CA2350608A1 - Water soluble fluorescent dyes free of aggregation and serum binding and related products and methods - Google Patents

Abstract

The present invention thus relates to marker components, fluorescent probes, oligonucleotides, hybridization assays, and immunoassays using such products and methods for making such products. According to the present invention, detectably labeled marker components are provided that comprise a fluorophore moiety coupled to two or more small solubilizing axial ligands, [define] which preferably reduce or remove the problems of solvent sensitivity and non-specific binding.

Description

DESCRIPTION
Water Soluble Fluorescent Dyes Free Of Aggregation And Serum Binding And Related Products And Methods Introduction The present invention relates generally to water soluble fluorescent dyes free of aggregation and serum binding and related products and methods. Preferred dyes include a luminescent substantially planar fluorophore linked to two or more solubilizing ligands.
l.0 Related Application This application claims the priority of Dtindliker, et al., United States Provisional 60/109,969, entitled "Water Soluble Fluorescent Dyes Free Of Aggregation And Serum Binding And Related Products And Methods", filed on November 3.5 25, 1998, which is incorporated by reference herein in its entirety including drawings.
Background Of The Invention Publications and other reference materials referred to herein are incorporated herein by this reference. The :?0 following description of the background of the invention is intended to aid in the understanding of the invention, but is not admitted to describe or constitute prior art to the invention.
The near infrared absorption and emission of porphyins, :?5 phthalocyanines and other azaporphyrins and certain other aromatic nitrogen cor..taining macrocycles have for some time made these compounds attractive candidates for use as fluorescence labels.
The phthalocyanines, particularly because of their :30 strong near infrared absorption (molar extinction coeffi ients about 200,000), their high quantum yields in 'organic solvents and the well known resistance to fading of common metallophthalocyanine dyes have given rise to many efforts to utilize them as fluorescence labels. However, earlier efforts along these lines did not yield entirely satisfactory products largely because of the unusually strong tendency of phthalocyanines to associate, particuarly by stacking in face to face aggregates and also to bind strongly to a variei~y of other molecular surfaces (non-specific binding).
As a result of intramolecular stacking unsubstituted phthalocyanines have very low solubilities in both organic and aqueous solvents. As is now well known, the tendency to stack can be dramatically decreased by the introduction of one or more charged groups, such as sulfonate. While phtha-locyanines with such substitutions may possess high solu-bility in water and in aqueous solutions of electrolytes, :L5 the tendency to bind nonspecifically largely persists.
Currently, much of the scientific interest in fluorescence labeling is focussed on applications involving biological materials such as tissue sections, cells, cell fragments, proteins, including glyco- and lipo- proteins, ?0 peptides oligo- and poly-saccharides, oligo- and poly-nucleotides and lipids. A tendency to bind nonspecifically in fluorescence assays involving these materials may interfere by partial:~y masking the specific interaction of interest.
25 The nonspecific binding as well as the tendency to stack can be reduced to levels negligible in assays for therapeutic drugs by coupling the phthalocyanines dye to one or more polyoxyhydroc:arbyl groups, typically methoxy-termi-nated polyethylene glycol) (PEG). At the same time the 30 attachment of such groups preserves the desirable absorption and emission characteristics. The same technology is also effective for a wide variety of other near infrared dyes.
See, U.S. Patent Application No. 08/476,544, filed June 7, 1995, entitled POL'fOXYHYDROCARBYL RELATED PRODUCTS AND
35 METHODS FOR FLUORESCENT ASSAY by Dandliker et al, Lyon &
Lyon Docket No. 211/167 (and the priority applications referred therein), which is incorporated herein by reference in its entirety, including any drawings.
Recently, significant advances have been made in the area of fluorescable dyes. In one aspect, dyes being excit able by longer wavelength radiation, such as in the red and infrared wavelengths, are now available. These dyes are described in two commonly assigned patent applications:
Arrhenius, U.S. Patent Application Serial No. 701,449, filed May 15, 1991, entitled, "Fluorescent Marker Components and 7_0 Fluorescent Probes," (which is a continuation-in-part of U.S. Patent Applicat~,_on No. 523,601, filed May 15, 1990), and Dandliker and ~-Lsu, U.S. Patent Application Serial No.
701,465, filed May 15, 1991, entitled "Fluorescent Dyes Free of Aggregation and Serum Binding" (which is a continuation-~LS in-part of U.S. Patent Application Serial No. 524,212, filed May 15, 1990). These applications are incorporated herein by reference, in their entirety, including any drawings.
Further significant advancements have been made in increasing sensitivity through data collection and analysis :?0 techniques. As disclosed in Dandliker et al., U.S. Patent No. 4,877,965, entitled "Fluorometer," which is incorporated herein by reference, in its entirety, including any draw-ings, time gating techniques are used in conjunction with data collection and, analysis techniques to obtain an ;?5 improved signal relative to the background. Generally, the '965 Patent consider~> the detected intensity as a function of time to be compc>sed of signals from various sources, including the desired signal source, and various undesired background sources. Optimization of the desired signal is :30 achieved through data collection and analysis techniques.
Further significant advancements have been made in the ability to measure relevant materials in immunoassays. For example, using the technique described in Dandliker, et al., U.S. Patent Applicat_Lon Serial No. 490,770, filed March 6, 35 1990, entitled "Transient State Luminescence Assays," (which is a continuation-in--part of U.S. Patent Application Serial No. 365,420, filed June 13, I_989) incorporated herein by reference, in its entirety, including any drawings, allows the bound and free form of materials in a homogeneous assay to be determined. Generally, the technique requires measurement of the tame-dependent decay of the intensity of parallel and perpendicular polarization components. By mea-suring the time-dependent decay of various polarization states, it is possible to determine the bound and free forms of materials such as haptens, peptides, or small proteins in a homogeneous analysis format. Significantly, no separation of the bound and free materials is required.
Despite the significant and promising improvements made in the field of fluorescable dyes, and in the data analysis aspects, there remair~:~ a need in the art for additional dyes with these and/or other advantages and which also have favorable chemical reactivity.
Summary Of The Invention The present invention exploits an unexpected result that even very small groups such as -OH can produce effect-ive protection againat non-specific binding and stacking in a planar molecule i:E two such groups are present, one on either side of the molecular plane. Increasing the net negative charge accentuates the favorable effect of the these ligands for many biological systems where most of the "biomolecules" themselves carry a negative net charge.
Thus, one aspect of this invention is that the desirable effects of engineering phthalocyanine and other fluorescent dyes by coupling to polyoxyhydrocarbyl groups can be accomplished instead by two very small axial ligands (such as -OH) provided that the net charge on the dye is sufficiently large. For most circumstances this net charge preferably is negative, since in the physiological pH range most biological materials including proteins and DNA will also carry a negative net charge. Thus, we have found that highly sulfonated dihydroxy-silicon-dicarboxy-phthalocyanine has nearly as low non-specific binding to serum proteins as the PEG-conjugated, unsulfonated dye.

