WO2000075237A2 - Colorants a base de cyanine sensibles au ph utilises comme reactifs fluorescents - Google Patents

Colorants a base de cyanine sensibles au ph utilises comme reactifs fluorescents Download PDF

Info

Publication number
WO2000075237A2
WO2000075237A2 PCT/US2000/015682 US0015682W WO0075237A2 WO 2000075237 A2 WO2000075237 A2 WO 2000075237A2 US 0015682 W US0015682 W US 0015682W WO 0075237 A2 WO0075237 A2 WO 0075237A2
Authority
WO
WIPO (PCT)
Prior art keywords
dye
straight
branched alkyl
cooh
integer
Prior art date
Application number
PCT/US2000/015682
Other languages
English (en)
Other versions
WO2000075237A3 (fr
WO2000075237A9 (fr
Inventor
Ratnakar Mujumdar
John Anthony Smith
Original Assignee
Carnegie Mellon University
Amersham Pharmacia Biotech Uk Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Carnegie Mellon University, Amersham Pharmacia Biotech Uk Limited filed Critical Carnegie Mellon University
Priority to AU57284/00A priority Critical patent/AU5728400A/en
Priority to CA002375740A priority patent/CA2375740A1/fr
Priority to EP00942696A priority patent/EP1212375A2/fr
Priority to JP2001502512A priority patent/JP2003501540A/ja
Publication of WO2000075237A2 publication Critical patent/WO2000075237A2/fr
Publication of WO2000075237A3 publication Critical patent/WO2000075237A3/fr
Publication of WO2000075237A9 publication Critical patent/WO2000075237A9/fr

Links

Classifications

    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • the present invention provides pH sensitive cyanine dyes and methods of using them as fluorescent reagents.
  • Cyanine and related polymethine dyes having light absorbing properties have been employed in the photographic films, optical recording media, and recently as luminescence (fluorescent and phosphorescent) dyes for biological investigations.
  • cyanine dyes F. Hamer "Cyanine Dyes and Related Compounds", A. Weissberger, Ed., Interscience Publishers, New York, (1964); D. M. Sturmer "Synthesis and Properties - Cyanine and Related Dyes," The Chemistry of Heteroaromatic Compounds, Vol. 30A. Weissberger and E. C. Taylor Eds., John Wiley & Son, New York, NY, (1977); G. E.
  • a typical cyanine may be defined as a molecule having two nitrogen containing heteroaromatic rings (A and A') joined by a polymethine chain as shown in formula 1.
  • Groups Rj and R are essentially alkyl or substituted alkyl groups and "m" is an integer selected from the group consisting of 0, 1, 2, 3, and 4. These dyes have cationic character due to delocalized positive charge on the chromophore. This delocalization also accounts for very high extinction coefficients of cyanine dyes.
  • Groups Ri and R are alkyl or substituted alkyl groups
  • Groups R , R 4 , R 5 , R 6 and R are H, water solubilizing groups, halogens, alkoxy, hydroxy, amine, amide, carboxyhc acid, or carboxyhc ester.
  • rings "A" and “A”' are selected from the following heteroaromatic rings.
  • R 3 , R 4 , R 5 , R 6 and X are hereinbefore defined.
  • W is selected from hydrogen
  • n is an integer from 1 to 26 and R is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, sulphonate, sulphate, carboxylate, substituted amino, nitro, primary amide, substituted amide and groups reactive with amino, hydroxyl, carbonyl, phosphoryl and sulphydryl groups.
  • cyanine dyes are only slightly sensitive to solvent changes and they are generally considered as pH insensitive dyes.
  • cyanines are known to show pH dependency.
  • Amine or carboxyhc acid groups directly attached to the aromatic ring of the heteroaromatic system affect the cation delocalization between the nitrogens.
  • Absorption bands of amino-cyanine dyes (formula 2) are very broad with almost total mixing of main electronic transition and vibronic side bands at neutral pH. In a mildly acidic solutions or when the amine is blocked by an acetyl group the transition becomes sharper, more intense and shifts to the blue.
