JP2002530662A - Water-soluble fluorescent dyes without aggregation and serum binding and related products and methods - Google Patents

Abstract

  (57) [Summary] The present invention relates to marker components, fluorescent probes, oligonucleotides, hybridization assays and immunoassays using such products, and methods for producing such products. In accordance with the present invention, there is provided a detectably labeled marker component comprising a fluorophore moiety coupled to two or more small solubilized axially binding ligands, which preferably has solvent sensitivity and non-specificity. Reduce or eliminate the problem of dynamic coupling.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates generally to water-soluble fluorescent dyes without aggregation and serum binding, and related products and methods. Preferred dyes include a luminescent, substantially planar fluorophore linked to two or more solubilizing ligands.

RELATED APPLICATIONS This application is based on Dandlicker et al.
oresent Dies Free Of Aggregation An
d Serum Binding And Related Products
U.S. Provisional Patent Application No. 60 / 109,969 (1) entitled "And Methods"
(Filed November 25, 998), which is hereby incorporated by reference in its entirety, including drawings.

BACKGROUND OF THE INVENTION [0003] Publications and other references referred to herein are hereby incorporated by reference. The following description of the background of the invention is intended to assist in understanding the invention and is not an admission that it describes or constitutes prior art to the present invention.

[0004] The near infrared absorption and emission of porphines, phthalocyanines and other azaporphyrins, and certain other aromatic nitrogen-containing macrocycles, may be
These compounds make them attractive as candidates for use as fluorescent labels.

[0005] Phthalocyanine has a particularly strong near-infrared absorption (molar quenching coefficient is about 200,0
Much effort has been put into using these as fluorescent labels due to their high quantum yield in organic solvents, and the resistance to the well-known fading of common metallophthalocyanine dyes. However, early efforts along this line did not result in a completely satisfactory product. This is mainly due to the strong tendency of phthalocyanines to associate, especially by stacking in opposing aggregates, and to bind strongly to various other molecular surfaces (non-specific binding).

Unsubstituted phthalocyanines have very low solubility in both organic and aqueous solvents as a result of intramolecular stacking. As is now well known, the tendency to stacking can be dramatically reduced by introducing one or more charged groups, such as sulfonates. Phthalocyanines with such substitutions may have high solubility in aqueous solutions of water and electrolytes, but the tendency to bind non-specifically remains.

[0007] At present, much of the scientific interest in fluorescent labels is for biological materials such as tissue sections, cells, cell fragments, proteins (including glycoproteins and lipoproteins), peptides, oligosaccharides and polysaccharides, oligonucleotides And applications involving polynucleotides and lipids. The tendency for non-specific binding in fluorescent assays containing these materials will interfere with the specific interaction of interest by partially masking.

[0008] The non-specific binding as well as the tendency to stacking is attributed to the coupling of phthalocyanine dyes to one or more polyoxyhydrocarbyl groups, typically methoxy-terminated poly (ethylene glycol) (PEG), to allow therapeutic agents to be used. In assays, it can be reduced to negligible levels. At the same time, the desired absorption and release properties are preserved by the attachment of such groups. The same technique is also valid for a wide variety of other near infrared dyes. "POLYOXY
HYDROCARBYL RELATED PRODUCTS AND MET
US Patent Application No. 08 / 476,544 entitled "HODS FOR FLUORESCENT ASSAY" (filed July 7, 1995, Lyon & Lyon).
Socket No. 211/167, which is hereby incorporated by reference in its entirety, including the drawings) and earlier applications cited herein.

[0009] Recently, significant progress has been made in the field of fluorescent dyes. In one aspect, dyes are currently available that can be excited by longer wavelength radiation, eg, red and infrared wavelengths. These dyes are described in two patent applications filed by the applicant: "Fluorescent Marker C" by Arrhenius.
U.S. Patent Application 701,449 entitled "components and Fluorescent Probes" (filed May 15, 1991), which is a continuation-in-part of U.S. Patent Application 523,601 (filed May 15, 1990), and "Fluorescent Dyes" by Dandliker and Hsu.
Free of Aggregation and Serum Bindi
ng ", filed May 15, 1991, which is a continuation-in-part of U.S. Patent Application 524,212, filed May 15, 1990. These applications are hereby incorporated by reference in their entirety, including the drawings.

[0010] Another important advance is the increased sensitivity of data collection and analysis techniques. U.S. Pat. No. 4, entitled "Fluorometer" to Dandliker et al.
877,965, which is incorporated by reference herein in its entirety, including drawings, uses time gating techniques in combination with data collection and analysis techniques to improve the signal. Against background is obtained. In general, the '965 patent contemplates that the detected intensity as a function of time is composed of signals from various sources, for example, signals from a desired signal source and various undesirable background sources. Optimization of the desired signal is achieved by data collection and analysis techniques.

[0011] Another important advance has been made with respect to the ability to measure relevant substances in immunoassays. For example, Dandliker et al., “Transient S
US Patent Application No. 4 entitled "State Luminescence Assays"
90,770 (filed March 6, 1990; U.S. Patent Application 365,420 (198
(Filed Jun. 13, 9), incorporated herein by reference in its entirety, including drawings, to obtain a homogeneous or free form of the material. It is possible to detect in the assay. In general, this technique requires the measurement of the time-dependent decay of the intensity of the parallel and perpendicular polarization components. By measuring the time-dependent decay for various polarization states, it is possible to determine bound and free forms of substances such as haptens, peptides, or small proteins in a homogeneous assay format. Importantly, no separation of bound and free material is required.

[0012] Despite significant and promising improvements made in the field of fluorescent dyes and in terms of data analysis, there is a need in the art for these and / or other advantages and desirable chemistry. There is a need for other dyes that are also reactive.

SUMMARY OF THE INVENTION [0013] The present invention is directed to non-specific binding and stacking of planar molecules, even for very small groups, such as -OH, when two such groups are present on either side of the molecular plane. To take advantage of the unpredictable consequences of providing effective protection. Increasing the negative net charge emphasizes the desirable effects of these ligands on many biological systems where most of the "biomolecules" themselves have a negative net charge.

That is, one aspect of the present invention is that the desired effect of phthalocyanine and other fluorescent dyes processed by coupling to polyoxyhydrocarbyl groups is that if the net charge on the dye is sufficiently large, This can be achieved with very small axial binding ligands (eg -OH). In most cases, this net charge is preferably negative. This is because in the physiological pH range, most biological substances, such as proteins and DNA, will also have a negative net charge. That is, we have found that highly sulfonated dihydroxy-silicon-dicarboxy-phthalocyanines have nonspecific binding to serum proteins that is nearly as low as PEG-conjugated non-sulfonated dyes.

[0015] Furthermore, there is an advantage that micelle formation by PEG does not occur. PEG-processed dyes behave much like macromolecules at dye concentrations of 10 ^-4 M and higher, with passage through membranes designed to prevent the passage of molecules of about 30 K daltons or more. Very disturbed. Dyes also move through voids in gel permeation chromatography designed to separate macromolecules from small molecules. In contrast, sulfonated dihydroxy-dicarboxy-silicon-phthalocyanine (SDDSiPc
) Behaves as expected for that formula weight numerator.

With respect to nonspecific binding as measured by changes in fluorescence polarization and intensity, when the dye is exposed to, for example, diluted human serum, SDDSiPc behaves much like a PEG-coupled dye (implementation). See Example 6). In this regard,
Non-sulfonated dihydroxy-dicarboxy-silicon-phthalocyanine (DDSi
It is important to note that the negative charge itself has a significant effect on reducing non-specific binding, given that Pc) exhibits considerable non-specific binding. Hydroxy-aluminum-phthalocyanine-trisulfonate is strongly sensitive to ionic strength and has strong non-specific binding. Although the phthalocyanine molecule appears to have to have axial binding ligands on both sides of the molecular plane, the -OH group is sufficient to effectively eliminate non-specific binding when the net charge is high enough. Big.

Another advantage of the present invention is that -OH or other small solubilized axial binding ligands and dyes processed with high charge make the molecules (eg, proteins and oligonucleotides) that are labeled themselves negatively charged. Even if it does, it appears to be much more chemically reactive (in the labeling reaction). This suggests that labeling of haptens and other small molecules usually proceeds easily, but that PEG ligands actually interfere in the labeling of macromolecules.

The present invention was completely unpredictable in view of the strong non-specific binding of hydroxy-aluminum-phthalocyanine-trisulfonate. From this it will be inferred that dihydroxy-dicarboxy-silicon-phthalocyanines with smaller negative net charges show stronger non-specific binding than aluminum dyes. The present invention is based, in part, on the findings that show the opposite (ie,
Figures 1 and 2). Based on the findings described herein, other small axial binding ligands, for example _{-OCH 3, -O-CH 2 OH} , -Cl, -Br and -F
It is expected that the effect of will also work.