In addition, an advantage emerges in that micelle formation by the PEG is absent. The PEG engineered dye at dye concentrations of 10-q M and above behaves largely like a macromolecule in that its passage through membranes designed 5 to prevent the passage of molecules of about 30K daltons or more is very impeded. Also, the dye moves in the void volume in gel permeation chromatography designed to separate macromolecules from small molecules. In contrast, the sulfonated, dilzydroxy-dicarboxy-silicon-phthalocyanine (SDDSiPc) behaves as expected for a molecule of its formula weight.
In regard to the non-specific binding as measured by changes in fluorescence polarization and intensity, when the dyes are exposed, i=or example, to diluted human serum, SDDSiPc behaves abouti the same as the PEG coupled dye (cf.
Example 6). In this connection it is important to note that negative charge in itself has a marked influence on decreas-ing non-specific binding considering that the unsulfonated dihydroxy-dicarboxy-silicon-phthalocyanine (DDSiPc) shows appreciable non-specific binding. Hydroxy-aluminum-phthalo-cyanine-trisulfonate shows both strong sensitivity to ionic strength and also has strong non-specific binding. It appears that the phthalocyamine molecule must have an axial ligand on both sides of the molecular plane, but that the -OH group is sufficiently large to virtually eliminate non-specific binding if the net charge is sufficiently high.
Another advantage of the present invention is that dyes engineered by -OH or other small solublizing axial ligands together with high charge appear to be much more reactive chemically (in labeling reactions) even though the molecules being labeled, e.g., proteins and oligonucleotides, are themselves negatively charged. This suggests that the PEG
ligands do interfere in labeling macromolecules, although labeling haptens and other small molecules usually proceeds easily.
This invention i_s very unexpected in view of the strong nonspecific binding of hydroxy-aluminum-phthalocyanine-tri-sulfonate, from which one might presume that dihydroxy-dicarboxy-silicon-phthalocyanine with a smaller negative net charge would show even stronger nonspecific binding than the aluminum dye. This invention is based in part on findings which show quite the' reverse (cf. Figures 1 and 2). The effects of other small axial ligands such as: -OCH3, -O-CH20H, -C1, -Br and -F are also expected to work based on the findings reported herein.
Many other nitrogen containing macrocycles can be metallated with atoms of Group 14 with similar results.
Such macrocycles inc7_ude derivatives and structural variants of porphyrins, azaporphyrins, corroles, sapphyrins, penta phyrins, porphycenes and other like macrocycles which have extensively delocaliz:ed pi electron systems. In view of the fact that they incorporate many desirable characteristics, an especially preferred class of macrocycles comprises aza-porphyrin derivative~> and structural variants. Azaporphyrin derivatives include derivatives of mono-, di- and triazaporphyrin and porphyrazine. Any of these macrocycles may optionally also have fused aromatic rings. Such azaporphyrin derivai=Ives and variants include phthalo-cyanine, benzotriazaporphyrin and naphthalocyanine and their derivatives as well His their oxa-, thia-, or aza- structural variants.
The present invention thus relates to marker components, fluorescent probes, oligonucleotides, hybridiza-tion assays, and immunoassays using such products and methods for making such products. According to the present invention, detectably labeled marker components are provided that comprise a fluorophore moiety coupled to two or more small solubilizing :Ligands usually axial, the axis being defined by the octahedral geometry of complexes formed by a central metal atom, which preferably reduce or remove the problems of solvent :sensitivity and non-specific binding.
Use of such detectable labels or marker components in immunoassays is advantageous in that these labels have substantially the same intensities of parallel and perpen-W O 00/3 t t 87 PCT/US99/26983 dicular components o:E transient state fluorescence emission in the presence and absence of biological fluids such as serum. Thus, assay methods using these labels are capable of detecting low ccncentrations of an analyte, a target analyte or analog thereof in biological fluids. The term "analyte" refers to 1=he compound or compound to be measured in an assay which may be any compound for which a receptor naturally exists or can be prepared which is mono- or polyepitopic, antigenic or haptenic, a single or plurality of compounds which share at least one common epitopic site or a receptor. The term "target analyte" refers to the compound or compound to be measured in an assay which may be any compound for which a receptor naturally exists or can be prepared which is mono- or polyepitopic, antigenic or haptenic, a single or plurality of compounds which share at least one common epitopic site or a receptor. By "analog"
of a target analyte i.s meant a compound or compounds capable of competing with t=he target analyte for binding to a receptor. The terra "receptor" refers to a molecule or molecular complex which is capable of specifically recognizing or being recognized by a target analyte or analog thereof. For example, an antibody may be a receptor for an antigen.
These marker components may be used as labels for labeling an analyte, antigen, antibody or other molecule.
These marker components may be optionally functionalized so as to include a linker arm which allows the marker component to be linked to the analyte, antigen, antibody or other molecule. A variety of linker arms which are suited to this purpose have been described. Kricka, J.J.; Ligand-Binder Assays; Labels and Analytical Strategies; pages 15-51;
Marcel Dekker, Inc.,, New York, NY (1985). The marker component is linked to the analyte, antigen, antibody or other molecule using conventional techniques.
In one aspect the present invention provides a detect-ably labeled marker component which comprises: (1) a fluorophore moiety comprising a luminescent substantially planar molecular structure, preferably having excitation wavelengths of at least about 500 nm and (2) coupled thereto two or more small solubilizing axial ligands. Examples of preferred fluorophores, small solubilizing axial ligands, and linkages of the l~wo are described in detail herein. In addition, evidence is provided demonstrating the effectiveness of the marker components at reducing solvent sensitivity and non-specific binding.
In especially preferred embodiments, the marker components of the present invention can be used to make probes as generally described in commonly owned U.S.
application Serial No. 08/051,446, filed April 21, 1993, and used in immunoassays as generally described in commonly owned U.S. application Serial No. 08/035,633, filed March 23, 1993, the disclo:>ures of both of which are incorporated herein by referencE: in their entirety, including any drawings.
The marker components of the present invention are useful as detectable labels, for example as detectable labels in diagnostic reagents and in assays such as fluor escence binding assays and other immunoassays. According to the present invention, detectably labeled marker components are provided that have a fluorophore moiety coupled to two small solubilizing axial ligands which in the presence of serum components in aqueous solutions are characterized by transient state fluorescence emission having parallel and perpendicular components of substantially the same intensi-ties as without serum. The term ~~axial ligand" refers to a substituent which, together with a macrocyclic ligand, forms a coordination comp7_ex with a central atom. The axial ligand lies normal to the plane described by the macrocyclic ligand.
such molecular components are also believed to be useful in methods such as those described in Walker, et al, Clinical Chemistry 42:1 (1996); Clinical Chemistry 39:9 (1993); U.S. Patent No. 5,593,867; and, European Patent Application 93117909.7, which are incorporated herein by reference in their en1=irety, including any drawings.
Surprisingly, it has been found that marker components of the present invention which have a macrocyclic multi dentate ligand with two small solubilizing axial ligands, one located on either side of the plane of the multidentate ligand, exhibit dramatically lowered nonspecific binding to serum components, and exhibit negligible solvent sensi-tivity. The term "solvent sensitivity" refers to changes in .LO the fluorescence behavior of a molecule depending on the solvent system in use, most notably referring to differences in fluorescence behavior in aqueous solution in comparison with organic solvents (such as DMF). Many fluorophores which exhibit high fluorescence intensity in organic .L5 solvents such as DM1~ show substantially decreased fluor-escence intensity in aqueous solution. Fluorescence intensity is related to sample concentration and the intensity of the exciting radiation. The fluorescence intensity of a particular dye can be correlated to its :?0 characteristic light absorptivity (extinction coefficient) and fluorescence quantum efficiency, as well as environ-mental factors. These marker components also exhibit enhanced decay times which approach their radiative or unquenched lifetimes. We use the term "decay time" generic-:?5 ally to indicate the time which must elapse in order for the concentration of excited molecules to decrease from its initial concentration to 1/e of that value. Usage of terms regarding lifetime varies, of, for example, Demos, J.N., Excited State Lifetime Measurements, Academic Press, New :30 York, N.Y. (1983), Pages 10, 35, 44 and 158.
These marker components are useful as fluorescent labels for incorporat=ion in fluorescent probes. The term "fluorescent probe" refers to a marker component comprising a fluorophore moiety which is bonded to or coordinates :35 either directly or vi.a a linker arm to an analyte, antigen, hapten, antibody o.r other molecule which is used in an assay, such as a fluoroimmunoassay to determine the presence of and/or quantitate a substance of interest. Some of these marker components are useful as phosphorescent labels. The components of the present invention are also useful as labels for agents for in vivo imaging and also as labels for 5 agents used in in viva tumor therapy.
Accordingly, ire general, preferred are fluorophores which efficiently produce fluorescence upon excitation with light whose wavelength falls in the range of about 200 to about 1000 nanometers, preferably in the range of about 600 10 to 800 manometers. Suitable fluorophores include those which absorb and/or emit at wavelengths which are distinguishable from the excitation and emission maxima of other solution components (such as proteins present in a sample) to minimize background fluorescence.
Since these marker components are particularly useful in assays using samples of biological fluids, preferred for those uses are fluorophores having excitation and/or emis-sion wavelengths of at least about 500 manometers which reduces interference from the ambient fluorescence of other sample components. Some samples, such as serum, may exhibit considerable interfering background fluorescence from flavins, flavoproteins, NADH, etc., when excitation wave-lengths less than 500 nm are used.
For certain applications, such as fluorescence polarization immunoassays, preferred fluorophores may also exhibit a high degrEye of fluorescence polarization when in the bound form, preferably greater than about loo of the theoretical maximum value for an observable polarization.
The term "bound" refers to the condition in which a binding interaction has been formed between a molecule and its specific binding partner. For certain applications such as fluorescence transient state assays, preferred fluarophores are also characterized by measured fluorescence decay times in the range of about 1 nanosecond to about 50 nanoseconds, preferably in the range of about 5 to about 20 nanoseconds .
For other applications, such as phosphorescent labels, fluorophores having Even longer decay times may be used.

