CN1344293A - 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

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

Introduction to

The present invention relates generally to non-aggregating and serum-binding water-soluble fluorescent dyes and related products and methods. Preferred dyes include a luminescentsubstantially planar fluorophore linked to two or more solubilizing ligands.

RELATED APPLICATIONS

This application claims the benefit of prior U.S. provisional application 60/109,969 entitled "non-aggregating and serum-binding water-soluble fluorescent dyes and related products and methods" filed on 25/11/1998 by Dandliker, incorporated herein by reference in its entirety, including the figures.

Background

The publications and other references referred to herein are incorporated by reference. The following description of the background of the invention is provided to aid in the understanding of the invention and is not intended to describe or constitute features of the invention in the prior art.

The near infrared absorption and emission of porphyrins (porphyins), phthalocyanines and other azaporphyrins (azaporphins) and certain other nitrogen-containing aromatic macrocyclic compounds sometimes make these compounds attractive candidates for use as fluorescent labels.

Phthalocyanines, especially due to their strong near infrared absorption (molar extinction coefficient of about 200,000), their high quantum yield in organic solvents, and their well-known ability to resist fading of common metal phthalocyanine dyes, have made many attempts to utilize them as fluorescent labels. However, early attempts along this line of thought have not resulted in a completely satisfactory product, largely due to the unusually strong tendency of phthalocyanines to associate, particularly in the form of face-to-face aggregates, to pack and also to bind strongly to various other molecular surfaces (non-specific binding).

The result of intramolecular stacking is that the unsubstituted phthalocyanine has very low solubility in both organic and aqueous solvents. As is now well known, the tendency to pile up can be significantly reduced by the introduction of one or more charged groups (e.g. sulfonate groups). Phthalocyanines having such substitutions may have high solubility in aqueous solutions of water and electrolytes, substantially retaining their tendency to bind non-specifically.

Currently, most scientific research on fluorescent labeling is focused on applications involving biological materials such as tissue sections, cells, cell debris, proteins, including glycoproteins and lipoproteins, peptide oligo-and poly-saccharides, oligo-and poly-nucleotides and lipids. The tendency for non-specific binding in fluorescence assays involving these materials can be perturbed by partially masking the specific interactions involved.

Non-specific binding and tendency to accumulate can be reduced to levels that are negligible in therapeutic drug detection by coupling the phthalocyanine dye to one or more polyoxyhydrocarbyl groups, typically methoxy-terminated poly (ethylene glycol) (PEG). At the same time, the attachment of such groups preserves the desired absorption and emission characteristics. This technique is also effective for a large number of other near infrared dyes. See U.S. patent application No. 08/476,544, Lyon&Lyon Docket 211/167 (and prior applications referred to therein), entitled "polyoxyhydrocarbyl-related products and methods for fluorescence detection", filed on 7.6.1995 by Dandliker et al, which is incorporated herein by reference in its entirety, including all figures.

Recently, there has been significant development in dyes that can fluoresce. On the one hand, dyes that are excited by radiation of longer wavelengths (e.g., red and infrared wavelengths) are currently available. These dyes are described in the following two commonly assigned patent applications. Arrhenius, U.S. patent application serial No. 701,449 entitled "fluorescent marker components and fluorescent probes" (which is a continuation of U.S. patent application No. 523,601 filed on 5/15/1990), U.S. patent application serial No. 701,465 entitled "fluorescent dyes without aggregation and serum binding" filed on 5/15/1990, and dardliker and Hsu, which is a continuation of U.S. patent application serial No. 524,212 filed on 5/15/1990. These applications are incorporated by reference herein in their entirety, including all drawings.

There has been a more significant improvement in increasing sensitivity through data collection and analysis techniques. The use of data collection and analysis techniques in combination with time gating techniques (time gating techniques) resulted in improved signals over background as described by Dandliker et al in U.S. patent No. 4,877,965 entitled "fluorometer" (which is incorporated herein by reference in its entirety, including all figures). Generally, the 4,877,965 patent recognizes that the detected intensity as a function of time consists of signals from different sources, including a desired signal source and various undesired background sources. Optimization of the desired signal can be achieved by data collection and analysis techniques.

Significant improvements have also been made in the ability to detect related substances in immunoassays. Bound and free forms of a substance can be detected in a homogeneous assay, for example, using the techniques described in U.S. patent application serial No. 490,770 entitled "transition state fluorescence assay," filed on day 6 of 1990 by Dandliker (which is a continuation of U.S. patent application serial No. 365,420 filed on day 13 of 1989), which is incorporated herein by reference in its entirety, including all figures. Typically, this technique requires the measurement of the decay of the intensities of parallel and perpendicular polarised components over time. By measuring the time-dependent decay of different polarization states, the bound and free form of a substance (such as a hapten, a peptide or a small protein) can be determined in a homogeneous assay format. Importantly, this technique does not require separation of bound and free species.

Despite significant and promising improvements in the area of fluorescing dyes and in data analysis, there remains a need in the art for additional dyes that have these and/or other advantages, and which also have favorable chemical reactivity.

Summary of the invention

The present invention develops an unexpected result: even very small groups, such as-OH, are effective in preventing non-specific binding and stacking of molecules if two such groups are present on a planar molecule, one on either side of the molecular plane. The beneficial effect of these ligands on many biological systems, in which most "biomolecules" carry a negative net charge on their own, is enhanced by increasing the net negative charge.

Accordingly, one aspect of the present invention is: the desired effect can be achieved by coupling the engineered phthalocyanine and other fluorescent dyes to the polyoxyalkyl group instead with two very small axial ligands (e.g., -OH) so long as the net charge on the dye is sufficiently large. In most cases, it is preferred that this net charge be negative, since most biological substances, including proteins and DNA, also carry a negative net charge over the physiological pH range. Thus, we have found that highly sulfonated dihydroxy-silicon-dicarboxy-phthalocyanines have almost as low non-specific binding to serum proteins as PEG conjugated non-sulfonated dyes.

Furthermore, one advantage of the present invention is represented by the absence of molecular groups formed by PEG. PEG engineering dye at a dye concentration of 10^-4M and above, it behaves very similar to a macromolecule and is therefore greatly hindered when it passes through a membrane designed to prevent the passage of molecules of about 30K daltons or higher. Moreover, in gel permeation chromatography, which is designed to separate macromolecules from small molecules, dyes move in the void volume of the chromatography. In contrast, sulfonated dihydroxy-dicarboxy-silicon-phthalocyanine (SDDSiPc) behaves as expected for molecules having this formula weight.

As for non-specific binding as measured by varying fluorescence polarization and intensity, SDDSiPc behaves substantially the same as PEG-coupled dyes (see example 6) when these dyes are exposed to, for example, diluted human serum. In this regard, given that unsulfonated dihydroxy-dicarboxy-silicon-phthalocyanine (DDSiPc) exhibits significant non-specific binding, it is important to note that its own negative charge has a significant effect in reducing non-specific binding. The hydroxy-aluminum-phthalocyanine trisulfonate exhibits a strong sensitivity to ionic strength and also has a strong nonspecific binding. It appears that the phthalocyanine molecule must have axial ligands on both sides of its molecular plane, but if the net charge is high enough, the-OH groups are also large enough to virtually eliminate unspecific binding.

Another advantage of the present invention is that dyes engineered with-OH or other small solubilizing axial ligands along with high charges appear to be more chemically active (in the labeling reaction) even if the labeled molecules (e.g., proteins and oligonucleotides) themselves carry negative charges. This suggests that although haptens and other small molecules are often easily labeled, PEG ligands do interfere with labeling of the molecules.

The present invention is highly unexpected because hydroxy-aluminum-phthalocyanine-trisulfonate has strong non-specific binding, from which one can speculate that dihydroxy-dicarboxy-silicon-phthalocyanine with a smaller net negative charge will exhibit stronger non-specific binding than aluminum dye. It is based, in part, on these findings that the present invention is directed to the exact opposite (see FIGS. 1 and 2). According to the findings reported here, other small axial ligands are expected such as: -OCH₃、-OCH₂OH, -Cl, -Br and-F have the same effect.

Many other nitrogen atom-containing macrocycles can be metallated with group 14 atoms with similar results. Such macrocycles include derivatives and structural variants of porphyrins, azaporhyrins, corrins, sapphyrins, pentaphyrins, porphycenes, and other similar macrocycles with broadly delocalized pi-electron systems. Due to the fact that macrocycles combine many desirable properties, particularly preferred types of macrocycles include azaporphyrin derivatives and structural variants. Azaporphyrin derivatives include mono-, di-and triaza-porphyrins and derivatives of porphyrazine. Any of these macrocycles may optionally also contain fused aromatic rings. Such azaporphyrin derivatives and variants include phthalocyanines, benzotriazaporphyrins and naphthalocyanines and their derivatives and their oxa, thia or aza structural variants.