  • These amino-cyanines are virtually non-fluorescent in neutral solution (R. B. Mujumdar, L. E. Ernst, S.R. Mujumdar, and A. S. Waggoner, "Cyanine Dye Labeling Reagents containing Isothiocyanate Groups", Cytometry vol. 10, pp 11-19 (1989)).
  • Squaraines usually are pH sensitive but its derivatives (formula 3) are insensitive in a biological pH range (pH 6-pH 9) and therefore, can not be used effectively as pH sensitive dyes for biological investigations (E. Te ⁇ etsching, H. Szmacinski, A. Ozinskas, and J. R. Lakowicz, "Synthesis of squaraine-N- Hydroxysuccinimide Esters and Their Biological Application as Long- Wavelength Fluorescent Labeling", Analytical Biochemistry vol. 217, pp 197-204 (1994)).
  • the dyes of formula 4 and 5 have an exchangeable hydrogen on one of the nitrogens, but they are water insoluble, lack reactive groups and are unstable, rendering them unsuitable for biological applications.
  • Fluorescent dyes are generally known and used for fluorescence labeling and detection of various biological and non-biological materials by procedures such as fluorescence microscopy, fluorescence immunology and flow cytometry. Fluorescent dyes may also be used as tracers to measure, for example, intracellular and extracellular ionic concentrations simultaneously. In this way ionic gradients across membranes may be measured. Moreover, it is possible to monitor two ions simultaneously using two different fluorescent probes.
  • fluorescent dyes include those based on fluorescein (green fluorescence) and rhodamine (orange fluorescence), coumarin and pyrene (blue fluorescence) chromophores.
  • Commonly available fluorescent pH indicators are generally either fluorescein or pyrene derivatives. Dyes based on fluorescein have a number of disadvantages, including their tendency to photobleach when illuminated with strong excitation sources. The resulting rapid loss of image with time makes detection and quantitation more difficult. Fluorescein derivatives also have a pH sensitive absorption spectrum and fluorescence yield decreases markedly below pH 8.
  • Pyrene derivative, such as pyrene trisulphonates have broad absorption and emission spectra and relatively low extinction coefficients.
  • LYSOSENSOR probes which are weak bases that become more fluorescent in acidic environments.
  • LYSOSENSOR YELLOW/BLUE which has the following formula
  • LYSOSENSOR pH sensitive fluorescent indicators include LYSOSENSOR BLUE DND-167 which is non-fluorescent in non-acidic environments, soluble in DMSO, has a pKa of 5.1, fluoresces at pH of 4.5-6 with an emission wavelength of 425 nm when excited at 373 nm and has the following structure
  • LYSOSENSOR GREEN DND-189 is also non-fluorescent in non-acidic environments, soluble in DMSO, has a pKa of 5.2, fluoresces at a pH of 4.5-6 with an emission wavelength of 505 nm when excited at 443 nm and has the following structure
  • LYSOSENSOR BLUE DND-192 fluoresces brightly at neutral pH, is soluble in DMSO, has a pKa of 7.5, fluoresces at a pH of 6.5-8 with an emission wavelength of 425 nm when excited at 374 nm and has the following structure
  • LYSOSENSOR GREEM DND-153 also fluoresces brightly at neutral pH, is soluble n DMSO, has pKa of 7.5, fluoresces at a pH of 6.5-8 with an emission wavelength of 505 nm when excited at 442 nm and has the following structure
  • LYSOSENSOR probes may localize in membranes rather than in an aqueous medium, it is probable that the pKa values noted above will be different in a cellular environment and that qualitative or semi-qualitative comparisons of cellular pH may be possible.
  • fluorescein derivatives wherein an electron- withdrawing group has been introduced to lower the pKa of the phenolic group to 5 or below.
  • the product literature reports that fluorinated fluorescein dyes are still pH sensitive in moderately acidic solutions, with pKa values of about 4.7.