Thus, preferred are fluorophores which produce fluorescent light efficiently, i.e., which are characterized by high absorbitivity at the appropriate wavelength and high fluorescence quantum yields. For certain applications, preferred fluorophores have measured fluorescence decay times on the order of at least 2 nanoseconds and exhibit a high degree of fluorescence polarization.
Preferred small solubilizing axial ligands include -OH, -0-t-butyl, -OCHzOf~, -OCH2CH20H, -OCH2CHOHCHzOH, -OCHzCH2-O-CHZCH20H, -OCH2CHz-CHz--C>-CHzCH20H, C1, Br and F. For any of these axial ligands lined by -O- to the central atom the stability towards hydrolysis probably would be improved by replacing the -O- link by a direct carbon to metal linkage, e.g., carbon to silicon In preferred embodiments, the fluorophore moiety has a substantially planar, multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two small solubilizing axial ligands. For use as marker components in fluorea~~ence binding assays, suitable central atoms are those to which may coordinate two axial ligands and which are not of_ high enough atomic number to cause extensive fluorescence quenching by transition to the triplet state. Preferred elements for the central atom include silicon, germanium, and tin, especially preferred are silicon and germanium.
The present invention is also directed to methods for determining the prese=nce or amount of a target analyte in a sample by using, as a label for the target analyte or a receptor which is capable of specifically recognizing the target analyte, a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.
Use of such detectable labels or marker components in immunoassays is advantageous in that these labels have substantially the same intensities of parallel and perpen dicular components of fluorescence emission in the presence and absence of biological fluids such as serum. Thus, assay methods using these labels are capable of detecting low concentrations of target analyte in biological fluids.
The methods of t:he present invention are particularly suitable for use with the improved fluorescence detection system described in commonly assigned U.S. Patent Application entitled "Fluorometer Detection System," Lyon &
Lyon Docket No. 195/129, Serial No. 07/855,238, filed March 23, 1992. The marker components and labels are also believed to be useful in methods described in Walker, et al, Clinical Chemistry 42:1 (1996); Clinical Chemistry 39:9 (1993); U.S. Patent No. 5,593,867; and, European Patent Application 93117909.7, all of which are incorporated herein by reference in their entirety, including any drawings.
In one aspect, the present invention is directed toward competitive inhibition assay procedures utilizing particular labels. In this aspect, the present invention is directed to a method of determining the presence or amount of a target analyte by contacting the sample suspected of containing the target. analyte with a known quantity of added target analyte or analog thereof linked to a fluorescent probe which includes a detectably labeled marker component made up of a fluorophore moiety which includes a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure; contacting the sample with a receptor capable of specifically recognizing the target ligand; and. determining either the amount of fluorescent probe bound to receptor or free fluorescent probe. The amount o:E bound or free fluorescent probe in the unknown samples may be compared with blank samples and samples containing known amounts of target analyte.
In a preferred embodiment, the resultant mixture of sample, fluorescent. probe and receptor is diluted immediately before measurement of the amount of bound and/or free fluorescent probe. The dilution step allows for greater sensitivity of the assay. Particularly preferred are dilutions of 2-fold to 100-fold, preferably about 7-fold to about 50-fold, and more preferably about 35-fold.
In one aspect, the present invention provides an improvement in immunoassay procedures which utilize a label for either the targf=_t analyte (or analog thereof) or the receptor. The improvement is the use of a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure. Assays u~:ing this type of label are advantageous in that they are free of serum binding and aggregation and are therefore, espec:~ally suitable for testing biological samples such as serum., plasma, whole blood and urine.
In another aspect, the present invention provides a method for performincr a "sandwich" or "two-site" immunoassay having the steps of: (a) contacting a sample suspected of containing a target analyte with a first receptor capable of specifically recognizing the target analyte to form a complex of the targe~t~ analyte and the first receptor, the first receptor being labeled with a fluorescent probe which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure; (b) contacting the complex with a second receptor capable of specifically recognizing the target analyte or the first receptor, the second receptor being bound to a solid carrier, to form a complex of the first labeled receptor, the target analyte and the second receptor bound to the solid carrier; and (c) measuring either the amount of labeled first receptor associated with the solid carrier or the amount of unreacted labeled first receptor.
A sandwich-type assay may be either a heterogeneous assay or a homogeneous assay. If it is heterogeneous, it may incorporate the additional step of separating the solid carrier from the unreacted labeled first receptor.

