WO1999031513A1

WO1999031513A1 - Method and composition for characterizing a sample containing a sample lipid and an assay kit

Info

Publication number: WO1999031513A1
Application number: PCT/US1998/026559
Authority: WO
Inventors: Joseph R. Lakowicz
Original assignee: Lakowicz Joseph R
Priority date: 1997-12-15
Filing date: 1998-12-14
Publication date: 1999-06-24
Also published as: AU1824899A

Abstract

A method of characterizing a sample containing a sample lipid includes the step of adding a metal-ligand complex to a lipid-containing sample to form a mixture containing labeled sample lipid. The labeled sample lipid includes the sample lipid labeled with the metal-ligand complex lipid probe. The mixture is exposed to an exciting amount of electromagnetic light energy which causes the labeled sample lipid to emit fluorescent light. The emitted light is measured. A composition for characterizing a lipid containing sample includes a fluorescent metal-ligand complex coupled to a lipid. An assay kit includes a fluorescent metal-ligand complex and instructions for coupling the fluorescent metal-ligand complex with a lipid.

Description

METHOD AND COMPOSITION FOR CHARACTERIZING A SAMPLE CONTAINING A SAMPLE LIPID AND AN ASSAY KIT

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is in the field of characterizing a sample containing a sample lipid.

Description of the Background Art Measurement of fluorescence is widely used in the biomedical sciences, in clinical chemistry, and diagnostic assays.

Cell membranes are predominantly composed of phospholipids, which are spectroscopically silent in the ultraviolet and visible regions of the spectrum. Consequently, extrinsic fluorophores embedded in the membranes or attached to the phospholipids are typically used to study the structure and dynamics of the membranes. The physical properties of membranes have been studied using energy transfer, solvent- sensitive fluorophores, and anisotropy measurements. However, the vast majority of membrane probes display decay times of 1-10 nanoseconds, which limits the information content of the measurements to phenomena which can affect the excited state on this timescale. Consequently, most fluorescence experiments are not able to reveal the rates of lipid diffusion in bilayers or phenomena on the microsecond timescale. The limitations of short decay times have been circumvented using phosphorescence. However, the use of phosphorescence requires the complete exclusion of oxygen, and the phosphorescence time-zero anisotropies are often low.

U.S. Patent No. 5,322,794 discloses coronene- labeled lipids and methods for characterizing lipids . However, coronene-labeled lipids can only exhibit single lifetimes.

Fluorescent liposomes have been used for immunoassays . Fluorescein-phosphatidyl ethanolamine has been used in heterogeneous immunoassays. Liposomes are useful in high sensitivity assays because many fluorophores can be bound to one liposome. However, fluorophore-derivatized liposomes are sensitive to concentration quenching.

Terpetschnig, E., Szmacinski, H., and Lakowicz, J.R., "Fluorescence Polarization Immunoassay of a High- Molecular-Weight Antigen Based on a Long-Lifetime Ru- Ligand Complex," Analytical Biochemistry 227:140-47 (1995), describes a known immunoassay method which utilizes detection of fluorescence. There remains a need in the art for improved methods of characterizing lipid containing samples.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method of characterizing a sample containing a sample lipid includes the step of adding a metal-ligand complex lipid probe to a lipid-containing sample to form a mixture containing labeled sample lipid. The labeled sample lipid is comprised of the sample lipid labeled with the metal-ligand complex lipid probe. The mixture is exposed to an exciting amount of electromagnetic light energy which causes the labeled sample lipid to emit fluorescent light. The emitted light is measured.

Also in accordance with the present invention a composition for characterizing a lipid containing sample includes a fluorescent metal-ligand complex coupled to a lipid.

Further in accordance with the present invention an assay kit includes a fluorescent metal-ligand complex and instructions for coupling the fluorescent metal-ligand complex with a lipid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting metal-ligand complex lipid probes made with rhenium, ruthenium, and osmium that have different emission wavelengths.

FIG. 2 shows long-lifetime rhenium carbonyl complexes that can be attached to lipids.

FIG. 3 graphically depicts absorption and emission spectra of Ru-PE and Ru-PE₂ in DPPG vesicles at 20°C, and the excitation anisotropy spectra of the parent compounds of Ru (bpy) ₂ (mcbpy) ⁺¹ and Ru (bpy) ₂ (dcbpy) in glycerol/water at -55°C.