  • rhodol derivatives which are structural hybrids of fluorescein and rhodamine dyes which have retained the pH-sensitive properties conferred by a phenolic hydroxy group while also containing an amine substituent to reduce the pKa values.
  • C-NERF which has the following structure
  • This dye permits ratiometric pH measurements using two principal visible lines of an argon-ion laser (514 nm and 488 nm) with emission reported at 540 nm.
  • the cyanines described in this invention preferably have functional groups to enhance aqueous solubility.
  • the cyanines preferably have at least one sulfonate group which may be an aryl sulfonate or aralkyl sulfonate.
  • the water solubilizing group of this invention may be a phosphonate or a phosphate.
  • Figure 1 shows the absorbance spectra at pH 3 to pH 9.5 of an exemplified compound, Cy3.39, according to the invention.
  • Figure 2 shows the emission spectra of an exemplified compound, Cy3.39, according to the invention over a range of pH from pH 4.0 to pH 9.5 with an excitation wavelength of 514 nm.
  • Figure 3 shows the excitation spectra of an exemplified compound, Cy3.39, over a range of pH from 4.0 to pH 9.5 measured at 564 nm emission.
  • Figure 4 shows the absorbance at pH 4 to 9 of exemplified compounds
  • Figure 5 shows the pKa of compound 7.
  • Figure 6 provides the RFU vs. wavelength for the pH sensitive tandem dye energy transfer cassette-Compound 11 with an excitation of 530 nm.
  • Figure 7 provides the details of Figure 6 at an excitation of 630 nm.
  • Figure 8 provides an example of the internalization of pH sensitive Cy5, as described herein.
  • the present invention provides a cyanine derivative of the following formula (I)
  • X and Y are selected from >C(C ⁇ -C alkyl) 2 , sulfur and oxygen;
  • R 1 and R " are independently selected from H, CH NH 2 , phosphate, phosphonate, quaternary ammonium, NO 2 , (CH 2 ) q COOH, SO 3 ⁇ CH 2 COOH, NCS, CH 2 NH- COR 7 where R 7 is C ⁇ -C 20 straight or branched alkyl or -(CH 2 ) q -COOH where q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • R 3 is H or is -L-P where L is selected from C C 20 straight or branched alkyl optionally containing 0, 1 or 2 unsaturated groups selected from alkenyl, alkynyl and aryl, and P is selected from a reactive group, H, C 1 -C 20 straight or branched alkyl, SO ⁇ , NH 2 , quaternary ammonium, CH 2 NH- COR 8 where
  • R 1 , R 2 or R 3 may be joined through a linker or spacer group, L, selected from a straight or branched . 20 alkyl chain, a C 2 - 20 monoether or polyether and a C 2 - 2 o atom containing up to four secondary amide linkages to a fluorescent partner in an energy transfer relationship.
  • L linker or spacer group
  • L may be attached to a target bonding group or reactive group or reactive moiety, P, as described herein, such as succinimidyl ester, isothiocyanate, thiocyanate, monochlorotriazine, dichlorotriazine, anhydride, haloacetamide, maleimide, sulphonyl halide, phosphoramidite, acid halide, alkylimidate, hydrazide and carbodiimide; and groups reactive with amino, hydroxyl, aldehyde, phosphoryl, or sulphydryl groups.
  • L may include up to two unsaturated groups selected from alkenyl, alkynyl and aryl which may be, for example, interspersed in the chain.
  • the target bonding group may be substituted directly on either of the fluorescent molecules, such as R'-R 6 , as defined herein.
  • the dye or derivative of the present invention is one dye of an energy transfer complex as described in European Patent Application No.747700, WO 99/39203 and WO 00/13026, the entire contents of each of which are incorporated herein by reference.
  • the complex comprises a first (or donor) fluorochrome, a second (or acceptor) fluorochrome and at least one linker for covalently attaching the donor and acceptor fluorochrome s.
