Fluorescent Probes Field Of The Invention This invention relates generally to fluorescencent materials and to their use in fluorescence immunoassays and more particularly to the use of such materials in fluorescence resonance energy transfer (FRET) based assays and in fluorescence polarization (FP) based immunoassays.

Background Of The Invention It is known that assays based on optical detection methods, especially fluorescence lifetime based assays, have certain advantages over other optical analytical methods because the fluorescence lifetime of the fluorescent molecule or material ('probe or dye) is independent of the probe concentration. Improved assay accuracy and minimized background can be achieved by also using filters to monitor only the desired signal. Spectroscopic methods for FRET and FP based immunoassays that measure the lifetime of excited fluorescencent probes have been developed in recent years and are described by ; . J. Ozinskas in'Principles of Fluorescence Immunoassays. Topic in Fluorescence Spectroscopy."Vol. 4. 1994. page 449-490.

Optimum use of this technology is enhanced when the fluorescence lifetime of the probe is sufficiently long that changes in it are readily determined. Most currently available commercial probes have short lifetimes. In general conventional probes based on organic compounds have lifetimes around 15 ns or lower. There is a need for probes having longer lifetimes, especially probes having lifetimes of 30 ns or more. There is also a need to develop probes, which have long lifetimes and also reasonable good polarization or anisotrop values, since This lifetime (>15 ns) is very short value for most analvtes of commercial interest.

Polarization values (P) and anisotropy (r) is already defined in US Patent 5. 660. 991. dated Aug 26. 1997. Lakowicz. J. R. et. al. The relationship between P and r can be defined as P = 3r/2+r and r = 2p/ (3-P).

In general. there are two types of metal compounds that are fluorescent and also have relatively long fluorescence lifetimes (more than 30 ns). One type includes lanthanide chelate compounds, and other type includes transition metal complexes. The lanthanide compounds have good emission, but they do not show polarized emission and, thus, are not useful as polarization probes. Transition metal complexes, however, show polarized emission and, thus. have potential application as polarization probes as demonstrated recently by Lakowicz., J. R. et. al. ;"Recent Development in Fluorescence Spectroscopy. Near Infrared Dyes for High

Technology Application."Edited by Daehne et. al. (eds.) 1998, Kluwer Academic Publishers, page 3-19. The emission lifetimes of these probes are relatively long, in most cases more than 30 ns and in some cases extending up to microseconds. Because emission is due to metal to ligand charge transfer (MLCT) in these compounds, delayed emission occurs. resulting in longer lifetimes. The long lifetime probes make possible new, improved and low cost methods for doing optical sensing using phase modulation and frequency modulation analysis as is described in US Patent 5, 281. 825. dated Jun. 25, 1994. Lakowicz. J. R., et. nljThere are several examples of the ruthenium metal complexes as shown by Juris, A. et. al.. Ru (II) Polypyridine Complexes : photophysics, photochemistry. electrochemistry and chemiluminescence. Coord.

Chem. Rev. 1988, 84, 85-277. and most of these complexes show MLCT and delayed emission and give usually long lifetime. The long lifetime emissive character of these materials has also been exploited for the use of lifetime based sensing for clinical chemistry as described by Murtaza. Z. et. al.. Long lifetime Metal-ligand pH Probes. Anal. Biochem. 1997. 217. 216, and by Lakowicz. J. R et. al. Development of Long lifetime Metal Ligand Probes for Biophyics and Cellular Imaging"J. Fluoresc. 1997. 7. 17. and in PCT W009838496A 1. (09/03/1998) ; Lakowicz. J. R. et. al. The fluorescence properties of these metal complexes usually differ from each other depending upon the ligand used and upon the group attached on the ligand. Most of these reported complexes can not be labeled to biomolecules as they do not have a group that can form a covalent bond with biomolecules.

