LONG LIFETIME ANISOTROPY (POLARIZATION) PROBES FOR CLINICAL CHEMISTRY, IMMUNOASSAYS, AFFINITY ASSAYS AND BIOMEDICAL RESEARCH

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. The measurement technique is schematically illustrated in the figure.

Description: LONG LIFETIME ANISOTROPY (POLARIZATION) PROBES FOR CLINICAL CHEMISTRY, IMMUNOASSAYS, AFFINITY ASSAYS AND BIOMEDICAL RESEARCH FIELD .OF THE INVENTION The present invention relates to the field cf fluorescent anisotrcpy (polarization) probes for immunoassays and the like and, more particularly, to a method of conducting an immunoassay Ξ using a fluorescent metal-iigand complex which can be coupled to proteins and which also displays a long lifetime and a high initial anisotrcpy or polarized fluorescence. BACKGROUND CF THE INVENTION ic Presently, fluorescence polarization .anisotropy) immunoassays, which are based en the polarization or anisotropy cf emitted light when a sample is excited with vertically polarized light, are widely used in clinical chemistry but are limited to the analysis cf low molecular weight antigens such as is drugs. This limitation exists because the short lifetime of the fluorescent probes preclude their use with larger, high molecular weight antigens which rotate more slowly in solution than do the smaller antigens. With respect to metal-Iigand complexes, there have been many 23 reports which attempt to determine whether the excited state is distributed among the organic iigands en a rather symmetric complex, or whether it is localized between the metal and one of the Iigands. This is an important distinction, because the former model predicts a low anisotropy, while the latter model 25 predicts a higher anisotropy. An expert in the field would predict a low anisotropy, as symmetric molecules typically display low anisotropies, and metal ions in solution typically display zero anisotropies. Given the possibility of different effects which could account for loss of anisotropy, it was not 30 clear that complexes such as Ru-metal-ligand complexes iRu-MLC) would display useful anisotropy values. Also, even if the anisotropies were non-zero, it was not clear whether complexes such as Ru-MLC would display anisotropies which depend on molecular size, as needed for a fluorescence polarization ;= immunoassav (FPI) , or whether they would become depolarized by transfer of the excited state energy among the Iigands, and thus would be independent of molecular size or rotational diffusion. Therefore, m regard to metal-Iigand complexes, tr.ere nas been no recognition of tr.eir use as fluorescent proDes for oio edical = applications. The following references represent tr.e state cf .ie art of fluorescent polarization immunoassay: Measurement of Anσiotensinoσen m Human Serum Bv Fluorescence Polarization Immunoassay, David B. Gordon, Clin. & 0 Exper. Hyper. - Theory and Practice, A10(3) , 1988, pages 485-503. Immunoassay - Innovations m Label Tecnnoio v. Joan H. Howanitz, M.D., Arcn. Patnci . Lab. Med., Volume 112, August 1988, pages 775-779. Four Fluorescent Polarization Immunoassays for Therapeutic 5 Druα Monitoring Evaluated, Virginia M. Havre et al , Clinical Chemistry, Volume 35, No. 1, 1989, pages 138-140. New Fluorescent Derivatives of Cyclosponr. for Use in Immunoassays, M. T. Frencn et al, Journal of Pharmaceutical & Biomed cal Analysis, Volume 10, No. 1, 1992, pages 23-30. 0 A Decade of Development of Immunoassay Methodology, James P. Gosling, Clinical Chemistry, Volume 35, No. S, 1990, pages 1408-1427. Fluoroimmunoassav: Present Status and Key Problems. Erkki Soir.i et al, Clinical Chemistry, Volume 25, No. 3, 1979, pages 5 353-361. Fluorescent Excitation Transfer Immunoassay, Edwin F. Ullman et al, The Journal of Biological Chemistry, Volume 251, No. 14, July 25, 1976, pages 4172-4178. Fluorescence Polarization in Immunochemistrv, W. B. o Dandliker et al, Immunochemistry, Volume 7, 1970, pages 799-828. Photophvsics of Ruthenium Complexes Bound to Double Helical DNA. Challa V. Kumar et al. Journal American Chemical Society, Volume 107, No. 19, 1985, pages 5518-5523. The following references represent the state of the art of 5 time-resolved immunoassay: Time-Resolved Fluorometrv m Immunoassay, T. Lovgren et al, Alternative Immunoassays, 1985, pages 203-217. Current Concepts and Future Developments. R.p. Ekins, Alternative Immunoassays, 1985, pages 219-237. Immunoassays wit Time-Resolved Fluorescence Soectroscopy: Principles and Applications. Eleftherios P. Diamanαis, Clinical Biochemistry, Volume 21, June 1988, pages 139-150. Eurooium Cheiate Labels in Time-Resoiveα Fluorescence Immunoassays and DNA Hybridization Assays. Eleftherios P. Diamandis et al, Analytical Chemistry, Volume 62, No. 22, November 15, 1990, pages 1149-1157. Europium as a Label m Time-Resolved Immunofluorometric Assays, Ilkka Hem ila et al, Analytical Biocnemistry, 137 (1984) , pages 335-343. Alternative to Rare Earth Fluorescent Labels?. A. P. Savitskii et al, Doklaαy Akademn Nau SΞSR, 1989, pages 48-51. Fiber-Qptic Time-Resolved Fluorimetry for Immunoassays. Randy D. Petrea et al, Talanta, Volume 35, No. 2, 1988, pages 139-144. Applications cf Lanthanide Chelates for Time-Resolved 0 Fluoroimmunoassay, Philip Mottram et al, American Chemical Laboratory, May/June 1990, pages 34-38. U.S. 4,374,120 2/15/83 Somi et al U.S. 4,745,076 5/17/88 Mullεr et al The following references disclose the Known spectral 5 properties of transition metal-ligand complexes: Design and Applications of Highly Luminescent Transition Metal Complexes, J. N. Demas et al, Analytical Chemistry, Volume 63, No. 17, Septemner 1, 1991, pages 829-837 Novel Fluorescent Label for Time-Resolved Fluorescence o Immunoassay, Richard B. Thompson et al, SPIE, Volume 909, Time- Resolved Laser Spectroscopy in Biochemistry, 1988, pages 426- 433. Redox Properties of Ruthenium(II) Tris Cheiate Complexes Containing the Ligands 2 , 2 ' -Bipyrazme, 2.2 ' -Bipyridme. and 5 2.2' -Bipynmidme, . D. Paul Rillema et al, Inorganic Chemistry, Volume 22, No. 11, 1983, pages 1617-1622. Localization cf Electronic Excitation Enerαv In Ru(2.2'- Bipyridinei - '2.2 ' -BiPyridine-4 , 4 ' -Dicarboxyiic Acid) 2 ' and Related Complexes, James Ferguson et al, Chemical Physics Letters, Volume 63, No. 1, pages 21-24. Energy Transfer from Luminescent Transition Metal Complexes to Oxygen, J. X. Demas et al , Journal cf the American Chemical Society, :* * .ay 25, 1977, pages 3547-3551. The following references are cf further background interest with respect to the present invention: Thin Layers cf Depolarizers and Sensitizers. Lasovsky et al , Chem. Abstract 106, 1987: 95354n. Time-Resolved Photoselection of ' Ru (bpy) '-exciton Hopping in the Excited State. Myrick et al, J. Amer. Chem. Soc. 109, 1987: 2841-2342. Circularly Polarized Luminescence of Tris-Bipyridine Ruthenium (II) Complexes at Low Temperature. Tsubomura et al, Chem. Abstract 112, 1990: 65776h. The present inventors wish to stress that none of the experts listed above have suggested complexes such as Ru or Os metal-ligand complexes for use in fluorescence polarization immunoassays. We refer to the emission from these complexes as fluorescence, primarily for convenience. The exact nature of the excited state is unknown, and the emission may be regarded as fluorescence or phosphorescence. As indicated above, FPIs are presently limited to low molecular weight analytes, such as drugs and hormones, as admitted in Urios et al, "Adaptation of Fluorescence Polarization Immunoassay to tne Assay of Macromoiecules" , Analytical Biochemistry, 185, 308-312 '1990) iwhich used F ab fragments having a molecular weignt near 40,000, as