WO2000031187A1 - Colorants fluorescents solubles dans l'eau sans agregation et sans liaison au serum, produits et procedes associes - Google Patents

Colorants fluorescents solubles dans l'eau sans agregation et sans liaison au serum, produits et procedes associes Download PDF

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Publication number
WO2000031187A1
WO2000031187A1 PCT/US1999/026983 US9926983W WO0031187A1 WO 2000031187 A1 WO2000031187 A1 WO 2000031187A1 US 9926983 W US9926983 W US 9926983W WO 0031187 A1 WO0031187 A1 WO 0031187A1
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Prior art keywords
marker component
target analyte
receptor
labeled
component according
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PCT/US1999/026983
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English (en)
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WO2000031187A9 (fr
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Walter B. Dandliker
Mao Lin Hsu
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Hyperion, Inc.
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Priority to MXPA01005243A priority Critical patent/MXPA01005243A/es
Priority to BR9915677-6A priority patent/BR9915677A/pt
Priority to EP99962769A priority patent/EP1133531A1/fr
Priority to JP2000584008A priority patent/JP2002530662A/ja
Priority to AU19140/00A priority patent/AU1914000A/en
Priority to CA002350608A priority patent/CA2350608A1/fr
Publication of WO2000031187A1 publication Critical patent/WO2000031187A1/fr
Publication of WO2000031187A9 publication Critical patent/WO2000031187A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B47/00—Porphines; Azaporphines
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B47/00—Porphines; Azaporphines
    • C09B47/04—Phthalocyanines abbreviation: Pc
    • C09B47/06—Preparation from carboxylic acids or derivatives thereof, e.g. anhydrides, amides, mononitriles, phthalimide, o-cyanobenzamide
    • C09B47/065—Preparation from carboxylic acids or derivatives thereof, e.g. anhydrides, amides, mononitriles, phthalimide, o-cyanobenzamide having -COOH or -SO3H radicals or derivatives thereof, directly linked to the skeleton
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B47/00—Porphines; Azaporphines
    • C09B47/04—Phthalocyanines abbreviation: Pc
    • C09B47/08—Preparation from other phthalocyanine compounds, e.g. cobaltphthalocyanineamine complex

Definitions

  • the present invention relates generally to water soluble fluorescent dyes free of aggregation and serum binding and related products and methods.
  • Preferred dyes include a luminescent substantially planar fluorophore linked to two or more solubilizing ligands.
  • phthalocyanines particularly because of their strong near infrared absorption (molar extinction coeffi- ients about 200,000), their high quantum yields in organic solvents and the well known resistance to fading of common metallophthalocyanine dyes have given rise to many efforts to utilize them as fluorescence labels.
  • earlier efforts along these lines did not yield entirely satisfactory products largely because of the unusually strong tendency of phthalocyanines to associate, particuarly by stacking in face to face aggregates and also to bind strongly to a variety of other molecular surfaces (nonspecific binding) .
  • phthalocyanines As a result of intramolecular stacking unsubstituted phthalocyanines have very low solubilities in both organic and aqueous solvents. As is now well known, the tendency to stack can be dramatically decreased by the introduction of one or more charged groups, such as sulfonate. While phthalocyanines with such substitutions may possess high solubility in water and in aqueous solutions of electrolytes, the tendency to bind nonspecifically largely persists.
  • the present invention exploits an unexpected result that even very small groups such as -OH can produce effective protection against non-specific binding and stacking in a planar molecule if two such groups are present, one on either side of the molecular plane. Increasing the net negative charge accentuates the favorable effect of the these ligands for many biological systems where most of the "biomolecules" themselves carry a negative net charge.
  • one aspect of this invention is that the desirable effects of engineering phthalocyanine and other fluorescent dyes by coupling to polyoxyhydrocarbyl groups can be accomplished instead by two very small axial ligands
  • the dye moves in the void volume in gel permeation chromatography designed to separate macromolecules from small molecules.
  • the sulfonated, dihydroxy-dicarboxy-silicon-phthalocyanine (SDDSiPc) behaves as expected for a molecule of its formula weight .
  • SDDSiPc behaves about the same as the PEG coupled dye (cf. Example 6) .
  • DDSiPc unsulfonated dihydroxy-dicarboxy-silicon-phthalocyanine
  • Hydroxy-aluminum-phthalo- cyanine-trisulfonate shows both strong sensitivity to ionic strength and also has strong non-specific binding. It appears that the phthalocyamine molecule must have an axial ligand on both sides of the molecular plane, but that the - OH group is sufficiently large to virtually eliminate nonspecific binding if the net charge is sufficiently high.
  • Another advantage of the present invention is that dyes engineered by -OH or other small solublizing axial ligands together with high charge appear to be much more reactive chemically (in labeling reactions) even though the molecules being labeled, e.g., proteins and oligonucleotides, are themselves negatively charged. This suggests that the PEG ligands do interfere in labeling macromolecules, although labeling haptens and other small molecules usually proceeds easily.
  • This invention is very unexpected in view of the strong nonspecific binding of hydroxy-aluminum-phthalocyanine-tri- sulfonate, from which one might presume that dihydroxy- dicarboxy-silicon-phthalocyanine with a smaller negative net charge would show even stronger nonspecific binding than the aluminum dye.
  • This invention is based in part on findings which show quite the reverse (cf . Figures 1 and 2) .
  • the effects of other small axial ligands such as: -OCH 3 , -0- CH 2 0H, -Cl, -Br and -F are also expected to work based on the findings reported herein.
  • macrocycles can be metallated with atoms of Group 14 with similar results.
  • Such macrocycles include derivatives and structural variants of porphyrins, azaporphyrins, corroles, sapphyrins, penta- phyrins, porphycenes and other like macrocycles which have extensively delocalized pi electron systems.
  • an especially preferred class of macrocycles comprises aza- porphyrin derivatives and structural variants.
  • Azaporphyrin derivatives include derivatives of mono-, di- and triazaporphyrin and porphyrazine . Any of these macrocycles may optionally also have fused aromatic rings.
  • Such azaporphyrin derivatives and variants include phthalo- cyanine, benzotriazaporphyrin and naphthalocyanine and their derivatives as well as their oxa-, thia-, or aza- structural variants .