The invention therefore relates to marker components, fluorescent probes, oligonucleotides, hybridization samples and immunoassays using these products and methods for producing these products. The present invention provides detectably labeled marker components comprising a fluorophore moiety coupled to two or more small solubilizing ligands, generally axial, the axes being determined by the octahedral geometry of the complex formed by the central metal atom, preferably such components that reduce or eliminate the problems of solvent sensitivity and nonspecific binding.

The advantage of using such a detectable label or label component in an immunoassay is that: the intensities of the parallel and perpendicular components of the transition state fluorescence emission of these labels are substantially the same in the presence and absence of biological fluids such as serum. Thus, assay methods using these labels are capable of detecting low concentrations of analytes, target analytes, or the like in biological fluids. The term "analyte" refers to a compound in an assay or compound being measured, and may be any compound whose receptor is naturally occurring or capable of being prepared, and may be a single-or multiple-epitope (polyepitopic), antigenic or hapten, single or multiple compounds sharing at least one common epitope (epitopic site) or receptor. The term "target analyte" refers to a compound in an assay or compound being measured, and may be any compound whose receptor is naturally occurring or capable of being prepared, which may be mono-or polyepitopic, antigenic or hapten, single or multiple compounds sharing at least one common epitope or receptor. An "analog" of a target analyte refers to a compound or compounds that are capable of competing with the target analyte for binding to a receptor. The term "receptor" refers to a molecule or molecular complex that is capable of specifically recognizing or being recognized by a target analyte or analog thereof. For example, antibodies may act as receptors for antigens.

These marker components may be used as labels to label analytes, antigens, antibodies or other molecules. These marker components may optionally be functionalized to include a linker arm to allow attachment of the marker component to an analyte, antigen, antibody or other molecule. Linker arms suitable for this purpose are described in Kricka, J.J., ligand-conjugate analysis, labeling and analytical strategies, pages 15-51 (Marcel Dekker Inc., N.Y., NY (1985)). The labeling component is linked to the analyte, antigen, antibody or other molecule using conventional techniques.

In one aspect, the invention provides a detectably labeled marker component comprising: (1) a fluorophore moiety comprising a luminescent substantially planar molecular structure, preferably having an excitation wavelength of at least about 500nm, and (2) two or more small solubilizing axial ligands coupled to the fluorophore moiety of (1). Examples of preferred fluorophores, small solubilizing axial ligands, and the attachment of both are described in detail herein. In addition, evidence is provided that demonstrates the effectiveness of the marker component in reducing solvent sensitivity and non-specific binding.

In a particularly preferred embodiment, the marker components of the invention can be used to make probes, as described primarily in commonly owned U.S. application Serial No. 08/051,446, filed on 3/21/1993, and can also be used in immunoassays, as described primarily in commonly owned U.S. application Serial No. 08/035,633, filed on 3/23/1993, the disclosures of which are incorporated herein by reference in their entirety, including all drawings.

The marker components of the present invention are useful as detectable labels, e.g., as diagnostic reagents and as detectable labels in assays, such as fluorescence binding assays and other immunoassays. The present invention provides a detectably labeled marker component having a fluorophore moiety coupled to two small solubilizing axial ligands, the marker component characterized by, in the presence of a serum component in an aqueous solution: the transition state fluorescence emission has parallel and perpendicular components of substantially the same intensity as in the absence of serum. The term "axial ligand" refers to a substituent that forms a coordination compound with the central atom along with the macrocyclic ligand. The axial ligands are perpendicular to the plane formed by the macrocycle.

Such molecular components are also believed to be useful in the methods described below. By Walker et al in 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 hereby incorporated by reference in their entirety, including all figures.

Surprisingly, we have found that the marker components of the present invention having two small solubilizing axial ligands coupled thereto, wherein one axial ligand is located on either side of the polydentate ligand plane, exhibit significantly reduced non-specific binding to serum components and exhibit negligible solvent sensitivity. The term "solvent-sensitive" means that the fluorescence properties of the molecule vary depending on the solvent system used, in particular the difference in fluorescence properties in aqueous solution compared to organic solvents (such as DMF). Many fluorophores that exhibit high fluorescence intensity in organic solvents (such as DMF) exhibit greatly reduced fluorescence intensity in aqueous solution. The fluorescence intensity is related to the sample concentration and the intensity of the excitation radiation. The fluorescence intensity of a particular dye can be related to its characteristic light absorption coefficient (extinction coefficient) and fluorescence quantum efficiency, as well as environmental factors. These marker components also exhibit enhanced decay times that approach their radiative or unquenched lifetimes. The term "decay time" as we use generally refers to the time that must be spent to reduce the concentration of excited atoms from their original concentration to 1/e of that value. The term "Lifetime" is used differently than, for example, Demos, j.n., Excited State Lifetime Measurements (Academic Measurements), Academic Press, new york, n.y. (1983), pages 10, 35, 44 and 158.

These marker components can be used as fluorescent labels incorporated into fluorescent probes. The term "fluorescent probe" refers to a label component containing a fluorophore moiety that is bound or coordinated, either directly or via a linker arm, to an analyte, antigen, hapten, antibody or other molecule used in an assay, such as a fluorescent immunoassay, to determine the presence and/or amount of a substance of interest. Some of these marking components may be used as phosphorescent markers. The components of the invention may be used as labels for agents used for in vivo imaging, and also as labels for agents used in vivo tumour therapy.

Thus, in general, preferred fluorophores are those that are effective to produce fluorescence upon excitation by light having a wavelength in the range of from about 200 to about 1000nm, preferably about 600 and 800 nm. Suitable fluorophores include those that absorb and/or emit at wavelengths different from the excitation and emission maxima of other solution components, such as proteins present in the sample, to minimize background fluorescence.

Since these marker components are particularly useful in assays of samples using biological fluids, preferred for those applications are fluorophores having excitation and/or emission wavelengths of at least about 500nm, which reduce interference from ambient fluorescence of other sample components. Some samples, such as serum, exhibit considerable interfering background fluorescence from flavins, flavoproteins, NADH, etc., when excitation wavelengths less than 500nm are used.

For certain applications, such as fluorescence polarization immunoassays, the preferred fluorophores also exhibit a high degree of fluorescence polarization in the bound form, preferably about 10% above the theoretical maximum observable polarization. The term "association" refers to the state of formation of an association between a molecule and its particular binding partner. For certain applications, such as fluorescence transition state assays, preferred fluorophores are also characterized by: the measured fluorescence decay time ranges from about 1 nanosecond to about 50 nanoseconds, and preferably ranges from about 5 to about 20 nanoseconds. For other applications, such as phosphorescent labels, fluorophores with longer decay times may be used.

Thus, those fluorophores which are efficient in generating fluorescence, i.e., which are characterized by a high absorption coefficient at the appropriate wavelength and a high fluorescence quantum yield, are preferred. For some applications, preferred fluorophores measure fluorescence decay times of at least about 2 nanoseconds and exhibit a high degree of fluorescence polarization.

Preferred small solubilizing axial ligands include: -OH, -oxy-tert-butyl-, -OCH₂OH、-OCH₂CH₂OH、-OCH₂CHOHCH₂OH、-OCH₂CH₂-O-CH₂CH₂OH、-OCH₂CH₂-CH₂-O-CH₂CH₂OH, Cl, Br and F. For any of these axial ligands attached to the central atom via-O-, the stability to hydrolysis may be improved by using a direct carbon to metal bond (e.g., carbon-silicon) instead of an-O-linkage.

In a preferred embodiment, the fluorescent moiety has one substantially planar multidentate macrocyclic ligand coordinated to a central atom that is capable of coordinating with two small solubilizing axial ligands. Suitable central atoms for use as label components in fluorescent binding assays are those to which two axial ligands can coordinate, which are not so high in atomic number as to cause substantial quenching of fluorescence by conversion to the triplet state. Preferred elements as central atoms include silicon, germanium and tin, with silicon and germanium being particularly preferred.

The invention also relates to a method for determining the presence or amount of a target analyte in a sample by using as a label for the target analyte or receptor a fluorophore moiety capable of specifically recognizing the target analyte, said fluorophore having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one axial ligand being located on either side of said planar molecular structure.