opposed to a full gG ( Immunoglobm G, human) molecule having a molecular weignt near 160,000) and Tsuruoka et al, "Fluorescence Polarization Immunoassay Employing Immobilized Antibody" , Biosensors & Bioeiectronics, 6, 501-505 ιl991) iwhich bound the Ab to colloidal gold to increase its molecular weight in an attempt to cnange the correlation time of the larger antigens) . W th respect to Tsuruoκa m particular, the present inventors do not consider its approacn to pe useful, since tne molecular weignt cf tne antioody is already too high for the lifetime of the labe . In tπis regard, it is noted tnat tne lifetimes of the propes are near 4 ns . The limitation to low molecular weignt anaiytes arises oecause tne FPIs depend on a change m the apparent molecular weight cf a fluorescently-labeled antigen upon binding to the antibody. A change in "apparent molecular weight" results upon binding to the large antibody molecule because tne smaller antigen is now bound to the larger antibody molecule. Typical molecular weights of antigen and antibody are 1,000 and 160,000 daltons, respectively. The limitation to low molecular weight antigens is due to the snort lifetimes of erodes used in present FPIs and can be circumvented by using longer lifetime fluoropnores. However, few such long-lived prooes are known. The review articles mention pyrene derivatives, whicn display lifetimes near 100 ns. However, pyrene requires UV excitation near 320 nm, is photosensitive, and displays a low polarization. UV excitation results m significant autofluorescence from biological samples. A further advantage of the RuMLCs is their high chemical and pnotocnemical stability. To assist m understanding the above-described limitation and the present invention, some examples are set forth below. The need for a change in apparent molecular weight can oe seen from the following example calculation. Suppose tne labeled antigen has a molecular weight cf 1,000 daltons, which results in a rotational correlation time cf aoout 0.5 ns. The molecular weight cf the antibody IgG is 160,000, resulting in a rotational correlation time near ICO ns iv + h =- l.Ξ, see eq. :r labeled macromolecule is given by :i) J I where r is a constant typically near 0.3, r is the lifetime and 9 is the rotational correlation time. For present immunoassays, the lifetimes cf the probes are near 4 ns . The anisotropy of the free and antibody-bound antigens are thus as follows: Free Ag : r = 2 . 2 = 2.033 1-4/0.5 3ound Ag: = 0.3 = C.288 '% change = 773%) Hence, a large change m anisotropy is found upon binding of Ag to Ab for low molecular weight antigens. The favorable change described above is not o tained for high molecular weight antigens. Suppose the molecular weight of tne antigen is accut 160,300, wi r. a correlation time of 100 ns, and the molecular weight of the antibody is about 450,000, with a correlation time of 300 ns . The correlation time of the antigen-antibody complex will be near 400 ns . For the presently-used short lifetime fluorophores, the anisotropy values will be as follows: Free Ag: r = 0.3 = 0.288 1+4/100 Bound Ag: r = 0.3 = 0.297 ( % change = 3%) 1+4/400 This change in anisotropy is small because the lifetime is much shorter than the correlation time of the antigen. The present invention provides a long lifetime label with good r„ values. In particular, data show lifetimes near 400 ns . r or tne larger molecular weight Ag-Ab complex ( 9 = 400 ns ) , tne expected aniεotropv values are as fellows : ( % change = 150% : The change in anisotropy for the 400 ns lifetime is 150%, which is much improved as compared to only 3% for the 4 ns lifetime. Also, many antigens of interest ιe.q. , IgM) are still larger (MW = 950,000, 6 ≥ 600 nsi , which will yield still higher anisotropy tr = 0.180, % change = 200%) . It snould be noted that the % change in anisotropy is more important than the aosoiute values . In order to avoid confusion, it should be noted that polarization (P) and anisotropy ir) describe the same phenomena, and are related as follows: (3) where I and I_ are the vertically and horizontally polarized components of the emission, when excited with vertically polarized light. The polarization and anisotropy are related by r = 2P (4) 3-P P = 3r (5) 2+r The parameters P and r are both in common use . The values of P are used more often in FPI because they are entrencned by tradition and are slightly larger than the anisotropy values. The parameter r is preferred on the oasis of theory. Both P and r are related to the correlation time and/or molecular volume as follows: θ τ) V . 6 ) η f θ In these equations : is tne 3oltzmann constant, T is the temperature (K) , 17 is tne viscosity and V is tne molecular volume. The correlation time is related to the molecular weight (M) of the protein as follows: θ = 'v+h) (8) RT where R is the ideal gas constant, * - is the specific volume of the protein and n is tne hydration, typically 0.2 g H : 0 per gram of protein. SUMMARY OF THE INVENTION We have syntnesizeα a new type of protein laoeimg reagent or prooe wnich has αesiraple features for use in immunoassays, clinical cnemistry and biomedical researcn, i.e., a fluorescent metal-ligand complex wnicn can pe coupled to proteins and which also displays a long lifetime and high anisotropy or polarization m tne aosence of rotational diffusion. The value is often referred to as the initial or funαamental anisotropy (r β ) or polarization (P 0 ) . This reagent or prope also has the advantage of having fluorescent emission m the reα region of the spectrum, and can pe excited with simple light sources or visible-wavelength lasers, both of whicn are αesiraole from the standpoint of reducing auto-fluorescence and instrument cost and complexity The spectral cnaracteristics and long lifetime of such proces also allow fluorescence lifetime measurements with simple exciting-iignt sources, such as a flasn lamp, laser diode, blue LED or a lue electroluminescent lamp. The long luminescence lifetime allows the prope to be used for lmmuno- cnemical polarization or intensity assays of r.ign moiecuiar weignt species. The use cf complexes sucn as Ru or Os metal- liganα complexes allows fluorescence polarization immunoassays to cs performed en nig -lolecuiar weignt antigens MW>1000) , wnicn is not routinely ossicle witn otner fluoropnores . ≡ Thus, our invention provides a long luminescence lifetime hici, m turn, allows tne assay cf higner molecular weight antigens oy tne polarization or anisotropy method. The use of luminescent metal-ligand complexes for fluorescence poiarizaticn assays nas , to our knowledge, not been earlier 0 proposed, oecause sucn complexes typically display low anisotropies (polarizations) . Also, it was not known wnether the luminescence from tnese complexes was αepolarizeα due to rotational motion, Λ/nicn is requireα for a fluorescence polarization immunoassay, cr due to internal randomization of 5 the excited state energy within tne complex. The latter behavior makes a rooe -nsuitabie for use m a fluorescence polarization immunoassay. In contrast, the asymmetric complex used m our invention displays a high polarization oecause of the presence of non-identical ligands. 