  • the present invention thus relates to marker components, fluorescent probes, oligonucleotides, hybridization assays, and immunoassays using such products and methods for making such products.
  • detectably labeled marker components comprise a fluorophore moiety coupled to two or more small solubilizing ligands usually axial, the axis being defined by the octahedral geometry of complexes formed by a central metal atom, which preferably reduce or remove the problems of solvent sensitivity and non-specific binding.
  • detectable labels or marker components in immunoassays is advantageous in that these labels have substantially the same intensities of parallel and perpen- dicular components of transient state fluorescence emission in the presence and absence of biological fluids such as serum.
  • assay methods using these labels are capable of detecting low concentrations of an analyte, a target analyte or analog thereof in biological fluids.
  • analyte refers to the compound or compound to be measured in an assay which may be any compound for which a receptor naturally exists or can be prepared which is mono- or polyepitopic, antigenic or haptenic, a single or plurality of compounds which share at least one common epitopic site or a receptor.
  • target analyte refers to the compound or compound to be measured in an assay which may be any compound for which a receptor naturally exists or can be prepared which is mono- or polyepitopic, antigenic or haptenic, a single or plurality of compounds which share at least one common epitopic site or a receptor.
  • analog of a target analyte is meant a compound or compounds capable of competing with the target analyte for binding to a receptor.
  • receptor refers to a molecule or molecular complex which is capable of specifically recognizing or being recognized by a target analyte or analog thereof.
  • an antibody may be a receptor for an antigen.
  • marker components may be used as labels for labeling an analyte, antigen, antibody or other molecule.
  • These marker components may be optionally functionalized so as to include a linker arm which allows the marker component to be linked to the analyte, antigen, antibody or other molecule.
  • linker arms which are suited to this purpose have been described. Kricka, J.J.; Ligand-Binder Assays; Labels and Analytical Strategies; pages 15-51; Marcel Dekker, Inc., New York, NY (1985).
  • the marker component is linked to the analyte, antigen, antibody or other molecule using conventional techniques.
  • the present invention provides a detectably labeled marker component which comprises: (1) a fluorophore moiety comprising a luminescent substantially planar molecular structure, preferably having excitation wavelengths of at least about 500 nm and (2) coupled thereto two or more small solubilizing axial ligands. Examples of preferred fluorophores, small solubilizing axial ligands, and linkages of the two are described in detail herein. In addition, evidence is provided demonstrating the effectiveness of the marker components at reducing solvent sensitivity and non-specific binding.
  • the marker components of the present invention can be used to make probes as generally described in commonly owned U.S. application Serial No. 08/051,446, filed April 21, 1993, and used in immunoassays as generally described in commonly owned U.S. application Serial No. 08/035,633, filed March 23, 1993, the disclosures of both of which are incorporated herein by reference in their entirety, including any drawings .
  • detectably labeled marker components are provided that have a fluorophore moiety coupled to two small solubilizing axial ligands which in the presence of serum components in aqueous solutions are characterized by transient state fluorescence emission having parallel and perpendicular components of substantially the same intensities as without serum.
  • axial ligand refers to a substituent which, together with a macrocyclic ligand, forms a coordination complex with a central atom. The axial ligand lies normal to the plane described by the macrocyclic ligand.
  • marker components of the present invention which have a macrocyclic multi- dentate ligand with two small solubilizing axial ligands, one located on either side of the plane of the multidentate ligand, exhibit dramatically lowered nonspecific binding to serum components, and exhibit negligible solvent sensitivity.
  • solvent sensitivity refers to changes in the fluorescence behavior of a molecule depending on the solvent system in use, most notably referring to differences in fluorescence behavior in aqueous solution in comparison with organic solvents (such as DMF) .
  • organic solvents such as DMF
  • Fluorescence intensity is related to sample concentration and the intensity of the exciting radiation.
  • the fluorescence intensity of a particular dye can be correlated to its characteristic light absorptivity (extinction coefficient) and fluorescence quantum efficiency, as well as environmental factors .
  • These marker components also exhibit enhanced decay times which approach their radiative or unquenched lifetimes.
  • fluorescent probe refers to a marker component comprising a fluorophore moiety which is bonded to or coordinates either directly or via a linker arm to an analyte, antigen, hapten, antibody or other molecule which is used in an assay, such as a fluoroimmunoassay to determine the presence of and/or quantitate a substance of interest.
  • fluorescent probe refers to a marker component comprising a fluorophore moiety which is bonded to or coordinates either directly or via a linker arm to an analyte, antigen, hapten, antibody or other molecule which is used in an assay, such as a fluoroimmunoassay to determine the presence of and/or quantitate a substance of interest.
  • Some of these marker components are useful as phosphorescent labels .
  • the components of the present invention are also useful as labels for agents for in vivo imaging and also as labels for agents used in in vivo tumor therapy.
  • fluorophores which efficiently produce fluorescence upon excitation with light whose wavelength falls in the range of about 200 to about 1000 nanometers, preferably in the range of about 600 to 800 nanometers.
  • Suitable fluorophores include those which absorb and/or emit at wavelengths which are distinguishable from the excitation and emission maxima of other solution components (such as proteins present in a sample) to minimize background fluorescence. Since these marker components are particularly useful in assays using samples of biological fluids, preferred for those uses are fluorophores having excitation and/or emission wavelengths of at least about 500 nanometers which reduces interference from the ambient fluorescence of other sample components.
  • Some samples, such as serum may exhibit considerable interfering background fluorescence from flavins, flavoproteins, NADH, etc., when excitation wavelengths less than 500 nm are used.
  • preferred fluorophores may also exhibit a high degree of fluorescence polarization when in the bound form, preferably greater than about 10% of the theoretical maximum value for an observable polarization.
  • bound refers to the condition in which a binding interaction has been formed between a molecule and its specific binding partner.
  • preferred fluorophores are also characterized by measured fluorescence decay times in the range of about 1 nanosecond to about 50 nanoseconds, preferably in the range of about 5 to about 20 nanoseconds.
  • fluorophores having even longer decay times may be used.
  • preferred fluorophores which produce fluorescent light efficiently, i.e., which are characterized by high absorbitivity at the appropriate wavelength and high fluorescence quantum yields.
  • preferred fluorophores have measured fluorescence decay times on the order of at least 2 nanoseconds and exhibit a high degree of fluorescence polarization.