The advantage of using such a detectable label or label component in an immunoassay is that: the intensities of the parallel and perpendicular components of the fluorescence emission of these labels are substantially the same in the presence and absence of biological fluids (e.g., serum). Thus, assay methods using these labels are capable of detecting low concentrations of target analytes in biological fluids.

The method of the present invention is particularly suited for use with a modified fluorescence detection system described in commonly assigned U.S. patent application Ser. No. 3/23 1992, entitled "fluorometer detection System", Lyon&Lyon Docket 195/129, Ser. No. 07/855,238. It is believed that the labeling components and labels may also be used in the following methods, including Walker et al in 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 all figures.

In one aspect, the invention relates to a competitive inhibition assay using a specific label. In this regard, the present invention relates to a method of determining the presence or amount of an analyte of interest, the method comprising: contacting a sample suspected of containing the target analyte with a known amount of added target analyte or analog thereof linked to a fluorescentprobe comprising a detectably labeled marker component formed by a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one ligand on either side of the planar molecular structure; contacting the sample with a receptor capable of specifically recognizing the target ligand; and determining the amount of fluorescent probe bound to the receptor or free fluorescent probe. The amount of bound or free fluorescent probe in an unknown sample can be compared to a blank sample and a sample containing a known amount of the analyte of interest.

In a preferred embodiment, the resulting mixture of sample, fluorescent probes and acceptors are diluted rapidly prior to measuring the amount of bound and/or free fluorescent probes. The dilution step allows for a higher sensitivity of the assay. Particularly preferred is a dilution of 2 to 100 times, preferably about 7 to about 50 times, more preferably about 35 times.

In one aspect, the invention improves immunoassay methods using labels for target analytes (or analogs thereof) or receptors. This improvement uses a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one on either side of the planar molecular structure. The advantage of using such labels for assays is that they are free of serum binding and aggregation and are therefore particularly suitable for the detection of biological fluids such as serum, plasma, whole blood and urine.

In another aspect, the invention provides a method of performing a "sandwich" or "two-site" immunoassay comprising the steps of: (a) contacting a sample suspected of containing a target analyte with a first receptor capable of specifically recognizing said target analyte to form a complex of said target analyte and first receptor, the first receptor being labeled with a fluorescent probehaving a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one axial ligand being located on either side of said planar molecular structure; (b) contacting the complex with a second receptor (bound to a solid support) capable of specifically recognizing the target analyte or first receptor to form a complex of a first labeled receptor, a target analyte, and a second receptor bound to a solid support; and (c) measuring the amount of labeled first receptor bound to the solid support or the amount of unreacted labeled first receptor.

The sandwich-type assay may be a heterogeneous assay or a homogeneous assay. In the case of a heterogeneous assay, an additional step of separating the solid support from the unreacted labelled first acceptor may be introduced. Homogeneous assays are generally preferred because they are faster.

In another embodiment, the assay may incorporate the additional step of correlating the amount of labeled first receptor measured in an unknown sample with the amount of labeled first receptor measured in a control sample that does not contain the analyte of interest, or with the amount of labeled first receptor measured in a sample containing a known amount of the analyte of interest.

In another aspect, the present invention provides a method of simultaneously performing a sandwich-type assay for determining the presence or amount of a target analyte in a sample, comprising the steps of: (a) simultaneously contacting a sample suspected of containing a target analyte with first and second receptors capable of specifically recognizing said target analyte to form a complex of said first receptor, target analyte and second receptor, said first receptor being labeled with a fluorescent probe having a fluorophore moiety having a luminescent substantially planar molecular structure coupledto two small solubilizing axial ligands, one axial ligand on either side of the planar molecular structure, the second receptor being bound to a solid support; (b) measuring the amount of labeled first receptor bound to the solid support or the amount of unreacted labeled first receptor.

In another aspect, the present invention provides a method for simultaneously performing sandwich-type assays, further comprising the steps of: correlating the measured amount of labeled first receptor with the amount of labeled first receptor measured in a control sample not containing the target analyte, or correlating the measured amount of labeled first receptor with the amount of labeled first receptor measured in a sample containing a known amount of target analyte.

In another aspect, the present invention provides a sandwich-type fluorescence immunoassay method for measuring a target analyte capable of independently recognizing two different receptors without interfering with each other. This method utilizes two receptors, each labeled with a different dye. For example, one receptor is labeled with a first dye having absorption and emission maxima at 680nm and 690nm, respectively, and the other receptor is labeled with a second dye having absorption and emission maxima at 695nm and 705nm, respectively. Analytes can be detected and quantified using steady state or transition state measurements. In either case, excitation at 680nm and detection at 705nm were given for the examples. The basis of such assays is energy transfer, which has the advantage that it is homogeneous.

In preferred embodiments, the present invention relates to immunoassays for biological fluids, including serum, plasma, whole blood, and urine. Preferably, the red blood cells in the whole blood are lysed prior to measuring the whole blood sample. A preferred method of lysing erythrocytes comprises adding stearoyl lysolecithin, palmitoyl lysolecithin and myristoyl lysolecithin.

Depending on the type of immunoassay used, the analyte of interest may be an antigen, a hapten or an antibody and the receptor may be an antigen or an antibody. The antibody may be polyclonal or monoclonal. Preferably, the antibody is a monoclonal antibody. The monoclonal antibodies used in the present invention can be obtained by the Kohler&Milstein method reported in Nature 256 at pages 495-497 (1975), or can be prepared by recombinant methods at pages 1275-1281 (1989) of Science 246.

In one embodiment, the target analyte is a drug or a metabolite of a drug. The drug may be a steroid, hormone, antibiotic, immunosuppressant, antiasthmatic, antineoplastic, antiarrhythmic, anticonvulsant, antiarthritic, antidepressant or cardiac glycoside. Examples of such drugs include: digoxin, digitoxin, theophylline, phenobarbital, thyroxine, N-acetylprocainamide, plinetron, amikacin, gentamicin, netilmicin, tobramycin, carbamazepine, ethosuximide, valproic acid, propiram, lidocaine, procainamide, quinidine, methotrexate, amitriptyline, mortriptyline, imipramine, desipramine, vancomycin and cyclosporine. In a preferred embodiment, the drug is digoxin.

In another embodiment, the analyte of interest is a peptide, for example a peptide hormone such as luteinizing hormone, follicle stimulating hormone, human chorionic gonadotropin, thyrotropin, angiotensin I, angiotensin II, prolactin, or insulin. The peptide may also be a tumor marker, such as a carcinoembryonic antigen. Alternatively, the peptide may be a virus or a portion of a virus, such as rubella virus or a portion thereof.

The method of the present invention provides a measured concentration of about 1X 10^-5M/L-about 1X 10^-13M, especially in the concentration range of about 1X 10^-9M/L-about 1X 10^-12M/L of target analytes. For the measurement ofdrugs and their metabolites, the range that can be measured by the method of the present invention is about 5X 10^-9M/L-about 5X 10^-12M/L, in particular about 1X 10^-10M/L-about 5X 10^-10Concentration of M/L. For the measurement of peptides, the method of the invention is capable of measuringHas an enclosure of about 1 × 10^-11M/L-about 1X 10^-12M/L。

Measurement of the amount of fluorescent probe (bound or free or both) can be determined by measuring steady state fluorescence or by measuring transition state fluorescence. In a preferred embodiment, the wavelength of the measuring light is greater than about 500nm, preferably greater than about 650nm, more preferably greater than about 680nm or 690 nm. Since the transition state detection system employs a laser diode, the dye must have an excitation maximum that matches the diode output wavelength. We enable the dye to be matched to other commercial laser diodes with output wavelengths of 680, 690, 720, 750 or 780 nm. Thus, the wavelength of the measurement light may be greater than about 680nm, 690nm, 720nm, 750nm, or 780 nm. The more the filter length is shifted into the red region of the spectrum, i.e. the greater the wavelength, the denser the signal on the background (attenuation).

In a preferred embodiment, the detection and quantification is performed by a transition state measurement method. The transition state energy transfer provides an improved measurement method due to the optimization of the absorption and emission wavelengths, and due to the optimization of the decay times of the first and second dyes. This optimization eliminates Rayleigh and Raman scattering and maximizes transfer efficiency and minimizes direct excitation of the second dye by the first dye.

It is therefore a primary object of the present invention to provide improved various FIAs with greatly enhanced sensitivity. It is another object of the present invention to provide a FIA method that enables rapid (typically within minutes) and accurate measurements. It is an object of the present invention to provide a FIA method that is capable of measuring very low concentrations of a fluorescing substance. It is an object of the present invention to provide a FIA method for Clinical Setting that is fast and accurate, relatively low cost, and capable of using unmodified biological samples, such as whole blood.