0 Another advantage cf our invention is that a fluorescence polarization immunoassay can ce accomplished _n a manner comparable to so-caileo "time-resolved immunoassays" Thus, bacKground auto-fluorescence is suppressed by gating the fluorescence detector en at long times, after αecav of the 5 initial autofluorescence, wnicn typically decays in 5 ns . With the shorter lived probes of the prior art, such gating is not technologically practical, and it is not advantageous because the probe decays on the same timescale as the autofluorescence. Gating is very advantageous and practical when using the long 0 lifetime luminescence metal complexes of our invention. Thus, our invention creates a new class of fluorescence polarization immunoassays for use with high molecular weight antigens, while allowing suppression of auto-fluorescence by time-gating of the fluorescence detector. These novel probes 5 find use in biocnemical and biomedical researcn for measuring the rotational dynamics of high molecular weight species, particularly membrane-pound proteins. Advantages include the use of an inexpensive Iignt source and simple instrumentation cecause cf the long lifetime, therepy allowing oedside clinical enemistry and tne assay of hign molecular weignt antigens. Accordingly, tne present invention provides a method of : conducting an immunoassay of a sampie of interest comprising tne steps of : coupling a luminescent asymmetric -.etal-iigand complex to tne sample cf interest to form a coupled sample ,* 10 exciting the coupled sample with linearly polarized electromagnetic energy to cause tne coupled sample to emit partially polarized fluorescent light; ana measuring tne polarization cf the fluorescent light emission as a measure cf a oioiogical characteristic of -5 tne sample cf interest. In addition, the present invention provides a fluorescence polarization assay for quantifying tne amount of an analyte in a sample, comprising the steps of: (a) mixing 1) an asymmetric metal-iiganα complex 20 conjugated to a molecule which specifically inds the analvte with 'I 1 tne sample, (b) exciting tne mixture of step { & > with linearly polarized Iignt to cause the complex to emit polarized Iignt ,- 25 (c) measuring the polarization of the _ιgnt emitted by tne complex; (d) calculating the amount of analyte in the sample by correlating the polarization measureα m step 'o with the polarization of light emitted from a control sample 30 containing a Known amount cf analyte. Also, the present invention provides a competitive fluorescence polarization immunoassay for quantifying tne amount of an analyte m a sample, comprising the steps of: (a) mixing (1) a control containing a known amount 35 of analyte conjugated to an asymmetric metal-ligand complex with '2) a molecule wnicn specifically Dinds the analyte ,* ι'b) exciting the mixture cf step ιa) with linearly polarized light to cause the complex to emit polarized light ,- c: measuring the polarization of the light emitted Ξ cv the complex; d) adding the sample to tne mixture to form a new mixture including analyte not conjugated which competes with the analyte conjugated to the asymmetric metal-ligand complex in binding to the molecule which specifically ic binds the analyte, thereby causing a change in polarization; e> measuring the cnange m polarization,* vf, calculating tne amount cf analyte m the sample by correlating tne change m polarization with the control is containing a Known amount of analyte. Additionally, the present invention provides a method of conducting an affinity polarization assay cf a sample of interest to quantify the amount of an analyte m the sample, comprising the steps of : 20 (a) mixing (1) a control containing a known amount cf analyte conjugated to an asymmetric metal-ligand complex with !2) a molecule wnicn has affinity for the analyte ,* (b) exciting the mixture of step ιa) with linearly 25 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 30 mixture including analyte not conjugated which competes with the analyte conjugated to the asymmetric metal-ligand complex in associating with the molecule which has affinity for the analyte, thereby causing a change in polarization,* 3 5 (e) measuring the change m polarization; (f) calculating tne amount of analyte m tr.e sample by correlating the change m polarization with tne control containing a -mown amount of analyte . BRIEF DESCRIPTION CF THE DRAWINGS Figure 1 is a scne atic clock diagram cf an apparatus for implementing tne present invention using steady-state polarization or anisotrcpy measurements. P0L1 and POL2 are polarizers. Figure 2 is a scnematic blocκ diagram of an apparatus for implementing the present invention using time-resolved measurements. Figure 2 αescrices tne syntnesis cf a Ru metal-Iigand complex wnich is suitaole for covaient attachment to proteins. Figure 4 snows aosorpticn spectra cf tne complex wnen covalently pound to numan serum albumin HSA) at pH 7.0, and complex free in solution at pH 0.1 and pH 7.0. "Bpy" refers to 2, 2' -bipyridine, and "dcbpy" refers to 4 , 4 ' -dιcarPoxy-2 , 2' - bipyridme. Figure 5 shows the emission spectra of the complex free in solution ,pH 0.1 and ~ : and pound tc HSA. For comparison, included are emission spectra of symmetrical Ru-compiexes . Figure S snows tne excitation anisotropy spectra of Ru (bpy) : (dcbpy) free and bound to HSA in glycerol/water (9:1, v/v) at -55°C. Also included for comparison are the anisotropy spectra of the symmetrical Ru-complexes Ru(bpy),-' and Ru (dcbpy) j " . Figure 7 snows the temperature-dependent emission anisotropy of the complex free and protein conjugates in glycerol/water r 6:l, v/v) . The emission wavelength for [Ru (bpy) ,] :* was 600 nm, and 650 nm for the Ru (bpy) - (dcbpy) and Ru-labeled proteins. Figure 8 snows the intensity decays cf [Ru (bpy; - (dcbpy) ] conjugated to ConA (Concanavalin A) . Similar intensity decays were obtained for [Ru (opy) - (dcbpyi ] free and conjugated to otner proteins . These data were cotamed using an apparatus similar to that snown ir. Figure 2. Figure 5 snows tne anisotropy decays cf free [Ru(bpy; - (dcbpy) ] m glycerol/water 60/40, vv) at the indicated temperatures, optamed using an apparatus similar to that snown in Figure 2. e Figure 13 snows the anisotropy decays of [Ru lopyi - idcbpy) ] free and conjugated to proteins in a puffer. The anisotropy decay is seen to ce slower for higher molecular weight proteins . Figures 11A and llB show the viscosity-dependent ID anisotropy decays of [Ru(bpy) - (dcbpyι ] conjugated to ConA and IgG, respectively. Increasing viscosity, or increasing % glycerol , results in slower anisotropy decays. Figures 12A and 12B show the temperature-dependent anisotropy decays cf [Ru(bpy) - (dcbpyι 1 conjugated to HSA and is Ferπtm, respectively. The anisotropy decays more slowly at lower temperatures due to slower rotational diffusion. Figure 13 shows the fluorescence polarization immunoassay of HSA. Figure 14 snows the time-resolved anisotropy decays of the 20 complex conjugated to HSA, m the absence and presence of various cencentraticns of HΞA-specific antibody. Figure 15 snows a competitive immunoassay fer HSA. In this case tne presence of uniapeled HSA in the sample decreases the fluorescence polarization ooserved for the mixture of 25 labeled HSA and antibody. Figure 16 snows the anisotropy decays of labeled HSA in the absence and presence of antibody, and with unlabeled HSA. The anisotropy decays more rapidly with xarger concentrations of unlabeled HSA in this competitive immunoassay, wnich is 30 consistent with data presented m Figure 15. Figure 17 snows the absorption and anisotropy spectrum of an Os complex, Os (bpy) 2 (dcbpy) . Figure 18 snows the emission spectrum of tne Os complex. Figure 19 snows the absorption spectrum of a Rhenium (Re) 35 complex. Figure 20 snows tne excitation anisotropy spectrum of a Re complex. Figure 21 snows tne emission spectra of a Re complex. Figure 22 snows a fluorescence polarization immunoassay oased on an osmium metal-Iigand complex. This complex can be executed Ξ with hign polarization