  • Preferred small solubilizing axial ligands include -OH, -O-t-butyl, -OCH 2 OH, -0CH 2 CH 2 0H, -OCH 2 CHOHCH 2 OH, -OCH 2 CH 2 -0- CH 2 CH 2 0H,-OCH 2 CH 2 -CH 2 -O-CH 2 CH 2 ⁇ H, Cl,Br and F.
  • the fluorophore moiety has a substantially planar, multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two small solubilizing axial ligands.
  • suitable central atoms are those to which may coordinate two axial ligands and which are not of high enough atomic number to cause extensive fluorescence quenching by transition to the triplet state.
  • Preferred elements for the central atom include silicon, germanium, and tin, especially preferred are silicon and germanium.
  • the present invention is also directed to methods for determining the presence or amount of a target analyte in a sample by using, as a label for the target analyte or a receptor which is capable of specifically recognizing the target analyte, a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.
  • detectable labels or marker components are advantageous in that these labels have substantially the same intensities of parallel and perpendicular components of fluorescence emission in the presence and absence of biological fluids such as serum.
  • assay methods using these labels are capable of detecting low concentrations of target analyte in biological fluids.
  • the methods of the present invention are particularly suitable for use with the improved fluorescence detection system described in commonly assigned U.S. Patent Application entitled “Fluorometer Detection System,” Lyon & Lyon Docket No. 195/129, Serial No. 07/855,238, filed March 23, 1992.
  • the marker components and labels are also believed to be useful in methods described in Walker, et al,
  • the present invention is directed toward competitive inhibition assay procedures utilizing particular labels.
  • the present invention is directed to a method of determining the presence or amount of a target analyte by contacting the sample suspected of containing the target analyte with a known quantity of added target analyte or analog thereof linked to a fluorescent probe which includes a detectably labeled marker component made up of a fluorophore moiety which includes a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure; contacting the sample with a receptor capable of specifically recognizing the target ligand; and determining either the amount of fluorescent probe bound to receptor or free fluorescent probe.
  • the amount of bound or free fluorescent probe in the unknown samples may be compared with blank samples and samples containing known amounts of target analyte.
  • the resultant mixture of sample, fluorescent probe and receptor is diluted immediately before measurement of the amount of bound and/or free fluorescent probe.
  • the dilution step allows for greater sensitivity of the assay. Particularly preferred are dilutions of 2-fold to 100-fold, preferably about 7-fold to about 50-fold, and more preferably about 35-fold.
  • the present invention provides an improvement in immunoassay procedures which utilize a label for either the target analyte (or analog thereof) or the receptor.
  • the improvement is the use of a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.
  • Assays using this type of label are advantageous in that they are free of serum binding and aggregation and are therefore, especially suitable for testing biological samples such as serum, plasma, whole blood and urine.
  • the present invention provides a method for performing a "sandwich" or "two-site” immunoassay having the steps of: (a) contacting a sample suspected of containing a target analyte with a first receptor capable of specifically recognizing the target analyte to form a complex of the target analyte and the first receptor, the first receptor being labeled with a fluorescent probe which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure; (b) contacting the complex with a second receptor capable of specifically recognizing the target analyte or the first receptor, the second receptor being bound to a solid carrier, to form a complex of the first labeled receptor, the target analyte and the second receptor bound to the solid carrier; and (c) measuring either the amount of labeled first receptor associated with the solid carrier or the amount of unreacted labeled
  • a sandwich-type assay may be either a heterogeneous assay or a homogeneous assay. If it is heterogeneous, it may incorporate the additional step of separating the solid carrier from the unreacted labeled first receptor. Homogeneous assays are generally preferred because they are more rapid.
  • the assay may incorporate the additional step of relating the amount of labeled first receptor measured in the unknown sample to the amount of labeled first receptor measured in a control sample free of the target analyte, or to the amount of labeled first receptor measured in samples containing known quantities of target analyte.
  • the present invention provides a method for a simultaneous sandwich-type assay for determining the presence or amount of a target analyte in a sample having the steps of: (a) simultaneously contacting a sample suspected of containing a target analyte with first and second receptors capable of specifically recognizing the target analyte, the first receptor being labeled with a fluorescent probe which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure, and the second receptor being bound to a solid carrier, to form a complex of the first receptor, the target analyte, and the second receptor; and (b) measuring either the amount of labeled first receptor associated with the solid carrier or the amount of unreacted labeled first receptor.
  • the present invention provides a method for a simultaneous sandwich-type assay having the further step of relating the amount of labeled first receptor measured to the amount of labeled first receptor measured for a control sample free of said target analyte, or relating to the amount of labeled first receptor measured with the amount of labeled first receptor measured in samples containing known amounts of target analyte.
  • the present invention provides a sandwich-type fluorescence immunoassay method for measurement of a target analyte which is capable of recognizing two different receptors independently without mutual interference.
  • the method utilizes two receptors, each of which is labeled with a different dye.
  • one receptor is labeled with a first dye having absorption and emission maxima of 680 nm and 690 nm, respectively, and the other receptor is labeled with a second dye having absorption and emission maxima of 695 and 705 nm, respectively.
  • Detection and quantitation of the analyte can be made using either steady state or transient state measurements. In either case, for the example given, excitation would be at 680 nm and detection would be at 705 nm.
  • This type of assay is based on energy transfer and is advantageous in that it is homogeneous.
  • the present invention is directed to immunoassay of biological fluids, including serum, plasma, whole blood and urine.
  • red blood cells in whole blood are lysed prior to assay of whole blood samples.
  • Preferred methods of lysing red blood cells include addition of stearoyl-lysolecithin, palmitoyl- lysolecithin and myristoyl lysolecithin.
  • the target analyte may be an antigen, a hapten or an antibody; and the receptor may be an antigen or antibody.
  • the antibody may be polyclonal or monoclonal.
  • the antibody is a monoclonal antibody.
  • Monoclonal antibodies useful in the present invention may be obtained by the Kohler & Milstein method reported in Nature 256:495-497 (1975). Alternatively, they may be produced by recombinant methods . Science 246:1275-1281 (1989).
  • the target analyte is a drug or a metabolite of a drug.
  • the drug may be a steroid, hormone, antibiotic, immunosuppressant, antiasthmatic, antineo- plastic, antiarrhythmic, anticonvulsant , antiarthritic, antidepressant, or cardiac glycoside.