[0019] Many other nitrogen-containing macrocycles can be metallated with group 14 atoms to achieve similar results. Such macrocycles include derivatives and structural variants of porphyrins, azaporphyrins, corroles, saphirins, pentaphyllins, porphycenes, and other similar macrocycles having a highly delocalized pi-electron system. Can be In view of the fact that they incorporate many desirable features, particularly preferred types of macrocycles include azaporphyrin derivatives and structural variants. Azaporphyrin derivatives include derivatives of monoazaporphyrin, diazaporphyrin and triazaporphyrin and porphyrazine. Any of these macrocyclic compounds may optionally have a condensed aromatic ring. Such azaporphyrin derivatives and variants include phthalocyanines, benzotriazaporphyrins and naphthalocyanines, and derivatives thereof, and oxa-, thia-, or aza-structural variants thereof.

Therefore, the present invention provides a marker component, a fluorescent probe, an oligonucleotide,
It relates to hybridization assays and immunoassays using such products, and to methods for producing such products. According to the present invention,
Provided is a detectably labeled marker component comprising a fluorophore moiety coupled to two or more small, usually axially binding, solubilizing ligands, wherein the axis is formed by a central metal atom. Is defined by the octahedral geometry of the resulting complex. This marker component preferably reduces or eliminates solvent sensitivity and non-specific binding problems.

[0021] The use of such detectable labels or marker components in immunoassays is such that these labels are capable of transition state fluorescence emission of substantially the same intensity in the presence and absence of biological fluids, such as serum. Has the advantage of having parallel and vertical components of That is, an assay method using these labels can detect a low concentration of a test substance, a target test substance, or an analog thereof in a biological fluid. The term "analyte" refers to a compound or a compound to be measured in an assay, which can be any compound for which a receptor for which naturally exists or can be produced, It may be epitopic or polyepitopic, antigenic or haptenic, and may be a single or multiple compounds sharing at least one common epitopic site or receptor. The term "target analyte" refers to a compound or a compound to be measured in an assay, which can be any compound for which a receptor for which naturally exists or can be produced, and which is a single epitope It may be sexual or polyepitopic, antigenic or haptenic, and may be a single or multiple compounds sharing at least one common epitope site or receptor. By "analog" of a target analyte is meant one or more compounds that can compete with the target analyte for binding to the receptor. The term "receptor" refers to a molecule or molecular complex that specifically recognizes or can be recognized by a target analyte or an analog thereof. For example, an antibody can be a receptor for an antigen.

These marker components can be used as labels for labeling test substances, antigens, antibodies or other molecules. These marker components may optionally be functionalized to include a linker arm that can link the marker component to a test substance, antigen, antibody or other molecule. Various linker arms suitable for this purpose have been described (Kricka, JJ; Ligand-Binde).
r Assay; Labels and Analytical Strategies
gies; pages 15-51; Marcel Dekker, Inc .; ,
New York, NY (1985)). The marker component can be linked to the test substance, antigen, antibody or other molecule using conventional techniques.

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

In a particularly preferred embodiment, the marker component of the present invention is used to produce a probe as generally described in US patent application Ser. No. 08 / 051,446 filed Apr. 21, 1993 by the present applicant. And can be used in immunoassays as generally described in US patent application Ser. No. 08 / 035,633, filed Mar. 23, 1993, the disclosure of which is hereby incorporated by reference. , Which is hereby incorporated by reference in its entirety).

The marker component of the present invention is useful as a detectable label, for example, a diagnostic reagent and a detectable label in assays such as fluorescence binding assays and other immunoassays. In accordance with the present invention, there is provided a detectably labeled marker component having a fluorophore moiety coupled to two small solubilized axial binding ligands. It is characterized by a transition state fluorescence emission in the presence of a serum component in an aqueous solution having parallel and vertical components of substantially the same intensity as without serum. The term "axial binding ligand" refers to a substituent that forms a coordination complex with a central atom with a macrocyclic ligand. The axial binding ligand lies perpendicular to the plane described by the macrocyclic ligand.

[0026] Such molecular components are also described by Walker, et al, Clinical.
Chemistry 42: 1 (1996); Clinical Chemis
try 39: 9 (1993); US Pat. No. 5,593,867; and European Patent Application 93117909.7, which is incorporated herein by reference in its entirety including the drawings. Deemed useful.

Surprisingly, the marker component of the present invention having a macrocyclic polydentate ligand with two small solubilized axial binding ligands flanking the plane of the polydentate ligand is a serum component It has been found to dramatically reduce non-specific binding to and exhibit negligible solvent sensitivity. The term “solvent sensitivity” describes the change in the fluorescence behavior of a molecule depending on the solvent system used, most notably the fluorescence in aqueous solutions when compared to that in organic solvents (eg DMF). Indicates a difference in behavior. Many fluorophores that exhibit high fluorescence intensity in organic solvents, such as DMF,
It shows a substantially reduced fluorescence intensity in aqueous solution. The fluorescence intensity is related to the sample concentration and the intensity of the excitation irradiation. The fluorescence intensity of a particular dye can be correlated with its characteristic light absorbance (extinction coefficient) and fluorescence quantum efficiency, as well as environmental factors. These marker components also exhibit an increased decay time, which is a time near the lifetime of luminescence or nonquenching. The term "decay time" is generally used herein to indicate the time that must elapse to decrease from its initial concentration to 1 / e of its value, depending on the concentration of the excited molecule. The use of terminology for life varies, and is described, for example, in Demos, J. et al. N. , Excited State
Lifetime Measurement, Academic Press,
New York, N.M. Y. (1983), Pages 10, 35, 44 and 158.

[0028] These marker components are useful as fluorescent labels for incorporation into fluorescent probes. The term "fluorescent probe" refers to a test substance, antigen, hapten, antibody or other molecule used in an assay, eg, a fluorescence immunoassay, to determine and / or quantify the presence of a substance of interest, directly or with a linker arm. Represents a marker component comprising a fluorophore moiety bound or coordinated through. Some of these marker components are useful as phosphorescent labels. The components of the present invention are also useful as labels for drugs for in vivo imaging and for drugs used in in vivo tumor therapy.

Therefore, generally preferred are fluorophores that efficiently generate fluorescence when excited with light whose wavelength is in the range of about 200 to about 1000 nanometers, preferably about 600 to 800 nanometers. It is. Suitable fluorophores include those that absorb and / or emit at wavelengths that can be distinguished from the maximum excitation and emission of other solution components (eg, proteins present in the sample) to minimize background fluorescence. included.

Because these marker components are particularly useful in assays using samples of biological fluids, preferred for these uses are at least about 500 nanometers that reduce interference from ambient fluorescence of other sample components. Fluorophore with meter excitation and / or emission wavelength. Certain samples, such as serum, will show considerable interference background fluorescence from flavin, flavoproteins, NADH, etc. when using excitation wavelengths shorter than 500 nm.

In certain applications, such as fluorescence polarization immunoassays, preferred fluorophores will also exhibit a high degree of fluorescence polarization in bound form. Preferably, it will exhibit a fluorescence polarization greater than about 10% of the theoretical maximum for observable polarization. The term "binds" refers to the state in which a binding interaction has been formed between a molecule and its specific binding partner. For certain applications, such as fluorescence transition state assays, preferred fluorophores also have measured fluorescence decay times ranging from about 1 nanosecond to about 50 nanoseconds, preferably from about 5 to about 20 nanoseconds. It is characterized by. In other applications, such as phosphorescent labels, fluorophores with longer decay times may be used.

Thus, preferred are fluorophores that produce fluorescence efficiently, ie, are characterized by high absorption at appropriate wavelengths 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.

[0033] Preferred small solubilizing axial binding ligand, -OH, -O-t- _{butyl, -OCH 2 OH, -OCH 2 CH} 2 OH, -OCH 2 CHOHCH 2 OH, -O
CH ₂ CH ₂ —OCH ₂ CH ₂ OH, —OCH ₂ CH ₂ —CH ₂ —O—CH ₂ CH ₂ OH
, Cl, Br and F. All of these axial binding ligands are arranged toward the central atom by -O-. The stability to hydrolysis is
Possibly, it would be improved by replacing the -O- bond with a direct carbon to metal bond, such as a direct carbon to silicon bond.

In a preferred embodiment, the fluorophore moiety has a substantially planar, multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two small solubilized axial binding ligands. . For use as a marker component in a fluorescence binding assay, a suitable central atom is an atom that is high enough that two axial binding ligands can coordinate and cause greater fluorescence quenching upon transition to the triple state. An atom that is not a number. Preferred elements for the central atom include silicon, germanium, and tin, particularly preferred are silicon and germanium.

The present invention also provides a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilized axial binding ligands flanking the planar molecular structure. The present invention relates to a method for detecting the presence or amount of a target test substance in a sample by using the test substance or a target test substance as a label of a receptor that can specifically recognize the test substance.

[0036] The use of such detectable labels or marker components in immunoassays is such that these labels have parallel and parallel fluorescence emission of substantially the same intensity in the presence and absence of biological fluids, such as serum. It is advantageous in that it has a vertical component. That is, assay methods using these labels can detect low concentrations of target analytes in biological fluids.

The method of the present invention is described in “Fluorometer Detecti” by the present applicant.
on System "(Lyon & Lyon Document No. 1)
95/129, Application No. 07 / 855,238, filed March 23, 1992). Marker components and labels are also available from Walker, et al, Clinical.
Chemistry 42: 1 (1996); Clinical Chemist
ry 39: 9 (1993); U.S. Pat. No. 5,593,867; and European Patent Application 93117909.7, all of which are hereby incorporated by reference in their entirety, including drawings. It is believed to be useful in the described method.