  • the energy transfer complex according to the invention contains a target bonding group which is capable of forming a covalent bond with a target material.
  • the energy transfer complex may include a functionality to facilitate cell permeability. Examples include acetoxymethyl ester.
  • the energy transfer complexes may be used in ratiometric measurements involving changes in pH of a system and in which either the donor or the acceptor species may be a pH sensitive cyanine dye described herein.
  • the pH sensitive cyanine dye is the donor
  • the intensity of fluorescence emission of the acceptor dye is measured after excitation of both the donor and the acceptor dye species independently.
  • the pH sensitive cyanine dye is the acceptor
  • the method therefore provides for a ratiometric measurement in which two dyes which are in energy transfer relationship can be used to provide concentration independent information relating to pH changes in biological and other systems.
  • the present invention provides a substituted cyanine dye which becomes fluorescent upon protonation of the indolic nitrogen under acidic or basic conditions, depending on the basicity of the dye. That is, for example, in acidic conditions, the protonated cyanine of formula (I) has the following structure
  • the present invention provides pH sensitive dyes of the following formula (la)
  • R is C] to C 2 o straight or branched alkyl
  • the invention provides derivatives of formulas (I) and (la) which contain a substituent to make them covalently reactive with amine, hydroxyl, aldehyde and sulfhydryl groups. These substituents are useful for covalently attaching the cyanine dyes of formulas (I) and (la) to biological materials, non-biological molecules and macromolecules and particles so that these materials may be fluorescently labeled; detected and/or quantified by fluorescent detection methods.
  • the derivatives of formulas (I) and (la) include compounds of the following formulas (Et), (EH) and (IV), including the protonated forms thereof, 16
  • X and Y are selected from >C(C ⁇ -C alkyl) 2 , sulfur, and oxygen;
  • Z a and Z b each independently represent the atoms necessary to complete 1 or 2 fused aromatic rings having 6 carbon atoms containing 0, 1 or 2 nitrogen atoms;
  • R 1 , R 2 , R 5 and R 6 are independently selected from H, CH 2 NH 2 , SO 3 " , phosphate, phosphonate, quaternary ammonium, NO 2 , (CH 2 ) q P, (CH 2 ) q COOH, CH 2 NH- COR 7 where R 7 is C]-C 2 o straight or branched alkyl or -(CH 2 ) m -COOH where q is an integer from 0-10;
  • R 3 is H or -L-P; where L is selected from C ⁇ -C 2 o straight or branched alkyl optionally containing 0, 1 or 2 unsaturated groups selected from alkenyl, alkynyl and
  • the reactive groups may attach the dye to a component to be labeled.
  • Any of R 1 , R 2 and R 4 may independently include a spacer or linker, L, as described above, and/or may include a target bonding group or reacting group or reactive moiety, as described herein, such as succinimidyl ester, isothiocyanate, anhydride, haloacetamide, maleimide, sulphonyl halide, phosphoramidite, acid halide, alkylimidate, hydrazide, and carbodiimide; and groups reactive with amino hydroxyl, aldehyde, phosphoryl, or sulphydryl groups.
  • At least one of P (where R is -(CH 2 ) m -P), R 1 , R 2 , and R 4 groups on each molecule may also be a group that increases the solubility of the chromophore or affects the selectivity of labeling of the labeled component or affects the position of labeling of the labeled component by the dye. More than one of each of R 1 , R", R 5 and R 6 may be contained on any ring and each substituent may be the same or different.
  • At least one of R 1 , R 2 or P contains a sulfonate.
  • At least one of R and R is at least one sulfonate group; where sulfonate includes sulfonic acid as the sulfonate group is merely an ionized sulfonic acid.
  • R or R may be reactive moieties, such as primary amines, for example, NHR 1 , where R 10 is C1-C 20 straight or branched alkyl.