  • Examples of such drugs include digoxin, digitoxin, theophylline, phenobarbital, thyroxine, N-acetylprocainamide, primidone, amikacin, genta- macn, netilmicin, tobramycin, carbamazepine, ethosuximide, valproic acid, disopyramide, lidocaine, procainamide, quini- dine, methotrexate, amitriptyline, mortriptyline, imipra- mine, desipramine, vancomycin, and cyclosporine .
  • the drug is digoxin.
  • the target analyte is a peptide, for example, a peptide hormone such as luteinizing hormone, follicle stimulating hormone, human choriogonadotropin, thyroid stimulating hormone, angiotensin I, angiotensin II, prolactin or insulin.
  • the peptide may also be a tumor marker such as carcinoembryonic antigen.
  • the peptide may be a virus or portion thereof, for example, rubella virus or a portion thereof.
  • the methods of the present invention provide ways of measuring target analytes in concentrations of from about 1 x 10 "5 M/L to about 1 x 10 "13 M, and particularly in the concentration range of from about 1 x 10 "9 M/L to about 1 x 10 ⁇ 12 M/L.
  • the present methods allow measurement in the range from about 5 x 10 ⁇ 9 M/L to about 5 x 10 "12 M/L, and particularly, concentrations of from about 1 x 10 ⁇ 10 M/L to about 5 x 10 ⁇ 10 M/L.
  • the present methods allow measurement in the range of from about 1 x 10 -11 M/L to about 1 x 10 "12 M/L.
  • the measurement of amount of fluorescent probe -- bound or free or both -- can be determined by measuring steady- state fluorescence or by measuring transient state fluorescence.
  • the wavelength of light measured is greater than about 500 nm, preferably greater than about 650 nm, and more preferably greater than about 680 nm or 690 nm. Because the transient state detection system utilizes a laser diode, it is necessary for the dyes to have excitation maxima matched to the diode output wavelengths. Dyes have been made available to match other commercially available laser diodes having output wavelengths of 680, 690, 720, 750, or 780 nm. Thus, the wavelength of the light measured may be greater than about
  • detection and quantitation is performed using transient state measurement.
  • Transient state energy transfer offers improved measurements due to optimization of the wavelengths of absorption and emission, as well as due to optimization of the decay times of the first and second dyes. Such optimization allows removal of Rayleigh and Raman scattering, and maximizes efficiency of transfer and minimizes direct excitation of the second dye by the first dye.
  • the present invention also provides a method of synthesizing a marker component by reacting the fluorophore moiety with a reactive form of the solubilizing axial ligands.
  • the invention also features a fluorescent probe having a marker component of the invention, linked to one member of a specific binding pair or a target analyte or an analog.
  • specific binding pair refers to two different molecules (or compositions) wherein one of the molecules has an area on the surface or in a cavity which specifically recognizes and binds to a particular spatial and polar organization of the other molecule or molecular complex involving other molecules.
  • a method synthesizing a fluorescent probe is also provided which includes the step of linking a marker component of the invention to two or more solubilizing axial ligands.
  • a kit useful for detecting a target analyte in a sample suspected of containing the target analyte is also provided, the kit including a marker component or probe of the invention.
  • the present invention is also directed to novel dye- oligonucleotide conjugates and methods of synthesizing them and methods of using them. Methods of using these conjugates or probes involve nucleic acid by hybridization methods, nucleic acid amplification methods and nucleic acid sequencing methods.
  • the dye portion of the dye-oligo- nucleotide conjugate is a detectably labeled marker component having a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.
  • the present invention is directed to a composition having an oligonucleotide linked to a detectably labeled marker component which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.
  • oligonucleotide is meant a chain of nucleotide residues. Typically, an oligonucleotide useful in the present invention has a length of from 5 to 50 nucleotides.
  • the oligonucleotide probes used in the method of the invention include polynucleotides of DNA, RNA or any other kind of sequence hybridizable to nucleic acid sequences. It will be appreciated that such nucleic acid sequences may include base analogues as well as the naturally occurring bases cytosine, adenine, guanine, thymine and uracil. Such base analogues include hypoxanthine, 2.6-diaminopurine and 8-azaguanine .
  • the probes may be in double stranded or single stranded form but are preferably in single stranded form. They may be prepared by direct synthesis, polymerase mediated extension reactions or by cloning or any other convenient method. By “linked” is meant combined chemically by an intermediate molecule which is connected to both moieties.
  • the present invention is directed to composition having an oligonucleotide linked to a detectably labeled marker component having a fluorophore moiety which has a substantially planar multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two axial ligands which are coordinated to the central atom on either side of the macrocyclic ligand.
  • the detectably labeled marker component has a decay time in the range of from about 1 nanosecond to about 50 nanoseconds, more preferably, the decay time is in the range of from about 5 nanoseconds to about 20 nanoseconds .
  • the caged dicarboxy silicon phthalocyanine dye may have a variety of functional groups available for coupling. These groups include free carboxyl, free amino and N-hydroxysuccimide ester (NHS ester) .
  • the oligonucleotide of the claimed compositions will have a length of about 5 to about 50 bases.
  • Linkage of the oligonucleotide or polynucleotide to the marker may be accomplished using condensation reactions leading, for example, to the formation of amide, ester, hydrazone, semicarbazone, thiosemicarbazone, urea, and thiourea bonds.
  • a linker may terminate in an amino group, preferably primary.
  • Other linkers may terminate in a carboxyl group.
  • the present invention provides methods for preparing certain dye-conjugated oligonucleo- tides.
  • such a method involves the steps
  • Attachment of a linker to the oligonucleotide can be accomplished by using a diamine or an amino alcohol.
  • the detectably labeled marker component comprises a caged dicarboxy silicon phthalocyanine dye.
  • preparation of the dye-conjugated oligonucleotides may be accomplished: reacting a detectably labeled marker component which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure, with a carbodiimide in the presence of hydroxy- benzotriazole and in the presence of an oligonucleotide or polynucleotide to form a conjugate; and separating the resulting conjugate from other components of the reaction mixture.
  • the detectably labeled marker component has a caged dicarboxy silicon phthalocyanine dye.