Homogeneous assays are generally preferred because they are more rapid.
In another embodiment, the assay may incorporate the additional step of relating the amount of labeled first receptor measured in the unknown sample to the amount of labeled first receptor measured in a control sample free of the target analyte, or to the amount of labeled first receptor measured in samples containing known quantities of target analyte.
:10 In another aspect, the present invention provides a method for a simultaneous sandwich-type assay for determin-ing the presence or amount of a target analyte in a sample having the steps of: (a) simultaneously contacting a sample suspected of containing a target analyte with first and second receptors capable of specifically recognizing the target analyte, the first receptor being labeled with a fluorescent probe which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure, and the second receptor being bound to a solid carrier, to form a complex of the first receptor, the target analyte, and the second receptor; anc~ (b) measuring either the amount of labeled first receptor associated with the solid carrier or the amount of unreacted labeled first receptor.
In another aspect, the present invention provides a method for a simultaneous sandwich-type assay having the further step of relating the amount of labeled first receptor measured to the amount of labeled first receptor measured for a control sample free of said target analyte, or relating to the amount of labeled first receptor measured with the amount of labeled first receptor measured in samples containing known amounts of target analyte.
In another aspect, the present invention provides a sandwich-type fluorescence immunoassay method for measure ment of a target ana7_yte which is capable of recognizing two different receptors independently without mutual interference. The method utilizes two receptors, each of which is labeled with a different dye. For example, one receptor is labeled with a first dye having absorption and emission maxima of 5Ec0 nm and 690 nm, respectively, and the 5 other receptor is labeled with a second dye having absorption and emission maxima of 695 and 705 nm, respectively. Detection and quantitation of the analyte can be made using either steady state or transient state measurements. In either case, for the example given, 10 excitation would be at; 680 nm and detection would be at 705 nm. This type of assay is based on energy transfer and is advantageous in that. it is homogeneous.
In preferred embodiments the present invention is directed to immunoassay of biological fluids, including 15 serum, plasma, whole blood and urine. Preferably, red blood cells in whole blood are lysed prior to assay of whole blood samples. Preferred methods of lysing red blood cells include addition of stearoyl-lysolecithin, palmitoyl-lysolecithin and myristoyl lysolecithin.
Depending on the type of immunoassay used, the target analyte may be an antigen, a hapten or an antibody; and the receptor may be an antigen or antibody. The antibody may be polyclonal or monoclonal. Preferably, the antibody is a monoclonal antibody. Monoclonal antibodies useful in the present invention may be obtained by the Kohler & Milstein method reported in Nature 256:495-497 (1975).
Alternatively, they may be produced by recombinant methods.
Science 246:1275-1281 (1989).
In one embodiment, the target analyte is a drug or a metabolite of a drug. The drug may be a steroid, hormone, antibiotic, immuno~~uppressant, antiasthmatic, antineo plastic, antiarrhythmic, anticonvulsant, antiarthritic, antidepressant, or cardiac glycoside. Examples of such drugs include digoxin, di.gitoxin, theophylline, phenobarbital, thyroxine, N-acetylpracainamide, primidone, amikacin, genta-micin, netilmicin, tobramycin, carbamazepine, ethosuximide, valproic acid, disopyramide, lidocaine, procainamide, quini-WO 00/31187 PC1'/US99/Z6983 dine, methotrexate, amitriptyline, mortriptyline, imipra-mine, desipramine, vancomycin, and cyclosporine. In a preferred embodiment, the drug is digoxin.
In another embodiment, the target analyte is a peptide, for example, a peptide hormone such as luteinizing hormone, follicle stimulating hormone, human choriogonadotropin, thyroid stimulating hormone, angiotensin I, angiotensin II, prolactin or insulin. The peptide may also be a tumor marker such as carcinoembryonic antigen. Or, the peptide may be a virus or portion thereof, for example, rubella virus or a portion thereof.
The methods of the present invention provide ways of measuring target ana7_ytes in concentrations of from about 1 x 10-5 M/L to about 1 x 10-13 M, and particularly in the concentration range of from about 1 x 10-9 M/L to about 1 x 10-12 M/L. For measurement of drugs and their metabolites, the present methods allow measurement in the range from about 5 x 10 9 M/L to about 5 x 10-12 M/L, and particularly, concentrations of from about 1 x 10-1° M/L to about 5 x 10-1°
M/L. For measurement of peptides, the present methods allow measurement in the range of from about 1 x 10-11 M/L to about 1 x 10-12 M/L .
The measurement of amount of fluorescent probe -- bound or free or both -- can be determined by measuring steady state fluorescence or by measuring transient state fluorescence. In a preferred embodiment, the wavelength of light measured is greater than about 500 nm, preferably greater than about 650 nm, and more preferably greater than about 680 nm or 690 nm. Because the transient state detection system utilizes a laser diode, it is necessary for the dyes to have e:~citation maxima matched to the diode output wavelengths. Dyes have been made available to match other commercially available laser diodes having output wavelengths of 680, 690, 720, 750, or 780 nm. Thus, the wavelength of the light measured may be greater than about 680 nm, 690 nm, 720 nm, 750 nm or 780 nm. The further into the red region of thw spectrum one moves, i.e., the greater the wavelength, the greater signal enrichment there is over background.
In a preferred embodiment, detection and quantitation is performed using transient state measurement. Transient state energy transfer offers improved measurements due to optimization of the wavelengths of absorption and emission, as well as due to optimization of the decay times of the first and second dyes. Such optimization allows removal of Rayleigh and Raman :scattering, and maximizes efficiency of transfer and minimizes direct excitation of the second dye by the first dye.
Accordingly, it is a principal object of this invention to provide improved FIAs with greatly enhanced sensitivity.
It is yet another object of this invention to provide FIA
methods which allow rapid and accurate determinations, often within a matter of minutes. It is an object of this invention to provide FIA methods which are capable of measuring extremely low concentrations of fluorescable material. It is an object of this invention to provide FIA
methods useful for the clinical setting in that they are rapid and accurate, of relatively low cost and capable of use with unmodified biological samples, such as whole blood.
It is a further object of this invention to provide FIA
methods particularly adapted to exploit the improved fluorescence detecti.an system, described in U.S. Patent Application entitled "Fluorometer Detection System,", Serial No. 07/855,238, filed March 23, 1992, referenced above. It is a further object of this invention to provide homogeneous "mix and read" therapeutic drugs assay methods which can be used to determine the level of digoxin in serum, plasma or whole blood. It is a further object of this invention to provide an assay for. peptides, for example, for a rubella virus or a portion thereof. The present invention also provides particular fluorescent markers and probes for use in immunoassays, as described herein.
The present invention also provides a method of synthesizing a marker component by reacting the fluorophore moiety with a reacaive form of the solubilizing axial ligands. The invention also features a fluorescent probe having a marker component of the invention, linked to one member of a specific binding pair or a target analyte or an analog. The term "specific binding pair" refers to two different molecules (or compositions) wherein one of the molecules has an area on the surface or in a cavity which specifically recognizes and binds to a particular spatial and polar organization of the other molecule or molecular complex involving other molecules.
A method synthesizing a fluorescent probe is also provided which inc_Ludes the step of linking a marker component of the invention to t:wo or more solubilizing axial ligands. A kit useful for detecting a target analyte in a sample suspected of containing the target analyte is also provided, the kit including a marker component or probe of the invention.
The present invention is also directed to novel dye oligonucleotide conjugates and methods of synthesizing them and methods of using them. Methods of using these conju gates or probes involve nucleic acid by hybridization methods, nucleic acid amplification methods and nucleic acid sequencing methods. The dye portion of the dye-oligo-nucleotide conjugate is a detestably labeled marker component having a :Eluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing a:~ial ligands, one located on either side of the planar molecular structure.
Thus, in one aspect, the present invention is directed to a composition having an oligonucleotide linked to a detestably labeled marker component which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.
By "oligonucleotide" is meant a chain of nucleotide residues. Typically, an oligonucleotide useful in the present invention has a length of from 5 to 50 nucleotides.
The oligonucleotide probes used in the method of the invention include polynucleotides of DNA, RNA or any other kind of sequence hybridizable to nucleic acid sequences. It will be appreciated that such nucleic acid sequences may include base analogues as well as the naturally occurring bases cytosine, adenine, guanine, thymine and uracil. Such base analogues include hypoxanthine, 2.6-diaminopurine and 8-azaguanine. The probes may be in double stranded or single stranded form but are preferably in single stranded form. They may be prepared by direct synthesis, polymerase mediated extension reactions or by cloning or any other convenient method. By "linked" is meant combined chemically by an intermediate molecule which is connected to both moieties.
In one preferred aspect, the present invention is directed to composition having an oligonucleotide linked to a detectably labeled marker component having a fluorophore moiety which has a substantially planar multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two axial ligands which are coordinated to the central atam on either side of the macrocyclic ligand.
Preferably, the detectably labeled marker component has a decay time in the range of from about 1 nanosecond to about 50 nanoseconds, more preferably, the decay time is in the range of from about 5 nanoseconds to about 20 nanoseconds.
Particularly preferred is a caged dicarboxy silicon phthalocyanine. The caged dicarboxy silicon phthalocyanine dye may have a variety of functional groups available for coupling. These groups include free carboxyl, free amino and N-hydroxysuccimid.e ester (NHS ester).
Preferably, the oligonucleotide of the claimed compositions will have a length of about 5 to about 50 bases.
Linkage of the oligonucleotide or polynucleotide to the marker may be accomplished using condensation reactions wo oom is~ Pc~nus~n69s3 leading, for example., to the formation of amide, ester, hydrazone, semicarbazone, thiosemicarbazone, urea, and thiourea bonds. For example, a linker may terminate in an amino group, preferably primary. Other linkers may 5 terminate in a carbo~!:yl group.
In another aspect, the present invention provides methods for preparing certain dye-conjugated oligonucleo-tides. In one embodiment, such a method involves the steps (a) of reacting an o:Ligonucleotide having an attached linker 10 terminating in an amino group with an N-hydroxy succinimide ester or an imidazolide of a detestably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on 15 either side of the planar molecular structure, to form a conjugate; and separating the conjugate formed in step (a) from unreacted oligonucleotide or polynucleotide and from unreacted dye. Attachment of a linker to the oligonucleo-tide can be accomplished by using a diamine or an amino 20 alcohol. Preferably, the detestably labeled marker compo-nent comprises a caged dicarboxy silicon phthalocyanine dye.
Alternatively, preparation of the dye-conjugated oligonucleotides may be accomplished: reacting a detestably labeled marker component which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure, with a carbodiimide in the presence of hydroxy-benzotriazole and in the presence of an oligonucleotide or polynucleotide to form a conjugate; and separating the resulting conjugate from other components of the reaction mixture. Preferably, the detestably labeled marker component has a caged dicarboxy silicon phthalocyanine dye.
In another aspect, the present invention is directed to a method for the detection of a target nucleic acid sequence in a sample comprising the steps of contacting sample nucleic acid with o7_igonucleotide-labeled marker component, preferably oligonucleotide caged dicarboxy silicon phthalo-cyanine dye conjugate capable of hybridizing with said target nucleic acid sequence in homogenous solution, and detecting the presence or amount of such hybridization by transient state polarized fluorescence.
In yet another_ aspect, the present invention is directed to a method for detection of a target nucleic acid sequence in a sample having the steps of contacting a sample suspected of containing a target nucleic acid sequence with a complementary oligonucleotide capable of hybridizing with said target sequence; contacting said sample with an oligonucleotide-labeled marker component, preferably oligonucleotide-caged dicarboxy silicon phthalocyanine dye conjugate capable of. hybridizing to said complementary oligonucleotide; and detecting the presence or amount of hybridization of said conjugate with said complementary oligonucleotide.
In a further aspect, the present invention is directed to methods for detection or quantification of a target nucleic acid wherein the target nucleic acid is a product of nucleic acid amplification. Nucleic acid amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR;, self-sustained sequence replication (3SR) and transcript-based amplification system (TAS), which are discussed herein.
In another aspects, the present invention is directed to an improvement in a nucleic acid hybridization method or nucleic acid amplification method employing as a label a fluorescent probe which has a detectably labeled marker component which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.
The methods of the present invention are particularly useful when used w_Lth a time-correlated transient state detection system, as described in commonly assigned Studholme, et al., U.S. Patent Application entitled "Fluorometer Detection System," Serial No. 07/855,238, filed March 23, 1992. 'that system features transient state detection permitting direct readout of the time-dependent polarization of the sample. The system uses a laser diode which can be modulated at very high frequencies, e.g., MHz rate, and exhibits high output power. Typically the laser "on" time is approximately 2-3 nanoseconds. Photons from the solution are detected using a photomultiplier tube (PMT) operating in a single photon counting mode. The 10 photon event along with the relative time of the photon event as compared with the laser pulse time is determined.
By storing the indiv_Ldual photon event times a histogram of frequency of photons as a function of time is generated.
In another aspect, the present invention provides a method for monitoring the kinetics of a nucleic acid amplification process;, and/or quantifying nucleic acid in a target sample. For example, during amplification by PCR, a probe consisting of an oligonucleotide which has been both "capped" and labeled with a composition having an oligonucleotide or polynucleotide linked to a detestably labeled marker component which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure, may be adlded directly to the PCR reaction. By "capped" is meant that the 3' end has been reacted dideoxynucleotide.
At each cooling phase, the hybridization with amplified product may be followed kinetically. As the concentration of amplified product increases, the rate of combination of probe with amplified product increases and quantitates the concentration of amplified product. This information together with the number of cycles quantitates the amount of DNA originally in the sample before amplification.
Use in sequencing. Another aspect of this invention is the applicaitonto DNA sequencing in which the detestably labeled marker compo:nenet .is incorporated either chemically or enzymatically into the DNA: the DNA is cleaved at numerous points and t he resulting collection of fragments is analyzed by gel electrophoresis. If desired, labeling can be done with different marker components, one for each of the bases to facilitate analysis. Another aspect of this invention is the broad scope of application to any design, variation or modification of fluorometers. This breadth of applicability is well illustrated in the following example of a specialized type=_ of instrument. In non-absorbing media a light wave traversing medium A surrounded by a second medium B of lower refractive index undergoes total internal reflection at the boundaries of medium A if the angle of incidence is greater than the critical angle. However, the electromagnetic field of the totally reflected light penetrates the boundaries for a short distance and there can produce physical effects such as the excitation of fluorophore molecules located near the interface between A
and B.
This effect enables homogeneous, fluorescence-based assays in which the specific reaction occurs with molecules immobilized on the surface of medium A and hence at the interface which is the only location where excitation of fluorescence can occur. A simple glass or plastic plate acts both as an opti~:al waveguide for the incident light and as a carrier for specific receptor previously deposited at known locations on t:he surface. This methodology has been termed "evanescent 7_ight fluoroimmunoassay" (Herron et al.
U.S. Patent No. 5,512,492).
Advantageously, the present invention incorporates the features of very large Stokes' shifts utilizing fluorescent dyes based upon N--containing macrocycles (classes listed below) which commonly have a near UV excitation region with emissions in the near infrared region of the spectrum. Such dyes are applicable to immuno/receptor assays in either steady state or transient state modes. Excitation sources include mercury arcs, nitrogen lasers and nitrogen laser pumped dye lasers. Alternatively, these same dyes can be excited in the near infrared with diode lasers allowing excellent results with pulsed excitation and transient state detection. The choice of source depends upon the position of the absorption band for the particular dye in question, the preferred mode of excitation and detection, whether steady state or transient state and upon space requirements.
The inclusion of these features in the chemistry and instrument design fo:r "evanescent light fluoroimmunoassay"
should lead to very low background together with high signal :LO levels and thus favor high assay sensitivity.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.
:L5 Brief Description Of 'rhe Drawings Figure 1 shows effects of nonspecific binding and solvent on fluorescence intensity, showing a protective effect of Si as compared to Al presumably because of the two axial ligands of S:i as compared to only one for Al.
20 Abbreviations: Al trisulf - hydroxyaluminumtrisulfonate; Si dicarb - bis-hydroxy(2,3-dicarboxyphthalocyanino)silicon IV;
Si dicarb sulf - ~;ulfonated Si dicarb; BBKC1 - borate buffered KC1 (cf. Materials, Example 6); NHS - pooled normal human serum; Cts - counts obtained during one 25 measurement cycle of the FAST 1 transient state fluorometer.
Cts is proportional to fluorescence intensity.
Figure 2 shows a comparison of silicon and aluminum phthalocyanine derivatives with respect to solvent effects 30 and nonspecific binding. The results indicate that the presence of Si (with two axial ligands) produces dramatic-ally better performance than having A1 as a central atom.
This is evidenced by a much smaller relative change in polarization for dih~rdroxydicarboxysiliconphthalocyanine (Si 35 dicarb) as compared to A1 trisulfonate (A1 trisulf). A
further improvement ~~s afforded by sulfonation as shown for dihydroxy dicaboxy silicon phthalocyanine sulfonate {Si dicarb, sulf) which has probably two or three sulfonate groups. The values for polarization in glycerol indicate approximately what the potential limits would be if the dye 5 were used as a labe:L ,. Polarization is in millipolarization units (mP). For further information on abbreviations see legend for Figure 1. Those in the art will recognize that the dyes of the invention can be used in a variety of fluorescence applications over a wide range of the visible :LO spectrum.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawings are not necessarily to scale. Certain :L5 features of the invention may be exaggerated in scale or shown in schematic form in the interest of clarity and conciseness.
Detailed Description Of The Invention The present invention relates generally to improved ;?0 fluorescent marker components having a substantially planar luminescent moiety linked to two or more small axial ligands, and related products and methods. The preferred fluorophore involves small solubilizing axial ligands, fluorescent properties, and methods of synthesis and use are ;?5 described below. The following description includes preferred modes of carrying out the invention and is made for the general purpose of illustrating the general principles of the invention rather than limiting the invention in any manner.
I. Preferred marker components The following i:~ a brief description of the preferred marker components to be used in the fluorescence immuno-assays of the present invention. A more complete discussion is found in common:Ly assigned U.S. Patent Applications wo oor~ i ~ s~ Pcrius~n69s3 Serial Nos. 701,449 and 201,465, which, as noted above, have been incorporated herein by reference.
A. Preferred Fluoro~hore Moieties Suitable fluorophore moieties comprise a luminescent substantially planar molecular structure. Preferred are fluorophore moieties in which the luminescent substantially planar molecular structure has a substantially planar macrocyclic multidentate ligand which coordinates a central atom which may coordinate with two axial ligands, one on LO either side of the macrocyclic ligand (i.e. having a trans orientation).
Preferred central atoms are elements which may form octahedral coordination complexes containing two ligands with a trans or a~:ial orientation, on either side and perpendicular to the planar macrocyclic ligand. For use as fluorescent marker components in certain applications the central atom should not have too high atomic number (about 30 or less) so that fluorescence is not diminished through coupling interaction with orbitals of the central atom.
:20 Preferred multidentate ligands include nitrogen-containing macrocycles which have conjugated ring systems with pi-electrons. These macrocycles may be optionally substituted, including substitution on bridging carbons or on nitrogens. Suitable macrocycles include derivatives structural variants of porphyrins, azaporphyrins, corrins, sapphyrins, pentaphyrins and porphycenes and other like macrocycles which contain electrons which are extensively delocalized. These macrocycles may optionally have fused aromatic rings with or without hetero atoms such as N, O or S. In view of the fact that they incorporate many of the above-noted characteristics, an especially preferred class of macrocycles comprise porphyrin derivatives, and azapor-phyrin derivatives (porphyrin derivatives wherein at least one of the bridging carbons is replaced by a nitrogen atom).
Azaporphyrin derivatives include derivatives of mono-, di-and triazaporphyrin and porphyrazine. These macrocycles may optionally have fused aromatic rings with or without hetero atoms such as 0., N or S. These azaporphyrin derivatives include phthalocyanine, benzotriazaporphyrin and naphthalo-cyanine and their derivatives. The preparation and fluor-escent qualities of many of these compounds are known and some are available commercially. See U.S. Patent Application Serial Nc>. 201,465 and references cited therein, particularly, references 2-5 in that application.
For certain applications, such as fluorescence polarization assays, preferred are azaporphyrin derivatives which exhibit a high degree of. polarization in the bound form, that is, those which emit strongly polarized light.
For these applications, preferred are macrocycles having lower degrees of symmetry, preferably having lower symmetry than D4h. One preferred group includes macrocycles having at least one fused aromatic ring. Thus, preferred macrocycles include azaporphyrin derivatives having fused aromatic rings at positions which result in decreased symmetry. Preferred classes of azaporphy:rin derivatives comprise derivatives of monoazaporphyrin, diazaporphyrin, and triazaporphyrin having lower than Dqh Symmetry. Other preferred fluorescent dyes are described on "Polyoxyhydrocarbyl Related Products and Methods for Fluorescence Assay", U.S. Application No.
08/476,544, filed June 6, 1995, Lyon & Lyon Docket no.
211/167, which is incorporated herein by reference in its entirety, including any drawings.
B. Preferred Small Solubilizing Axial Ligands Preferred small. solubilizing axial ligands include -OH, -0-t-butyl, -OCHZOH, -OCHZCH20H, -OCH2CHOHCH20H, -OCHZCHZ-0-CHZCHZOH, -OCHZCHZ-CHZ-O-CHZCHzOH, C1, Br and F.
C. Absorbance and Polarization Behavior of Preferred Marker Components These marker components which comprise a central atom (for example, silicon) coupled to two small solubilizing axial ligands may be characterized by measurements of transient state fluorescence. In such measurements the intensity of the two components polarized either parallel or perpendicular to the direction of polarization of the exciting pulse is monitored over a time period equal to about 3 times the decay time of the marker component. Such curves reflect extinction coefficient, quantum yield, decay time and state of polarization and supply sensitive indications on the chemical and physical condition of the marker component. For example, if the excited state is being deactivated o:r converted to the triplet state the overall intensities are lowered and the decay times shortened. If the rotary brownian motion of the molecule is being altered by an increase in viscosity or by being bound to a large molecule', the ratio of the intensity of the parallel to the perpendicular component is increased.
Some marker camponents according to the present invention show, within experimental error of about 50, the same intensities, decay time and polarization in DMF (an organic solvent) as in SAP (saline azide phosphate, an aqueous neutral buffer). To some extent these properties are shared by other marker component preparations. A
distinctive and important property of the marker components of the present invention is a insensitivity to (and lack of binding to) the components in serum which is evidenced by a lack of any significant measured effect of serum on the intensities, decay time or relative magnitudes of the polarized components of the fluorescence. This property is crucial for the marker components to be useful for applications such as assays using biological materials.
II. Preparation Of Preferred Marker Components According to one method of preparing the preferred marker components of the present invention, the appropriate fluorophore moiety having hydroxy or halide groups as axial ligands is reacted with a reactive form of the solubilizing moiety in a ligand. exchange reaction according to the general reaction scheme:

Mcl-CA-(X)2 + 2(SM) --> Mcl-CA-(SM)z + 2X
wherein Mcl denotes the macrocyclic ligand, CA the central atom, X the displaced ligand and SM the solubilizing moiety.
This reaction may be carried out neat or, if desired, in solvent. Suitable solvents include quinoline, THF, DMF, imidazole when dissolved itself in one of the other listed solvents and the lil~:e. Suitable reaction temperatures may vary, depending on 'the nature of the macrocyclic starting material and the s.alubilizing group. The reaction is generally complete in about 2 minutes to about 24 hours.
The reaction mixture can be conveniently heated under reflux or by means such a,s a sand bath. For convenience, the reaction may be carried out at ambient pressure.
It is believed that this reaction takes place in two steps, with one pol.yoxyhydrocarbyl group coordinating as an axial ligand at a time.
When used as fluorescent labels in fluorescence immunoassays, these marker components may be linked to one member of a specific binding pair ("labeled binding partner") or an analog of such a member. The term "binding partner" refers to a molecule or molecular complex which is capable or specifics:L:Ly recognizing or being recognized by a particular molecule or molecular complex. The marker component may be directly attached or conjugated thereto or attached or conjugated via a linker arm.
III. Utility The marker components of the present invention are useful as fluorescent labels for fluorescent probes and in fluorescence binding assays and also in as labels for in vivo imaging and in vivo tumor therapy.
These marker components may be advantageously used as fluorescent labels in conventional fluorescence binding assays, including fluorescence polarization immunoassays.
When so used, these marker components may be linked to one member of a specific binding pair ("labeled binding partner") or an analog of such a member. The marker component may be directly attached or conjugated thereto or attached or conjugated via a linker arm.
These labeled binding partners are useful in assays having a variety of formats, such as assays which involve 5 competition for anal_yte or analyte binding partner (if a labeled analyte or analyte-analog as used) and may be used in either homogeneous or heterogeneous assays.
In view of their advantageous freedom from aggregation in aqueous solution and lack of solvent sensitivity 10 (indicating no detectable aggregation) in combination with their lack of nonspecific binding to serum components and other biological macromolecules, these markers are especially suited for use in assays for detecting an analyte in a sample containing a biological fluid such as serum.
15 Thus, these marker components may be used as labels for fluorescent probes far detecting analytes in solutions where non-specific binding by serum components would severely compromise sensitivity of an assay, affecting both its accuracy and precision.
20 Alternatively, these marker components may be used as agents for in vivo imaging. When used as imaging agents, these marker components are conjugated to one member of a specific binding pair to give a labeled binding partner.
The labeled binding partner is introduced into an animal.
25 If the other member of the specific binding pair is present, the labeled binding partner will bind thereto and the signal produced by the marker component may be measured and its localization identified.
These marker components may also be used in in vivo 30 tumor therapy. For example, photodynamic therapy involves using the marker component as a photosensitizing agent. The marker component (fluorescent label) is conjugated to a binding partner which may specifically recognize and bind to a component of a tumor cell.
The present invention provides nucleic acid probes and methods of making arid using the probes. Methods of using the novel nucleic acr d probes include various nucleic acid hybridization sequencing techniques now known or later developed, and various nucleic acid amplification techniques now known or later developed. The probes (also referred to as conjugates herein) and methods of the present invention allow the achievement of 7_ fmole sensitivity in a homogeneous hybridization assay. As demonstrated herein, this sensitivity is comparable to the sensitivity achieved by current heterogeneous hybridization measurement techniques. As noted above, however, current heterogeneous assays have several disadvantages, which result from the many steps involved _~n the assays, including increased risk of contamination and increased time required to perform the assays. Other advantages of the compositions and methods of the present invention will be apparent to those in the art upon review of the examples provided herein.
Examples To assist in understanding the present invention, the following Examples are included which describe the results of a series of experiments. The following Examples relating to this invention should not, of course, be construed in specifically limiting the invention and such variations of the invention, now know or later developed, which would be within the purview of' one skilled in the art are considered to fall within the scope of the invention as described herein and herein aft~'r claimed.
Example 1: Synthesis of tetradiiminopyromellitic acid diimide from 1,2,4,5-tetracyanobenzene (TCNB).
TCNB, 20.0 g (0.112 moles) in a 3-neck, 1 1 flask was dried in vacuo about 1 hr. The flask was fitted with a :30 slow, high torque, Teflon vane stirrer, an inlet for bubbling in ammonia ar adding liquid, and a water cooled condenser. After flushing the entire apparatus with nitrogen, 400 ml of methanol was added, stirring was begun at room temperature and ammonia was bubbled in slowly.