It is another object of the present invention to provide a FIA method that is particularly well suited for utilizing an improved fluorescence detection system described in the above-referenced U.S. patent application entitled "fluorometric detection System" entitled "Serial No. 07/855,238, filed 3/22 1992. It is yet another object of the present invention to provide a homogeneous "mix and read" therapeutic drug assay method that can be used to determine digoxin levels in serum, plasma, or whole blood. It is a further object of the invention to provide assays for peptides, such as rubella virus or portions thereof. The invention also provides specific fluorescent labels and probes for use in immunoassays, as described herein.

The invention also provides a method of synthesizing a marker component by reacting a fluorophore moiety with an activated form of a solubilizing axial ligand. The invention also features fluorescent probes having a marker component of the invention attached to one member of a specific binding pair or to a target analyte or analog. The term "specific binding pair" refers to two different molecules (or compositions), one of which has an area on the surface or in the cavity that specifically recognizes and binds to a specific spatial and polar organization of the other molecule or molecular complex comprising the other molecule.

The invention also provides a methodof synthesizing a fluorescent probe comprising the step of linking a marker component of the invention to two or more solubilizing axial ligands. The invention also provides a kit (kit) useful in the detection of a target analyte in a sample suspected of containing the target analyte, comprising a marker component or probe of the invention.

The invention also relates to novel dye oligonucleotide conjugates, methods of synthesizing them, and methods of using them. Methods using these conjugates or probes include nucleic acid hybridization methods, nucleic acid amplification methods, and nucleic acid sequencing methods. The dye moiety of the dye-oligonucleotide conjugate is a detectably labeled marker component having a fluorophore moiety with a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one on either side of the planar molecular structure.

Thus, in one aspect, the invention relates to a composition having an oligonucleotider linked to a detectably labeled marker component having a fluorophore moiety with a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one on either side of the planar molecular structure.

The term "oligonucleotide" refers to a chain of nucleotide residues. Oligonucleotides generally useful in the present invention are 5-50 nucleotides in length. Oligonucleotide probes used in the methods of the invention include polynucleotides of DNA, RNA, or any other sequence species that can hybridize to a nucleic acid sequence. It will be appreciated that such nucleic acid sequences may include base analogues in addition to the naturally occurring bases cytosine, adenine, guanine, thymine and uracil. Such base analogs include hypoxanthine, 2, 6-diaminopurine and 8-azaguanine. The probe may be in double-stranded or single-stranded form, but is preferably in single-stranded form. Probes may be prepared by direct synthesis, polymerase mediated extension reactions, or by cloning or other conventional methods. The term "linked" refers to chemical bonding through an intermediate molecule that is linked to two moieties.

In a preferred aspect, the invention relates to a composition having an oligonucleotide attached to a detectably labeled marker component having a fluorophore moiety having a substantially planar multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two axial ligands coordinated to the central atom on either side of the macrocyclic ligand.

Preferably, the detectable labeled marker component has a decay time in the range of about 1 nanosecond to about 50 nanoseconds, and more preferably a decay time in the range of about 5 nanoseconds to about 20 nanoseconds.

Particularly preferred is caged dicarboxy silicon phthalocyanine. The caged dicarboxysilicophthalocyanine dye may have a variety of functional groups available for coupling. These groups include free carboxyl, free amino and N-hydroxysuccinimide ester (NHS ester).

Preferably, the oligonucleotides of the claimed compositions are from about 5 to about 50 bases in length.

The linkage between the oligonucleotide or polynucleotide and the label may be achieved using a condensation reaction resulting in the formation of, for example, amides, esters, hydrazones, semicarbazones, thiosemicarbazones, ureas, and thioureas. For example, the linker may be terminated with an amino group, preferably a primary amino group. Other linkers may terminate in a carboxyl group.

In another aspect, the invention provides methods for preparing certain dye conjugated oligonucleotides. In one embodiment, such a method comprises the steps of: (a) reacting an oligonucleotide having an amino-terminal attachment linker with N-hydroxysuccinimide ester, or a detectable imidazolide of a labeled marker component containing a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one on either side of the planar molecular structure, to form a conjugate; and separating the conjugates formed in step (a) from the unreacted oligonucleotide or polynucleotide and from the unreacted dye. Attachment of the linker to the oligonucleotide may be achieved by using a diamine and an amino alcohol. Preferably, the detectably labeled marker component comprises a caged dicarboxysilicophthalocyanine dye.

Alternatively, the preparation of dye conjugated oligonucleotides can be achieved by: reacting a detectably labeled marker component having a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one axial ligand on either side of the planar molecular structure, with a carbodiimide in the presence of hydroxybenzotriazole and an oligonucleotide or polynucleotide to form a conjugate; the resulting conjugates are separated from the other components of the reaction mixture. Preferably, the detectably labeled marker component comprises a caged dicarboxysilicophthalocyanine dye.

In another aspect, the invention relates to a method for determining a target nucleic acid sequence in a sample, comprising the steps of: contacting the sample nucleic acid with an oligonucleotide-labeled marker component, preferably an oligonucleotide caged dicarboxy silicon phthalocyanine conjugate capable of hybridizing in homogeneous solution to the target nucleic acid sequence; the presence and amount of such hybridization is detected by transition state polarized fluorescence.

In yet another aspect, the present invention relates to a method for detecting a target nucleic acid sequencein a sample, comprising the steps of: contacting a sample suspected of containing a target nucleic acid sequence with a complementary oligonucleotide capable of hybridizing with the target sequence; contacting said sample with an oligonucleotide-labeled marker component, preferably an oligonucleotide caged dicarboxysilicon phthalocyanine dye conjugate capable of hybridizing to said complementary oligonucleotide; detecting the presence and amount of hybridization of the conjugate to the complementary oligonucleotide.

In another aspect, the invention relates to a method of detecting or quantifying 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 amplification (3SR), and transcription-based amplification system (TAS), which are discussed herein.

In another aspect, the invention relates to the use of fluorescent probes as labels to improve nucleic acid hybridization methods or nucleic acid amplification methods. The fluorescent probe has a detectably labeled marker component comprising a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one axial ligand on either side of the planar molecular structure.

The method of the present invention is particularly useful when used in conjunction with a time-dependent transition state detection system, as described in commonly assigned student's name, filed on 23/3 1992, entitled "fluorometric detection System," U.S. patent application Ser. No. 07/855,238. The system is characterized by a transient state detection that allows direct readout of the time-dependent polarization of the sample. The laser diodes used in this system can be tuned at very high frequencies, such as 10MHz rates, and exhibithigh output power. Typically the laser "ON" time is about 2-3 nanoseconds. Photons from the solution are detected using a photomultiplier tube (PMT) operating in single photon counting mode. The relative time of the photon event to the photon event is determined compared to the laser pulse time. By storing the individual photon event times, a histogram of the photon frequency as a function of time can be obtained.

In another aspect, the invention provides methods of monitoring the kinetics of a nucleic acid amplification process and/or methods of quantifying nucleic acid in a target sample. For example, during PCR amplification, a probe consisting of an oligonucleotide is "capped" and labeled with a composition comprising an oligonucleotide or polynucleotide linked to a detectably labeled labeling component having a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one on either side of the planar molecular structure. Such probes can be added directly to the PCR reaction. The term "capped" means that the 3' end has undergone a dideoxynucleotide reaction.

Hybridization with the amplification product can be carried out kinetically (kinetically) in each cooling phase. As the concentration of amplification product increases, the rate of binding of probe to amplification product also increases and the concentration of amplification product can be quantified. This information together with the cycle number quantifies the amount of original DNA in the sample prior to amplification.

Use in sequence determination. Another aspect of the invention is the use in DNA sequencing, wherein a detectably labeled marker component is introduced into the DNA either chemically or enzymatically: the DNA was fragmented at many points and the collected fragments were analyzed by gel electrophoresis. If desired, the labeling can be carried out using different labeling components, one of which aids in the analysis of the various bases. Another aspect of the invention is the broader application of the various designs, variations or modifications to fluorometers. This broad application will be described in detail in the following examples of specific types of devices. In a non-absorbing medium, if the angle of incidence is greater than the critical angle, total internal reflection occurs at the boundary of medium a when a light wave traverses medium a surrounded by a second medium B having a lower refractive index. The electromagnetic field of the total reflected light penetrates the boundary for a short distance and can produce physical effects such as excitation of fluorophore molecules near the interface of a and B.