In one aspect, the present invention relates to a competitive inhibition assay method using a specific label. In this regard, the present invention provides a method for combining a sample suspected of containing a target analyte with a luminescent, substantially planar, coupled to two small solubilized axial binding ligands located on either side of a planar molecular structure. Contacting with a known amount of an added target analyte or analog thereof linked to a fluorescent probe, comprising a detectably labeled marker component, to create a fluorophore moiety comprising the molecular structure of The sample is contacted with a receptor that can specifically recognize the target ligand; and the presence or amount of the target analyte is determined by determining either the amount of fluorescent probe bound to the receptor or the amount of free fluorescent probe. About the method. The amount of bound or free fluorescent probe in the unknown sample can be compared to a blank sample and a sample containing a known amount of the target analyte.

In a preferred embodiment, the resulting mixture of sample, fluorescent probe and receptor is diluted just before measuring the amount of bound and / or free fluorescent probe.
The dilution step allows for a more sensitive 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 present invention provides an improved immunoassay method comprising using a label for either a target analyte (or analog thereof) or a receptor. An improvement is to use a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilized axially bound ligands located on either side of the planar molecular structure. Assays using this type of label are beneficial in that they are free of serum binding and aggregation and are therefore particularly suitable for testing biological samples such as serum, plasma, whole blood and urine.

In another aspect, the invention provides a method for performing a “sandwich” or “two-site” immunoassay. The method includes the following steps: (a) A sample suspected of containing a target test substance is identified as a first substance capable of specifically recognizing the target test substance.
To form a complex between the target analyte and the first receptor, wherein the first receptor comprises two small solubilized axial binding ligands located on either side of the planar molecular structure. Being labeled with a fluorescent probe having a coupled fluorophore moiety having a luminescent substantially planar molecular structure; (b) allowing the complex to specifically target a target analyte or first receptor. Contacting a recognizable second receptor, wherein the second receptor is bound to a solid support and comprises a complex of the first labeled receptor, the target analyte and the second receptor bound to the solid support. Bodies are formed; and (c) measuring either the amount of labeled first receptor associated with the solid support or the amount of unreacted labeled first receptor.

[0042] The sandwich type assay can be a heterogeneous assay or a homogeneous assay. In the case of a heterologous component, this may incorporate an additional step of separating the solid support from unreacted labeled first receptor. Homogeneous assays are generally preferred because they are faster.

[0043] In another embodiment, the assay comprises the step of measuring the amount of labeled first measured in the unknown sample.
The amount of the labeled first receptor, measured in a control sample without the target analyte, or the amount of the labeled, measured in the sample with a known amount of the target analyte. Additional steps may be incorporated that correlate with the amount of the first receptor made.

In another aspect, the invention provides a method for a simultaneous sandwich-type assay for determining the presence or amount of a target analyte in a sample. The method comprises the steps of: (a) simultaneously contacting a sample suspected of containing a target analyte with a first receptor and a second receptor capable of specifically recognizing the target analyte; Here, the first receptor is a fluorescent probe having a luminescent, substantially planar molecular structure fluorophore moiety coupled to two small solubilized axial binding ligands located on either side of the planar molecular structure. The labeled and the second receptor are bound to the solid support to form a complex of the first receptor, the target analyte, and the second receptor; and (b) associating with the solid support Either the amount of labeled first receptor or the amount of unreacted labeled first receptor is determined.

In another aspect, the present invention relates to a method of measuring the amount of labeled first receptor measured with respect to a control sample not containing said target analyte. Associating or measuring the amount of labeled first receptor with the amount of labeled first receptor measured in a sample containing a known amount of the target analyte. A method for a sandwich type assay is provided.

In another aspect, the present invention provides a sandwich-type fluorescent immunoassay method for measuring a target analyte capable of independently recognizing two different receptors without mutual interference. This method utilizes two receptors, each of which is labeled with a different dye. For example, one receptor may be 680 nm and 6
Label the first dye with a maximum absorption and emission of 90 nm and the other receptor with a second dye having a maximum absorption and emission of 695 and 705 nm, respectively. Detection and quantification of the test substance can be performed using either steady state or transition state measurements. In each case, for the given example, the excitation would be 680 nm and the detection would be 705 nm. This type of assay is based on energy transfer and has the advantage of being homogeneous.

In a preferred embodiment, the present invention relates to immunoassays for biological fluids such as serum, plasma, whole blood, and urine. Preferably, red blood cells in whole blood are lysed prior to assaying the whole blood sample. Preferred methods for lysing erythrocytes include the addition of stearoyl-lysolecithin, palmitoyl lysolecithin and myristoyl lysolecithin.

[0048] Depending on the type of immunoassay used, the target analyte can be an antigen, hapten or antibody; the receptor can be an antigen or antibody.
The antibodies may be polyclonal or monoclonal. Preferably, the antibodies are monoclonal antibodies. Monoclonal antibodies useful in the present invention can be obtained using the Kohler & Milstein method reported in Nature 256: 495-497 (1975). Alternatively, they may be produced using recombinant methods (Science 246: 1275).
-1281 (1989)).

[0049] In one embodiment, the target analyte is a drug or a metabolite of a drug. The drug can be a steroid, hormone, antibiotic, immunosuppressant, asthma sedative, antineoplastic, arrhythmic, antispasmodic, antiarthritic, antidepressant, or cardiac glycoside. Examples of such agents include digoxin, digitoxin, theophylline, phenobarbital, thyroxine, N-acetylprocainamide, primidone, amikacin, gentamicin, netilmicin, tobramycin, carbamazepine, ethosuximide, valproic acid, disopyramide, lidocaine, procainamide, Includes quinidine, methotrexate, amitriptyline, maltriptyline, imipramine, dicipramine, vancomycin and cyclosporine. In a preferred embodiment, the drug is digoxin.

In another embodiment, the target analyte is a peptide, eg, a peptide hormone such as luteinizing hormone, follicle stimulating hormone, human chorionic gonadotropin, thyroid stimulating hormone, angiotensin I, angiotensin II, prolactin or insulin. The peptide may be a tumor marker, such as an oncofetal antigen. Alternatively, the peptide may be a virus or a part thereof, for example, a rubella virus or a part thereof.

The process according to the invention is carried out at a concentration of about 1 × 10 ⁻⁵ M / L to about 1 × 10 ⁻¹³ M, in particular at about 1
Provided is a method for measuring a target analyte in a concentration range of x10 ^-9 M / L to about 1 x 10 ^-12 M / L. For the determination of drugs and their metabolites, the method of the present invention may be in the range of about 5 × 10 ⁻⁹ M / L to about 5 × ^{10 −12} M / L, especially about 1 × ^{10 −10} M / L—
It allows measurement of a concentration of about 5 × ^{10 −10} M / L. For peptide measurement,
The method of the present invention allows for measurements in the range of about 1 × 10 ⁻¹¹ M / L to about 1 × 10 ⁻¹² M / L.

Measurement of the amount of a fluorescent probe (bound and / or released) can be determined by measuring steady state fluorescence or by measuring transition state fluorescence. In a preferred embodiment, the wavelength of the light to be measured is longer than about 500 nm, preferably longer than about 650 nm, more preferably about 680 nm or 69 nm.
Longer than 0 nm. Since the transition state detection system uses a laser diode,
The dye must have a maximum excitation matched to the diode output wavelength. 680,6
Dyes are available that are compatible with other commercially available laser diodes having output wavelengths of 90, 720, 750, or 780 nm. That is, the wavelength of the light to be measured may be longer than about 680 nm, 690 nm, 720 nm, 750 nm, or 780 nm. The further into the red region of the spectrum, ie the longer the wavelength, the higher the signal enrichment relative to the background.

In a preferred embodiment, the detection and quantification is performed using a transition state measurement. Transition state energy transfer provides improved measurements by optimizing the wavelength of absorption and emission, and by optimizing the decay time of the first and second dyes. Such an optimization can eliminate Rayleigh Raman scattering, maximize the efficiency of the transfer and minimize the direct excitation of the second dye by the first dye.

Therefore, it is a primary object of the present invention to provide an improved FIA with greatly increased sensitivity. It is another object of the present invention to provide a FIA method that allows quick and accurate decisions, often within minutes. It is an object of the present invention to provide a FIA method capable of measuring substances capable of emitting fluorescence at very low concentrations. SUMMARY OF THE INVENTION It is an object of the present invention to provide a FIA method useful in a clinical setting that is fast, accurate, and can use unmodified biological samples, such as whole blood, at relatively low cost. is there.

Particularly adapted to utilize the improved fluorescence detection system described in US patent application Ser. No. 07 / 855,238, filed Mar. 23, 1992, supra, entitled “Fluorometer Detection System”. It is another object of the present invention to provide a FIA method. A homogeneous "mix and readout" that can be used to determine the level of digoxin in serum, plasma or whole blood
It is another object of the present invention to provide a method for assaying a therapeutic agent. It is another object of the present invention to provide an assay for a peptide, such as rubella virus or a portion thereof. The invention also provides certain fluorescent markers and probes for use in immunoassays, as described herein.