  • the present invention is directed to a method for the detection of a target nucleic acid sequence in a sample comprising the steps of contacting sample nucleic acid with oligonucleotide-labeled marker component, preferably oligonucleotide caged dicarboxy silicon phthalocyanine dye conjugate capable of hybridizing with said target nucleic acid sequence in homogenous solution, and detecting the presence or amount of such hybridization by transient state polarized fluorescence.
  • oligonucleotide-labeled marker component preferably oligonucleotide caged dicarboxy silicon phthalocyanine dye conjugate capable of hybridizing with said target nucleic acid sequence in homogenous solution
  • the present invention is directed to a method for detection of a target nucleic acid sequence in a sample having the steps of contacting a sample suspected of containing a target nucleic acid sequence with a complementary oligonucleotide capable of hybridizing with said target sequence; contacting said sample with an oligonucleotide-labeled marker component, preferably oligonucleotide-caged dicarboxy silicon phthalocyanine dye conjugate capable of hybridizing to said complementary oligonucleotide; and detecting the presence or amount of hybridization of said conjugate with said complementary oligonucleotide .
  • an oligonucleotide-labeled marker component preferably oligonucleotide-caged dicarboxy silicon phthalocyanine dye conjugate capable of hybridizing to said complementary oligonucleotide
  • the present invention is directed to methods for detection or quantification of a target nucleic acid wherein the target nucleic acid is a product of nucleic acid amplification.
  • Nucleic acid amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR) , self-sustained sequence replication
  • the present invention is directed to an improvement in a nucleic acid hybridization method or nucleic acid amplification method employing as a label a fluorescent probe which has a detectably labeled marker component which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure.
  • the methods of the present invention are particularly useful when used with a time-correlated transient state detection system, as described in commonly assigned Studholme, et al . , U.S. Patent Application entitled “Fluorometer Detection System,” Serial No. 07/855,238, filed March 23, 1992. That system features transient state detection permitting direct readout of the time-dependent polarization of the sample.
  • the system uses a laser diode which can be modulated at very high frequencies, e.g., 10 MHz rate, and exhibits high output power. Typically the laser "on" time is approximately 2-3 nanoseconds.
  • Photons from the solution are detected using a photomultiplier tube (PMT) operating in a single photon counting mode. The photon event along with the relative time of the photon event as compared with the laser pulse time is determined. By storing the individual photon event times a histogram of frequency of photons as a function of time is generated.
  • PMT photomultiplier tube
  • the present invention provides a method for monitoring the kinetics of a nucleic acid amplification process, and/or quantifying nucleic acid in a target sample.
  • a probe consisting of an oligonucleotide which has been both "capped” and labeled with a composition having an oligonucleotide or polynucleotide linked to a detectably labeled marker component which has a fluorophore moiety having a luminescent substantially planar molecular structure coupled to two small solubilizing axial ligands, one located on either side of the planar molecular structure, may be added directly to the PCR reaction.
  • capped is meant that the 3' end has been reacted dideoxynucleotide .
  • the hybridization with amplified product may be followed kinetically. As the concentration of amplified product increases, the rate of combination of probe with amplified product increases and quantitates the concentration of amplified product. This information together with the number of cycles quantitates the amount of DNA originally in the sample before amplification. Use in sequencing.
  • Another aspect of this invention is the applicaitonto DNA sequencing in which the detectably labeled marker componenet is incorporated either chemically or enzymatically into the DNA: the DNA is cleaved at numerous points and the resulting collection of fragments is analyzed by gel electrophoresis . If desired, labeling can be done with different marker components, one for each of the bases to facilitate analysis.
  • Another aspect of this invention is the broad scope of application to any design, variation or modification of fluorometers .
  • This breadth of applicability is well illustrated in the following example of a specialized type of instrument.
  • a light wave traversing medium A surrounded by a second medium B of lower refractive index undergoes total internal reflection at the boundaries of medium A if the angle of incidence is greater than the critical angle.
  • the electromagnetic field of the totally reflected light penetrates the boundaries for a short distance and there can produce physical effects such as the excitation of fluorophore molecules located near the interface between A and B .
  • the present invention incorporates the features of very large Stokes' shifts utilizing fluorescent dyes based upon N-containing macrocycles (classes listed below) which commonly have a near UV excitation region with emissions in the near infrared region of the spectrum.
  • fluorescent dyes are applicable to immuno/receptor assays in either steady state or transient state modes .
  • Excitation sources include mercury arcs, nitrogen lasers and nitrogen laser pumped dye lasers. Alternatively, these same dyes can be excited in the near infrared with diode lasers allowing excellent results with pulsed excitation and transient state detection. The choice of source depends upon the position of the absorption band for the particular dye in question, the preferred mode of excitation and detection, whether steady state or transient state and upon space requirements .
  • Figure 1 shows effects of nonspecific binding and solvent on fluorescence intensity, showing a protective effect of Si as compared to Al presumably because of the two axial ligands of Si as compared to only one for Al .
  • Al trisulf hydroxyaluminumtrisulfonate
  • Si dicarb bis-hydroxy (2, 3-dicarboxyphthalocyanino) silicon IV
  • Si dicarb sulf sulfonated Si dicarb
  • BBKC1 borate buffered KC1 (cf. Materials, Example 6)
  • NHS pooled normal human serum
  • Cts counts obtained during one measurement cycle of the FAST 1 transient state fluorometer.
  • Cts is proportional to fluorescence intensity.
  • Figure 2 shows a comparison of silicon and aluminum phthalocyanine derivatives with respect to solvent effects and nonspecific binding.
  • the results indicate that the presence of Si (with two axial ligands) produces dramatically better performance than having Al as a central atom. This is evidenced by a much smaller relative change in polarization for dihydroxydicarboxysiliconphthalocyanine (Si dicarb) as compared to Al trisulfonate (Al trisulf) .
  • Si dicarb dihydroxydicarboxysiliconphthalocyanine
  • Al trisulf Al trisulf
  • the present invention relates generally to improved fluorescent marker components having a substantially planar luminescent moiety linked to two or more small axial ligands, and related products and methods.
  • the preferred fluorophore involves small solubilizing axial ligands, fluorescent properties, and methods of synthesis and use are described below. The following description includes preferred modes of carrying out the invention and is made for the general purpose of illustrating the general principles of the invention rather than limiting the invention in any manner.