near 500 or 700 nm. DETAILED DESCRIPTION OF THE INVENTION There are a r.umoei of metal-iigand complexes wnich display luminescence, including complexes containing Co, Cr, Cu, Mo, Ru, Rh, w, Re, Cs, Ir, and P . In particular, transition metal c complexes, especially those with Ru, Cs, Re, Rh, Ir, W and Pt, can De used. The metal in tne metal-ligand complex is particularly preferably selected from tne group consisting of ruthenium, osmium, ano rnenium. A suitaole iigand in tne metal-iigand complex can be poiypyridme, bipvridme, or a related compound, and the Iigand can s contain a reactive group commonly used for linkage to biological molecules, such as a N-nyαroxysuccmimide ester of a carooxylic acid, haloacetyl groups, maieimides, sulfonyl chlorides, and lsotniocyanates. Other ligands for sucn metal-ligand complexes are bipyrazyl, pnenantnrciine, and related suostituted derivatives, or 0 inorganic ligands such as CO, Cl, nitriie and isonitrile. Suitable metal-ligand complexes 'MLCs) for use m fluorescence polarization immunoassays and affinity assays according to the present invention are set forth below. MLC-: MLC-2 MLC-3 MLC-4 MLC- MLC- MLC-7 MLC-8 MLC-15 M C-16 MLC- MLC-18 MLC-19 The complexes used m the present invention can be synthesized according to the scheme set forth in Figure . A discussion of this figure and the ether figures is set forth below. Figure 1 shows a schematic diagram for L-format measurements cf fluorescent anisotrcpy. In Figure 1, P0L1 and P0L2 represent polarizers. Figure 2 shows a typical time-correlated single-photon arrangement . Figure 2 illustrates ow the reactive metal-ligand complexes used in the present invention can be syntnesized. Figure 4 snows adsorption spectra of [Ru (bpy; - idcbpy) ] at pH C.l and 7 and wnεn conjugated to HSA. Similar absorption spectra were found for other protein conjugates . Figure 5 shows emission spectra of [Ru(bpy) .; ' and [Ru (dcbpyj ,] ''" at pH " .0 and Ruibpyi- dcbpy)] at pH 0.1 and 7 and when conjugated to HSA. Similar emission spectra were found for other protein conjugates. Figure 6 shows excitation anisotropy spectra cf metal- Iigand complexes in glycerol/water (9:1, v/v) at -55°C. Figure 6 shows that in frozen solutions, where rotational motion does not occur, the anisotropy of the invention complex is higher than for a symmetric [Ru(bpy)-. - " and [Ru (dcbpyi .] " " complexes. Figure 7 illustrates the temperature-dependent emission anisotropy of metal-ligand complexes and protein conjugates. The emission wavelength for [Ru (bpy) .] * was 600 nm. Figure 7 shows that the anisotropy cf Ru (bpy) - (dcbpy) is higher when bound to proteins, which indicates the anisotropy will depend on the molecular weight. Figure 8 illustrates the intensity decays of [Ru (bpy) - (dcbpy) ] conjugated to ConA. Similar intensity decays were obtained for [Ru (bpy) : (dcbpy) ] conjugated to other proteins. Figure 8 shows that the lifetime of the complex when bound to a protein (Concanavalin A) is near 400 ns, and thus is suitable for use in FPI of high molecular weight antigens. Figure 9 illustrates anisotropy decays of free [Ru (bpy) : (dcbpy) ] in glycerol/water (60/40, v/v) at various temperatures indicated thereon. Figure 9 shows that the anisotropy decay of the complex depends on the rotational rate of the probe, as needed for FPI. Figure 10 illustrates anisotropy decays of [Ru (bpy; - (dcbpy) ] m a buffer, and Figure 10 shows that the anisotropy of [Ru (bpy; - j decays more slowly with higher molecular weight proteins. This sensitivity to molecular weight is essential for use in FPI . Figures 11A and I1B illustrate viscosity-dependent anisotropy decays cf [Ru (bpy) - (dcbpy) ] conjugated to ConA and IgG, respectively. Figures 11A and I1B show that the anisotropy decays more slowly upon increasing viscosity by adding giycerol . This result again shows that the anisotropy of the complex depends on rotational rate and thus molecular weight . Figures 12A and 12B illustrate temperature-dependent anisotropy decays of [Ru (bpy) 2 (dcbpy) ] conjugated to HSA and Ferritin, respectively. Figures 12A and 12B again demonstrate that anisotropy decays more slowly as the rotational rate decreases, in this case by decreasing the temperature. Figure 13 illustrates a fluorescence polarization immunoassay of HSA using the [Ru(bpyj - (dcbpy) j complex with the addition of HSA-specific antibody (closed circles) and with the addition of nonspecific antibody (open circles) . A significant increase in polarization is observed upon binding of HSA labeled complex to anti-HSA. Figure 14 illustrates the time-resolved anisotropy decays of the complex conjugated to HSA, in the absence and presence of HSA-specific antibody. It is shown that the correlation decay strongly depends on amount of anti-HSA. The increase of the correlation time observed from anisotropy decays confirms the increase of polarization observed in Fig. 13. Figure 15 illustrates a competitive immunoassay for HSA. In this case the presence of unlabeled HSA in the sample decreases the fluorescence polarization observed for the mixture of labeled HSA and antibody. The decrease in polarization is due to competitive binding of labeled HSA and unlabeled HSA to anti-HSA. Figure 16 illustrates the anisotropy decays of labeled HSA in the absence and presence of antibody, and with unlabeled HSA. The anisotropy decays more rapidly with larger concentrations of unlabeled HSA m this competitive immunoassay, wnicn is consistent with data presented in Figure 15. Figure 17 illustrates the absorption and anisotropy spectrum of an Os complex, Os (bpy) - (dcbpy) . This complex displays nigh anisotropy in frozen solution. This indicates that this compound can also oe useful as a prooe of protein rotation, i.e., affinity assays. Figure 13 illustrates tne emission spectrum of the Os complex. An important characteristic cf the Os complex is its long wavelength absorpticn and emission. It can be excited with laser diodes from 600 to over 700 nm, cr possibly a light emitting diode or an electroluminescent device. The extent of autofluorescence decreases at longer wavelengths. The lifetime cf the Os complex can be near 50 ns. This lifetime may be better than the Ru complex ,400 ns) for substances like serum albumin (MW == 70,000) with correlation times near 50 ns . The Ru complex may be better for higher molecular weight antigens. However, it snould be noted that some osmium Iigand complexes are known to display longer lifetimes near 400 ns . In this case the use of osmium metal- ligand complexes wiil have the combined advantages of the long lifetimes described for the ruthenium metal-ligand complexes, and in addition will display long wavelength absorption and emission. The long wavelength absorption allows excitation with laser diodes and other simple light sources. Figures 19 and 20 illustrate the absorption and anisotropy spectra of a Rhenium (Re) complex. The Re complex displays good polarization at a wide range of excitation wavelengths, and it should be useful in immunoassays and affinity assays. Figure 21 snows the emission spectra for an Re complex. The quantum yield and lifetime of an Re complex depend on temperature. Figure 22 snows immunoassay cased on a long wavelength absorbing osmium complex. As shown m Figure l' 7 , this complex displays hign anisotropy when excited near 500 or 700 nm. In Figure 22 we snow an immunoassay oetween tne osmium metal-Iigand complex labeled HSA, and monoclonal ana polycionai anti odies. The data in Figure 22 were cotamed with 500 nm excitation. We have obtained similar data with long wavelength excitation near 600 and 700 nm. Sucn wavelength