The present invention also provides a method for synthesizing a marker component by reacting a fluorophore moiety with a reactive form of a solubilized axial binding ligand. The invention also features a fluorescent probe having a marker component of the invention linked to a specific binding pair or one member of a target analyte or analog. The term "specific binding pair" refers to two different molecules (or compositions), where one of the molecules is a molecule or complex comprising another molecule on its surface or cavity. Have specific regions that recognize and bind to specific spatial and polarization configurations of

[0057] Also provided is a method of synthesizing a fluorescent probe, comprising linking a marker component of the invention to two or more solubilized axial binding ligands. Also provided is a kit useful for detecting a target analyte in a sample suspected of containing the target analyte, the kit comprising a marker component or probe of the invention.

The present invention also relates to novel dye-oligonucleotide conjugates, and methods for synthesizing and using the same. 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 has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilized axially bound ligands flanking the planar molecular structure. , A marker component that is detectably labeled.

That is, in one aspect, the present invention provides a fluorescent light having a luminescent substantially planar molecular structure coupled to two small solubilized axially bound ligands located on either side of the planar molecular structure. Compositions comprising an oligonucleotide linked to a detectably labeled marker component having a moiety.

“Oligonucleotide” means a chain of nucleotide residues. Typically, oligonucleotides useful in the present invention have a length of 5-50 nucleotides. Oligonucleotide probes used in the method of the present invention include D
Polynucleotides of any other type of sequence capable of hybridizing to a NA, RNA or nucleic acid sequence are included. It will be appreciated that such nucleic acid sequences may include base analogs as well as 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. These can be produced by direct synthesis, a polymerase-mediated extension reaction, or by cloning, or by any other convenient method.

“Linked” means chemically joined by an intermediate molecule that is connected to both parts.

In one preferred aspect, the invention relates to a composition comprising an oligonucleotide linked to a detectably labeled marker component having a fluorophore moiety, wherein the fluorophore moiety comprises a macrocycle. It has a substantially planar multidentate macrocyclic ligand coordinated to a central atom capable of coordinating to two axial binding ligands coordinated to a central atom on both sides of the ligand.

[0063] Preferably, the detectably labeled marker component has a decay time in the range of about 1 ns to about 50 ns, more preferably, the decay time is from about 5 ns to about 20 ns. Within the nanosecond range.

Particularly preferred are cage dicarboxysilicon phthalocyanines. The cage dicarboxysilicon phthalocyanine dye may have various functional groups available for coupling. These groups include free carboxyl, free amino and N-hydroxysuccinimide esters (NHS esters).

[0065] Preferably, the oligonucleotide of the composition of the invention will have a length of about 5 to about 50 bases.

The linkage of an oligonucleotide or polynucleotide to a marker can be performed using a condensation reaction, for example, forming amide, ester, hydrazone, semicarbazone, thiosemicarbazone, urea, and thiourea linkages. For example,
The linker can have an amino terminal, preferably a primary amino terminal. Other linkers can have a carboxyl end.

In another aspect, the invention provides a method of making certain dye-conjugated oligonucleotides. In one embodiment, such a method comprises the steps of coupling an oligonucleotide having an attached linker with an amino terminus to two small solubilized axial binding ligands flanking a planar molecular structure. Reacting with a N-hydroxysuccinimide ester or imidazolide of a detectably labeled marker component comprising a fluorophore moiety comprising a substantially planar molecular structure of (a) to form a conjugate; and Separating the conjugate formed in step (a) from unreacted oligonucleotides or polynucleotides and from unreacted dyes. Attachment of the linker to the oligonucleotide can be performed by using a diamine or an amino alcohol. Preferably, the detectably labeled marker component comprises a caged dicarboxysilicon phthalocyanine dye.

Alternatively, the production of the dye-conjugated oligonucleotide can be performed as follows. A detectably labeled marker component having a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilized axially bound ligands flanking the planar molecular structure. Is reacted with carbodiimide in the presence of hydroxybenzotriazole and in the presence of an oligonucleotide or polynucleotide to form a conjugate, and the resulting conjugate is separated from other components of the reaction mixture. Preferably, the detectably labeled marker component comprises a caged dicarboxysilicon phthalocyanine dye.

In another aspect, the present invention relates to a method for detecting a target nucleic acid sequence in a sample, comprising the steps of: synthesizing a sample nucleic acid in a homogeneous solution with an oligonucleotide-label capable of hybridizing to said target nucleic acid sequence. Marker components, preferably oligonucleotides
Contacting the caged dicarboxysilicon phthalocyanine dye conjugate and detecting the presence or amount of such hybridization by transition state polarization fluorescence.

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

In yet 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-retention (self-
Sustained sequence replication (3SR) and transcript-based amplification system (TAS), which are discussed herein.

In another aspect, the invention is directed to a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilized axially bound ligands flanking the planar molecular structure. The present invention relates to an improvement in a nucleic acid hybridization method or a nucleic acid amplification method using a fluorescent probe having a detectably labeled marker component as a label.

The method of the present invention is described in Applicant's “Fluorometer Detect”.
St. Holme et al., US patent application Ser.
855, 238 (filed March 23, 1992) is particularly useful when used with the time-related transition state detection system. This system features transition state detection that allows direct reading of the time-dependent polarization of the sample. This system uses a laser diode that can modulate at very high frequencies, for example, 10 MHz, and exhibits high 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 photon event is determined along with the relative time of the photon event compared to the laser pulse time. By storing individual photon event times, a histogram of photon frequencies is obtained as a function of time.

In another aspect, the invention provides a method for monitoring the kinetics of a nucleic acid amplification process and / or a method for quantifying nucleic acids in a target sample. For example, PC
During amplification by R, the fluorophore moiety has a luminescent substantially planar molecular structure coupled to two small solubilized axially bound ligands flanking the planar molecular structure, so as to be detectable. Probes consisting of oligonucleotides that are "capped" and labeled with a composition having an oligonucleotide or polynucleotide linked to a labeled marker component can be added directly to the PCR reaction. "Capped" means that the 3 'end has been reacted with a dideoxynucleotide.

At each cooling step, hybridization with the amplification product can be kinetic tracked. As the concentration of the amplification product increases, the speed of the combination of probe and amplification product increases, and the concentration of the amplification product is quantified. This information is combined with the number of cycles to quantify the amount of DNA originally present in the sample before amplification.

Use in sequencing. Another aspect of the invention is the application to DNA sequencing. Here, a detectably labeled marker component is chemically or enzymatically incorporated into the DNA. The DNA is cut at a number of points and the resulting assembly of fragments is analyzed by gel electrophoresis. If desired, each base can be labeled with a different marker component, respectively, to facilitate analysis. Another aspect of the invention is its broad application to any design, change or modification of a fluorometer. This breadth of applicability is best illustrated in the following examples for specific types of equipment. In a non-absorbing medium, a second medium B having a lower refractive index
The light wave passing through the medium A surrounded by is completely reflected inward at the boundary of the medium A when the incident angle is larger than the critical angle. However, the electromagnetic field of the fully reflected light penetrates the boundary at a short distance, where it can produce physical effects such as excitation of fluorophore molecules present near the interface between A and B.

This effect allows a homogeneous, fluorescence-based assay in which a specific reaction occurs between the molecules immobilized on the surface of the medium A and thus at the interface, where fluorescence excitation is the only place where excitation can occur. It becomes possible. A simple glass or plastic plate acts as an optical waveguide for the incident light and as a carrier for specific receptors pre-positioned at known locations on the surface. This methodology is described as "weak (ev
anescent) photofluorescence immunoassay "(Herron et al., US Pat.
12, 492).

Advantageously, the present invention relates to N-containing macrocycles, the types of which are listed below, which have in common the emission in the near-ultraviolet excitation and near-infrared regions of the spectrum.
It incorporates a very large Stokes shift feature that uses a fluorescent dye based on FT. Such dyes are applicable to immuno / receptor assays in either steady state or transition state modes. Excitation light sources include mercury arcs, nitrogen lasers and nitrogen laser pumping dye lasers. Alternatively, these same dyes can be excited with near-infrared light using a diode laser to achieve better results with pulsed excitation and transition state detection. The light source is selected according to the location of the absorption band for the particular dye in question, the preferred excitation and detection modes, whether steady or transition, and the space required.

The incorporation of these features into the “light fluorescence immunoassay” chemistry and instrument design results in very low background and high signal levels, thus favoring 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.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the effect of non-specific binding and solvent on fluorescence intensity. In FIG.
The protective effect of Si compared to Al is shown, probably because Si has two axial binding ligands but Al has only one. Abbreviation: Al trisulf = hydroxyaluminum trisulfonate; Si
dicarb = bis-hydroxy (2,3-dicarboxyphthalocyanino) silicon IV; Si dicarbsulf = sulfonated Si dicarb; BB
KCl = borate buffered KCl (Material, see Example 6); NHS = pooled normal human serum; Cts = counts obtained during one measurement cycle of the FAST 1 transition state fluorometer. Cts is proportional to the fluorescence intensity.