  • Reactive groups that may be attached directly or indirectly to the chromophore to form any of P (where R 3 is -(CH2) m -P), R 1 , R 2 , and R 4 groups which may include reactive moieties such as, for example, groups containing isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di- halogen, substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxy succinimide ester, hydroxy sulfosuccinimide ester, imido ester, alkylimidate, hydrazide carbodiimide hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)- propri on amide, glyoxal and aldehyde.
  • R 3 is -(CH2)
  • the target bonding group, reactive group or reactive moiety such as may be any of R 1 , R 2 and R 4 , as described herein, may be a group reactive with an amine, hydroxy and/or sulfhydryl groups which are, for example, covalently attached to proteins, nucleic acids, carbohydrates, sugars, cells and combinations thereof, and other biological and non-biological materials, to make these materials fluorescent and detectable, as described in U.S. Patent No. 6,048,982.
  • the dyes of the present invention may be used therefore, to label, for example, avidin, antibodies, DNA, RNA or lectins to detect, measure and/or quantify, for example, biotinylated materials, antigens, haptens, carbohydrate groups, DNA, RNA and complimentary DNA or RNA, such as described therein.
  • P, R 1 , R 2 , and R 4 groups that are especially useful for labeling components with available amino, hydroxyl and sulfhydryl group include:
  • Q or W is a leaving group such as I, Br, Cl.
  • R 3 is -(CH ) m -P
  • R 1 , R 2 , and R 4 groups that are especially useful for labeling components with available sulfhydryls which can be used for labeling antibodies in a two step process include:
  • n is 0 or an integer from 1-8.
  • R 3 is -(CH 2 ) m -P
  • R 1 , R 2 , and R 4 groups that are especially useful for labeling components by light-activated cross linking
  • the P (where R 3 is -(CH 2 ) m -P), R 1 , R 2 , and R 4 groups can be selected from well known and electrically charged chemical groups.
  • R 3 is -(CH 2 ) m -P
  • R 1 , R 2 , and R 4 groups can be selected from well known and electrically charged chemical groups. Examples are E-F where F is hydroxyl, phosphate, phosphonate, sulfonate, sulfate, carboxylate, substituted amino or quaternary amino and where E is a spacer group such as -(CH 2 ) t - where t is 0, 1, 2, 3, 4, or 5.
  • Useful examples include lower alkyl sulfonate, for example, — ⁇ CH 2 ) 4 -SO 3 ' .
  • the present invention provides pH sensitive dyes of the following formulas (JHa) and (ECTb)
  • the quaternary nitrogen is removed and are also included in the present invention.
  • R is a straight or branched Ci to C 20 alkyl.
  • the present investigation provides pH sensitn e dyes of the following formulas (EVa) and (EVb)
  • nprotonated forms of Formulas are those wherein hydrogen on quaternary nitrogen is removed and are included as embodiments of the present invention.
  • R is straight or branched C1-C20 alkyl.
  • the invention provides acetoxymethyl ester and acetate esters derivatives of formulas (I), (la), (II), (HI), (THa), (JTfb), (IV), (EVa) and (IVb).
  • esters are permeant to membranes, such as biological membranes, and are hydrolyzed by esterases, such as are found in cells.
  • the compounds of the present invention may not require derivatization or modification to be cell permeable.
  • One of ordinary skill will appreciate the variability of the cell permeability of the compounds of the present invention and be able to routinely test for the same.
  • the polymethine chain of the dyes of the present invention may also contain one or more cyclic chemical groups that form bridges between two or more of the carbon atoms of the polymethine chain. These bridges might serve to increase the chemical or photostability of the dye and might be used to alter the absorption and emission wavelength of the dye or change its extinction coefficient or quantum yield. Improved solubility properties in aqueous environments may be obtained by this modification.