are available from laser αioαes and other simpie light sources. The data in Figure 22, combmeα with the competitive assay m Figure 15, demonstrate that the competitive immunoassay is possible based on this osmium metal-Iiganα complex or similar metal-ligand complexes using long wavelength excitation. In a direct polarization assay of the present invention, the asymmetric metal-ligand complex can oe conjugated to a receptor, antibody or lectm. A receptor, antibody or lectm can also be used m a competitive immunoassay of the present invention, i.e., as the molecule which specifically binds the analyte. In the present invention, the sample of interest can be an antigenic suostance or other analyte with a molecular weight over 2,000. The antigenic substance can pe a protein, nucleic acid, polysaccharide, or compination of these substances Also, the antigenic suϋstance can be a cellular or cell surface antigen, glycopeptide, lipoprotem, or glycoiipid, whicn can be naturally present or due to a foreign object (such as bacteria or a virus) The exciting electromagnetic energy used in the present invention can be a linearly polarized light pulse, and the method can further comprise the step of measuring the polarization of the fluorescent light only after background autofluorescence of the coupled sample has subsided. The exciting step can implemented by a light source selected from the group consisting of a flash lamp, a modulated lamp, an electroluminescent device, a light-emitting diode and a laser (such as a diode laser or an amplitude modulated laser) . The light pulse and the fluorescent light can be transmitted througn optical fibers. The measuring step in the present invention can be performed using an implanted patch containing the coupled sample . In the methoα of the present invention, the steady state of linear polarized light can be dependent on a characteristic of the coupled sample or any uncoupled analyte wnicn is present. Also, the intensity decay cr polarization decay can be dependent on the coupled sample. The Ξ amount cf analyte can oe estimated from time-dependent anisotropy decay as measured following pulsed excitation. Also, the amount of analyte can be determined from the emission anisotropy decay measured with amplitude-modulated excitation by pnase-modulation fluorometry. 0 In a particularly preferred e oodiment of the present invention, the immunoassay is a competitive immunoassay. Another preferred emoodiment of tne present invention is an affinity assay. In tne affinity assay, the molecule which has affinity for the analyte can be selected from the group consisting s of strepavidin, avidm, oiotm, and lectms. A desirable type of affinity assay uses proteins 'e.g. , proteins wnicn bind glucose and polysaccharides, like Ccncanavalm A) . The invention can also pe used in affinity assays based on specific binding oetween macromoiecules . For instance, 0 Concanavalin A has affinity for dextran, and is displaced by glucose. The polarization ef a mixture of labeled Con A and dextran can be expected to display polarization values which depend on glucose concentration. The present invention will now oe described in further detail 5 by way of the following experimentation. Unless otherwise indicated, all parts, percents, ratios and the like are by weight. EXPERIMENTATION MATERIALS AND METHODS 0 RuCl,, Ru(bpy) : C1 : and Ru (bpy) -,Cl were purcnased from the Aldrich Chemical Company. Chemical synthesis of the NHS-ester of [Ru (bpy) 2 (dcbpy) ] 2* and of the more symmetric complex [Ru (dcbpy) 3 ] 2* was carried out as described in Figure 3. Synthesis of Ru bis (2 , 2 ' -bipyridine) (2 , 2 ' -bιpyrιdine-4 , 4 ' - 5 dicarboxylic acid) bis (hexafluorophosphate) (1) : Ru(bpy) 2 Cl 2 (0.4 g) , NaHCO, (0.4 g) and 2, 2' -bιpyrιdine-4 , 4 ' -dicarboxylic acid (0.3 g) were heated in MeOH:H : 0 = 4:1 for 8 to 10 hours. The solution was cooled in an ice bath for 2 hours, and the pH was adjusted with concentrated H-SO. tc 4.4. The fcrmed precipitate was filtered and 24 then washed with MeOH, tne filtrate was treated with 5 g NaPF c in 25 mi H 2 0 and then cooled in an ice path, and the precipitate was collected by filtration. Yield: 3.6 g (77%) . Synthesis of Ru tris (2 , 2 ' -bιpyrιdme-4 , 4 ' -dicarboxylic acid) Ξ bis nexafluoropnospnate) !2) : RuCl, 0.1 gi and 2 , 2' -bipyridine- 4 , 4 ' -dicarooxylic acid '3.67 gj were suspended in 15 ml ethylene glycci and refluxeα for 2 hours. The solution was cooled to room temperature and filtered. After the addition of 2.5 g NaPF in 25 ml H-,0, the pH cf the filtrate was adjusted to 1.0 with 0 concentrated H-,SO„, and the solution was cooled for a few hours. The precipitate was coliecteα and resuspended in MeOH, filtered and dried over P^O,,. Yield: 0.38 g (68%) . Syntnesis of the NHS esters. Ru tris (2 , 2 ' -bιpyrιdme-4 , 4 ' - dicarooxylic acid) N-hydroxysuccinimide ester (4) 0.46 g DCC and s 0.238 g N-hydroxysuccinimioe were dissolved in 3 ml DMF with stirring and cooled m an ice bath. A solution of 0.38 g Ru tris ι2 , 2 ' -bιpyrιdme-4 , 4 ' -dicarboxylic acid) (2) was added, and the mixture stirred for a few hours. The formed precipitate was removed by filtration tnrough a syringe filter, and the filtrate 0 containing the active Ru-complex was used for labeling the substrates . The proteins HSA, IgG, ConA ana Ferntin were ootained from Sigma Chemical Company and used without furtner purification. The proteins (10 mg portions) were labeled by adding a 100-fold molar 5 excess of the Ru-NHS ester m 50 μi of DMF to 1 ml of stirred protein solution (0.2 M caroonate buffer, pH 8.3 - 9.1) , followed by a 2-6 hour incuoation and purification of the laoeled protein by gel filtration cnromatography on Sephadex G-25 or G-50, using 0.1 M PBS, pH 7.2. 0 Fluorescence intensity and anisotropy decays were measured by time-correlated single photon counting (TCSPC) The primary light source was a cavity-dumped (1 MHz) pyπdme 1 dye laser, with the frequency doubled to 360 nm. This dye laser was pumped by a mode-locked Nd:YAG laser. The 360 nm output was 5 less useful for excitation of the Ru-complex because of the lower anisotropy at tnis excitation wavelength. Hence, the 360 nm laser pulses were generally used to illuminate a nearly saturated solution of peryiene in cyclohexane and a 483 nm interference filter to isolate the perylene emission, which was used to excite the Ru-complexes . The approximate 5 ns decay time of the "lamp" was easily short enougn for the 200 - 500 ns decay times displayed by the invention samples. Detection of the emission was accomplished with a Hammamatsu R2809 microchannel plate (MCP) PMT and the usual electronics for TCSPC. Some of the time-resolved intensity decays (Figs. 14 and 16) were obtained using 360 nm excitation. The time-resolved intensity decays (I(t)) were fit to the single and double exponential models, I < t) = ∑ a exp(-t/τ_) (9) - 1 where a are the pre-exponential factors and T are the decay times using software from IBH Software (Edmburgn, Scotland) . The "lamp" function was taken as the response observed from a scattering solution at 483 nm illustrated with the perylene "lamp" . The time-resolved anisotropy decays were obtained by measuring the time-dependent decays of the vertically ι.I«{t)) and norizontally I . t) components cf the emission: r ) ~ W - 1 - ^ (10) J,(t) +2J_(t) These data were fit to a single and double correlation time model, again using stanαard software. 2 r(t) = ∑ r 0 ,exp(-τ/θ.) ( ID ι=ι where r 0l are the amplitudes and 9_ are the rotational correlation times. Steady-state fluorescence data were obtained using a spectrofluoro eter from SLM Instruments, with magic-angle polarizer conditions and a Hamamatsu R-928 detector. The emission spectra are uncorrected. 