This effect makes possible a homogeneous, fluorescence-based assay in which excitation of fluorescence can occur at the interface (which is the only location) due to a specific reaction with molecules immobilized on the surface of medium a. Ordinary glass or plastic plates not only act as optical waveguides for the incident light, but also as carriers for specific receptors that have been previously deposited at known locations on the surface. This methodology is known as "evanescent light fluorescence immunoassay" (Herron et al, U.S. Pat. No. 5,512,492).

Advantageously, the present invention combines the characteristics of very large Stokes shifts (Stokes' shifts) with emission in the near infrared region of the spectrum, which utilize fluorescent dyes based on N-containing macrocycles (listed below) typically having a near UV excitation region. Such dyes are suitable for use in immuno/receptor assays performed in steady state or transition state mode. Excitation sources include mercury arcs, nitrogen lasers, and nitrogen laser pumped dye lasers. Alternatively, these same dyes can be excited by a diode laser in the near infrared, resulting in excellent pulse excitation and transition state detection results. The choice of excitation source depends on the location of the absorbance band for the particular dye described above, the preferred excitation and detection modes (whether steady state or transition state), and the spatial requirements.

These features of the summarized chemistry and the device designed for use in "evanescent light fluorescence immunoassay" should give rise to very low background and very high signal levels, thereby facilitating high assay sensitivity.

The foregoing summary of the invention is not limiting, and other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.

Brief Description of Drawings

FIG. 1 shows the effect of nonspecific binding and solvent on fluorescence intensity, and shows the protective effect of Si compared to Al, presumably because Si has two axial ligands, while Al has only one ligand. Abbreviations: al trisulf-aluminum hydroxytriphthalate, Si dicarb-bis-hydroxy (2, 3-dicarboxy phthalocyanine) silicon IV, Si dicarb sulf-sulfonated bis-hydroxy (2, 3-dicarboxy phthalocyanine) silicon IV, BBKCl-borate buffered KCl (see example 6), NHS-mixed normal human serum, Cts-readings obtained during one measurement cycle of FAST1 transition state fluorometer. Cts is proportional to fluorescence intensity.

FIG. 2 shows a comparison of solvent effect and nonspecific binding of silicon and aluminum phthalocyanine derivatives. The results show that the presence of Si (with two axial ligands) as the central atom has significantly better performance than Al. This is evidenced by the very small relative change in polarization of dihydroxydicarboxysilicophthalocyanine (Si dicarb) compared to aluminum trisulfonate (Al trisulf). Further enhancement is obtained by sulfonation, as shown by dihydroxydicarboxysilicophthalocyanine sulfonate (Si dicarb, sulf), which may have two or three sulfonate groups. If a dye is used as a label, the polarization value in glycerol indicates about the potential limit. The unit of polarization is millipolarization (mP). Further abbreviated information is described with reference to fig. 1. One skilled in the art will recognize that the dyes of the present invention can be used in a wide range of fluorescent applications over the visible spectrum.

Other features and advantages of the invention will become apparent from the following description of the preferred embodiments of the invention and from the claims.

The drawings are not necessarily to scale. Certain features of the invention may be exaggerated in scale or in schematic form for clarity and conciseness.

Detailed description of the invention

The present invention relates generally to improved fluorescent marker compositions having a substantially planar light-emitting portion attached to two or more small axial ligands and related products and methods. Preferred fluorophores contain small solubilizing axial ligands and fluorescent properties, the synthesis and application of which are described below. The following description includes preferred modes of carrying out the invention, and is made primarily for the purpose of illustrating the general principles of the invention and not in any way limiting the invention.

I. Preferred marker Components

The following is a summary of preferred marker components for use in the fluorescence immunoassay of the present invention. A more complete discussion may be found in commonly assigned U.S. patent application serial nos. 701,449 and 201,465, which are incorporated herein by reference as if set forth above.

A. Preferred fluorophore moieties

Suitable fluorophore moieties comprise a luminescent, substantially planar molecular structure. The preferred substantially planar molecular structure emitting light in the fluorophore moiety has a substantially planar macrocyclic multidentate ligand complexed to a central atom that can be complexed with two axial ligands, one of which is located on either side of the macrocyclic ligand (i.e., has a trans-orientation).

The preferred central atom is an element capable of forming an octahedral coordination complex comprising two ligands with trans or axial orientation, located on either side of and perpendicular to a planar macrocyclic ligand. In some applications, the central atom should not have an atomic number that is too high (about 30 or less) for use as a fluorescent labeling component, so that fluorescence is not diminished by intercoupling with the orbital of the central atom.

Preferred multidentate ligands include nitrogen-containing macrocycles having a conjugated ring system with pi electrons. These macrocycles may be optionally substituted, including substitutions on bridging carbon or nitrogen atoms. Suitable macrocycles include derivatives structural variants of porphyrins, azaporphyrins, corrins, sapphyrin, pentaphyrin, and porphycene, and other similar macrocycles containing broadly delocalized electrons. These macrocycles may optionally have fused aromatic rings with or without heteroatoms such as N, O or S. Due to the fact that they include many of the above properties, a particularly preferred class of macrocycles includes porphyrin derivatives and azaporphyrin derivatives (porphyrin derivatives wherein at least one bridging carbon atom is replaced by a nitrogen atom). Azaporphyrin derivatives include mono-, bis-and trisazaporphyrins and derivatives of porphyrazine. These macrocycles may optionally have fused aromatic rings with or without heteroatoms such as O, N or S. These azaporphyrin derivatives include phthalocyanines, benzotriazaporphyrins and naphthalocyanines and their derivatives. Many methods of preparation and fluorescent qualities of such compounds are known, and some of such compounds are commercially available. See U.S. patent application serial No. 201,465 and references cited therein, particularly references 2-5 in that application.

For certain applications, such as fluorescence polarization detection, azaporphyrin derivatives that exhibit a high degree of polarization in the bound form, i.e., those that emit strongly polarized light, are preferred. For these applications, macrocycles with a low degree of symmetry, preferably with a degree of symmetry lower than D, are preferred_4hA macrocycle of degree of symmetry. A preferred group includes macrocycles having at least one fused aromatic ring. Thus, preferred macrocycles include those having reduced symmetry at positions that reduce symmetryFused aromatic ring azaporphyrin derivatives. Preferred classes of azaporphyrin derivatives include those having a degree of substitution below D_4hMono-, di-and tri-azaporphyrins of degree of symmetry. Other preferred fluorescent dyes are those disclosed at 6.6.1995, U.S. patent application No. 08/476,544 entitled "Polyoxyalkyl-related products and methods for fluorescence determination", Lyon&Lyon Docket 211/167, which is hereby incorporated by reference in its entirety, including all drawings.

B preferred Small solubilizing axial ligands

Preferred small solubilizing axial ligands include-OH, -oxy-t-butyl, -OCH₂OH、-OCH₂CH₂OH、-OCH₂CHOHCH₂OH、-OCH₂CH₂O-CH₂CH₂OH、-OCH₂CH₂-CH₂-O-CH₂CH₂OH, Cl, Br and F.

C absorbance and polarization Properties of preferred marker Components

These marker components containing a central atom (e.g., silicon) coupled to two small solubilizing axial ligands can be characterized by transition state fluorescence measurements. In this measurement, the intensity of two polarized components, parallel or perpendicular to the polarization direction of the excitation pulse, is monitored for about 3 times the decay time of the marker component. This curve reflects the extinction coefficient, quantum yield, decay time and polarization state and provides a sensitive indication in terms of the chemical and physical state of the marker components. For example, if the excited state is passivated or converted to a triplet state, the overall intensity will be reduced and the decay time will be shortened. If the rotational Brownian motion of the molecules is altered by increasing the viscosity or by binding to macromolecules, the ratio of the intensities of the parallel component to the perpendicular component will increase.

Some marker components of the invention exhibit the same intensity, decay time and polarization in DMF (organic solvent) as in SAP (phosphate salt of a salt azide, a neutral aqueous buffer) with about 5% experimental error. To some extent, these properties are also present in the preparation of other marker components. The distinctive and important property of the marker components of the present invention is the insensitivity to (and lack of binding to) components in the serum, which has been demonstrated by the serum not having any significant measured effect on the intensity, decay time or relative amount of the fluorescence-polarized components. This is of crucial importance for marker components used in assay applications, such as those using biological materials.