FIG. 4 graphically depicts the frequency-domain intensity decays of MLC-PE and MLC-PE₂ in DPPG vesicles at various temperatures.

FIG. 5 graphically depicts temperature dependent steady state anisotropies of DPPG vesicles labeled with Ru-PE or Ru-PE₂.

FIG. 6 shows structures of two MLC lipid probes: Ru (bpy) ₂ (mcbpy) -PE (Ru-PE) and Ru (bpy) ₂ (dcpby) -PE₂ (Ru- PE,) . FIG. 7 graphically depicts frequency-domain anisotropy decays of Ru-PE in DPPG vesicles.

FIG. 8 graphically depicts frequency-domain anisotropy decays of Ru-PE₂ in DPPG vesicles.

DETAILED DESCRIPTION OF THE INVENTION In accordance with one embodiment of the present invention, a method is disclosed for characterizing a sample lipid by measuring the light emitted by the sample lipid labeled with a metal-ligand complex lipid probe .

In accordance with another embodiment of the present invention, a composition is provided, which includes a fluorescent metal-ligand complex, for characterizing a lipid containing sample.

In accordance with another embodiment of the invention, an assay kit is provided, which includes a metal-ligand complex and instructions for coupling the metal-ligand complex with a lipid. Metal-ligand complex lipid probes are particularly useful because they display decay times longer than any available lipid probe. Additionally, the use of metal- ligand complexes provides probes with a range of lifetimes. This is in contrast to the single lifetime available from coronene-labeled lipids.

Fluorescence detection has previously been limited by the ability to detect signal over background. Metal-ligand complex lipid probes allow increased high sensitivity detection by gating off the prompt autofluorescence from the samples. An advantage of using metal-ligand complex lipid probes in immunoassays is that metal-ligand complexes are less sensitive to the concentration quenching that occurs with fluorescein. Lipid probes in accordance with the invention that contain covalently linked metal-ligand complexes have been synthesized. The inventive long lifetime metal- ligand complex lipid probes can be used to characterize lipid-containing samples . The change in fluorescent light emission of the probe and fluorescent light emission of the sample lipid labeled with the metal- ligand complex lipid probe is utilized to characterize the lipid.

The intensity of the emitted light can be measured and used to characterize the sample lipid. The lifetime of the emitted light can also be measured and used to characterize the sample lipid. If linearly polarized light energy is used to excite the mixture of the sample lipid labeled with the metal-ligand complex lipid probe, then the lipid is characterized by measuring the polarization of the emitted light.

Metal-ligand complex lipid probes can be used to characterize the membrane dynamics of lipid-containing samples. Metal-ligand complex lipid probes can be used to measure the rotational hydrodynamics of phospholipid vesicles . These probes can be used to measure microsecond rotational motions of lipid vesicles or within lipid membranes.

Metal-ligand complex lipid probes can also be used to measure two-dimensional diffusion in membranes.

These probes can be used in studies of energy transfer in which the purpose is to determine two dimensional diffusion coefficients in membranes.

The probe lipid can be any suitable lipid including, but not limited to, phospholipids and phosphatidyl ethanolamine .

Metal-ligand complex lipid probes can also be used in clinical assays based on liposomes. Metal-ligand complex lipid probes can be used in homogeneous and heterogeneous assays. Metal-ligand complex lipid probes can be used in homogeneous assays because of the long lifetime of the metal-ligand complexes, which allows gating and using fluorescence polarization or anisotropy which is independent of intensity. Examples of sample and probe lipids include, but are not limited to, liposomes and phospholipids, respectively.

The metal-ligand complex lipid probes of the present invention may be used in place of the known fluorophores in any suitable assay method.

There are a number of metal-ligand complexes which display luminescence, including complexes containing Co,^' Cr, Cu, Mo, Ru, Rh, W, Re, Os, Ir, or Pt . In particular, transition metal complexes, especially those with Ru, Os, Re, Rh, Ir, W or Pt, can be used in accordance with the invention. The metal in the metal- ligand complex is particularly preferably selected from the group consisting of ruthenium, osmium, and rhenium. A suitable ligand in the metal-ligand complex can be polypyridine, bipyridine, or a related compound, and the ligand can contain a reactive group commonly used for linkage to biological molecules, such as a N- hydroxysuccinimide ester of a carboxylic acid, haloacetyl groups, maleimides, sulfonyl chlorides, and isothiocyanates . Other ligands for such metal-ligand complexes are bipyrazyl, phenanthroline, and related substituted derivatives, or inorganic ligands such as CO, Cl, nitrile and isonitrile.