The absorption of ammonia is very efficient and after a few minutes the suspension becomes a clear pale green solution. A f.ew minutes later (with constant addition of ammonia) the solution becomes turbid and the temperature rises slightly. After 40 minutes from the beginning of addition of ammoni<~ the reaction mixture had become difficult to stir and 200 ml more methanol was added while stirring and ammonia addition were continued. At this point ammonia absorption was still very efficient as evidenced by the lack of bubbles emerging from the surface of the suspension. After 100 minutes it was necessary to add an additional 175 ml of methanol to enable stirring. After 125 min large amounts of ammonia began to appear at the exit from the condenser and reaction mixture was put into water bath at 45 deg. c~ with continued mixing, heating and ammonia addition for an additional 240 minutes. After cooling, the reaction mixture was stored at + 4 deg. C for 24 hrs. The solid was then filtered by suction on Whatman #42 paper and dried in vacua. Meld 23.2 g (0.109 moles).
Example 2: Synthesis of bis-chloro(2,3-dicarboxyphthalo-cyanino)silicon(IV) Diiminoisoindoline (30.0 g; 0.207 moles) and tetra-iminopyromellitic acid diimide (10.5 g, 0.050 moles) were pulverized together and dried in vacuo overnight in a one liter, 3 neck flask. The flask was fitted with a Teflon vane mixer, septum, thermometer and reflux condenser with a silica gel drying tube. The apparatus with the stirred reactants was flushed with dry nitrogen and under nitrogen, 600 ml quinoline was added and mixed for 30 min. under nitrogen flow. A uniform, fluid suspension resulted.
Thereafter, over a 5 min period f0 ml silicon tetrachloride was added slowly through the septum. The solution darkened and without heating stirring was continued for 15 min.
Then, with continuous stirring, an oil bath preheated to 195 deg. C was raised into position to immerse the flask to a level above it.s contents. After 5 min the bath temperature had dropped to 175 deg. C and after another 15 min the bath stabilized at 185-190 deg. C where it was maintained for an additional 60 min. The bath was then lowered and the reaction mixture was allowed to cool for about 15 min. Nitrogen flow was then started to remove unreacted silicon tetrachloride which was detected by moist pH paper at the condenser outlet. After about 45 min of ventilation the bath now at 100 deg. C was replaced to continue heating slowly to about 130 deg. C to facilitate 7.0 removal of silicon tetrachloride which was complete by the above test after an additional period of 70 min. when only quinoline fumes were evident.
The bath was them removed and when the reaction mixture had cooled to ca. 80 deg. C a mixture of 424 ml water and 7.5 424 ml concentrated hydrochloric acid was added with mixing.
Heat was evolved and the final mixture was acidic. The reaction products stood at room temperature overnight. The next day an additional 424 ml water and 424 ml concentrated hydrochloric acid was added with mixing, and the mixture was 20 allowed to settle at :room temperature overnight.
The reaction mi=xture was then filtered on a Buchner funnel (24 cm paper), washed with water and air dried in the hood overnight. The moist filter cake was stirred in one liter of acetone and filtered. The washed material was 25 dried in the hood for 2 days, The dried material (50 g) was pulverized in a mort=ar with acetone and the mixture was stirred, filtered and dried in vacuo leaving 47.9 g of a dark finely divided solid.
Example 3: Hydrolysis of bis-chloro(2,3-dicarboxy phthalo-30 cyanino)silicon (IV) (dicarboxydichloro dye).
Concentrated sulfuric acid (98 ml) was placed in a 250 ml round bottom fla:~k using a long stem funnel to avoid wetting the neck of t=he flask. With magnetic stirring 16.3 g of dicarboxydichloro dye was added in small portions 35 through a funnel with a shorter stem. The additions were extended over period of about an hour to allow lumps of dye to disperse before adding more solid. A drying tube was attached to the flask and the mixture was heated in an oil bath and maintained at 50 deg. C for 24 hr.
The reaction flask was removed from the oil bath and cooled in ice. Water (75 ml) was added cautiously in small portions and without cooling, the mixture was heated with stirring in an oil bath at 80 deg. C for 20 hr. After cooling, the mixture was poured into ice in a one liter beaker and stirred.
The mixture was centrifuged at room temperature at 2000 x g for 30 min. The sediment was suspended in water (ca.
250 ml) and again centrifuged. This washing was repeated once more, the sediment was collected and suspended in 300 ml 1M K2C03. The mixture was heated with stirring in a beaker covered with a watch glass. In 10 min the temperature reached 90 deg. C and heating was continued at about 93 deg. for 50 min more. While still hot the mixture was acidified with concentrated HCl and allowed to cool and stand at room temperature for 2 days.
The solid was then collected on a Buchner funnel, 11 cm with Whatman #42 paper, the filtration taking ca 1 hr. The solid was washed on the funnel with 3x100 ml portions of water and air dried in the hood. The solid was then broken up and dried in vacuo over P205 and KOH. Yield 13.3 g (870).
Example 4: Purification of bis-hydroxy (2,3-dicarboxy-phthalocyanino) silicon IV (dicarboxy dye) by adsorption chromatography on silica.
Crude dicarboxy dye, 3.0 g, produced as in Example 3, was placed in a 250 ml bottle to which was added 100 ml MeOH
containing 20 (v/v) ethyldiisopropyl amine (DIEA) and the mixture was stirred for 30 min. After this time 43 g silica (EM Science) was added and the mixture was shaken by hand to form a dark paste. After 20 min an additional 100 ml MeOH
with 2o DIEA was added, the bottle was inverted a few times and the contents were stirred for 20 min. Adjustment of the solvent characterist_Lcs by adding EtOH in addition to MeOH