II preparation of preferred marker Components

According to one method of preparing the preferred marker components of the present invention, a suitable fluorophore moiety with a hydroxyl or halogen group as axial ligand is reacted with an activated form of the solubilizing moiety in a ligand exchange reaction, which proceeds according to a conventional reaction scheme:

where Mcl represents a macrocyclic ligand, CA is the central atom, X is the displaced ligand, and SM is the solubilizing moiety. The reaction can be carried out in the neat phase (neat) or, if desired, in a solvent. Suitable solvents include quinoline, THF, DMF, imidazole (when dissolved in itself in one of the other above-mentioned solvents), and the like. Suitable reaction temperatures may vary depending on the nature of the macrocyclic starting materials and the solubilizing group. The reaction is generally complete in about 2 minutes to about 24 hours. The reaction mixture may conveniently be heated under reflux conditions or in a sand bath, for example. For convenience, the reaction may be carried out at ambient pressure.

We believe that this reaction is carried out in two steps, each time with one conjugated polyoxyalkylene group as the axial ligand.

When used as fluorescent labels in a fluoroimmunoassay, these label components may be linked to one member of a particular binding pair (the "labeled binding partner") or an analog of that member. The term "binding partner" refers to a molecule or molecular complex that is capable of specifically recognizing or being recognized by a particular molecule or molecular complex. The labeling component can be attached or conjugated directly to the binding pair or via a linker arm.

III utilization of

The marker components of the invention can be used as fluorescent markers for fluorescent probes, in fluorescence binding assays, and as markers in vivo imaging and in vivo tumor therapy.

These marker components may advantageously be 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 particular binding pair (the "labeled binding partner") or an analog of that member. The labeling component can be attached or conjugated directly to the binding pair or via a linker arm.

These labeled binding partners are useful in assays having a variety of formats, such as assays involving competition for the analyte or analyte binding partner (if an analog of the labeled analyte or analyte is used), and can also be used in homogeneous or heterogeneous assays.

Because the marker components do not aggregate in aqueous solution, are not solvent sensitive (indicating no detectable aggregation), and do not specifically bind serum components and other biological macromolecules, these markers are particularly suitable for use in assays that detect analytes in samples containing biological fluids, such as serum. Thus, these marker components can be used as labels for fluorescent probes to detect analytes in solution, where nonspecific binding by serum components would severely impair assay sensitivity, affecting accuracy and precision.

Alternatively, these marker components may be used as reagents in vivo imaging. When used as imaging agents, these labeling components are conjugated to a member of a specific binding pair to become a labeled binding partner. Such a labeled binding partner is introduced into the animal. If the other member of the particular binding pair is present, the labelled binding partner will bind to the other member and the signal generated by the labelled component can be measured to identify its position.

These marker components may also be used in vivo tumor therapy. For example, photodynamic therapy (photodynamic therapy) involves the use of a marker component as a photosensitizer. The labeling component (fluorescent label) is conjugated to a binding partner that specifically recognizes and binds to the cancer cell component.

The invention provides nucleic acid probes and methods of making and using such probes. Methods of using the novel nucleic acid 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 herein as conjugates) and methods of the invention are capable of achieving a sensitivity of 1 femtomole (fmole) in a homogeneous hybridization assay. As explained herein, this sensitivity is comparable to that achieved by current heterogeneous hybridization measurement techniques. However, as mentioned above, current multiphase assays have several disadvantages resulting from the multiple steps involved in the measurement, including increased risk of contamination and increased time required to perform the assay. Other advantages of the compositions and methods of the present invention will be apparent to those skilled in the art from a reading of the examples provided herein.

Examples

To aid in understanding the invention, the following examples are included herein to describe the results of a series of experiments. Of course, the following examples of the present invention should not be construed as specifically limiting the invention and variations of the invention, now known or later developed, which are within the purviewof one skilled in the art are considered to be within the scope of the invention described herein and claimed below.

Example 1: synthesis of tetrakis (diimino) 1, 2,4, 5-benzenetetracarboxylic acid diimide from 1, 2,4, 5-Tetracyanobenzene (TCNB)

20.0g (0.112mol) of TCNB in a 1L 3-necked flask was dried in vacuo for about 1 hour. The flask was equipped with a slow, high torque, Teflon blade stirrer, an inlet for bubbling or adding liquid to the ammonia and a water condenser. After flushing the whole apparatus with nitrogen, 400ml of methanol were added, stirring was started at room temperature and ammonia was bubbled slowly.

The absorption of ammonia is very efficient and the suspension turns into a transparent pale green solution after a few minutes. After a few minutes (with constant addition of ammonia) the solution became turbid and the temperature slightly increased. After 40 minutes from the start of the addition of ammonia, the reaction mixture became difficult to stir, and 200ml of methanol was added with continued stirring and addition of ammonia. At this point, the absorption of ammonia was still very efficient, as evidenced by the absence of foam emerging from the surface of the suspension. After 100 minutes, it was necessary to add 175ml more methanol to enable stirring. After 125 minutes, a large amount of ammonia appeared at the outlet of the condenser, and the reaction mixture was placed in a water bath at 45 ℃ and further stirred, heated and added with ammonia for 240 minutes. After cooling, the reaction mixture was stored at 4 ℃ for 24 hours. The solid was then filtered by suction on Whatman #42 filter paper and dried under vacuum. Yield 23.2g (0.109 mol).

Example 2: synthesis of bis-chloro (2, 3-dicarboxy phthalocyanine) silicon (IV)

Diiminoisoindoline (30.0g, 0.207mol) and tetramino 1, 2,4, 5-benzenetetracarboxylic diimide (10.5g, 0.050mol) were ground together and dried under vacuum overnight in a 1 liter 3-necked flask. The flask was equipped with a Teflon blade mixer, septa, thermometer, and reflux condenser with a silica gel drying tube. The apparatus with the stirred reaction was flushed with dry nitrogen, 600ml of quinoline were added under nitrogen and stirred under a nitrogen stream for 30 minutes. A homogeneous, fluid suspension is obtained. Thereafter, 60ml of silicon tetrachloride were slowly added over 5 minutes via a septum. The solution turned black and stirring was continued for 15 minutes without heating.

Then, an oil bath preheated to 195 ℃ was raised to a position to submerge the contents of the flask with continuous stirring. After 5 minutes, the oil bath temperature was reduced to 175 ℃ and after an additional 15 minutes the oil bath was stabilized at 185 ℃ and 190 ℃ and maintained for an additional 60 minutes. The oil bath was then lowered and the reaction mixture was allowed to cool for about 15 minutes. A nitrogen flow was then started to remove unreacted silicon tetrachloride, which could be detected with wet pH paper at the condenser outlet. After about 45 minutes of aeration, the now 100 ℃ oil bath was returned to its original position, and slow heating continued to about 130 ℃ to facilitate removal of silicon tetrachloride, which had been completely removed when there was only significant quinoline smoke after 70 minutes of the above test.

The oil bath was then removed and a mixture of 424ml water and 424ml concentrated hydrochloric acid was added with mixing as the reaction mixture cooled to about 80 ℃. Heating was continued and the final mixture was acidic. The reaction product was kept at room temperature overnight. The next day, with mixing, 424ml of water and 424ml of concentrated hydrochloric acid were added, and the mixture was left at room temperature overnight.

The reaction mixture was then filtered on a Buchner funnel (24cm filter paper), washed with water, and air dried overnight in a fume hood. The wet cake was stirred in 1 liter of acetone and filtered. The washed material was dried in a fume hood for 2 days, the dried material (50g) was triturated in a mortar with acetone, the mixture was stirred, filtered and dried under vacuum leaving 47.9g of a black finely divided solid.

Example 3: hydrolysis of bis-chloro (2, 3-dicarboxy phthalocyanine) silicon (IV) (dicarboxy dichloro dye)

Concentrated sulfuric acid (98ml) was charged to a 250ml round bottom flask using a long neck funnel to avoid wetting the flask neck. 16.3g of dicarboxyldichloride dye was added in small portions through a short-necked funnel with magnetic stirring. The addition process lasted about 1 hour to disperse the dye cake before more solids were added. The dry tube was attached to the flask, and the mixture was heated in an oil bath and held at 50 ℃ for 24 hours.

The reaction flask was removed from the oil bath and cooled in ice. Water (75ml) was carefully added in small portions without cooling and the mixture was heated in an oil bath at 80 ℃ for 20 hours with stirring. After cooling, the mixture was poured into ice in a 1 liter beaker and stirred.