Preferred metal-ligand complexes include [Ru(2,2'- bipyridine) ₂ (4-carboxy-4 ' -methyl-2, 2 ' -bipyridine) ] ; [Ru (2,2' -bipyridine) ₂ (4, 4 ' -dicarboxy-2, 2 ' -bipyridine) ] ; [Re (2, 9-dimethyl-4,7-diphenyl-l, 10- phenanthroline) (CO) ₃ (monosubstituted pyridine)]*; [Re (3, 4,7, 8-tetramethyl-l, 10- phenanthroline) (CO) ₃ (monosubstituted pyridine)]⁺; [Re(5- phenyl-1, 10-phenanthroline) (CO) ₃ (monosubstituted pyridine) ]⁺; [Re (4 , 7-diphenyl-l, 10- phenanthroline) (CO) ₃ (monosubstituted pyridine)]*; [Re ( 1 , 10-phenanthroline) (CO) ₃ (monosubstituted pyridine) ]⁺; and [Re (2,2'- bipyridine) (CO) ₃ (monosubstituted pyridine) ] ⁺ .

One embodiment of the present invention utilizes phospholipid analogues of phosphatidyl ethanolamine which contain a ruthenium metal-ligand complex covalently bound to the amino group. A detailed description of the synthesis and fluorescence characterization is'^'disclosed in Li, L., Szmacinski, H., and Lakowicz, J.R., "Synthesis and Luminescence

Spectral Characterization of Long-Lifetime Lipid Metal- Ligand Probes," Analytical Biochemistry 244:80-85 (1997) and Li, L., Szmacinski, H., and Lakowicz, J.R., "Long-Li etime Lipid Probe Containing a Luminescent Metal-Ligand Complex," Biospectroscopy 3:155-159 (1997), incorporated herein by reference. Another embodiment of the invention utilizes metal-ligand complex lipid probes in assays. A description of a prior art use of fluorescent liposomes for immunoassay is disclosed in Singh, A.K., Kilpatrick, P.K., and Carbonell, R.G., "Application of Antibody and Fluorophore-Derivatized Liposomes to Heterogeneous Immunoassays for D-dimer, " Biotechnol . Prog-,. 12:272-80 (1996).

In another embodiment of the present invention, a metal-ligand complex lipid probe is utilized in a fluorescence polarization immunoassay of a high molecular weight antigen. The present invention thus can be incorporated into the method disclosed in Terpetschnig, E., Szmacinski, H., and Lakowicz, J.R., "Fluorescence Polarization Immunoassay of a High- Molecular-Weight Antigen Based on a Long-Lifetime Ru- Ligand Complex," Analytical Biochemistry 227:140-47 (1995), incorporated herein by reference.

The invention is further illustrated by the following examples, which are not intended to be limiting .

Example 1 :

Ru (bpy) ₂ (mcbpy) (360 mg) and 51 mg of N- hydroxysuccinimide (ΝHS) were dissolved in 1.2 ml of acetonitrile at room temperature. The synthesis of Ru (bpy) ₂ (mcbpy) is described in Szmacinski, H., Terpetschnig, E., and Lakowicz, J.R. (1996) Biophys . Chem.. in press. N, N'-Dicyclohexylcarbodiimide (DCC, 120 mg) was then added. The mixture was sealed and stirred for a few hours. The formed precipitate was removed by filtration through a syringe filter (Nylon Acrodish, 0.45-um pore size). The filtrate was added to a stirring solution of 2-propanol. The mixture was kept at -4°C for 1 hour. The precipitate, Ru (bpy) ₂ (mcbpy) -NHS (120 mg) was collected by filtration and washed with dry ether (3 * 5.5 ml) with an approximate 70% yield. The same procedure was used for the preparation of NHS-ester of Ru (bpy) ₂ (dcbpy) . Ru (bpy) ₂ (mcbpy) -NHS (120 mg) was dissolved in 4.5 ml of dry DMF and slowly added to a stirring solution of PE 980 mg in 10 ml of CHC1₃) and triethylamine (6.0 ml) under an argon atmosphere. The mixture was stirred for 20 hours in the dark. The solvents were removed under vacuum and the product was redissolved in 2.5 ml of CHCl₃/MeOH (2/1, v/v) . The pure Ru-PE was obtained by TLC on K6F silica gel plates using CHCl₃/MeOH/H₂0

(65/25/4, v/v/v) as the developing solvent with a 15% yield. The JR_f value of the product is near 0.6, relative to that of PE (0.78). Ru-PE₂ was prepared in analogy to Ru-PE. The same developing solvent, CHCl₃/MeOH/H₂0 (65/25/4), was used to purify Ru-PE₂, whose R_f value is near 0.70. The purification by TLC was repeated twice to obtain pure Ru-PE₂, with a final, yield of about 2.3%.