26 RESULTS Absorption spectra of [Ru(bpy) dcbpy)] , here called the Ru-comp ex, are snown _n Figure -= These spectra are normalized to unity to facilitate comparison. The aosorption spectra of the Ru-compiex αepenαs on pH. At pH , the net charge on the complex is expected to oe zero, with two positive charges on the Ru ana two negative charges from the two dcbpy ligands The long-wavelength absorption spectra of the Ru- labeled proteins are similar, ana appear to be intermediate to that ooserved for tne Ru-complex at pH 7 and 0.1. These aosorption wavelengths allow excitation using simple blue LED, blue electroluminescent light sources, cr frequency-doubled laser diodes. Emission spectra cf [Ru(bpy; (dcbpy)] m aqueous solution are snown m Figure 5 The emission spectrum of tne Ru-complex at pH 7 0 is comparable to that ooserved for [Ru (dcbpy) ,] 4 with a small red-shift ' 5 nm) and significantly red shifted relative to [Ru(bpy) ] 2* by 28 nm. This suggests that the spectral properties of the Ru-complex are determined by the presence of a single dcbpy Iigand. Consequently, the anisotropy of [Ru(bpyι (dcbpy)] mav oe higner than that of more symmetrical complexes, oecause the excited state may oe localized between the metal and a single Iigand, rather tnan being delocalized among the three ligands. The emission spectra of the Ru- labeled proteins are similar and also appear to be intermediate to that observed for Ru-complex at pH 7 and 0.1 (see Figure 5) . Similar spectra and quantum yields were found for all the labeled proteins. A somewhat lower quantum yield was found for labeled Ferritin, which is probably due to the long wavelength absorption of Ferritin and the possibility of Forster and/or Dexter transfer from the Ru to the protein. The effect of oxygen quenching on quantum yields was also investigated. In the absence of oxygen, air equilibrated and oxygen equilibrated buffer solutions, the relative fluorescent intensities were 1, 0.77, 0.44 and 1, 0.89, 0.65 for [Ru(bpy) j (dcbpy) ] and Ru-HSA, respectively. While tnis probe is sensitive to dissolved oxygen, the sensitivity of Ru- complex-labeled proteins _s modest and will not require elimination of oxygen to ooserve the emission. The steaαy-state excitation anisotropy spectra were examined for [Rufppy ]- * , L Ru (dcbpy) ] ' , and [Ru (bpyj - >dcopy) ] Ξ free and labeled to HSA (see Figure 6; m vitrified solution where rotational diffusion αoes not occur curing tne excited state lifetime. Importantly, tne asymmetric complex [Ru (bpy) : (dcbpy; ] and its protein conjugates displayed anisotropies from 0.25 to 0.3 for excitation near 480-490 nm. In contrast, the anisotrcpy spectra of Rulbpy) ] - * and [Ru (dcbpy) ] * ' displayed consioeraoly smaller values at all excitation wavelengths aoove 450 nm. Evidently, tne presence of a non-identical liganα is important for ootain g a useful anisotropy probe. The steady-state anisotropy cr the -.aoeleα proteins ana of the Ru-compiex was examined ever a range of temperatures and/or viscosities (see Figure 7^ The solvent was 60% glycerol/40% buffer, which formed an optically clear glass at -55°C. At low temperatures (-55°C , the anisotropies were nearly identical 0 for the free Ru-complex and for the Ru-labeled proteins. The anisotropy values was about 0.25, which is close to 0.28 obtained at -70°C. In contrast, tne steady-state anisotropies of [Ru(bpy),]- * and [Ru(bpy) ] ■ " remained low at all temperatures . 5 For the Ru-complex and tne Ru-labeled proteins, the temperature-dependent anisotropies indicate that the anisotropies are sensitive to rotational motions isee Figure 7) . The steady-state anisotropy cf the free Ru-complex decreased rapidly aoove -50°C, whereas the anisotropies of the 0 Ru-labeled proteins decreased more slowly with temperature, and remained relatively nigh even at 20°C. The steady-state values were only moderately dependent on the molecular weight: Ferritin = 500,000, IgG = 160,000; ConA = 102,000; HSA = 65,000 daltons. As will be snown below, some of the anisotropy of the 5 Ru-protem complexes is lost by fast motions of the probe m addition to rotational motion of the proteins. Importantly, the anisotropies of the labeled proteins are always larger than tnat of the free Ru-compiex (see Figure ' , which indicates that protein hydrodynamics contributes to the anisotropy. The detection of rotational motions using these complexes is not an obvious result. A large number of published reports have suggested that the anisotropy and anisotropy decay cf the Ru metal-ligand complexes is due to intermoiecuiar processes such as randomization of the excited state among the three organic iigands and/or interactions with the solvent which result in localization of the excited state after randomization. The time-range cf anisotropy decay measurements is determined by the lifetime of the excited state. We used TCSPC to determine the luminescence lifetimes of the Ru-compiex and the Ru-labeled proteins. The intensity decays were closely approximated by a single decay time isee Figure 8) . The decay times of the labeled proteins were comparable to that of the Ru-compiex alone under a comparable experimental condition, as shown below in Table I . Table -. Fluorescence lifetime cf [Ru(bpy) - (dcbpy) ] and the labeled proteins.- Excitation 483 nm, emission aoove 540 nm, (Corning 3-67 filter), air equilibrated. % giycerol DV volume with puffer. Ru-free refers to xutopy, , cppy The decay times creaseα somewhat in the presence of giycerol, and at lower temperatures, but the overall range was only about two-fold (250 to 500 ns ) As might be expected, the lifetime of Ru-labeled Ferritin was somewhat smaller than tnat of the other proteins, wnich was prooably due to energy transfer to the long-wavelength aosorption of Ferritin. The long lifetime of tnese labels suggest that the Ru-complex can oe used to measure rotation correlation times as long as 1.5 μs, about three times the lαminescence lifetime. One may notice that the signal/noise ratio is only modest m tnese data (see Figure 8), which is αue to a comomation of factors including the inefficient "perylene lamp" and the slow emission rate cf tne complexes, which resulted a relatively low number of counts per timing channel f from about 1000 to 3000 counts) Nonetheless, these data are aαequate for these studies to determine tne usefulness of these metal-ligand complexes as anisotropy probes. It is noted that while the number of photon counts per channel is low, the total number of counts is high, near 10", and the decay times are well defined from these data. To demonstrate that the time-dependent anisotropy depends on rotational diffusion, the anisotropy of free [Ru (bpy) - (dcbpy) ] was examined in 60% giycerol-water (v/v) at varying temperatures ana viscosities (see Figure 9' At 20°C, the anisotropy decayed rapidly with a correlation time near 8 ns . As the temperature decreased, the anisotropy decayed more slowly, with the correlation time increasing to 240 ns at -30°C, and to over 1 μs at -51°C (see Figure 9) . Since the lifetime of the Ru-complex is near 500 ns, the intensity only decayed to about 60% of the initial value at 240 ns . Hence, it should be possible to measure still longer correlation times. At -51°C, the correlation time was longer than 1 μs, with some evidence of a more rapid component near 115 ns. The origin of this shorter component is unknown and may reflect the role of solvent relaxation localization of the excited state within the complex. Nonetheless, the near single exponential