FIG. 2 shows a comparison of phthalocyanine derivatives of silicon and aluminum with respect to solvent effects and non-specific binding. The results show that the presence of Si (with two axial binding ligands) results in dramatically better properties than those with Al as the central atom. This is because Al trisulfonate (Al tris)
ulf) compared to dihydroxydicarboxysilicon phthalocyanine (Sid
It is evident from the fact that the relative change in polarization of icarb) is much smaller. A further improvement is the dihydroxydicarboxysilicon phthalocyanine sulfonate, possibly having two or three sulfonate groups (Sidicarb, sul)
Provided by sulfonation as seen for f). The values for polarization in glycerol approximately indicate potential limitations when using dyes as labels. Polarization is expressed in millipolarization units (mP). See the legend in Figure 1 for more information on abbreviations. Those skilled in the art will appreciate that the dyes of the present invention can be used in a variety of fluorescent applications over a wide range of the visible spectrum.

[0083] Other features and advantages of the invention will be apparent from the following description of preferred embodiments of the invention, and from the claims.

The drawings need not be to scale. Certain features of the invention will be enlarged or shown schematically for clarity and brevity.

DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to improved fluorescent marker components having a luminescent substantially planar portion linked to two or more small axial binding ligands, and related products and About the method. Preferred fluorophores include small solubilized axial binding ligands, whose fluorescent properties and methods of synthesis and use are described below. The following description, including the preferred modes of carrying out the invention, is provided for the general purpose of illustrating the general principles of the invention and is not intended to limit the invention in any way. Absent.

I. Preferred marker components The following is a brief description of preferred marker components to be used in the fluorescent immunoassays of the present invention. For a more complete discussion, see US patent application Ser.
, 449 and 201, 465 (as noted above, these are hereby incorporated by reference as part of this specification).

A. Preferred Fluorophore Moieties Suitable fluorophore moieties include luminescent, substantially planar molecular structures. Preferably, the luminescent substantially planar molecular structure is coordinated to a central atom capable of coordinating with two axially bound ligands on either side of the macrocyclic ligand (ie having a trans orientation). , A fluorophore moiety having a substantially planar macrocyclic polydentate ligand.

Preferred central atoms are those elements which are capable of forming an octahedral coordination complex containing the two ligands in a trans or axial bond orientation perpendicular to both sides of the planar macrocyclic ligand. For use as a fluorescent marker component in certain applications, the central atom must not have an atomic number that is too high (approximately 30 or less) so that the coupling interaction with the orbital of the central atom does not reduce fluorescence. Less than).

Preferred polydentate ligands include nitrogen-containing macrocycles having a conjugated ring system with pi electrons. These macrocycles may be optionally substituted, for example, on bridged carbon or on nitrogen. Suitable macrocycles include derivatives and structural variants of porphyrins, azaporphyrins, choline, sapphirines, pentaphyrins and porphycenes, and other similar macrocycles containing highly delocalized electrons. These macrocycles can optionally be N, O or S
May have a condensed aromatic ring containing or not containing a hetero atom. Particularly preferred types of macrocycles include porphyrin derivatives and azaporphyrin derivatives (porphyrin derivatives in which at least one of the bridging carbons has been replaced by a nitrogen atom) in view of the fact that they incorporate many of the features described above.
Azaporphyrin derivatives include derivatives of monoazaporphyrin, diazaporphyrin and triazaporphyrin and porphyrazine. These macrocycles may optionally have fused aromatic rings with or without heteroatoms such as O, N or S. These azaporphyrin derivatives include phthalocyanine, benzotriazaporphyrin and naphthalocyanine, and derivatives thereof. The preparation and fluorescence quality of many of these compounds is known, and some are commercially available. See U.S. Patent Application 201,465 and references cited therein, particularly references 2-5 of this application.

For certain applications, such as fluorescence polarization assays, preference is given to azaporphyrin derivatives which exhibit a high degree of polarization in bound form, ie emit strongly polarized light. For these applications, preference is given to macrocycles having a low degree of symmetry, preferably less than D _4h . One preferred group includes macrocycles having at least one fused aromatic ring. That is, preferred macrocyclic compounds include azaporphyrin derivatives having fused aromatic rings at positions that reduce symmetry. Preferred types of azaporphyrin derivatives include _{monoazaporphyrins} , _{diazaporphyrins} having a symmetry lower than D _4h ,
And derivatives of triazaporphyrin. Another preferred fluorescent dye is "
Polyoxyhydrocarbyl Related Products
and Methods for Fluorescence Assay, "US Patent Application 08 / 476,544 (filed June 6, 1995, Lyon & Lyo).
n, document number 211/167, which is hereby incorporated by reference in its entirety, including drawings.

B. Preferred small solubilizing axial binding ligand preferably smaller solubilizing axial binding ligand, -OH, -O-t- _{butyl, -OCH 2 OH, -OCH 2 CH} 2 OH, -OCH 2 CHOHCH 2 OH, -O
_{CH 2 CH 2 -O-CH 2} CH 2 OH, -OCH 2 CH 2 -CH 2 -O-CH 2 CH 2 O
H, Cl, Br and F are included.

C. Absorbance and Polarization Behavior of Preferred Marker Components These marker components containing a central atom (eg, silicon) coupled to two small solubilized axial binding ligands can be characterized by measuring transition state fluorescence. In such a measurement, the intensity of the two components polarized either parallel or perpendicular to the direction of polarization of the excitation pulse is monitored for a time approximately three times the decay time of the marker component. Such curves are: extinction coefficient, quantum yield,
It reflects the state of decay time and polarization and provides a sensitive indicator of the chemical and physical state of the marker component. For example, if the excited state is being deactivated or converted to a triple state, the overall intensity will be reduced and the decay time will be shorter. If the rotational Brownian motion of a molecule changes due to an increase in viscosity or binding to a large molecule, the ratio of the intensity of the parallel component to the vertical component increases.

[0093] Certain marker components according to the present invention can be used in DMF (organic solvents) in SAP.
(Sarin azide phosphate, aqueous neutral buffer) exhibit the same strength, decay time and polarization within about 5% of experimental error. These properties are also shared to some extent by other marker component preparations. A characteristic and important property of the marker component of the present invention is that it is not sensitive to (and does not bind to) components in serum, which may be the intensity, decay time or the relative magnitude of the polarized components of fluorescence. It is evident from the fact that serum has no significant measurable effect on
This property is very important for the marker component to be useful for applications such as assays using biological substances.

II. Preparation of a Preferred Marker Component According to one method of preparing a preferred marker component of the present invention, a suitable fluorophore moiety having a hydroxy or halide group as an axial binding ligand is subjected to a ligand exchange reaction according to the following general reaction scheme. In the reaction with the solubilized part of the reactive form.

Mcl-CA- (X) ₂ +2 (SM) → Mcl-CA- (SM) ₂ + 2X where Mcl is a macrocyclic ligand, CA is a central atom, X is a substituted ligand, SM Represents a solubilized portion. The reaction can be carried out neat or in a solvent if desired. Suitable solvents include quinoline, THF, DM
F, imidazole (when dissolving itself in one of the other solvents mentioned) and the like. Suitable reaction temperatures may vary depending on the nature of the macrocyclic starting material and the solubilizing groups. The reaction is generally complete in about 2 minutes to about 24 hours. The reaction mixture can be appropriately heated by means such as reflux or a sand bath. For convenience, the reaction can be performed at atmospheric pressure.

This reaction occurs in two steps, and it is believed that one polyoxyhydrocarbyl group coordinates at a time as an axial binding ligand.

When used as fluorescent labels in a fluorescent immunoassay, these marker components can 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 that specifically recognizes or can be recognized by a particular molecule or molecular complex. The marker component may be directly linked or conjugated to it, or may be linked or conjugated via a linker arm.

III. Utility The marker component of the present invention is useful as a fluorescent label for fluorescent probes and in fluorescent binding assays and as a label for in vivo imaging and in vivo tumor therapy.

[0099] These marker components can advantageously be used as fluorescent labels in conventional fluorescent binding assays, such as fluorescence polarization immunoassays. When used as such, these marker components can 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 to it, or may be attached or conjugated via a linker arm.

These labeled binding partners are useful in assays having a variety of formats, for example, assays involving competition for the analyte or analyte binding partner (the labeled analyte or analyte). (If an analog is used), it can be used in either a homogeneous or heterogeneous assay.

These markers have the advantage of no aggregation in aqueous solutions and no solvent sensitivity (indicating no detectable aggregation), and non-specific binding to serum components and other biological macromolecules Based on their absence, they are particularly suitable for use in assays for detecting analytes in samples containing biological fluids, such as serum. That is, the accuracy and precision of using these marker components as labels for fluorescent probes to detect analytes in solutions where non-specific binding by serum components would severely reduce assay sensitivity. It can affect both sexes.

Alternatively, these marker components can be used as agents for in vivo imaging. When used as an imaging agent, these marker components can be conjugated to one member of a specific binding pair to provide a labeled binding partner. A labeled binding partner is introduced into the animal. If other members of the specific binding pair are present, the labeled binding partner will bind to it and the signal generated by the marker component can be measured to identify its location.