Emission spectra were recorded on a SLM AB2 spectrofluorometer . The frequency-domain instrumentation (ISS) was used for measurements of the fluorescence intensity and anisotropy decays. The excitation source was a CW air-cooled argon ion laser (543-AP, Omnichrome) . The laser was amplitude modulated by the electrooptical low-frequency modulator (K2.LF from ISS) and was tuned at 488 nm as the excitation wavelength. In those measurements a 610-nm cutoff filter (Corning 2-61) was also used to isolate the emission of Ru complexes.

The frequency-domain intensity and anisotropy data were fit to single and double-exponential decay laws.

The intensity decays were described by

I (t) = ∑ α^N , [1] i

where x are the amplitudes of the intensity decay times τ_if with Σot_L = 1.0. The anisotropy decays were fit to r (t) = ∑ q_{t 0}e-^t/θ _{i f} [ 2 ] j

where g_ir₀ is the amplitude of the anisotropy decaying with the correlation time θ_j, and r₀ is the anisotropy in the absence of rotational diffusion. The amplitudes of the anisotropy decay components ( r₀g_L ) are restricted so that _g^ = 1.0. The parameter values were recovered as described in Lakowicz, J.R., Gratton, E., Laczko,

G., Cherek, H., and Limkeman, M. (1984) Biophys . J. 46, 463-477; Gratton, E., Lakowicz, J.R., Maliwal, B., Cherek, H., Laczko, G., and Limkeman, M. (1984) Biophys. J. 46, 479-486; Lakowicz, J.R., Cherek, H., Kusba, J., Gryczynski, I., and Johnson, M.L. (1993) J. Fluoresc. 3, 103-116.

For vesicle preparation, lipids with a Ru- lipid/DPPG mole ratio ranging from 1:20 to 1:100 were dissolved in CHCl₃/MeOH (95/5, v/v) . The lipid- containing solution was kept in a water bath at a constant temperature (55°C) , and the solvent was removed by a stream of argon. Vesicles were prepared by sonication in 10 mM Tris and 50 mM KCl, pH 7.5, at a final lipid concentration of 2 mg/ml. The DPPG vesicles in the absence of Ru-lipid did not display significant signals (<1.5%) under the present experimental conditions. Unless indicated otherwise, all measurements were performed in the presence of dissolved oxygen from equilibrium with the air.

Absorption and emission spectra of DPPG vesicles labeled with Ru-PE and Ru-PE₂ are shown in Fig. 3. These spectra are similar except for a slightly longer absorption and emission spectra for Ru-PE₂, which contains 4 , 4'-dicarboxy-2, 2'-bipyridine as one of the ligands. These results are in agreement with previous studies of the mono- and dicarboxy derivative of Ru(bpy)₃ ²⁺. Szmacinski, H., Terpetschnig, E., and Lakowicz, J.R. (1997) Biophys. Chem. 62:109-120. Similar emission spectra were observed independent of temperature from 2 to 53°C, except for a progressive decrease in intensity with increasing temperature. At 25°C the quantum yields of Ru-PE and Ru-PE₂ in DPPG vesicles, in air equilibrium, appear to be about 0.044 and 0.034, respectively, as seen by comparison with a deoxygenated aqueous solution of Ru(bpy)₃ ²⁺ with an assumed quantum yield of 0.042. Van Houten, J., and Watts, R.J. (1975) J. Am. Chem. Soc. 97(13), 3843-3844. For the metal-ligand complex lipid probe to be useful as hydrodynamic probes, the metal-ligand complexes need to display polarized emission. The excitation anisotropy spectra of the parent compounds Ru(bpy) (mcbpy) ⁺¹ and Ru(bpy) (dcbpy) are shown in Fig. 3. In the absence of rotational diffusion, in glycerol/water (6/4, v/v) at -55°C, these complexes display maximal anisotropies of 0.17 and 0.23, respectively. These values are adequate for measurement of steady state and time resolved anisotropies. The lower anisotropy of Ru (bpy) ₂ (mcbpy) ⁺¹ is consistent with earlier reports of this complex coupled to proteins. Szmacinski, H., Terpetschnig, E., and Lakowicz, J.R. (1996) Biophys. Chem. 62:109-120. These studies showed that the excitation anisotropy spectra of the MLC probes covalently linked to the amino groups of proteins are similar to the spectra of the free carboxylic acids. Terpetschnig, E., Szmacinski, H., Malak, H., and Lakowicz, J.R. (1995) Biophys. J. 68, 342-350; Szmacinski, H., Terpetschnig, E., and Lakowicz, J.R. (1996) Biophys. Chem. 62:109- 120. Hence we expect^' the anisotropy spectra of the parent metal-ligand complexes to reflect that of the lipid probes, which could not be measured directly due to low solubility in glycerol/water. Frequency-domain intensity decays of the two lipid probes are shown in Fig. 4. These decays are closely approximated by a single exponential decay. The mean decay times of the two lipid probes are comparable and range from 682 ns at^' 2°C to 357 ns at 53°C.