and DIEA alters the composition of the dye extracted and enables extraction of a single component. The solid was then filtered on a sintered glass funnel (fine porosity, 6.5 cm diameter) under reduced pressure. To prevent too great 5 loss of MeOH the reduced pressure was maintained by connection to a partially evacuated tank. After filtering overnight, the residue was washed with 2 x 50 ml portions of MeOH + DIEA which required about 30 hr. The filtrate (230 ml) was concentrated in a rotary evaporator to near dryness.
10 The residue was dissolved in 14 ml MeOH +DIEA and the solution was divided equally and put into two 40 ml conical centrifuge tubes.
The contents of- each tube was acidified with 200 ul concentrated HC1 and water was added to nearly fill the 15 tubes. The contents were mixed by inverting and shaking few times, and centrifuged at about 650 x g for 30 min. The brown supernatant liquid was discarded and the sediment was washed three times with 0.01 M HC1. The sediment was transferred to a 100 ml round bottom flask and the mixture 20 was dried by rotary evaporation and then in vacuo over HZSO9 and KOH. Wt dry was 304 mg (purified dicarboxy dye).
Example 5: Sulfonation of purified dicarboxy dye by chloro-sulfonic acid.
Purified dicarboxy dye (Example 4), 161mg, was weighed 25 into a 50 ml long neck, round bottom flask with a magnetic mixer. At room tempf~rature 3.4 ml of C1S03H was added under NZ. A small air condenser with N2 balloon was attached and the flask and contents was heated in an oil bath at 110 deg.
for about 3.7 hr. at which point a 50 ml sample was 30 withdrawn for testing. Heating was then continued for an additional 3.3. hr. at 110 deg. whereupon heating was stopped and a second sample was taken out. Ice was added to both samples and each was diluted with water to a weight of 390 mg. 2 ml 1 M NaHf.03 was then added to each sample and the 35 absorbance of each was measured by diluting 10 ul of diluted sample with 2 ml of: neutral buffer to make the measurement.

For both samples the Amax was at 690nm, the 3.7 hr. sample reading 0.650 and the 7 hr sample reading 0.490 indicating about 25o destruction of the dye in the last 3.3 hr heating period.
The main reaction mixture was added in small portions to ice in a beaker, and the cold mixture was centrifuged at about 700 x g for 30 min. The very faintly colored supernatant liquid was discarded. The sediment was suspended in 30 ml oi= ice cold water, transferred with water to a 250 ml Erlenmey~=r flask, made basic with about 40 ml 1 M KHC03 and stirred at room temperature overnight. The reaction mixture was transferred to a beaker, acidified with concentrated HCl and stirred at room temperature for 6 hr and stored at room temperature for 48 hr.
The mixture was centrifuged at ca. 700 x g; the colored supernatant fluid was retained and the sediment was dissolved in 1 M Na.HC03 and stirred for 2 hr. The dark greenish solution was passed through a Sep Pak (2 g size, Rainin) and the filtrate after acidification was combined with the supernatant fluid from the centrifugation. Total volume was ca. 400 rnl. The dark blue acidic solution was adsorbed on a Sep Pad:, washed on the column with 3 N HCl and eluted with MeOH. The Sep pak after washing with MeOH and 3 N HCl can be used ovE:r and over. The MeOH eluate containing the dye was dried by rotary evaporation and over H2S04 and KOH in vacuo. Yield 7.58 mg.
Example 6: Measurement of the Nonspecific Binding and Characterization of the Solution Properties of Silicon and Aluminum Phthalocyanines Materials:
1) Borate buffered KCl (BBKC1): This buffer is made by mixing: 33.1 ml of 0.70 M boric acid; 4.0 ml of 0.50 M
KZB90~; 75 ml of 4.0 N.l KC1; H20 to make one liter; pH ca 8.1.
2) Sulfonated dicarboxysiliconphthalocyanine (Example 5), dicarboxysiliconphthalocyanine (Example 4), and aluminum trisulfonic acid, sodium salt (Porphyrin Products) were dissolved in BBKC1. The concentrations of these solutions were determined by absorbance measurements at the NIR
maximum (ca 680nm) of a dilution of each sample in methanol containing 5% (v/v) o:~ ethyldiisopropyl amine and assuming the value of the molar extinction coefficient to be 2x105 liter mole-1 for all samples. Dilutions were then made in BBKC1 to give solutions of each dye at a concentration of 5x10-$M.
Fluorescence Measurements. Measurements of intensity 1.0 and polarization were made in a transient state polarization fluorometer (FAST l, Hyperion, Inc. Miami, FL) by, in each case, diluting 10 microliters of a 5x10-8 M solution of the dyes (item 2, Materials) with lml of either BBKC1, BBKC1+
1$(v/v) pooled norma.l_ human serum (L3833, 10/18/84) or glycerol.
The results are shown in Figures 1 and 2 and characterize the nonspecific binding and solvent sensitivity of the three dyes as assessed by fluorescence intensity and polarization measurements. With regard to intensity, the properties desired are a constancy independent of solvent constituents together with a high fluorescence output. On the other hand, the polarization ideally should remain low in media of low viscosity and be as high as possible in a viscous solvent, such as glycerol.
Figure 1 shows that the fluorescence intensity from aluminumphthalocyaninetrisulfonate is very solvent-sensi-tive. In comparison, dihydroxy dicarboxy silicon phthalo-cyanine sulfonate shows a dramatically improved performance having almost the same fluorescence output in buffer, buffer plus serum or glycerol alone. Some of this improvement can be seen to be due to sulfonation by comparison with dihydroxy-dicarboxy-silicon-phthalocyanine (no sulfonate groups) which itself is less sensitive to serum and solvent than is the Al compound.
Figure 2 indicates even more strongly that the mere presence of the central Si atom results in a lowering of the sensitivity to the environment when compared to A1 as a central atom. This difference is most likely due to the fact that Si has two axial ligands and can hence "protect"
the planar structure of the dye from solvent effects since a "protecting group" is then present on each side of the molecular plane. In the case of A1 only one axial ligand is present and hence one side of the molecular plane is freely accessible to solvent effects. In Figure 2 the result of this interaction is clearly seen in the very large increase in polarization of the A1 dye nearly to the maximum 7_0 attainable by putting the dye into glycerol (which indicates the approximate limit for the polarization if rotational motion is nearly stopped).
Conclusion The above example applications, relating to the present J.5 invention, should not, of course, be construed as limiting the scope of the invention. Such variations of the invention, now known or later developed, which would fall within the purview o:f those skilled in the art are to be considered as falling within the scope of the invention as ~'.0 hereinafter claimed.
All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the 2.5 same extent as if: each individual publication was specifically and individually indicated to be incorporated by reference.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, ?'.0 limitation or limil~ations which is not specifically disclosed herein. 'Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions 35 which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the ;scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art., and that such modifications and :'_0 variations are considered to be within the scope of this invention as defined by the appended claims, Other embodiments are within the following claims.

Claims (63)

Claims

1. A detectably labeled marker component which comprises a luminescent substantially planar fluorophore moiety coupled to two or more small solubilizing axial ligands, one located on either side of the fluorophore moiety.

2. A marker component according to claim 1, wherein the fluorophore moiety comprises a macrocyclic multidentate ligand coordinated to a central atom and the solubilizing axial ligands are independently selected from the group consisting of hydroxyl, chlorine, bromine, fluorine, OCH3, and O-CH2OH.

3. A marker component according to claim 2, wherein the marker comprises two solubilizing hydroxyl moieties, one solubilizing hydroxyl moiety linked on either side of the macrocyclic ligand too the central atom.

4. A marker component according to claim 3, wherein the two hydroxyl moieties comprise axial ligands which coordinate to the central atom.

5. A marker component according to claim 4, wherein said central atom is capable of forming octahedral coordination complexes.

6. A marker component according to claim 5, wherein said macrocyclic ligand has a conjugated .pi.-electron system.

7. A marker component according to claim 6, wherein said macrocyclic ligand comprises a nitrogen containing macrocycle.

8. A marker component according to claim 7, wherein said macrocyclic ligand is selected from a porphyrin derivative, or a porphyrin derivative having one or more bridging carbons replaced by nitrogen, a corrin derivative, a sapphyrin derivative or a porphycene derivative.

9. A marker component according to claim 8, wherein said central atom is selected from silicon, germanium, phosphorus and tin.

10. A marker component according to claim 9, wherein said macrocyclic ligand has a low degree of symmetry so as to enhance the polarization of emission parallel to polarization of absorption.

11. A marker component according to claim 10, wherein said central atom is silicon or germanium.

12. A marker component according to claim 11, wherein said macrocyclic ligand has a lower symmetry degree of symmetry than D4h.

13. A marker component according to claim 12, wherein said macrocyclic ligand has at least one fused aromatic ring.

14. A marker component according to claim 9, wherein said macrocycle comprises a porphyrin derivative wherein from 1 to 4 of the bridging carbon atoms is replaced by nitrogen.

15. A marker component according to claim 14, wherein said macrocycle comprises a tetrabenzotriazaporphyrin derivative.

16. A marker component according to claim 15, wherein said macrocycle is selected from tetrabenzatriazaporphyrin, 27-phenyltetrabenzotriazaporphyrin, and 27-(p-methylphenyl) tetrabenzotriazaporphyrin.

17. A marker component according to claim 16, wherein the central atom is silicon.

18. A marker component according to claim 13, wherein said macrocyclic ligand comprises a phthalocyanine derivative.

19. A marker component according to claim 1, wherein the fluorophore moiety is a naphthalocyanine or a naphthalocyanine derivative.