anisotropy decays and the apparent activation energy for rotation diffusion near 9.46 kcai/moie using data from Figure 9) supports the use of tne Ru-compiex as a rotational diffusion probe . Time-depenαent anisotropy decays of the tree Ru-complex Ξ and the Ru-labeled proteins are snown m Figure 10. For the Ru-compiex alone in a cuffer i.e., not coupled to proteins) , tne anisotropy decayed witnm tne 5 ns pulse width of the "perylene lamp". In contrast, the anisotropy decayed much more slowly for the Ru-labeled proteins. Importantly, the time- 10 dependent decreases m anisotropy oεcame slower as the molecular weignt of the labeled protein increased. Specifically, Ru-iabeied Ferritin displayed tne slowest anisotropy decay, ConA displayed the most rapid anisotropy decay, and IgG displayed an intermediate decay. While one IΞ mignt expect the anisotropy αecay of ConA tMW 1C2,000 for the tetramer) to be slower than HSA MW 65,000) , it is not known whether the ConA subunits dissociate on this timescaie, and the shapes of these two proteins may differ. In any event, the data m Figure 10 demonstrate that the anisotropy decays of the 20 Ru-labeled proteins are sensitive to the size and/or shape of the proteins. In fact, these data have already suggested the presence of a multi-exponential anisotropy decay for IgG, in contrast to the single exponential anisotropy decays of HSA and ConA. 25 Additional anisotropy decays are snown in Figures 11A, 11B, 12A and 12B. The data for ConA and IgG demonstrate that the Ru-compiex displays a slower anisotropy decay as protein rotational diffusion is slowed by adding giycerol (Figures 11A and 11B) . At a given giycerol concentration, the anisotropy 30 decay is slower at lower temperatures (Figures 12A and 12B) . The longest measured correlation time was 807 ns, as estimated from the anisotropy decay of Ferritin 30% giycerol at 5°C. Correlation times longer than 1 us were ooserved, as set forth below in Table II, Put they were not well resolved. 35 Figure 13 snows a polarization immunoassay of human serum albumin. In this case the HSA was labeled with [Ru(bpy) -dcbpy] . The labeled HSA was titrated with HSA- specific IgG. The polarization increased by approximately 200% upon binding to antibody. The open circles show the polarization measured wnen tne laoeied HSA was titrated with nonspecific antiboαy. In this case the fluorescence polarization remameα uncnangeα. Figure 14 snows tne time-αepenαent anisotropy αecays of labeled HSA m the aosence and presence cf HSA-specific antioody One notices that the anisotropy decay was much slower m the presence of HSA-specific IgG than for labeled HSA alone. This ooservation indicates that binding of IgG to HSA was slowing tne rotational motions of the Ru-compiex. Figure 15 snows a competitive immunoassay for HSA. In this case, HSA was labeled with tne Ru-complex, and this labeled HSA was partially saturated with antibody. The presence of unlabeled HSA in the sample was observed by a decrease the fluorescence polarization. The decrease in polarization resulted from the competitive omdmg of labeled and unlabeled HSA to the antibody. Figure 16 snows the time-dependent anisotropy decay for the competitive immunoassay. The anisotropy decayed more rapidly as the concentration of analyte (unlabeled HSA) was increased. This effect was observed because the unlabeled HSA competes for mdmg to the antibody, preventing the binding of labeled HSA to the antibody. Figure 17 snows the aosorption and anisotropy spectrum of an Os complex, Os (bpy) - (dcbpy) This complex displays high anisotropy in frozen solution. This indicates that this compound can also oe useful as a probe for measurement of protein rotations, i.e. , affinity assays. An important characteristic of the Os complex is its long wavelength absorption and emission. It can be excited with laser diodes from 600 to over 700 nm, or possibly a light emitting diode or an electroluminescent device. The extent of autofluorescence from biological samples decreases at longer excitation wavelengths. Figure 18 shows the emission spectrum of the Os complex. The lifetime of the Os complex is near 50 ns . This lifetime may oe better tr.an tne Ru-compiex 400 ns, for sucstances like serum albumin (MW == 70,030) with correlation times near 50 ns. The Ru-complex -.ay ee cetter for igner -.oiecuiar weight antigens. Again, we note that there are Os complexes with decay times near 400 ns , wnicn nave the comomeo advantages of long wavelength excitation ana emission and long decay time. Figures 1? and 13 snow tne aosorption and anisotropy spectra cf a Rhenium Re) complex. Figure 21 shows its emission spectrum. The Re ccmpiex displays αooα polarization, and it sncu-d be useful immunoassays and affinity assays. Table Anisotropy decays c: L ΛU ■ cpyj dcbpy; ] and Ru-labeled proteins. ** a Excitation 483 nm, emission above 540 nm, iCorning 3-67 filter), air equilibrated. The viscosities at 20°C as estimated to be 1.02, 3.0 and 17 cP for buffer, 30% and 60% giycerol, respectively. Does not fit two components Ru-free refers to Ru(bpyj - (dcbpy) . As can ce seen rrcm the aoove, cr.e peiaπzeα emission from metal-Iigand complexes offers numerous experimental opportunities in ciepnysics and clinical chemistry. A wide range cf lifetimes, aosorpoicr. and emission maxima can be ≡ obtained cy careful selection of one metal ano one Iigand. Absorption wavelengths as _cng as 730 nm can ce cotained using osmium, and lifetimes as long as 130 us can oe cotained using rhenium as tne metal sucn complexes. The rnenium cempiexes also display good quantum yields in aqueous solution. i: The anisotropy decays snown aoove indicate a considerable mopility of the present .Ru-complex wnicn is independent of overall rotational diffusion. If tne independent motions can pe decreased m amplitude, then a nigner fraction cf one total anisotropy will oe avaiiaoie to detect tne overall -Ξ Hydrodynamics cf tne proteins. This could ce accomplished by structural variants cf tne .Ruippy- dcbpy) complex. It snouid also oe noted that such long-lived probes can be useful for studies of diffusive processes a timescale presently not accessible cy tne usual fluorescence prooes . For 0 instance, there is consideraoie interest m the rates and amplitudes cf domain-to-dc am motions in proteins, and there have been repeated attempts to study sucn motions cy time- resoived fluorescence resonance energy transfer FRET! . These measurements have ceen mostly unsuccessful due to the 5-10 ns is decay times and the limited extent of mterdomam motions on this timescale. The use cf longer lived MLC emission can allow measurement of these motions . Finally, it is noted that the MLC can provide considerable information en rotational processes using only steady-state so data. The emission cf these complexes can be quenched by a variety cf molecules and ions, typically oy pnoto-induced electron transfer to the quencher. The long lifetimes of these complexes suggests that the lifetimes of tne labeled macromolecules can oe varied over a wide range with modest 35 concentrations of quencher. Steady-state anisotropy measurements, as a function of lifetime or quencher concentrations, can oe used to determine tne anisotropy decay law of membrane and protein-bound fiuorophores . While the invention has oeen described in detail and with reference to specific examples thereof, it will be apparent to one skilled in tne art that various changes and modifications can oe made therein without departing from tne spirit and scope th r o