TABLE 1

Fluorescence Lifetimes (ns) of Ru (bpy) ₂ (mcbpy) -PE and Ru (bpy) ₂ (dcbpy) -PE₂ in DPPG Vesicles at Various Temperatures³

Temperature (°C)

Ligand 2 10 20 31 35 41 53 mcbpy 682 593 518 463 357 dcbpy 616 559 487 456 408 405

Solutions were in equilibrium with the air

These long decay times suggest that these probes can be used to measure rotational motions in membranes to nearly 2 μs, or about three times the mean decay time. Such long correlation times are typically measured from the phosphorescence anisotropy decay of eosin or erythrosin. Burkli, A., and Cherry, R.J. (1981) Biochemistry 20, 138-145; Mersol, J.V., Kutchai, H., Mahaney, J.E., and Thomas, D.D. (1995) Biophys. J. 68, 208-215; Gonzalez-Rodriguez, J. , Acun, A.U., Alvarez, M.V., and Jovin, T.M. (1994) Biochemistry 33, 266-274;^" Voss, J.C., Mahaney,,. J.E. , Jones, L.R., and Thomas, D.D. (1995) Biophys. J. 68, 1787-1795. However, the use of phosphorescence requires the rigorous exclusion of oxygen. In contrast, the metal-ligand complex lipid probes are only moderately sensitive to dissolved oxygen. For instance, at 4 and 45°C the decay times increased by about 8 and 25%, respectively, upon removal of dissolved oxygen. Hence, exclusion of oxygen is not needed when using these probes.

The steady state anisotropies of DPPG vesicles labeled with Ru-PE or Ru-PE₂ were studied over a range of temperature spanning the transition temperature of 41°C. The anisotropies decrease progressively with increasing temperature as expected for thermally activated motions (Fig. 5) . However, there is no sharp phase transition of the type seen with DPH-labeled membranes. This suggests that the metal-ligand complex lipid of the probe is localized at the lipid-water interface, as suggested from the chemical structure (Fig. 6). The anisotropies of Ru-PE₂ are consistently larger than those of Ru-PE, as expected from the higher fundamental anisotropy of Ru (bpy) ₂ (dcbpy) and possibly due to less segmental motion of the MLC covalently bound to two PE molecules.

To determine the usefulness of these probes for micro-second membrane hydrodynamics the frequency- domain anisotropy decays of DPPG vesicles labeled with Ru-PE (Fig. 7) or Ru-PE₂ (Fig. 8) were examined. Analysis of these data in terms of a double-exponential anisotropy decay are summarized in Table 2.

TABLE 2

Rotational Correlation Times and Amplitudes for Ru- (bpy) ₂ (mcbpy) -PE and Ru (bpy) ₂ (dcbpy) -PE₂ in DPPG Vesicles at Various Temperatures

Ru (bpy) ₂ (mcbpy) -PE Ru (bpy) ₂ (dcbpy) -PE₂

T ( °c; c9_R(ns) ₀9i 6>_R(ns) r_Qg_±

22 116633 00..1100 113333 0.062

5795 0.083 1761 0 . 145

10 181 0.085 135 0 . 064

>14410 0.063 1337 0 . 112

20 107 0.081 99 0 . 087

>>1155445500 00..003388 11339933 0.071

31 — 117 0.082 1569 0.041

35 124 0.061 -

>10000 0.001

4411 -- 9922 0.080

53 100 0.033 84 0.055

For Ru-PE₂, the anisotropy decays display both a short { - 100 NS) and long [ ~ 1.5 μs) correlation time. These longer correlation times are consistent with those expected for overall rotational diffusion of phospholipid vesicles with diameter from 200 to 300 A: For instance, vesicles with a diameter of 250 A are expected to display a correlation time near 2020 ns