20. A detestably labeled marker component according to claim 1, wherein said fluorophore moiety coupled to two or more small solubilizing axial ligands is characterized by having, in the presence of serum components in aqueous solution, transient state fluorescence emission having parallel and perpendicular components of substantially the same intensities as without serum.

21. A detestably labeled marker component according to claim 1, wherein said luminescent substantially planar molecular structure has an excitation wavelength of at least about 500 nanometer and said marker component has decreased non-specific binding to components of serum as compared to the bare fluorophore moiety when it is not coupled to two or more hydroxyl moieties.

22. A marker component according to claim 21, wherein said fluorophore moiety has an excitation wavelength of about 600 to 800 nanometers.

23. A marker component according to claim 22, wherein said fluorophore moiety has an excitation wavelength of at least 650 manometers.

24. A marker component according to claim 21, wherein said marker component has substantially similar intensity, decay time and relative magnitudes of the polarized components in the presence and absence of serum.

25. A marker component according to claim 21, wherein said fluorophore moiety alone has a binding constant that is at least 60 times greater than the binding constant of the complete marker component.

26. A marker component according to claim 21, wherein said fluorophore is selected from the group consisting of:
(1) aryl terminated polymethine dyes; (2) quinoid dyes; (3) indanthrene dyes; (4) 1,4- diaminoanthraquinone-2,3-dicarboxamides; (5) tetraaminoanthraquinones; (6) azine dyes; (7) pyrylium or thiopyrylium dyes; and (8) naphthoquinone methides.

27. A method of synthesizing a marker component according to any one of claims 1-26, comprising the step of reacting the fluorophore moiety with a reactive form of said solubilizing axial ligands.

28. A method of detecting a target analyte in a sample suspected of containing said target analyte, the method comprising the steps of:
(a) linking a marker component of any one of claims 1-26 to a target analyte or analog thereof;
(b) contacting said sample suspected of containing said target analyte with a known quantity of marker component linked to target analyte or analog thereof;
(c) contacting said sample suspected of containing said target analyte with a receptor which specifically binds to said target analyte;
(d) determining either the amount of said marker component linked to target analyte or analog thereof bound to said receptor or the amount of unbound target analyte or analog thereof.

29. A fluorescent probe comprising a marker component of any of claims 1-26, linked to one member of a specific binding pair or a target analyte or an analog.

30. The probe of claim 29, wherein said receptor target analyte or analog thereof is attached directly to said fluorophore moiety.

31. The probe o.f claim 29, wherein said receptor target analyte or analog thereof is conjugated via a linker arm to said fluorophore moiety.

32. The probe of claim 29, wherein said receptor target analyte or analog thereof is selected from the group consisting of digoxin, digitoxin, theophylline, phenobarbital, acetylprocainamide, primidone, phenytoin, rubella antibody, and derivatives of each.

33. A fluorescent probe according to claim 29, wherein said member of a specific binding pair has at least one sterically tolerant marker component attachment site capable of permitting said probe to form a specific binding pair.

34. A method synthesizing a fluorescent probe which comprises the step of linking a marker component of any one of claims 1-26 to two or more solubilizing axial ligands.

35. A method of detecting a target analyte in a sample suspected of containing said target analyte, the method comprising the steps of:
(a) contacting a sample suspected of containing said target analyte with a first receptor capable of specifically binding to said target analyte to form a complex of said target analyte and said first receptor, said first receptor being labeled with a fluorescent probe comprising a marker component of any one of claims 1-26;

(b) contacting said complex with a second receptor capable of specifically binding to said target analyte, said second receptor being bound to a solid carrier, to form a complex of said first labeled receptor, said target analyte, and said second receptor bound to said solid carrier; and (c) measuring either the amount of labeled first receptor bound to said solid carrier or the amount of unbound labeled first receptor.

36. The method of claim 35, further comprising the step of relating the amount of said first receptor labeled with a fluorescent probe measured in said sample suspected of containing said target analyte to the amount of said first receptor labeled with a fluorescent probe measured in a control sample which contains no target analyte, or to the amount of first receptor labeled with a fluorescent probe measured in samples containing known quantities of said target analyte .

37. The method of claim 35, further comprising the step of separating the solid carrier from the unbound labeled first receptor.

38. A method for determining the presence or amount of a target analyte in a sample suspected of containing a target analyte comprising the steps of:
(a) simultaneously contacting said sample suspected of containing a target analyte with first and second receptors capable of specifically recognizing said target analyte, said first receptor being labeled with a fluorescent probe comprising a marker component of any one of claims 1-26, and said second receptor being bound to a solid carrier, to form a complex of said first receptor, said target analyte, and said second receptor; and (b) measuring either the amount of said labeled first receptor associated with said solid carrier or the amount of unreacted labeled first receptor.

39. The method of claim 38 further comprising the step of relating the amount of labeled first receptor measured in the sample suspected of containing a target analyte to the amount of labeled first receptor measured in a control sample which contains no target analyte, or to the amount of labeled first receptor measured in samples containing known quantities of said target analyte.

40. A method for measuring a target analyte capable of binding to two different receptors in a sample suspected of containing said target analyte comprising the steps of:
(a) contacting said sample suspected of containing said target analyte with a first receptor capable of specifically binding to said target analyte to form a complex of said target analyte and said first receptor, said first receptor being labeled with a fluorescent probe comprising a marker component of any one of claims 1-26; and (b) contacting said complex with a second receptor capable of specifically binding to said target analyte, said second receptor having absorption and emission maxima which are not identical to those of said first receptor.

41. A kit useful for detecting a target analyte in a sample suspected of containing said target analyte, the kit comprising a marker component of any one of claims 1-26.

42. A kit useful for detecting a target analyte in a sample suspected of containing said target analyte, the kit comprising a fluorescent probe of any one of claims 27-31.

43. A composition comprising an oligonucleotide linked to a detectably labeled marker component of any of claims 1-26.

44. The composition of claim 43 wherein said detectably labeled marker component has a decay time in the range of from about 1 nanosecond to about 50 nanoseconds.

45. The composition of claim 44 wherein said decay time is in the range of from about 5 nancseconds to about 20 nanoseconds.

46. The composition of claim 43 wherein said oligonucleotide having a length of between 5 to 50 bases.

47. The composition of claim 43 wherein said linkage comprises (CH2) 6O.

48. The composition of claim 43 wherein said linkage comprises (CH2)2NH.

49. A method for preparing a marker component-conjugated oligonucleotide comprising the steps of:
(a) reacting an oligonucleotide having an attached linker terminating in an amino group with an N-hydroxy-succinimide ester or in an imidazolide of a marker component a composition comprising an oligonucleotide linked to a detectably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structures to form a conjugate; and (b) separating the conjugate formed in step (a) from unreacted oligonucleotide and from unreacted marker component.

50. The method of claim 49 further comprising performing before step (a), the step of: attaching to an oligonucleotide a linker terminating in an amino group.

51. A method for preparing marker component-conjugated oligonucleotide comprising the steps of:
(a) reacting a marker component a composition comprising an oligonucleotide linked to a detectably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar-molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure, with a carbodiimide in the presence of hydrobenzotriole and in the presence of an oligonucleotide, to form a conjugate; and (b) separating the resulting conjugate formed in step (a) from other components of the reaction mixture.

52. A method for determining the presence or amount of a target nucleic arid sequence in a sample comprising the steps of:
(a) contacting sample nucleic acid with a composition of claim 43 wherein said composition is capable of hybridizing with said target nucleic acid sequence in homogenous solution; and (b) detecting the presence or amount of such hybridization by transient state polarized fluorescence.

53. A method for detection of a target nucleic acid sequence in a sample camprising the steps of:
(a) contacting a sample suspected of containing a target nucleic acid sequence with a complementary oligonucleotide capable of hybridizing with said target sequence;
(b) contacting said sample with a composition of claim 1 wherein said composition is capable of hybridizing to said complementary oligonucleotide or polynucleotide; and (c) detecting the presence or measuring the amount of hybridization of said conjugate with said complementary oligonucleotide or polynucleotide.

54. A method according to any of claims 51-53 wherein said target nucleic acid sequence is selected from the group consisting of a product of nucleic acid amplification, DNA, and RNA.

55. A method according to claim 54 wherein said nucleic acid amplification is performed by a method selected from the group consisting of polymerase chain reaction (PCR), ligand chain reaction (LCR), self-sustained sequence replication (3SR), and transcription-based amplification system (TAS).

56. In a nucleic acid amplification process, the improvement comprising employing as a label a fluorescent probe which comprises a detestably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.

57. In a nucleic acid amplification hybridization process, the improvement comprising employing as a label a fluorescent probe which comprises a detestably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.

58. The composition of claim 43 wherein said composition is thermostable.

59. The composition of claim 58 wherein said composition is stable after exposure to 90°C for 1 hour.

60. The composition of claim 59 wherein said composition is stable after exposure to 100°C for minutes.

61. A kit comprising the composition of claim 43 and a label, package or instructions.

62. A method of using the kit of claim 62 comprising using the composition in accordance with the label, package or instructions.

63. A method of making the kit of claim 61 comprising the step of combining said composition and said label, package or instructions.