  • Suitable fluorophore moieties comprise a luminescent substantially planar molecular structure.
  • Preferred central atoms are elements which may form octahedral coordination complexes containing two ligands with a trans or axial orientation, on either side and perpendicular to the planar macrocyclic ligand.
  • the central atom should not have too high atomic number (about 30 or less) so that fluorescence is not diminished through coupling interaction with orbitals of the central atom.
  • Preferred multidentate ligands include nitrogen- containing macrocycles which have conjugated ring systems with pi-electrons. These macrocycles may be optionally substituted, including substitution on bridging carbons or on nitrogens.
  • Suitable macrocycles include derivatives structural variants of porphyrins, azaporphyrins, corrins, sapphyrins, pentaphyrins and porphycenes and other like macrocycles which contain electrons which are extensively delocalized. These macrocycles may optionally have fused aromatic rings with or without hetero atoms such as N, 0 or S. In view of the fact that they incorporate many of the above-noted characteristics, an especially preferred class of macrocycles comprise porphyrin derivatives, and azaporphyrin derivatives (porphyrin derivatives wherein at least one of the bridging carbons is replaced by a nitrogen atom) .
  • Azaporphyrin derivatives include derivatives of mono-, di- and triazaporphyrin and porphyrazine . These macrocycles may optionally have fused aromatic rings with or without hetero atoms such as 0., N or S. These azaporphyrin derivatives include phthalocyanine, benzotriazaporphyrin and naphthalo- cyanine and their derivatives. The preparation and fluor- escent qualities of many of these compounds are known and some are available commercially. See U.S. Patent Application Serial No. 201,465 and references cited therein, particularly, references 2-5 in that application.
  • azaporphyrin derivatives which exhibit a high degree of polarization in the bound form, that is, those which emit strongly polarized light.
  • macrocycles having lower degrees of symmetry preferably having lower symmetry than D 4h .
  • One preferred group includes macrocycles having at least one fused aromatic ring.
  • preferred macrocycles include azaporphyrin derivatives having fused aromatic rings at positions which result in decreased symmetry.
  • Preferred classes of azaporphyrin derivatives comprise derivatives of monoazaporphyrin, diazaporphyrin, and triazaporphyrin having lower than D 4h symmetry.
  • Preferred small solubilizing axial ligands include -OH, -O-t-butyl, -OCH 2 OH, -OCH 2 CH 2 OH, -OCH 2 CHOHCH 2 OH, -OCH 2 CH 2 -0- CH 2 CH 2 0H,-OCH 2 CH 2 -CH 2 -O-CH 2 CH 2 ⁇ H, Cl,Br and F.
  • the intensity of the two components polarized either parallel or perpendicular to the direction of polarization of the exciting pulse is monitored over a time period equal to about 3 times the decay time of the marker component.
  • Such curves reflect extinction coefficient, quantum yield, decay time and state of polarization and supply sensitive indications on the chemical and physical condition of the marker component. For example, if the excited state is being deactivated or converted to the triplet state the overall intensities are lowered and the decay times shortened. If the rotary brownian motion of the molecule is being altered by an increase in viscosity or by being bound to a large molecule, the ratio of the intensity of the parallel to the perpendicular component is increased.
  • marker components according to the present invention show, within experimental error of about 5%, the same intensities, decay time and polarization in DMF (an organic solvent) as in SAP (saline azide phosphate, an aqueous neutral buffer) . To some extent these properties are shared by other marker component preparations.
  • a distinctive and important property of the marker components of the present invention is a insensitivity to (and lack of binding to) the components in serum which is evidenced by a lack of any significant measured effect of serum on the intensities, decay time or relative magnitudes of the polarized components of the fluorescence. This property is crucial for the marker components to be useful for applications such as assays using biological materials.
  • the appropriate fluorophore moiety having hydroxy or halide groups as axial ligands is reacted with a reactive form of the solubilizing moiety in a ligand exchange reaction according to the general reaction scheme: Mcl-CA-(X) 2 + 2(SM) ⁇ Mcl-CA-(SM) 2 + 2X wherein Mel denotes the macrocyclic ligand, CA the central atom, X the displaced ligand and SM the solubilizing moiety.
  • This reaction may be carried out neat or, if desired, in solvent. Suitable solvents include quinoline, THF, DMF, imidazole when dissolved itself in one of the other listed solvents and the like.
  • Suitable reaction temperatures may vary, depending on the nature of the macrocyclic starting material and the solubilizing group.
  • the reaction is generally complete in about 2 minutes to about 24 hours.
  • the reaction mixture can be conveniently heated under reflux or by means such as a sand bath.
  • the reaction may be carried out at ambient pressure.
  • these marker components When used as fluorescent labels in fluorescence immunoassays, these marker components may be linked to one member of a specific binding pair ("labeled binding partner") or an analog of such a member.
  • binding partner refers to a molecule or molecular complex which is capable or specifically recognizing or being recognized by a particular molecule or molecular complex.
  • the marker component may be directly attached or conjugated thereto or attached or conjugated via a linker arm.
  • the marker components of the present invention are useful as fluorescent labels for fluorescent probes and in fluorescence binding assays and also in as labels for in vivo imaging and in vivo tumor therapy.
  • marker components may be advantageously used as fluorescent labels in conventional fluorescence binding assays, including fluorescence polarization immunoassays.
  • these marker components may be linked to one member of a specific binding pair ("labeled binding partner") or an analog of such a member.
  • the marker component may be directly attached or conjugated thereto or attached or conjugated via a linker arm.
  • labeled binding partners are useful in assays having a variety of formats, such as assays which involve competition for analyte or analyte binding partner (if a labeled analyte or analyte-analog as used) and may be used in either homogeneous or heterogeneous assays.
  • these markers are especially suited for use in assays for detecting an analyte in a sample containing a biological fluid such as serum.
  • these marker components may be used as labels for fluorescent probes for detecting analytes in solutions where non-specific binding by serum components would severely compromise sensitivity of an assay, affecting both its accuracy and precision.
  • these marker components may be used as agents for in vivo imaging. When used as imaging agents, these marker components are conjugated to one member of a specific binding pair to give a labeled binding partner. The labeled binding partner is introduced into an animal. If the other member of the specific binding pair is present, the labeled binding partner will bind thereto and the signal produced by the marker component may be measured and its localization identified.