  • the present invention provides a component- labeled complex wherein the label is a compound of formulas (I), (IE), (HI) and (TV) and the component is an antibody, protein, peptide, enzyme substrate, hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin, avidin, streptavidin, toxin, carbohydrate, oligosaccharide, polysaccharide, nucleic acid, derivatized deoxy nucleic acid, DNA fragment, RNA fragment, derivatized DNA fragment, derivatized RNA fragment, natural drug, synthetic drug, virus particle, bacterial particle, virus component, yeast component, blocd cell, blood cell component, plasma component, serum component, biological cell, noncellular blood component, bacteria, bacterial component, natural or synthetic lipid vesicle, poison, environmental pollutant, polymer, polymer particle, glass particle, glass surface, plastic particle, plastic surface, polymer membrane, conductor or semiconductor.
  • the component is an antibody, protein, peptide, enzyme substrate,
  • a cyanine dye or its derivative of the present invention preferably absorbs maximally at a pH of 3-10, more preferably, at a pH of 3-6, in the range of 400- 800 nm, alternatively, in the ranges of 520-580 nm, or 650-670 nm, preferably in the range of 530-550 nm, more preferably in the range of 540-550 nm, most preferably about 548 nm with an isosbestic point in the range of 495-500 nm, preferably about 498 nm.
  • a cyanine dye or its derivative of the present invention fluoresces maximally in acidic conditions (pH of 4-7, preferably a pH of 4-5, most preferably a pH of 4), however, some compounds of the present invention fluoresce under basic conditions, such as at a pH in the range of 7.2-10, at about 560-580 nm, preferably 560-565, most preferably at 564 nm; when excited by a wavelength of 505-520 nm, preferably 510-515; most preferably 514 nm.
  • acidic conditions pH of 4-7, preferably a pH of 4-5, most preferably a pH of 4
  • some compounds of the present invention fluoresce under basic conditions, such as at a pH in the range of 7.2-10, at about 560-580 nm, preferably 560-565, most preferably at 564 nm; when excited by a wavelength of 505-520 nm, preferably 510-515; most preferably 514 nm.
  • Some dyes or derivatives of the present invention may preferably be maximally excited with 514 nm spectral line of an argon-ion laser. Dyes may be designed, however, within the scope of the present invention which may be excited and emit fluorescence over a range of wavelengths.
  • the extinction coefficient of dyes or derivatives of the present invention is preferably greater than about 130,000 cm “1 M "1 .
  • the fluorescence of the dyes and derivatives of the present invention are preferably sensitive to pH between pH 4 and pH 10 and are water soluble.
  • the present invention provides a fluorescent method of qualitative and/or quantitative detection of pH.
  • Such methods include contacting or mixing at least one dye of the present invention with a composition of cells, tissues or biological fluid wherein the presence of fluoresence indicates an acidic or, possibly basic, environment.
  • the present invention present invention provides a method of detecting intracellular acid environments, such as may be contained in subcellular compartments, or organells or structures. The compounds or derivatives of the present invention may be actively or passively adsorbed or absorbed into cells, where they may be detected by fluorescence detection.
  • the present invention provides a method of fluorescent detection wherein the dye or derivative of the present invention is detected in acidic conditions, such as in a differential, or when multiple fluorescent dyes are used, a dye or derivative of the present invention may be used in conjunction with dyes which do not either fluoresce in acidic conditions and/or at the wavelengths of the presently provided dyes or derivatives.
  • the present invention also provides a pH sensitive, concentration independent, ratiometric measurement method wherein at least a pair of fluorescent dyes are linked in an energy- transfer arrangement, preferably through a linker, L, with optional target bonding groups contained thereon, as described herein. At least one of the dyes of such a conjugate is one of the pH sensitive dyes of the present invention.
  • the pH sensitive dye of the present invention is the energy donor
  • the ratiometric measurement method of the present invention involves determining the ratio of fluorescence intensity of t e acceptor emission after excitation of each of the donor and acceptor. When the pH sensitive dye of the present invention is the energy acceptor, then the ratio of the maximum fluorescence intensity of both the donor and acceptor is determined from excitation at the maximum absorption of the donor species.
  • the methods according to the present invention may employ known devices for illumination and detection at separate defined wavelengths, such as first and second wavelengths, respectively.