The mixture was centrifuged at 2000Xg for 30 minutes at room temperature. The sediment was suspended in water (about 250ml) and centrifuged again. The washing process was repeated and the sediment was collected and suspended in 300ml of 1M K₂CO₃In (1).The mixture was heated with stirring in a beaker covered with a watch glass. The temperature reached 90 ℃ in 10 minutes and heating was continued at about 93 ℃ for 50 minutes. The mixture was acidified with concentrated HCl while still hot, allowed to cool and held at room temperature for 2 days.

The solids were then collected on an 11cm Buchner funnel (using Whatman #42 filter paper) and filtered, which took approximately 1 hour. The solid was washed on the funnel with 3X 100ml of water in portions and then air dried in a fume hood. The solid is then broken up and treated with P under vacuum₂O₅And KOH drying. Yield 13.3g (87%).

Example 4: purification of bis-hydroxy (2, 3-dicarboxy phthalocyanine) silicon IV (dicarboxy dye) by chromatographic adsorption on silica

3.0g of the crude dicarboxy dye prepared in example 3 was charged into a 250ml bottle, 100ml of MeOH containing 2% by volume of ethyldiisopropylamine (DIEA) was added thereto, and the mixture was stirred for 30 minutes. Thereafter, 43g of silica (EM Science) was added and the mixture was shaken by hand to form a black paste. After 20 minutes, a further 100ml of 2% DIEA in MeOH was added, the bottle was inverted for a few minutes, and the contents were stirred for 20 minutes. The extraction of the single component is made possible by the addition of EtOH in addition to MeOH and DIEA to adjust the nature of the solvent, which changes the composition of the extracted dye. The solid was then filtered under reduced pressure on a sintered glass plate funnel (microporosity, diameter 6.5 cm). To prevent excessive loss of MeOH, the reduced pressure was maintained by connecting to a partial vacuum tank (parallel evacuatantank). After overnight filtration, the residue was washed with 2X 50ml MeOH + DIEA in portions for about 30 hours. The filtrate (230ml) was concentrated to near dryness in a rotary evaporator. The residue was dissolved in 14ml MeOH + DIEA and the solution was divided into aliquots and loaded into two 40ml conical centrifuge tubes.

The contents of each tube were acidified with 200. mu.l concentrated HCl and the tube was nearly filled with water. The contents of the tube were mixed by inversion and shaking several times and centrifuged at about 650Xg for 30 minutes. The brown supernatant liquid was then discarded and the sediment was washed 3 times with 0.01 MHCl. Will sinkThe deposit was transferred to a 100ml round bottom flask, the mixture was dried by rotary evaporation and then under vacuum with H₂SO₄And KOH drying. The dry weight was 304mg (pure dicarboxy dye).

Example 5: sulfonation of pure dicarboxy dyes with chlorosulfonic acid

161mg of pure dicarboxy dye (example 4) were weighed into a 50ml long-necked round-bottom flask with a magnetic mixer. At room temperature under N₂Then 3.4ml of ClSO were added₃H. Is provided with N₂A small air condenser in a bulb, the flask and contents were heated in an oil bath at 110 ℃ for about 3.7 hours. At this point, a 50ml sample was taken for testing. Heating was continued at 110 ℃ for an additional 3.3 hours, whereupon heating was stopped and a second sample was taken. Ice was added to both samples and each sample was diluted with water to a weight of 390 mg. Then 2ml of 1M NaHCO was added to each sample₃The absorbance of each sample was measured by diluting 10. mu.l of the diluted sample with 2ml of a neutral buffer. Amax of both samplesNow 690nm, the sample reading at 3.7 hours was 0.650 and at 7 hours was 0.490, indicating that about 25% of the dye was destroyed during the last 3.3 hours of heating.

The main reaction mixture was added in small portions to ice in a beaker and the cold mixture was centrifuged at about 700Xg for 30 minutes. The very slightly colored supernatant liquid was discarded. The sediment was suspended in 30ml of ice cold water, transferred with water into a 250ml conical flask and treated with about 40ml of 1M KHCO₃It was made basic and stirred at room temperature overnight. The reaction mixture was transferred to a beaker, acidified with concentrated HCl, stirred at room temperature for 6 hours, and stored at room temperature for 48 hours.

The reaction mixture was centrifuged at about 700Xg, the colored supernatant liquid was retained and the precipitate was dissolved in 1M NaHCO₃Neutralized and stirred for 2 hours. The dark green solution was passed through Sep Pak (2g size, Rainin) and the acidified filtrate was combined with the supernatant liquid from the centrifugation. The total volume was about 400 ml. The dark blue acidic solution was blotted on Sep PakThe column was washed with 3N HCl and eluted with MeOH. And the Sep Pak washed with MeOH and 3N HCl can be used repeatedly. By rotary evaporation and under vacuum with H₂SO₄The MeOH eluate containing the dye was dried with KOH. The yield was 158 mg.

Example 6: measurement of non-specific binding and characterization of solution properties of silicon and aluminum phthalocyanines

Materials:

1) borate buffered kcl (bbkcl): the buffer was prepared by mixing: 33.1ml of 0.70M boric acid, 4.0ml of 0.50M K₂B₄O₇75ml of 4.0MKCl and H₂O (bring the solution to 1 liter), pH about 8.1.

2) Sulfonated dicarboxy silicon phthalocyanine (example 5), dicarboxy silicon phthalocyanine (example 4) and aluminum trisulfonate, sodium salt (porphyrin product) were dissolved in BBKCl. The concentrations of these solutions were determined as follows: the absorbance was measured at the NIR maximum (about 680nm) of a dilution of each sample in methanol containing 5% by volume of ethyldiisopropylamine, and assuming a molar extinction coefficient value of 2X 10 for all samples⁵L/mol. Dilutions were then made in BBKCl to give a concentration of 5X 10^-8M of each dye.

And (4) measuringfluorescence. In a transition state polarization fluorometer (FAST1, hyperson, inc. miam,FL) were measured by diluting 10. mu.l in each case with 1ml of BBKCl, BBKCl + 1% by volume of pooled normal human serum (L3833, 10/18/84) or glycerol at a concentration of 5X 10^-8M (material of item 2) in solution.

Results non-specific binding and solvent sensitivity of the three dyes were characterized as assessed by fluorescence intensity and polarization measurements, as shown in figures 1 and 2. With respect to intensity, the desired properties are steadily dependent on the solvent composition and high fluorescence output. On the other hand, the polarization ideally should remain low in low viscosity media and should be as high as possible in viscous solvents such as glycerol.

FIG. 1 shows that the fluorescence intensity from the aluminum phthalocyanine trisulfonate is very sensitive to solvent. In contrast, dihydroxydicarboxysilicophthalocyanine sulfonate shows significantly improved performance with nearly identical fluorescence output in buffer, buffer plus serum or glycerol alone. In part this improvement is seen to be due to sulfonation as compared to dihydroxydicarboxy silicon phthalocyanine (no sulfonate groups), which itself is less sensitive to serum and solvents than the aluminum compound.

Figure 2 more strongly indicates that the presence of only central silicon atoms also results in reduced sensitivity to the environment when compared to aluminum as the central atom. This difference is most likely due to the fact that: si has two axial ligands and therefore the planar structure of the dye can be "protected" from the solvent by the presence of "protecting groups" on each side of the molecular plane. In the case of aluminum, there is only one axial ligand, so that one side of the molecular plane can be acted upon by the solvent without constraints. In fig. 2, it is evident that the result of this interaction is a very large increase in the polarization of the Al dye, approaching the maximum obtainable by placing the dye in glycerol (indicating the approximate limit of polarization if the rotational motion is nearly stopped).

Conclusion

Of course, the above applications of the embodiments of the invention should not be considered as limiting the scope of the invention. Such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to be within the scope of the invention as 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 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, limitation or limitations which is not specifically disclosed herein. Thus, for example, in various examples herein, any of the terms "comprising," "consisting essentially of …," and "consisting of …" can be substituted for either of the other two terms. The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention, 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 ofthe concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and 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)

1. A detectably labelled marker component comprising a luminescent substantially planar fluorophore moiety coupled to two or more small solubilizing axial ligands (axialigands), one on either side of the fluorophore moiety.

2. A marker component according to claim 1, wherein said fluorophore moiety comprises a macrocyclic multidentate ligand coordinated to a central atom, said solubilizing axial ligand being independently selected from the group consisting of hydroxyl, chloro, bromo, fluoro, OCH₃And O-CH₂OH。

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

4. A marker component according to claim 3, wherein said two hydroxyl moieties comprise axial ligands coordinated to said central atom.

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

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 the group consisting of porphyrin derivatives, or porphyrin derivatives, corrin derivatives, sapphyrin derivatives or porphycene derivatives having one or more bridging carbon atoms replaced by a nitrogen atom.