  • marker components may also be used in in vivo tumor therapy.
  • photodynamic therapy involves using the marker component as a photosensitizing agent.
  • the marker component fluorescent label
  • the marker component is conjugated to a binding partner which may specifically recognize and bind to a component of a tumor cell.
  • the present invention provides nucleic acid probes and methods of making and using the probes. Methods of using the novel nucleic acid probes include various nucleic acid hybridization sequencing techniques now known or later developed, and various nucleic acid amplification techniques now known or later developed.
  • the probes (also referred to as conjugates herein) and methods of the present invention allow the achievement of 1 fmole sensitivity in a homogeneous hybridization assay.
  • Example 1 Synthesis of tetradiiminopyromellitic acid diimide from 1, 2 , 4 , 5-tetracyanobenzene (TCNB) .
  • Diiminoisoindoline (30.0 g; 0.207 moles) and tetra- iminopyromellitic acid diimide (10.5 g, 0.050 moles) were pulverized together and dried in vacuo overnight in a one liter, 3 neck flask.
  • the flask was fitted with a Teflon vane mixer, septum, thermometer and reflux condenser with a silica gel drying tube.
  • the apparatus with the stirred reactants was flushed with dry nitrogen and under nitrogen, 600 ml quinoline was added and mixed for 30 min. under nitrogen flow. A uniform, fluid suspension resulted. Thereafter, over a 5 min period 60 ml silicon tetrachloride was added slowly through the septum. The solution darkened and without heating stirring was continued for 15 min.
  • an oil bath preheated to 195 deg. C was raised into position to immerse the flask to a level above its contents . After 5 min the bath temperature had dropped to 175 deg. C and after another 15 min the bath stabilized at 185-190 deg. C where it was maintained for an additional 60 min. The bath was then lowered and the reaction mixture was allowed to cool for about 15 min. Nitrogen flow was then started to remove unreacted silicon tetrachloride which was detected by moist pH paper at the condenser outlet. After about 45 min of ventilation the bath now at 100 deg. C was replaced to continue heating slowly to about 130 deg. C to facilitate removal of silicon tetrachloride which was complete by the above test after an additional period of 70 min. when only quinoline fumes were evident.
  • the bath was then removed and when the reaction mixture had cooled to ca . 80 deg. C a mixture of 424 ml water and 424 ml concentrated hydrochloric acid was added with mixing. Heat was evolved and the final mixture was acidic. The reaction products stood at room temperature overnight. The next day an additional 424 ml water and 424 ml concentrated hydrochloric acid was added with mixing, and the mixture was 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 the hood overnight.
  • the moist filter cake was stirred in one liter of acetone and filtered.
  • the washed material was dried in the hood for 2 days,
  • the dried material (50 g) was pulverized in a mortar with acetone and the mixture was stirred, filtered and dried in vacuo leaving 47.9 g of a dark finely divided solid.
  • Example 3 Hydrolysis of bis-chloro (2, 3-dicarboxy phthalo- cyanino) silicon (IV) (dicarboxydichloro dye).
  • Concentrated sulfuric acid (98 ml) was placed in a 250 ml round bottom flask using a long stem funnel to avoid wetting the neck of the flask.
  • 16.3 g of dicarboxydichloro dye was added in small portions through a funnel with a shorter stem. The additions were extended over period of about an hour to allow lumps of dye to disperse before adding more solid.
  • a drying tube was attached to the flask and the mixture was heated in an oil bath and maintained at 50 deg. C for 24 hr.
  • the reaction flask was removed from the oil bath and cooled in ice. Water (75 ml) was added cautiously in small portions and without cooling, the mixture was heated with stirring in an oil bath at 80 deg. C for 20 hr. After cooling, the mixture was poured into ice in a one liter beaker and stirred. The mixture was centrifuged at room temperature at 2000 x g for 30 min. The sediment was suspended in water (ca. 250 ml) and again centrifuged. This washing was repeated once more, the sediment was collected and suspended in 300 ml 1M K 2 C0 3 . The mixture was heated with stirring in a beaker covered with a watch glass. In 10 min the temperature reached 90 deg. C and heating was continued at about 93 deg.
  • Example 4 Purification of bis-hydroxy (2, 3-dicarboxy- phthalocyanino) silicon IV (dicarboxy dye) by adsorption chromatography on silica.
  • the solid was then filtered on a sintered glass funnel (fine porosity, 6.5 cm diameter) under reduced pressure. To prevent too great loss of MeOH the reduced pressure was maintained by connection to a partially evacuated tank. After filtering overnight, the residue was washed with 2 x 50 ml portions of MeOH + DIEA which required about 30 hr . The filtrate (230 ml) was concentrated in a rotary evaporator to near dryness. The residue was dissolved in 14 ml MeOH +DIEA and the solution was divided equally and put into two 40 ml conical centrifuge tubes.
  • each tube was acidified with 200 ul concentrated HCl and water was added to nearly fill the tubes.
  • the contents were mixed by inverting and shaking few times, and centrifuged at about 650 x g for 30 min.
  • the brown supernatant liquid was discarded and the sediment was washed three times with 0.01 M HCl.
  • the sediment was transferred to a 100 ml round bottom flask and the mixture was dried by rotary evaporation and then in vacuo over H 2 S0 4 and KOH .
  • Wt dry was 304 mg (purified dicarboxy dye) .
  • Example 5 Sulfonation of purified dicarboxy dye by chloro- sulfonic acid.
  • Purified dicarboxy dye (Example 4), l ⁇ lmg, was weighed into a 50 ml long neck, round bottom flask with a magnetic mixer. At room temperature 3.4 ml of C1S0 3 H was added under N 2 . A small air condenser with N 2 balloon was attached and the flask and contents was heated in an oil bath at 110 deg. for about 3.7 hr . at which point a 50 ml sample was withdrawn for testing. Heating was then continued for an additional 3.3. hr . at 110 deg. whereupon heating was stopped and a second sample was taken out. Ice was added to both samples and each was diluted with water to a weight of 390 mg .
  • the sediment was suspended in 30 ml of ice cold water, transferred with water to a 250 ml Erlenmeyer flask, made basic with about 40 ml 1 M KHC0 3 and stirred at room temperature overnight.