(Table 3), which are similar to those obtained from the FD anisotropy data in Fig. 8 (Table 2). TABLE 3

Calculated Rotational Correlation Times for Membrane Vesicles of Various Diameters³ o

Diameter (A)

200 250 3C ) 400 500 600 ff(ns) 1034 2020 3490 8272 16156 27918 ^a Rotational correlation times ( θ) were calculated from the Stokes Einstein equation θ = τ V/RT, where η = l,cP is the viscosity, T = 297°K, and V is the volume.

The amplitude of the longer correlation time decreases above the phase transition temperature, suggesting free motion of the probe. The fractional amplitude of the short correlation time remains relatively constant with temperature, suggesting that local probe motions contribute to the anisotropy decay at all temperatures. Similar but less definitive results were obtained for DPPG vesicles labeled with Ru-PE (Fig. 7 and Table 2). The results are shown in Table 2. The anisotropy decays again show a short component near 150 ns and a longer correlation time from 6 to 15 μs . There is considerable uncertainty in these longer correlation times because of the smaller fundamental anisotropy of Ru (bpy) ₂ (mcbpy) ⁺¹ (Fig. 3), the smaller fractional amplitude of the long component to the anisotropy decay (Table 2), and the difficulty of measuring a 10 μs correlation time with a 400 ns decay time. Nonetheless, these long correlation times are consistent with those expected for phospholipid o vesicles with diameters of 400-500 A, which may be present in sonicated lipid dispersions. The contribution of a long component in the anisotropy decay has been observed for Ru-PE and described as a nonzero anisotropy at long times (r_∞) . Li, L. and Lakowicz, J.R. (1997) Biospectroscopy 3:155-159.

Another important characteristic of a lipid probe is the extent of labeling possible without spectral changes due to probe-probe interactions. The emission spectra, anisotropies, intensity, and anisotropy decays of labeled DPPG vesicles where the probe-to-lipid molar ratio was varied from l-to-20, l-to-50, and l-to-100 were examined. No significant changes in any of these spectral properties at these molar ratios were found. This suggests that the metal-ligand complex lipid probes display the favorable property of minimal interactions. This absence of probe-probe interaction is consistent with the large Stokes' shift of the emission seen in Fig. 3.

Metal-ligand complex lipid probes can be regarded as the first of many such probes with different spectral properties. For instance, it is known that the decay time of ruthenium metal-ligand complexes can be increased to several microseconds by the use of diphenyl-phenanthroline ligands, Lakowicz, J.R., Murtaza, Z., Jones, W.E., Kim, K., and Szmacinski, H. (1996) J. Fluoresc. 6:245-249; Demas, J.N., Harris, E.W., and McBridge, R.P. (1977) J. Am. Chem. Soc . 99(11), 3547-3551, and that the absorption maximum can be shifted to 650 nm by the use of osmium in place of ruthenium. Pankuch, B.J., Lacky, D.E., and Crosby, G.A. (1980) J. Phvs. Chem. 84, 2061-2067; Lacky, D.E., Pankuch, B.J., and Crosby, G.A. (1980) J. Phys . Chem. 84, 2068-2074; Lin, T-C, and Sutin, N. (1976) J. Phys. Chem. 80, 97-104; Kober, E.M., Marshall, J.L.,

Dressick, W.J., Sullivan, B.P., Caspar, J.V., and Meyer, T.J. (1985) Inorσ. Chem. 24, 2755-1763; Anderson, P. A., Strouse, G.F., Treadway, J.A., Keene, F.R., and Meyer, T.J. (1994) Inorσ. Chem. 33, 3863- 3864. It is also possible to increase the quantum yield of such complexes to near 0.5 by the use of osmium coupled to phosphine and arsine ligands, Kober, E.M., Marshall, J.L., Dressick, W.J., Sullivan, B.P., Caspar, J.V., and Meyer, T.J. (1985) Inorσ. Chem. 24, 2755-1763; Sacksteder, L., Lee, M., Demas, J.N., and