[0103] These marker components can also be used for tumor therapy in vivo.
For example, photodynamic therapy involves using the marker component as a photosensitizer. The marker component (fluorescent label) is conjugated to a binding partner capable of specifically recognizing and binding to components of the tumor cell.

The present invention provides nucleic acid probes and methods for making and using the probes. Methods using the novel nucleic acid probes include a variety of currently known or later developed nucleic acid hybridization sequencing techniques, and a variety of currently known or later developed nucleic acid amplification techniques. The probes (also referred to herein as conjugates) and methods of the present invention make it possible to achieve a sensitivity of 1 fmole in a homogeneous hybridization assay. As described herein, this sensitivity is comparable to that achieved by current heterogeneous hybridization assay techniques. However, as noted above, current heterogeneous assays have several disadvantages due to the many steps involved in the assay, such as an increased risk of contamination and an increase in the time required to perform the assay. Have. Other advantages of the compositions and methods of the present invention will become apparent to those skilled in the art by reference to the examples described herein.

Examples The following examples describe the results of a series of experiments to aid in understanding the present invention.
The following examples relating to the present invention should not, of course, be construed as limiting the invention to any particular one, but rather presently known or later developed inventions within the purview of those skilled in the art. Variations are considered to be within the scope of the invention as described herein and in the claims.

Example 1 Synthesis of tetradiiminopyromellitic acid diimide from 1,2,4,5-tetracyanobenzene (TCNB) 20.0 g (0.112 moles) of TCNB in a 1 L three-necked flask was placed under vacuum. For about 1 hour. A low-speed, high-torque Teflon impeller stirrer into the flask, inlet for bubbling with ammonia or adding liquid,
And a water-cooled condenser. After flushing the entire system with nitrogen, 400
ml of methanol was added, stirring was started at room temperature, and ammonia was slowly added by bubbling.

The absorption of ammonia is very efficient and after a few minutes the suspension becomes a clear blue-green solution. After a few minutes (with constant addition of ammonia), the solution becomes cloudy and the temperature rises slightly. Forty minutes after the start of the ammonia addition, the reaction mixture became difficult to stir, and an additional 200 ml of methanol was added, and stirring and addition of the ammonia were continued. At this point, ammonia absorption was still very efficient, as evidenced by the absence of bubbles emanating from the surface of the suspension. After 100 minutes, an additional 175 ml of methanol had to be added to stir. 125
After a minute, a large amount of ammonia began to appear at the outlet of the condenser and the reaction mixture was placed in a 45 ° C. water bath and mixing, heating and ammonia addition continued for another 240 minutes. After cooling, the reaction mixture was stored at + 4 ° C for 24 hours. Next, the solid is
Suction filtered through # 42 filter paper and dried under vacuum. Yield 23.2 g (0.109 mo
les).

Example 2: Synthesis of bis-chloro (2,3-dicarboxyphthalocyanino) silicon (IV ) diiminoisoindoline (30.0 g; 0.207 moles) and tetrimino pyromellitic diimide (10. 59, 0.050 moles) were ground together and dried under vacuum in a 1 L 3-neck flask overnight. The flask was fitted with a Teflon blade mixer, septum, thermometer, and reflux condenser equipped with a silica gel drying tube. The apparatus with the stirred reactants was flushed with dry nitrogen, 600 ml of quinoline was added under nitrogen and mixed for 30 minutes under a stream of nitrogen. A homogeneous liquid suspension was obtained. Thereafter, 60 ml of silicon tetrachloride was slowly added through the partition over 5 minutes. The solution turned dark and stirring was continued for 15 minutes without heating.

Next, with continuous stirring, the oil bath preheated to 195 ° C. was raised to a position where the flask was immersed to a level higher than its contents. After 5 minutes, the bath temperature dropped to 175 ° C, and after a further 15 minutes, the bath stabilized at 185-190 ° C and was maintained there for a further 60 minutes. The bath was then lowered and the reaction mixture was allowed to cool for about 15 minutes. A nitrogen flow is then started to remove unreacted silicon tetrachloride, which is wetted at the cooler outlet at a wet pH.
Detected on paper. After aeration for about 45 minutes, replace the bath that has reached 100 ° C.
Continued slow heating to 30 ° C. facilitated the removal of silicon tetrachloride. This was completed after an additional 70 minutes according to the test described above. At this time, only quinoline vapor was evident.

The bath was then removed and when the reaction mixture had cooled to about 80 ° C., a mixture of 424 ml of water and 424 ml of concentrated hydrochloric acid was added with mixing. Heat was generated and the final mixture was acidic. The reaction product was stored at room temperature overnight. The next day, an additional 424 ml of water and 424 ml of concentrated hydrochloric acid were added with mixing and the mixture was allowed to settle at room temperature overnight.

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

Example 3: Hydrolysis of bis-chloro (2,3-dicarboxyphthalocyanino) silicon (IV ) (dicarboxydichloro dye) concentrated sulfuric acid (98 ml) to avoid wetting the mouth of the flask Was placed in a 250 ml round bottom flask using a long leg funnel. With magnetic stirring, 16.3 g of dicarboxydichloro dye was added in portions through a shorter foot funnel. After the addition was extended for about one hour to disperse the dye mass, more solid was added. A drying tube was attached to the flask and the mixture was heated in an oil bath and kept at 50 ° C. for 24 hours.

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

The mixture was centrifuged at 2000 × g for 30 minutes at room temperature. The precipitate is washed with water (about 250
ml) and centrifuged again. This washing was repeated once more, and the precipitate was collected and suspended in 300 ml of 1M K ₂ CO ₃ . The mixture was heated with stirring in a beaker covered with a watch glass. The temperature reached 90 ° C. in 10 minutes and heating was continued at about 93 ° C. for another 50 minutes. While still hot, acidify the mixture with concentrated HCl,
It was allowed to cool and left at room temperature for 2 days.

The solid was then collected on a Buchner funnel (11 cm) using Whatman # 42 filter paper. Filtration took about 1 hour. The solid was washed on a funnel with 3 × 100 ml of water and air dried in a hood. Next, the solid was broken up and dried over P ₂ O ₅ and KOH under vacuum. Yield 13.3 g (87%).

Example 4: Purification of bis-hydroxy (2,3-dicarboxyphthalocyanino) silicon IV (dicarboxy dye) by silica adsorption chromatography 3.0 g of crude dicarboxy dye (prepared according to Example 3) ) Was placed in a 250 ml bottle, to which was added 100 ml of MeOH containing 2% (v / v) ethyldiisopropylamine (DIEA) and the mixture was stirred for 30 minutes. Thereafter, 43 g of silica (EM Science) was added and the mixture was shaken by hand to form a dark paste. After 20 minutes, an additional 100 ml of MeOH containing 2% DIEA was added, the bottle was inverted several times, and the contents were stirred for 20 minutes. By adjusting the solvent properties by adding EtOH in addition to MeOH and DIEA, the composition of the dye to be extracted was changed and a single component could be extracted. Next, the solid is poured into a sintered glass funnel (pore,
(6.5 cm diameter) under reduced pressure. The vacuum was maintained by connecting to a partially evacuated tank to prevent large loss of MeOH. After filtration overnight, the residue was washed with 2 × 50 ml of MeOH + DIEA, which took about 30 hours.
The filtrate (230 ml) was concentrated on a rotary evaporator to near dryness. 14 ml of residue
In MeOH + DIEA and the solution was aliquoted into two 40 ml conical centrifuge tubes.

The contents of each tube were acidified with 200 ul of concentrated HCl and water was added to nearly fill the tubes. Mix the contents by inverting and shaking several times and mix about 650x
Centrifuged at g for 30 minutes. Discard the brown supernatant liquid and remove the precipitate with 0.01 M HCl.
And washed three times. The precipitate was transferred to a 100 ml round bottom flask and the mixture was dried by rotary evaporation, then dried under vacuum over H ₂ SO ₄ and KOH. Dry weight is 30
4 mg (purified dicarboxy dye).

Example 5 Sulfonation of Purified Dicarboxy Dye with Chlorosulfonic Acid 161 mg of purified dicarboxy dye (Example 4) was added to a 50 equipped with a magnetic mixer.
Weighed into a ml long-necked round bottom flask. At room temperature, 3.4 ml of ClSO ₃
The H was added under N _2. A small air cooler with a N ₂ balloon was fitted and the flask and contents were heated in an oil bath at 110 ° C. for about 3.7 hours. At this point, 50
A ml sample was removed and tested. Next, after heating was continued at 110 ° C. for another 3.3 hours, the heating was stopped, and a second sample was taken out. Ice was added to both samples and each was diluted to 390 mg with water. Next, 2 ml of 1M NaHCO ₃ was added to each sample, and 10 ul of the diluted sample was diluted with 2 ml of neutral buffer, and the measurement was performed to measure each absorption. Amax was 690 nm for both samples. The reading for the 3.7 hour sample is 0.650 and the reading for the 7 hour sample is 0.4.
90, indicating that about 25% of the dye had been destroyed during the last 3.3 hours of heating time.