  • Such devices include, but are not limited to, fluorescence spectrometers, absorption spectrophotometers, fluorescence microscopes, transmission light microscopes, flow cytometers, fiberoptic sensors, immunoassay instruments and video fluorescent imaging devices.
  • Methods of the present invention include those described n the "Handbook of Fluorescent Probes and Research Chemicals" published by Molecular Probes, Inc. And “Fluorescent Probes in Cellular and Molecular Biology” by Slavik (1994) CRC Press, Inc., especially methods where measurement of acidic conditions are preferred, wherein the dye and/or derivative of the present invention is employed.
  • the method of this invention can also employ chemical analysis methods to detect attachment of the dye or derivative of the present invention to the labeled component or components.
  • Chemical analysis methods can include infrared spectrometry, NMR spectrometry, absorption spectrometry, fluorescence spectrometry, mass spectrometry and chromatographic methods.
  • the dyes and derivatives of the present invention may be made by methods well known in the art, such as by methods described in U.S. Patent No. 5,486,268, U.S. Patent No. 5,486,616, and U.S. Patent No, 4,981,977, the entire contents of which are inco ⁇ orated wherein by reference.
  • the potassium salt of sulfoindolenine was dissolved in water and passed through a column of Dowex, strongly acidic hydrogen ion exchange resin. Potassium free sulfoindolenine was obtained as brown powder. This is essential to ensure that the sulfoindolenine is an internal salt and the nitrogen is protonated.
  • N,N'diphenylformamidine (3.3 g, 0.0 17 mol) in acetic acid (20 mL) was heated to reflux.
  • the completion of the reaction was monitored by abso ⁇ tion spectra in methanol which showed that as the reaction progressed the abso ⁇ tion maximum at 286 nm declined almost to the vanishing point and the abso ⁇ tion at 415 nm rose correspondingly (about 3-4 h). Extended heating produced some symmetrical dye (>5%).
  • the unconjugated dye was separated from the protein by gel permeation chromatography over Sephadex G-50 (0.7 X 20cm column) using pH 7 buffer solution as eluent.
  • the dye was obtained by heating sulfoindolenine (I) with ethyl orthoformate in acetic acid for 2 hr. Acetic acid was removed on a rotary evaporator under reduced pressure and the residue was triturated with anhydrous ethyl ether to obtained red solid, which was purified by C18 reversed phase flash column chromatography (yield 25%).
  • the dye and its succinimidyl ester were prepared according to the procedure described for Cy3.39.OH.
  • the dye is water insoluble. Its water soluble dextran conjugate was prepared as described below.
  • aminodextran Mr 40,000, approx. 7.2 amino groups per dextran molecule
  • 0.02 M carbonate buffer 0.02 M carbonate buffer
  • active ester 2 mg
  • the dye-dextran conjugate was separated from nonconjugated dye by sephadex G-50 gel permeation chromatography using ammonium acetate (50mM) as elution buffer. An average of 2.2 dye molecules was covalently linked to each dextran molecule.
  • the reaction was monitored by UV until the absorbance of the anil intermediate (454nm) was no longer observed.
  • the solution was cooled and the solvent removed in vacuo.
  • the column retention time was 33 minutes (UV at 650nm).
  • 5-Sulfo-2,3,3-trimethylindolenine (potassium salt) (l lg, 0.04mol) was suspended in ethyl iodide (40ml) and the reaction refluxed for 24 hours. The mixture was cooled and the excess ethyl iodide was decanted. The light pu ⁇ le solid was triturated with acetone (3 x 50ml) to remove potassium iodide. The powder was dried.