Polarization immunoassays that also make use of the lifetime of'the probe have been used in assays to study the biological characterization of a sample of interest (US Patent 5, 660, 991, issued Aug 26, 1997. Lakowicz. J. R. et. al.) This method is very dependent on the selection of the probe. Proper selection can give better sensitivity and good result. The selection of the probe is depending upon the size of the analyt. For example. if one wishes to measure larger molecule, such as a large protein, one needs a longer lifetime probe. Normally, the larger the protein molecule to be detected in an assay. the longer the desired fluorescence lifetime of the probe. When FP is used as the assay method. a higher value of'anisotropy is needed. A useful probe should have 0. 3 or higher value of'anisotropy. The new invented FP dye is a good to study molecule having molecular weight 101-106 range A major problem for commercial success in using fluorescence techniques for immunoassays with conventional probes is that their relatively short lifetimes make their use in assays for larger bio-molecules commercially unattractive because of the relatively expensive high frequency instrumentation needed to measure short fluorescent lifetimes. A further problem with conventional probes is photostability. Many of the conventional dyes decomposes on exposure to high intensity light, creating errors in assays and also creating problems with analysis where longer exposing time is required. Another problem is that, there

are very limited number of long lifetime FP probes available, due to that the FP based immunoassay are limited for very low molecular weight antigen.

SUMMARY OF INVENTION The main object of the present invention is to circumvent problems described above.

It is also an object of this invention to provide new materials that are commercially attractive for use as fluorescent materials in assays requiring a longer fluorescent lifetime, a high anisotropy value, an ability to be attached to bio-molecules and resistance to photobleaching.

These and other objects are accomplished by the present invention which relates to a fluorescent metal ligand complex having ligands that are capable of attachment to a biomolecule and that have an asymetric charge state, the complex being capable of'emitting polarized radiation of a sufficient lifetime to be useful in FP assays with large molecules and as a donor in FRET assays.

The present invention provides a probe based on a new ruthenium complex for use in FP immunoassays as well as FRET based assays. As shown below, it provides a 5% improvement in quantum yield, a 70% improvement in lifetime values and a 50% improvement in polarization values when compared to an existing well documented probe that is also based on a ruthenium (II) metal complex with diimine ligands. [Ru (bpy) 2 (dcbpy)] (PF ( ;) 2.'I'ci-petschnig, E. et. al. 1995. Biophys. J. 68. 342-350. The present probe is also very photostable. as compare to the conventional organic compound based fluorescence probes. which are widely use in fluorescence based immunoassays.

BRIEF DESCRIPTION OF THE FIGURE FIG. 1 is an absorption and emission spectra of the [Ru (aphen) (phen) (dpphenS) cl probe in aqueous solution.

SCHEME I is the representation of the fluorescence dye [Ru (aphen) (phen) (dpphenS)] Cl2.

DETAILED DESCRIPTION OF THE INVENTION This invention stemmed from the discovery that the complex shown in Scheme 1, below, exhibits an unexpectedly long fluorescence lifetime. It is believed that in probes useful in FP assays the polarization value of the polarized emission is dependent on the arrangement

of ligands used and on their relative energy state : the ligand which is lowest in energy contributes the maximum in the emission process. A large energy difference between a single ligand and the other ligands is believed to result in a high polarization value. The unusually long lifetime and resistance to photobleaching of the present probe is not yet fully understood.

The complex of the present invention was synthesized and characterized as a probe for use in a fluorescence polarization immunoassay. The synthesized probe has improved photophysical properties and anisotropy values as compared to the well-known ruthenium based FP probe as is demonstrated in connection with FIG. 1..

Synthesis of (1, 10-phenanthroline) (5-amino-1. 10-p henanthroline) (4. a-dip henyl- disulphonic acid-1. 10-phenanthroline ruthenium (II) dichloride, [Ru (phen) (aphen) (dpphenS)] Cl2 (Ru-40) The target compound. [Ru (phen) (aphen) (dpphenS)] Cl2 was synthesized in three steps.

In first step compound Ru (phen) Cl., was prepared. The intermediate compound Ru (phen) Ci. tuas obtained by mixing equal molar solution of RuCl : 3 and l. 10-phenathroline in 3N HC1 (both chemicals were purchased from Aldrich Chemical Co. Inc.). The reaction mixture was stirred at room temperature for seven days after that black color precipitates form, which were separated by filtration and washed with water and dried in air.