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

10. A marker component according to claim 9, wherein said macrocyclic ligand has a low degree of symmetry, thereby enhancing the emission polarisation parallel to the absorption polarisation.

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

12. The labeling component of claim 11 in which said macrocyclic ligand has a ratio of D_4hLow pair ofDegree of symmetry.

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 in said porphyrin derivative are replaced by nitrogen atoms.

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

16. A marker component according to claim 15, wherein said macrocycle is selected from the group consisting of tetrabenzotriaza-porphyrin, 27-phenyltetrabenzotriaza-porphyrin and 27- (p-methylphenyl) tetrabenzotriaza-porphyrin.

17. A marker component according to claim 16, wherein said 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 said fluorophore moiety is a naphthalocyanine or a naphthalocyanine derivative.

20. The detectable labeled marker component of claim 1, wherein the fluorophore moiety conjugated to the two or more small solubilizing axial ligands is characterized by: the transition state fluorescence emission in the presence of serum components in aqueous solution has parallel and perpendicular components of substantially the same intensity as in the absence of serum.

21. The detectable labeled marker component of claim 1, wherein the luminescent substantially planar molecular structure has an excitation wavelength of at least about 500nm, the marker component reducing non-specific binding to serum components when compared to a naked fluorophore moiety that is not coupled to two or more hydroxyl moieties.

22. The labeling component of claim 21 in which the excitation wavelength of said fluorophore moiety is about 600 and 800 nm.

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

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

25. A marker component according to claim 21, wherein said fluorophore moiety alone has an association constant at least 60 times greater than the association 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) a polymethine dye with an aryl end; (2) quinoid dyes; (3) indanthrene dyes; (4)1, 4-diaminoanthraquinone-2, 3-dicarboximide; (5) tetraaminoanthraquinone; (6) an azine dye; (7) a pyrylium or thiopyrylium dye and (8) naphthoquinone methide.

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

28. A method of detecting a target analyte in a sample suspected of containing said target analyte, the method comprising the steps of:

(a) attaching the marker component of any one of claims 1-26 to a target analyte or analog thereof;

(b) contacting the sample suspected of containing the target analyte with a known amount of a labeling component attached to the target analyte or analog thereof;

(c) contacting the sample suspected of containing the target analyte with a receptor that specifically binds to the target analyte;

(d) determining the amount of the label component attached to the target analyte or analog thereof bound to the receptor, or the amount of unbound target analyte or analog thereof.

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

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

31. The probe of claim 29, wherein the acceptor target analyte or analog thereof is conjugated to the fluorophore moiety via a linker arm.

32. The probe of claim 29, wherein the receptor target analyte or analog thereof is selected from the group consisting of digoxin, digoxigenin, theophylline, phenobarbital, acetyl procainamide, plinetide, phenytoin, rubella antibodies, and derivatives of each.

33. The fluorescent probe of claim 29, wherein a member of the specific binding pair has at least one steric tolerant (steric tolerant) marker attachment site that allows the probe to form a specific binding pair.

34. A method of synthesizing a fluorescent probe comprising the step of linking the labeling component of any 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 labeling component of any of claims 1-26;

(b) contacting the complex with a second receptor capable of specifically binding to the analyte of interest, the second receptor being bound to a solid support to form a complex of the first labeled receptor, the analyte of interest, and the second receptor bound to the solid support; and

(c) measuring the amount of labeled first receptor bound to the solid support or the amount of unbound labeled first receptor.

36. The method of claim 35, further comprising the steps of: correlating the amount of said first acceptor labeled with a fluorescent probe measured in said sample suspected of containing said target analyte with the amount of said first acceptor labeled with a fluorescent probe measured in a control sample not containing said target analyte or with the amount of said first acceptor labeled with a fluorescent probe measured in a sample containing a known amount of said target analyte.

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

38. A method for determining the presence or amount of a target analyte in a sample suspected of containing the target analyte, comprising thesteps of:

(a) simultaneously contacting said sample suspected of containing a target analyte with first and second receptors capable of specifically recognizing said target analyte to form a complex of said first receptor, said target analyte and said second receptor, said first receptor being labeled with a fluorescent probe comprising a labeling component of any of claims 1-26, said second receptor being bound to a solid support; and

(b) measuring the amount of the labeled first receptor bound to the solid support or the amount of unreacted labeled first receptor.

39. The method of claim 38, further comprising the steps of: correlating the amount of labeled first receptor measured in the sample suspected of containing the target analyte with the amount of labeled first receptor measured in a control sample not containing the target analyte, or with the amount of labeled first receptor measured in a sample containing a known amount of the target analyte.

40. A method of measuring a target analyte capable of binding to two different receptors in a sample suspected of containing the target analyte, comprising the steps of:

(a) contacting the sample suspected of containing the target analyte with a first receptor capable of specifically binding to the target analyte to form a complex of the target analyte and the first receptor, the first receptor being labeled with a fluorescent probe comprising a labeling component of any of claims 1-26; and

(b) contacting the complex with a second receptor capable of specifically binding to the target analyte, the second receptor having a maximum of absorbance and emission that is different from the value of the first receptor.

41. A kit for detecting a target analyte in a sample suspected of containing said target analyte (kit), said kit comprising a marker component according to any of claims 1-26.

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

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

44. The composition of claim 43, wherein the decay time of the detectably labeled marker component ranges from about 1 nanosecond to about 50 nanoseconds.

45. The composition of claim 44, wherein the decay time is in a range of about 5 nanoseconds to about 20 nanoseconds.

46. The composition of claim 43, wherein the oligonucleotide has a length of 5-50 bases.

47. The composition of claim 43, wherein the linkage Comprises (CH)₂)₆O。

48. The composition of claim 43, wherein the linkage Comprises (CH)₂)₂NH。

49. A method for producing a labeling-component-conjugated oligonucleotide, comprising the steps of:

(a) reacting an amino-terminated adaptor-attached oligonucleotide with an N-hydroxy-succinimide ester, or with an imidozolide of a label component to form a conjugate, the label component being a composition comprising an oligonucleotide attached to a detectably labeled label component comprising a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one on either side of the planar molecular structure; and

(b) separating the conjugate formed in step (a) from the unreacted oligonucleotide and from the unreacted labeling component.

50. The method of claim 49, further comprising, prior to step (a), performing the steps of: an amino-terminated linker is attached to the oligonucleotide.

51. A method of preparing a labeling component conjugated oligonucleotide comprising the steps of:

(a) reacting a labeling component with carbodiimide in the presence of hydrobenzotriole and an oligonucleotide to form a conjugate, the labeling component being a composition comprising an oligonucleotide attached to a detectably labeled labeling component, the labeled labeling component comprising a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one axial ligand located on either side of the planar molecular structure; and

(b) separating the conjugates formed in step (a) from the other components of the reaction mixture.

52. A method for determining the presence and quantity of a target nucleic acid sequence in a sample comprising the steps of:

(a) contacting a sample nucleic acid with a composition of claim 43, wherein the composition is capable of hybridizing to the target nucleic acid sequence in a homogeneous solution; and

(b) the presence and amount of such hybridization is detected by transition state polarized fluorescence.

53. A method for detecting a target nucleic acid sequence in a sample, comprising the steps of:

(a) contacting a sample suspected of containing a target nucleic acid sequence with a complementary oligonucleotide capable of hybridizing to 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 of hybridization of the conjugate to the complementary oligonucleotide or polynucleotide or measuring the amount of hybridization.

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

55. The method of claim 54, wherein the 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 amplification (3SR), and transcription-based amplification system (TAS).

56. In a method of nucleic acid amplification, the improvement comprising using as a label a fluorescent probe comprising a detectably labeled marker component comprising a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one axial ligand on either side of the planar molecular structure.

57. In a nucleic acid amplification hybridization method, the improvement comprising using as a label a fluorescent probe comprising a detectable label tag component comprising a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one axial ligand on either side of the planar molecular structure.

58. The composition of claim 43, wherein the composition is thermally stable.

59. The composition of claim 58, wherein the composition is stable after 1 hour of exposure at 90 ℃.

60. The composition of claim 59, wherein the composition is stable after 10 minutes of exposure at 100 ℃.

61. A kit comprising the composition of claim 43 and indicia, packaging, or instructions.

62. A method of using the implement of claim 62, comprising using the composition in accordance with the marking, packaging, or instructions.

63. A method of making the implement of claim 61 comprising the step of combining said composition with said indicia, packaging or instructions.

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