified;

and

wherein said binding is quantified by a method from the group consisting of intensity, lifetime, and polarization, thereby quantifying the analyte.

10. A method as defined by claim 1, wherein fluorescence lifetime is measured.

11. A method as defined by claim 1, wherein said metal-ligand complex is coupled to said analyte of interest.

12. A method as defined by claim 1, wherein said metal-ligand complex is substituted with a functional group that can be directly conjugated to macromolecules selected from the group consisting of amine reactive N-hydroxysuccimide, isothiocyanate, sulfonyl chloride containing ligands, sulfhydryl reactive iodoacetadmide, and maleimide containing ligands.

13. A method as defined by claim 1,

wherein said electromagnetic light energy is linearly polarized light energy;

and

wherein polarization of the emitted light is measured.

14. A method as defined by claim 13, wherein said linearly polarized light energy has a wavelength of about 400.

15. A method as defined by claim 13, wherein said linearly polarized high energy has a wavelength from 280 to 1100 nm.