  • the reaction mixture was transferred to a beaker, acidified with concentrated HCl and stirred at room temperature for 6 hr and stored at room temperature for 48 hr .
  • the mixture was centrifuged at ca . 700 x g; the colored supernatant fluid was retained and the sediment was dissolved in 1 M NaHC0 3 and stirred for 2 hr .
  • the dark greenish solution was passed through a Sep Pak (2 g size, Rainin) and the filtrate after acidification was combined with the supernatant fluid from the centrifugation .
  • BBKC1 Borate buffered KC1
  • This buffer is made by mixing: 33.1 ml of 0.70 M boric acid; 4.0 ml of 0.50 M K 2 B 4 0 7 ; 75 ml of 4.0 M KC1; H20 to make one liter; pH ca 8.1.
  • Fluorescence Measurements Measurements of intensity and polarization were made in a transient state polarization fluorometer (FAST 1, Hyperion, Inc. Miami, FL) by, in each case, diluting 10 microliters of a 5x10-8 M solution of the dyes (item 2, Materials) with 1ml of either BBKC1, BBKC1+ 1% (v/v) pooled normal human serum (L3833, 10/18/84) or glycerol.
  • Figures 1 and 2 The results are shown in Figures 1 and 2 and characterize the nonspecific binding and solvent sensitivity of the three dyes as assessed by fluorescence intensity and polarization measurements.
  • intensity the properties desired are a constancy independent of solvent constituents together with a high fluorescence output.
  • the polarization ideally should remain low in media of low viscosity and be as high as possible in a viscous solvent, such as glycerol.
  • Figure 1 shows that the fluorescence intensity from aluminumphthalocyaninetrisulfonate is very solvent-sensitive.
  • dihydroxy dicarboxy silicon phthalocyanine sulfonate shows a dramatically improved performance having almost the same fluorescence output in buffer, buffer plus serum or glycerol alone.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

La présente invention concerne des composants marqueurs, des sondes fluorescentes, des oligonucléotides, des dosages d'hybridation, et des immunodosages utilisant de tels produits et des procédés de fabrication de tels produits. Cette invention concerne des composants marqueurs, marqués de manière détectable, qui comprennent une entité fluorophore couplée à un ou deux petits ligands axiaux solubilisants, qui permettent préférablement de réduire ou d'éliminer les problèmes de sensibilité au solvant et de liaisons non spécifiques.
PCT/US1999/026983 1998-11-25 1999-11-12 Colorants fluorescents solubles dans l'eau sans agregation et sans liaison au serum, produits et procedes associes WO2000031187A1 (fr)

Priority Applications (6)

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MXPA01005243A MXPA01005243A (es) 1998-11-25 1999-11-12 Colorantes fluorescentes solubles en agua libres de enlaces de agregacion y suero y metodos y productos relacionados.
BR9915677-6A BR9915677A (pt) 1998-11-25 1999-11-12 Corantes fluorescentes solúveis em água isentos de agregação e ligações séricas e produtos e métodos afins
EP99962769A EP1133531A1 (fr) 1998-11-25 1999-11-12 Colorants fluorescents solubles dans l'eau sans agregation et sans liaison au serum, produits et procedes associes
JP2000584008A JP2002530662A (ja) 1998-11-25 1999-11-12 凝集および血清結合のない水溶性蛍光染料および関連する生成物および方法
AU19140/00A AU1914000A (en) 1998-11-25 1999-11-12 Water soluble fluorescent dyes free of aggregation and serum binding and relatedproducts and methods
CA002350608A CA2350608A1 (fr) 1998-11-25 1999-11-12 Colorants fluorescents solubles dans l'eau sans agregation et sans liaison au serum, produits et procedes associes

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US60/109,969 1998-11-25

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2420784A (en) * 2004-11-25 2006-06-07 Pci Biotech As Phototherapeutic amphiphilic phthalocyanine-based compounds where 1 peripheral ring system is more hydrophilic & has more hydrophilic groups than the other 3
EP1758917A1 (fr) * 2004-06-14 2007-03-07 Hereth, Hannjorg Azaporphyrines substituees en tant que marqueurs de fluorescence

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988004777A1 (fr) * 1986-12-15 1988-06-30 Ultra Diagnostics Corporation Reactifs de phtalocyanine monomeres
WO1990002747A1 (fr) * 1988-09-08 1990-03-22 Ultra Diagnostics Corporation Reactifs de phtalocyanine et tetrabenztriazaporphyrine decales vers le rouge
WO1993019366A1 (fr) * 1992-03-23 1993-09-30 Diatron Corporation Immuno-essais par fluorescence au moyen de matrices fluorescentes exemptes d'agregat et de liant de serum
WO1996041144A2 (fr) * 1995-06-06 1996-12-19 Hyperion, Inc. Produits de marquage a base de polyoxyhydrocarbyle et dosages immunologiques en fluorescence

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988004777A1 (fr) * 1986-12-15 1988-06-30 Ultra Diagnostics Corporation Reactifs de phtalocyanine monomeres
WO1990002747A1 (fr) * 1988-09-08 1990-03-22 Ultra Diagnostics Corporation Reactifs de phtalocyanine et tetrabenztriazaporphyrine decales vers le rouge
WO1993019366A1 (fr) * 1992-03-23 1993-09-30 Diatron Corporation Immuno-essais par fluorescence au moyen de matrices fluorescentes exemptes d'agregat et de liant de serum
WO1996041144A2 (fr) * 1995-06-06 1996-12-19 Hyperion, Inc. Produits de marquage a base de polyoxyhydrocarbyle et dosages immunologiques en fluorescence

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1758917A1 (fr) * 2004-06-14 2007-03-07 Hereth, Hannjorg Azaporphyrines substituees en tant que marqueurs de fluorescence
EP1758917A4 (fr) * 2004-06-14 2009-11-11 Hereth Hannjorg Azaporphyrines substituees en tant que marqueurs de fluorescence
GB2420784A (en) * 2004-11-25 2006-06-07 Pci Biotech As Phototherapeutic amphiphilic phthalocyanine-based compounds where 1 peripheral ring system is more hydrophilic & has more hydrophilic groups than the other 3

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AU1914000A (en) 2000-06-13
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BR9915677A (pt) 2001-08-14
JP2002530662A (ja) 2002-09-17
CN1344293A (zh) 2002-04-10

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