Claims: WHAT Ξ CLAIMED IS: 1. A method of conducting an immunoassay of a sample of interest, comprising tne steps of: coupling a luminescent asymmetric metal-ligand complex to tne sample of interest to form a coupled sample; exciting tne coupled sample with linearly polarized electromagnetic energy to cause the coupled sample to emit polarized fluorescent Iignt ; and measuring tne polarization cf tne fluorescent light emission as a measure cf a ciolcgical characteristic of one samp-e cf interest . Z . A metnod ;: ciai _, wnere tne metax _n said metal- Iigand complex is selectee from the group consisting of ruthenium, osmium, rnenium, rnodium, _πdιum, tungsten and platinum. 3. A method of claim 1, wnerem the sample of interest is an antigenic suostance - ~ r ether analvte with a molecular weight ever 2,000. 4. A method of c-.aιm 1, wnerem tne exciting electromagnetic energy is a linearly polarized iignt pulse, and the method further comprises the step of measuring the polarization of tne fluorescent light only after oackground autofluorescence of tne coupled sample nas suosiαed. Ξ . A method of claim 4 , wnerem the exciting step is implemented by a light source selected from the group consisting of a flasn lamp, a modulated lamp, an electroluminescent device, a light-emitting diooe, a diode laser, and an amplitude modulated laser. 6. A metnod of claim -±, wnerem the light pulse and the fluorescent light are transmitted throuαn cotical fibers. 7. A method cf claim 1, wnerem tne measuring step is performed using an implanted paten containing the coupled sample . 8. A metnod cf claim 1, wnere a steady state of polarization is dependent on a characteristic cf the coupled sample or any uncoupled analyte which is present . 9. A method of claim 1, wherein intensity decay or polarization decay is dependent on tne coupled sample. 10. A method of claim 3, wnerem tne antigenic suostance is a protein, nucleic acid, or pciysaccnaπαe. 11. A metneα of claim 3, wnerem tne antigenic suostance is a cellular or cell surface antigen, glycopeptide, lipoprotem, cr glycoiipid, which can be naturally present or due to a foreign object. 12. A method of claim 1, wnerem the amount cf analyte is estimated from ime-dependent anisotrcpy decay as measured following pulsed excitation. 13. A method cf claim 1, wnerem tne amount of analyte is determined from emission anisotropy decay measured with amplitude-modulated excitation byphase-modulation fluorometry. 14. A method of claim 1, wnerem 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 (I; an asymmetric metal-Iigand complex conjugated to a molecule which specifically binds said analyte with (2) said sample,* P) exciting tne -ixture cr step a; with linearly polarized light tc cause the ccmpiex to emit polarized light ; c » measuring tne polarization of the _ιgnt emitted py said complex; d 1 oaicu_atmg tne amount of anaiyte tne sample oy correlating tne polarization measured step \ c ι with the polarization cf Iignt emitted from a control sample containing a kncwn amount cf anaiyte. 16. A fluorescence pc_arιzatιcr. assay or claim 15, wnerem tne metal _n said asymmetric -etal-Iigand complex is se_ected from the croup consisting of rutnenium, osmium, rr.emurr, mooium, orieium, tungsten and platinum. 17. A fluorescence polarization, assay of claim 15, wnerem tne Iigand in said metal-ligand complex comprises poiypyπdme, oipyrid e or a related ccmpound and can further comprise C , Cl, pnospnme, nitriie or isonitrile groups. 13. -. fluorescence polarization assav of claim 17, wnerem said Iigand contains a reactive group selectee from the group consisting cf a -nydrcxysuccmimide ester of a carooxylie acid, r.aioacetyl groups, suifonyi cnlorides, maieimides, and isotniocyanates . 19. A fluorescence polarization assay of claim 17, wherein said molecule is a receptor, antibooy or lectm. 20. A fluorescence pciarizaticn assay of claim 15, wnerem the Iigand m said metal-Iigand complex comprises a bipyrazyl group, a pnenanthrolme group, or a related compound. 21. A competitive fluorescence polarization immunoassay for quantifying the amount of an analyte a sample, comprising tne steps cf : .a) mixing ,_: a control containing a Known amount cf anaiyte conjugated to an asymmetric metal-ligand complex with Z a molecule wnich specifically binds the analyte; (b) exciting the mixture cf step .ai with linearly polarized light to cause tne ccmplex to emit polarized light ; 'c) measuring the polarization of the light emitted oy the complex; id; adding the sample to the mixture to form a new mixture including anaiyte not conjugated wnich competes with the anaiyte conjugated to the asymmetric metal-ligand complex binding to tne molecule wnich specifically binds the anaiyte, thereby causing a change m polarization,* (e) measuring tne change m polarization; (f) calculating the amount cf analyte in the sample oy correlating the cnange in polarization with the control containing a known amount of analyte. 22. A competitive fluorescence polarizaticn immunoassay of claim 21, wherein the metal said asymmetric metal-Iigand complex is selected from tne group consisting of ruthenium, osmium, rhenium, medium, iridium, tungsten and platinum. 23. A competitive fluorescence polarization immunoassay of claim 21, wherein the Iigand in said metal-ligand complex comprises polypyridine, bipvridme, or a related compound. 24. A competitive fluorescence polarization immunoassay of claim 23, wnerem said Iigand contains a reactive group selected from the group consisting of a N-hydroxysuccmimide ester cf a carbcxylic acid, aioacetyl groups, sulfonyi chlorides, maleimides, and isothiocyanates . 25. A competitive r_uorescencε polarization immunoassay claim 23, wnerem said molecule is a receptcr, antioodv or 26. A competitive riuorescence polarization immunoassay of claim 21, wnerem the Iigand m said metal-liganα complex comprises a pipyrazyi, a pnenanthrolme, or a related cempound and can further comprise CO, Cl , phospnme, nitriie or : isonitrile groups. 27. A metnod of conducting an affinity polarization assay of a sample of interest to quantify tne amount cf an analyte in tne sample, comprising tne steps cf : ai -lxmg 1, a control containing a Known amount _ of anaiyte conjugated to an asymmetric metal-ligand complex v.itn I a molecule wnicn has affinity for the anaiyte ; (b) exciting the mixture of step ιa) with linearly polarized Iignt to cause the complex to emit polarized ic light; ] -easurir.g tne polarization cf the light emitted oy the compIex,* 'd) adding tne sample to the mixture to fcrm a new mixture including anaiyte not conjugated wnicn competes is with tne anaiyte conjugated to tne asymmetric metal-ligand complex m associating with the molecu.e wnich has affinity for the analyte, tnereoy causing a change polarization; le) measuring the cnange m polarized emission,* 20 (f) calculating the amount of analyte m the sample by correlating the change m polarization with the control containing a Known amount of analyte. 28. A method of claim 27, wnerem the molecule wnich has affinity for the anaiyte is selected from the group consisting of streoavidm, avidin, iotin and iectins. 29. A method cf claim 27, wherein tne molecule which has affinity for the analyte is a protein. 30. A method of claim Z ~ , wnerem the metal in said metal-Iigand complex is selected frcm tne group ccnsisting of ruthenium, osmium, rnenium, rnodiu , indium, tungsten and oiatmum.

Inventors: LAKOWICZ JOSEPH R, SZMACINSKI HENRYK, TERPETSCHNIG EWALD, LAKOWICZ, JOSEPH, R., SZMACINSKI, HENRYK, TERPETSCHNIG, EWALD
Applicants: LAKOWICZ JOSEPH R, SZMACINSKI HENRYK, TERPETSCHNIG EWALD, LAKOWICZ, JOSEPH, R., SZMACINSKI, HENRYK, TERPETSCHNIG, EWALD
Application year: 1995
Main classification: G01N33
IPC codes: G01N33