The main reaction mixture was added portionwise to ice in a beaker and the cold mixture was centrifuged at about 700 × g for 30 minutes. The supernatant liquid having a very faint color was discarded. The precipitate was suspended in 30 ml of ice-cold water and transferred with water to a 250 ml Erlenmeyer flask.
It was made basic with about 40 ml of 1M KHCO ₃ 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 mixture is centrifuged at about 700 × g, the colored supernatant liquid is saved, and the precipitate is
Dissolved in NaHCO ₃ and stirred for 2 hours. The dark green solution was passed through SepPak (2 g size, Rainin) to acidify the filtrate, and then combined with the supernatant liquid of centrifugation. Total volume was about 400 ml. The dark blue acidic solution was adsorbed on SepPak, washed on the column with 3N HCl and extracted with MeOH. SepPak is M
After washing with MeOH and 3N HCl, it can be used repeatedly. The MeOH extract containing the dye was dried by rotary evaporation and dried under vacuum over H ₂ SO ₄ and KOH. Yield 158 mg.

Example 6: Measurement of non-specific binding and characterization of the solution properties of silicon and aluminum phthalocyanine Materials: 1) Borate buffered KCl (BBKCl): 33.1 ml of 0 buffer
. 70M borate; 4.0 ml of _{0.50M K 2 B 4 0 7;} 75ml 4.0M of
Make by mixing KCl and making up to 1 L with H ₂ O; l.

2) Sulfonated dicarboxysilicon phthalocyanine (Example 5), dicarboxysilicon phthalocyanine (Example 4), and aluminum trisulfonic acid,
The sodium salt (porphyrin product) was dissolved in BBKCl. The concentration of these solutions was determined by measuring the absorbance of each sample diluted in methanol containing 5% (v / v) of ethyldiisopropylamine at the maximum NIR (about 680 nm), and calculating the molar extinction coefficient value for all the samples by 2 × 10 5. Determined by assuming ⁵ liter mole ^-1 . Next, dilution was performed in BBKCl to obtain a solution having a concentration of 5 × 10 ⁻⁸ M for each dye.

Fluorescence Measurements: Intensity and polarization measurements were made using a transition state polarimeter (FAST 1, Hyperi).
on, Inc. Miami, FL), and in each case, 10 microliters of a 5 × 10 ^-8 M solution of dye (item 2, material) was added to 1 ml of BBK
Cl, BBKCl + 1% (v / v) pooled normal human serum (L3833, 10/1
8/84) or glycerol.

The results are shown in FIGS. 1 and 2 and characterize the non-specific binding and solvent sensitivity of the three dyes as assessed by fluorescence intensity and polarization measurements. With respect to intensity, desirable properties are steadiness and high fluorescence output independent of the solvent composition. On the other hand, the polarization should ideally remain low in low-viscosity media and be as high as possible in viscous solvents such as glycerol.

FIG. 1 shows that the fluorescence intensity from aluminum phthalocyanine trisulfonate is very solvent sensitive. In contrast, dihydroxydicarboxysilicon phthalocyanine sulfonate shows dramatically improved properties with almost the same fluorescence output in buffer, buffer plus serum or glycerol alone. From a comparison with dihydroxy-dicarboxy-silicon-phthalocyanine (no sulfonate group) which is itself less sensitive to serum and solvents than Al compounds, this improvement can be attributed to some extent to sulfonation.

FIG. 2 shows more strongly that the susceptibility to the environment is lower than in the case where Al is present as the central atom, simply because the central Si atom is present. This difference is probably due to the fact that Si has two axial binding ligands, and therefore, "protecting groups" on each side of the molecular plane, thus "protecting" the planar structure of the dye from solvent effects. Probably because of the fact that they can. For Al, 1
There are only two axial binding ligands, so one side of the molecular plane is freely accessible to solvent effects. In FIG. 2, the polarization of the Al dye is very large, near the maximum achievable when the dye is placed in glycerol, which indicates the approximate limit of polarization when the rotational movement is almost stopped. In the increase, the result of this interaction is clearly seen.

Conclusion The exemplary applications associated with the present invention described above should not, of course, be construed as limiting the scope of the invention. Such changes now known or later developed that are within the purview of those skilled in the art should be considered to be within the scope of the present invention as set forth in the appended claims.

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 cited as part of this specification to the same extent as each publication is specifically and individually cited herein as part of this specification.

The invention exemplarily described herein may be suitably practiced without any element or limitation not specifically disclosed herein. That is, for example, in each example in this specification, the terms “including”, “consisting essentially of” and “consisting of” are replaced with either of the other two. be able to. The terms and expressions used herein are used as terms of description and are not limiting. The use of such terms and phrases is not intended to exclude the equivalents of the features shown or described, or some of them, but is intended to be within the scope of the present invention, which is set forth in the following claims. It is understood that various modifications are possible. That is, although the present invention has been specifically disclosed by the preferred embodiments and optional features, those skilled in the art can make modifications and variations to the concept disclosed in the present specification, and such modifications and variations are also claimed. It is to be understood that this is considered to be within the scope of the present invention as defined in

Other embodiments are within the scope of the following claims.

[Brief description of the drawings]

FIG. 1 shows the effect of non-specific binding and solvent on fluorescence intensity.

FIG. 2 shows a comparison of phthalocyanine derivatives of silicon and aluminum with respect to solvent effects and non-specific binding.

[Procedure amendment]

[Submission date] June 11, 2001 (2001.6.11)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 49

[Correction method] Change

[Correction contents]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Claim 51

[Correction method] Change

[Correction contents]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) // G01N 21/64 G01N 21/64 F (81) Designated country EP (AT, BE, CH, CY, DE) , DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML) , MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, D , DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF term (reference) 2G043 AA01 BA16 CA03 EA01 GA08 GB16 KA01 2G045 AA35 BB25 DA12 DA13 DA14 DA36 FB01 FB02 FB03 FB12 GC15 4C050 PA

Claims (63)