DeGraff, B.A. (1993) J. Am. Chem. Soc . 115, 8230-8238, and that such complexes display lifetimes of several microseconds. Additionally, lifetimes as long as 14 μs have been reported for rhenium complexes with suitable ligands. Zipp, A. P., Sacksteder, L., Streich, J.,

Cook, A., Demas, J.N., and DeGraff, B.A. (1993) Inorσ. Chem. 32, 5629-5632; Shaver, R.J., Rillema, D.P., and _ Woods, C. (1990) J. Chem. Soc. 179-180; Wallace, L., and Rillema, D.P. (1993) Inorα. Chem. 32, 3836-3843. Rhenium complexes also display polarized emission,

Lakowicz, J.R., Terpetschnig, E., and Szmacinski, H. (1996) J. Fluoresc. 6:245-249, and can thus be expected to be useful for measurement of microsecond anisotropy decays . The long lifetime of the metal-ligand complex lipid probes may also allow measurement of lateral diffusion of lipids in membranes. Such measurements are not possible with ns probes due to the limited extent of diffusion during the excited state lifetime. While translational diffusion in membranes is often measured using fluorescence recovery after photobleaching, there has been controversy about the measured values, Gilmanshin, R., Creutz, C.E., and Tamm, L.K. (1994) Biochemistry 33, 8225-8232; Centonze, V.E., and Borisy, G.G. (1990) , Optical Microscopy for Biology p. 658, Wiley-Liss, New York, and independent measurements of lipid diffusion would be valuable. Lateral diffusion in membranes should be measurable by the use of acceptor-labeled lipids. In such measurements the diffusion coefficient can be recovered from the intensity decay of the donor, as described previously for linked and unlinked donor-acceptor pairs. Lakowicz, J.R., Szmacinski, H., Gryczynski, I., Wiczk, W., and Johnson, M.L. (1990) J. Phys. Chem. 94, 8413-9416; Lakowicz, J.R., Kusba, J. , Wiczk, W., and Gryczynski, I. (1990) Chem. Phys. Lett. 173(4), 319-

326. The rates of lipid diffusion can thus be compared with theoretical predictions. Almeida, P.F., Vaz, W.L., and Thompson, T.E. (1993) Biophys. J. 64(2), 399- 402; Almeida, P.F., ^'Vaz, W.L., and Thompson, T.E. (1992) Biochemistry 31 (31) , 198-210; Almeida, P.F., Vaz, W.L., and Thompson, T.E. (1992) Biochemistry 31(29), 6739-6747.

Metal-ligand complex lipid probes can be designed with a range of emission wavelengths, lifetimes, and quantum yields, and can have wide ranging applications for studies of the dynamics of model and cell membranes .

Since many modifications, variations, and changes in detail may be made to the described embodiments, it is intended that all matter in the foregoing description and shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense .

Claims

1. A method of characterizing a sample containing a sample lipid, comprising the steps of: adding a metal-ligand complex lipid probe to a lipid-containing sample to form a mixture containing labeled sample lipid comprising said sample lipid labeled with said metal-ligand complex lipid probe; exposing said mixture to an exciting amount of electromagnetic light energy which causes said labeled sample lipid to emit fluorescent light; and measuring the emitted light.

2. A method as defined in claim 1, wherein said light energy is linearly polarized light energy, and wherein polarization of the emitted light is measured.

3. A method as defined in claim 1, wherein intensity of the emitted light is measured.

4. A method as defined in claim 1, wherein lifetime of the emitted light is measured.

5. A method as defined in claim 1, wherein the lipid-containing sample is used to characterize membrane dynamics.

6. A method as defined in claim 5, wherein said membrane dynamics is rotational hydrodynamics.

7. A method as defined in claim 5, wherein said membrane dynamics is two dimensional diffusion.

8. A method as defined in claim 1, wherein the metal in said fluorescent metal-ligand complex is a transition metal.

9. A method as defined in claim 1, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Co, Cr, Cu, Mo, Ru, Rh, W, Re, Os, Ir, and Pt .