In the second step Ru (phen) (aphen) Cl2 was prepared. This intermediate, Ru (phen) (aphen) Cl2 was prepared by reaction of 5-amino-1, 10-phenanathroline (aphen) (purchased from Polysciences Inc.) with Ru (phen) Cl4 in 1 : 1 ratio respectively in refluxing DMF for 24 hr. The black color compound was separated from reaction mixture by evaporating solvent and washed with water and dried.

The third and final step was the synthesis of [Ru (phen) (aphen) (dpphenS) lCl : The target compound, [Ru (phen) (aphen) (dpphenS)] C12, was prepared by reaction of' Ru (phen) (aphen) Cl2with 4. 7-diphenyldisulphonic acid-1, 10-phenanthroline (dpphenS) in 1 : 1 ratio respectively in refluxing 1 : 1 ethanol water mixture for 24 hrs. After 24-hr. color of the reaction mixture changes from purple brown to orange red. At this time the reaction mixture was brought down to room temperature and solvent was reduced to less than half and product was crystallized by adding large amount of acetone. It was further purified by passing through silica column using acetone water mixture.

The absorption and emission spectra, quantum yield and lifetime of'synthesized [Ru (phen) (aphen) (dpphenS)] Cl2 were measured on a K-2 Spectrophotometer (ISS. Urbana, Champaign, IL, US), figurel. and the results are summarized in the table 1. The structure of

the compound is represented in scheme 1. The spectral properties of the compound were also compared with those of the well know compound. Ru (bpy) 2 (dcbpy)] (PFc) 2 which is generally accepted as the best MLC compound for FP immunoassays is known till now. The spectral data suggests that the newly synthesized compound has better properties than the known compound. and is a suitable candidate for use as a FP probe. A surprising absence of photobleaching was noted during the testing reported below.

Table 1. Photophysical parameters of probes at room temperature in water. Compounds #ab #em #n #h #(air) @0@ (nm) I (nm) (µs) ! (us) [Ru (phen)(aphen)(dpphenS)]Cl2 430- 630 0. 655 ! 3. 543 0. 28 (0. 052) 0. 345 (480- 470510nom) [Ru ((bpy) 2 (dCbpy)] (PFG) 2'1 460 650 0. 37 0.45 0.042(0.039) 0.24 (435- 1 i I a) Measured in aerated sample. b) Measured in nitrogen flushed sample c) measured in PVA. d) previously reported dye has best polarization value.

Scheme I [Ru (phen) (aphen) (dpphenS)] (PFG) 2 IMMUNOASSAY USING FP FLUORESCENCE DYE To study polarization immunoassay of a protein, one can attach this dye tO an antibody that is specific to an antigen of interest (a target analyt), after labeling to the antibody its anisotropy value can measured. which correspondsto the rotational motion of the antibody labeled. On conjugating this labeled antibody to target antigen, its anisotropy changes further, as it's rotation is further slowed down because of the increase size of the resulting system (labeled antibody and antigen). The change in the anisotropy value can be use to study information about the target analyt. such as in an assay.

The anisotropy of labeled analyte can be determined by the Perrin equation. r = rn-/1+ (/). where r is anisotropy of the analyt, rn = anisotropy of'dye (in the absence of rotational diffusion), is lifetime of the probe and is rotational correlation time. (Lakowicz. J. R.,"Principles of Fluorescence Spectroscopy Second edition. Kluwer Academic Press. 1999. page 583.) For FRET based sandwich immunoassay dyes according to the present invention can be use as a donor, and an appropriate acceptor can be selected based on the emission maximum of'the dye.

These dyes. in their role as a donor, can be attached to an antibody, and an acceptor dye can be attached to a second antibodv. Upon completion of the sandwich in the well know-n sandwich immunoassay format, a detectable FRET incident will occur upon excitation of the donor (Lakowicz. J.

R.."Principles of Fluorescence Spectroscopy'Second edition. Kluwer Academic Press. 1999. page 368- 391.) Other useful fluorescent materials and uses. which will be apparent to those skilled in the art after reading the disclosure above. are intended to be encompassed by the appended claims.