[Claims] 1. A detectably labeled fluorophore moiety comprising a luminescent substantially planar fluorophore moiety coupled to two or more small solubilized axial binding ligands flanking the fluorophore moiety. Marker components. 2. The method of claim 2, wherein the fluorophore moiety comprises a macrocyclic polydentate ligand coordinated to a central atom, and wherein the solubilized axial binding ligand comprises hydroxyl, chlorine, bromine,
Fluorine, OCH _3, and OCH ₂ OH are independently selected from the group consisting of marker component according to claim 1, wherein. 3. The marker comprises two solubilized hydroxyl moieties, each solubilized hydroxyl moiety being linked to a central atom on each side of the macrocyclic ligand.
The marker component according to claim 2. 4. The marker component of claim 3, wherein said marker component comprises an axial binding ligand wherein two hydroxyl moieties are coordinated to a central atom. 5. The marker component according to claim 4, wherein said central atom is capable of forming an octahedral coordination complex. 6. The marker component according to claim 5, wherein said macrocyclic ligand has a conjugated pi-electron system. 7. The marker component of claim 6, wherein said macrocyclic ligand comprises a nitrogen-containing macrocycle. 8. The macrocyclic ligand of claim 7, wherein the macrocyclic ligand is selected from a porphyrin derivative or a porphyrin derivative, a choline derivative, a saphyrin derivative or a porphycene derivative in which one or more bridging carbons have been replaced by nitrogen. Marker component. 9. The marker component according to claim 8, wherein said central atom is selected from silicon, germanium, phosphorus and tin. 10. The marker component of claim 9, wherein said macrocyclic ligand has a low degree of symmetry to promote emission polarization parallel to absorption polarization. 11. The marker component according to claim 10, wherein said central atom is silicon or germanium. 12. The marker component of claim 11, wherein said macrocyclic ligand has a degree of symmetry lower than D _4h . 13. The marker component of claim 12, wherein said macrocyclic ligand has at least one fused aromatic ring. 14. The marker component of claim 9, wherein said macrocycle comprises a porphyrin derivative, wherein 1-4 bridging carbon atoms have been replaced by nitrogen. 15. The marker component according to claim 14, wherein the macrocyclic compound includes a tetrabenzotriazaporphyrin derivative. 16. The method of claim 15, wherein said macrocyclic compound is selected from tetrabenzotriazaporphyrin, 27-phenyltetrabenzotriazaporphyrin, and 27- (p-methylphenyl) tetrabenzotriazaporphyrin. Marker component. 17. The marker component according to claim 16, wherein the central atom is silicon. 18. The marker component according to claim 13, wherein said macrocyclic ligand comprises a phthalocyanine derivative. 19. The marker component according to claim 1, wherein the fluorophore moiety is naphthalocyanine or a naphthalocyanine derivative. 20. The fluorophore moiety coupled to two or more small solubilized axial binding ligands, wherein the fluorophore moiety in an aqueous solution in the presence of a serum component has a parallel and substantially the same intensity as without serum. The detectably labeled marker component according to claim 1, characterized by having a transition state fluorescence emission having a vertical component. 21. The luminescent substantially planar molecular structure has at least about 5
Reduced non-specific binding to components of serum as compared to a bare fluorophore moiety having an excitation wavelength of 00 nanometers and wherein said marker moiety is not coupled to two or more hydroxyl moieties The detectably labeled marker component according to claim 1, comprising: 22. The marker component of claim 21, wherein said fluorophore moiety has an excitation wavelength of about 600-800 nanometers. 23. The marker component of claim 22, wherein said fluorophore moiety has an excitation wavelength of at least 650 nanometers. 24. The marker component having substantially similar intensity, decay time, and relative magnitude of the polarization component in the presence and absence of serum.
2. The marker component according to 1. 25. The marker component of claim 21, wherein the fluorophore moiety alone has a binding constant at least 60 times higher than the binding constant of the complete marker component. 26. The method according to claim 26, wherein the fluorophore comprises: (1) an aryl-terminated polymethine dye;
) Quinoid dyes; (3) indanthrene dyes; (4) 1,4-diaminoanthraquinone-2,3-dicarboxamides; (5) tetraaminoanthraquinones;
(6) azine dyes; (7) pyrylium or thiopyrylium dyes; and (8)
22. The marker component according to claim 21, which is selected from the group consisting of naphthoquinone methides. 27. A method of synthesizing a marker component according to any of claims 1-26, comprising the step of reacting a fluorophore moiety with a reactive form of the solubilized axial binding ligand. 28. A method for detecting a target analyte in a sample suspected of containing the target analyte, comprising: (a) subjecting the marker component according to any one of claims 1-26 to a target analyte; (B) contacting the sample suspected of containing the target analyte with a known amount of a marker component linked to the target analyte or an analog thereof; (c) Contacting said sample suspected of containing said target analyte with a receptor that specifically binds to said target analyte; (d) linked to a target analyte bound to said receptor or an analog thereof Determining either the amount of said marker component or the amount of unbound target analyte or analog thereof. 29. A fluorescent probe comprising a marker component according to any of claims 1-26 linked to one member of a specific binding pair or a target analyte or analog. 30. The probe of claim 29, wherein said receptor target analyte or analog thereof is directly bound to said fluorophore moiety. 31. The probe of claim 29, wherein said receptor target analyte or analog thereof is conjugated to said fluorophore moiety via a linker arm. 32. The receptor target analyte or analog thereof is selected from the group consisting of digoxin, digitoxin, theophylline, phenobarbital, acetylprocainamide, primidone, phenytoin, rubella antibody, and derivatives thereof. Item 30. The probe according to Item 29. 33. The fluorescence of claim 29, wherein said member of a specific binding pair has at least one sterically tolerant marker component binding site capable of allowing said probe to form a specific binding pair. probe. 34. A method for synthesizing a fluorescent probe, comprising the step of linking the marker component according to any one of claims 1-26 to two or more solubilized axial binding ligands. 35. A method for detecting a target test substance in a sample suspected of containing the target test substance, the method comprising: (a) detecting a sample suspected of containing the target test substance in the target test substance; The target analyte and the first analyte are brought into contact with a first receptor capable of specifically binding to a substance.
Forming a complex with the receptor of claim 1 wherein said first receptor is
(B) contacting the complex with a second receptor capable of specifically binding to the target analyte, wherein the complex is labeled with a fluorescent probe comprising the marker component according to any one of (26) to (26). A second receptor bound to a solid support, forming a complex of the first labeled receptor, the target analyte, and the second receptor bound to the solid support; and (c) Measuring either the amount of labeled first receptor bound to the solid support or the amount of unbound labeled first receptor. 36. The amount of said first receptor labeled with a fluorescent probe, measured in said sample suspected of containing said target analyte, in a control sample containing no target analyte. The first, labeled with a fluorescent probe
36. The method of claim 35, further comprising the step of correlating the amount of the first receptor with the amount of the first receptor labeled with a fluorescent probe, as measured in a sample containing a known amount of the target analyte. the method of. 37. The method of claim 35, further comprising the step of separating the solid support from unbound labeled first receptor. 38. A method for determining the presence or amount of a target test substance in a sample suspected of containing a target test substance, the method comprising: (a) the method comprising: Contacting the target analyte with the first receptor and the second receptor capable of specifically recognizing the target analyte;
Here, the first receptor is labeled with a fluorescent probe containing the marker component according to any one of claims 1-26, and the second receptor is bound to a solid support, Forming a complex of said receptor, said target analyte, and said second receptor; and (b) the amount of said labeled first receptor associated with said solid support, or unreacted labeled. Measuring any of the amounts of the first receptor. 39. The amount of labeled first receptor measured in a sample suspected of containing a target analyte is determined by measuring the amount of labeled first receptor measured in a control sample containing no target analyte. 39. The method of claim 38, further comprising the step of correlating the amount of the first receptor or the amount of the labeled first receptor measured in a sample containing a known amount of the target analyte. 40. A method for measuring a target test substance capable of binding to two different receptors in a sample suspected of containing the target test substance, comprising the steps of: (a) determining whether the target test substance is contained; Contacting said sample with a first receptor capable of specifically binding to said target analyte to form a complex between said target analyte and said first receptor, wherein said complex comprises One receptor is labeled with a fluorescent probe comprising a marker component according to any of claims 1-26; and (b) a second ligand capable of specifically binding said complex to said target analyte. Contacting the receptor of claim 2, wherein the second receptor has a maximum absorption and a maximum release that are not the same as the first receptor. : 41. A kit useful for detecting a target test substance in a sample suspected of containing the target test substance, the kit comprising the marker component according to any one of claims 1-26. . 42. A kit useful for detecting a target test substance in a sample suspected of containing the target test substance, the kit comprising the 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 of claims 1-26. 44. The composition of claim 43, wherein said detectably labeled marker component has a decay time ranging from about 1 nanosecond to about 50 nanoseconds. 45. The decay time ranges from about 5 nanoseconds to about 20 nanoseconds.
A composition according to claim 44. 46. The oligonucleotide has a length of 5-50 bases.
44. The composition of claim 43. 47. The composition of claim 43, wherein said linkage comprises (CH ₂ ) ₆ O. 48. The composition of claim 43, wherein said linkage comprises (CH ₂ ) ₂ NH. 49. A method for producing an oligonucleotide conjugated to a marker component, comprising: (a) reacting an oligonucleotide having a linked linker having an amino group at an end with an N-hydroxy-succinimide ester. Or a fluorophore moiety containing a luminescent, substantially planar molecular structure coupled to two small solubilized axially binding ligands flanking the planar molecular structure in the imidazolide, or marker component, detectable Reacting with a composition comprising an oligonucleotide linked to a marker component so labeled to form a conjugate; and (b) combining the conjugate formed in step (a) with unreacted oligonucleotide and unreacted oligonucleotide Separating from the marker component. 50. The method according to claim 49, further comprising a step of attaching a linker having an amino group terminus to the oligonucleotide before step (a). 51. A method for producing an oligonucleotide conjugated to a marker component, comprising: (a) a luminescent substantially coupled to two small solubilized axial binding ligands located on either side of a planar molecular structure. A marker component composition comprising an oligonucleotide linked to a detectably labeled marker component comprising a fluorophore moiety comprising a planar molecular structure in the presence of hydrobenzotriol and the presence of the oligonucleotide Reacting with the carbodiimide below to form a conjugate; and (b) separating the resulting conjugate formed in step (a) from other components in the reaction mixture. 52. A method for determining the presence or amount of a target nucleic acid sequence in a sample, comprising: (a) contacting a sample nucleic acid with the composition of claim 43, wherein the composition comprises a homogeneous solution. And (b) detecting the presence or amount of such hybridization by transition state polarization fluorescence. 53. A method for detecting a target nucleic acid sequence in a sample, comprising: (a) contacting a sample suspected of containing the target nucleic acid sequence with a complementary oligonucleotide capable of hybridizing with the target sequence; b) contacting said sample with a composition according to claim 1, wherein said composition is capable of hybridizing with said complementary oligonucleotide or polynucleotide; and (c) said conjugate and said complementary Detecting the presence or determining the amount of hybridization with the oligonucleotide or polynucleotide. 54. The method of claim 54, wherein the target nucleic acid sequence is a nucleic acid amplification product, DNA, and R
54. The method of any of claims 51-53, wherein the method is selected from the group consisting of NA. 55. The method of claim 55, wherein the nucleic acid amplification is selected from the group consisting of polymerase chain reaction (PCR), ligand chain reaction (LCR), self-sustaining sequence replication (3SR), and transcription-based amplification system (TAS). Claim 5
4. The method according to 4. 56. A method for amplifying a nucleic acid, comprising the steps of:
The use of a fluorescent probe containing a detectably labeled marker component as a label, comprising a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilized axial binding ligands And how. 57. The method of nucleic acid amplification hybridization, comprising a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two small solubilized axially binding ligands flanking the planar molecular structure. A method comprising using, as a label, a fluorescent probe containing a detectably labeled marker component. 58. The composition of claim 43, wherein said composition is heat stable. 59. The composition of claim 58, wherein said composition is stable to exposure at 90 ° C. for 1 hour. 60. The composition of claim 59, wherein said composition is stable after exposure to 100 ° C. for 10 minutes. 61. A kit comprising the composition of claim 43 and a label, package or instructions for use. 62. A method for using the kit according to claim 62, comprising the steps of:
A method comprising using the composition according to the packaging or instructions for use. 63. A method of making a kit according to claim 61 comprising combining the composition and the label, packaging or instructions.