10. A method as defined in claim 1, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Ru, Os, Re, Rh, Ir, W, and Pt .

11. A method as defined in claim 1, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Ru, Os, and Re.

12. A method as defined in claim 1, wherein the fluorescent metal-ligand complex is selected from the group consisting of^' [Ru (2, 2 ' -bipyridine) ₂ (4-carboxy-4 ' - methyl-2, 2 '-bipyridine) ] ; [Ru (2, 2 ' -bipyridine) ₂ (4 , 4 ' - dicarboxy-2, 2 ' -bipyridine) ] ; [Re (2, 9-dimethyl- , 7- diphenyl-1, 10-phenanthroline) (CO) ₃ (monosubstituted pyridine) ] ⁺; [Re (3, 4 , 7, 8-tetramethyl-l, 10- phenanthroline) (CO) ₃ (monosubstituted pyridine)]^*; [Re(5- phenyl-1, 10-phenanthroline) (CO) ₃ (monosubstituted pyridine) ] ⁺; [Re (4, 7-diphenyl-l , 10- phenanthroline) (CO) ₃ (monosubstituted pyridine)]^*; [Re (1, 10-phenanthroline) (CO) ₃ (monosubstituted pyridine)]*; and [Re (2,2'- bipyridine) (CO) ₃ (monosubstituted pyridine)]*.

13. A method as defined in claim 1, wherein said probe lipid is a phospholipid.

14. A method as defined in claim 1, wherein said probe lipid is phosphatidyl ethanolamine .

15. A method as defined in claim 1, wherein said characterizing of said lipid containing sample utilizes a change in fluorescent light emission of the probe compared to fluorescent light emission of the mixture.

16. A composition for characterizing a lipid containing sample, comprising a fluorescent metal- ligand complex coupled to a lipid.

17. A composition as defined by claim 16, wherein the lipid containing sample is used to characterize membrane dynamics .

18. A composition as defined by claim 17, wherein said membrane dynamics is rotational hydrodynamics.

19. A composition as defined by claim 17, wherein said membrane dynamics is two dimensional diffusion.

20. A composition as defined by claim 16, wherein the metal in said fluorescent metal-ligand complex is a transition metal.

21. A composition as defined by claim 16, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Co, Cr, Cu, Mo, Ru, Rh, W, Re, Os, Ir, and Pt .

22. A composition as defined by claim 16, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Ru, Os, Re, Rh, Ir, W, and Pt .

23. A composition as defined by claim 16, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Ru, Os, and Re.

24. A composition as defined by claim 16, wherein said fluorescent metal-ligand complex is selected from the group consisting of [Ru (2, 2 ' -bipyridine) ₂ (4-carboxy- ' -methyl-2, 2 ' -bipyridine) ] ; [Ru (2, 2 ' -bipyridine) ₂ (4 , 4 ' - dicarboxy-2, 2 ' -bipyridine) ] ; [Re (2, 9-dimethyl-4 , 7- diphenyl-1, 10-phena╬«throline) (CO) ₃ (monosubstituted pyridine)]*; [Re (3, 4 , 7, 8-tetramethyl-l, 10- phenanthroline) (CO) ₃ (monosubstituted pyridine)]^*; [Re (5- phenyl-1, 10-phenanthroline) (CO) ₃ (monosubstituted pyridine) ] *; [Re (4, 7-diphenyl-l, 10- phenanthroline) (CO) ₃ (monosubstituted pyridine)]*; [Re (1, 10-phenanthroline) (CO) ₃ (monosubstituted pyridine)]*; and [Re (2,2'- bipyridine) (CO) ₃ (monosubstituted pyridine)]^*.

25. A composition as defined by claim 16, wherein said probe lipid is a phospholipid.

26. A composition as defined by claim 16, wherein said probe lipid is phosphatidyl ethanolamine .

27. The method of claim 1 wherein said method comprises an assay.

28. The method of claim 27 wherein said assay is an immunoassay.

29. A method as defined in claim 27, wherein the sample lipid is present in a liposome.

30. A method as defined in claim 27, wherein the probe lipid is a phospholipid.

31. An assay kit which contains a fluorescent metal-ligand complex and instructions for coupling said fluorescent metal-ligand complex with a lipid.

32. An assay kit as defined in claim 31, wherein the metal in said fluorescent metal-ligand complex is a transition metal.

33. An assay kit as defined in claim 31, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Co, Cr, Cu, Mo, Ru, Rh, W, Re, Os, Ir, and Pt .

34. An assay kit as defined in claim 31, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Ru, Os, Re, Rh, Ir, W, and Pt .

35. An assay kit as defined in claim 31, wherein the metal in said fluorescent metal-ligand complex is selected from the group consisting of Ru, Os, and Re.

36. An assay kit as defined in claim 31, wherein the lipid is a phospholipid.