  • Example 7 3-(5-Carboxypentyl)-2-nE.3E)-5-(3.3-dimethyl-5-sulfo-1.3- dihydro-2H-indol-2-ylidene)-l,3-pentadienyl1-l,3-benzothiazol-3- ium (Compound 7)
  • Example 8 5-(Aminomethyl)-2-[lE,3E)-5-(3,3-dimethyl-5-sulfo-1.3-dihvdro- 2H-indol-2-ylidene)-l,3-pentadienyl1-l,3,3-trimethyl-3H-indolium
  • Example 9 1 -(5-Carboxy ⁇ entyl)-2-r lE.3E)-5-(l .1 -dimethv1-6-8-disuldo- 1 ,3- dihvdro-2H-benzo[elindol-2-ylidene)-1.3-pentadienyll-3.3- dimethyl-5-sulfo-3H-indolium (Compound 9)
  • 6-Hydrazinonaphthalene 1,3-disulfonate (6.2g, 0.02mol), methyl isopropyl ketone (6.4ml O.O ⁇ mol), potassium acetate (5.88g, O.O ⁇ mol) and glacial acetic acid (35ml) were refluxed at 135°C for 24hr. The acetic acid was removed in vacuo and the solid stirred with 2-propanol to yield a buff powder.
  • l,l,2-Trimethyl-6-8-disulfo-benzo[e]indole (20mg, 0.054mmol), l-(5- Carboxypentyl)-5-sulfo-2,3,3-trimethylindole (19mg, 0.054mmol) and malonaldehyde bis(phenylimine) (15mg, O.O ⁇ mmol) were stirred in a 1: 1:0.2 mixture of acetic acid, pyridine and acetic anhydride at 70°C for 4 hours to yield a dark blue solution. This solution was cooled. The dye was purified by reverse phase analytical (lml/min. flow) HPLC.
  • Example 10 2- ⁇ E,3E)-5-(3.3-Dimethyl-5-sulfo-l,3-dihydro-2H-indol-2- ylidene)-1.3-pentadienyl1-l-(6-hvdrazino-6-oxohexyl)-3.3- dimethyl-5-suIfo-3H-indolium ( " Compound 10)
  • Example 11 pH Sensitive Cy3-Cy5 Energy Transfer Cassette (Compound 11).
  • Cy5 was prepared according to the method described in Bioconjugate Chemistry, Volume 4, Number 2, page 105, (1993). l- ⁇ thyl-2,3,3-trimethyl-3H- indolium-5-sulfonate (4g, 0.015mol) and malonaldehyde bis (phenylimine) monohydrochloride (4.4g, 0.017mol) were refluxed in acetic acid (20ml) and acetic anhydride (20ml) for 4 hours. Pyridine (20ml) and l-(5-carboxypentyl- 1,3,3-trimethyl indoleninium-5-sulphonate (5.3g, 0.015mol) were then added to the solution and the reaction was heated for a further 3 hours.
  • N-diisopropylethylamine 40ml
  • O,-N-succinimidyl-N,N,N',N'- bis(tetramethylene)-uronium hexafluorophosphate 74mg, 0.18mmol
  • the reaction mixture was then quenched with diethyl ether, filtered and triturated with ethyl acetate to yield the product as a fine blue powder.
  • Example 12 Fluorescent Characteristics of pH Sensitive Cyanine Dyes.
  • the pKa was measured as the ratio of emission intensity at the pH measured and the emission intensity for maximum emission, or I/L,. This is illustrated in Figure 5 for Example 7 using a sigmoidal curve fit.
  • Example 13 Fluorescent Properties of a pH Sensitive Energy Transfer Probe
  • CHOM1 cells were seeded onto 3cm glass bottomed tissue culture coated dishes at approximately 50% confluency.
  • the cells were grown in F12 Ham's media containing 10% foetal calf serum and 2mM glutamine. The cells were then incubated for 20mins on ice, washed twice in serum free F12 and then incubated with O.lmg/ml of Compound 4 N-hydroxysuccinimidyl ester for 1.5 hours at 4°C.
  • the cells were then washed with serum free media and analysed immediately using a Zeiss LSM410 confocal microscope x 63 oil objective, 633nm excitation and 665nm emission (before internalisation).
  • the cells were then stimulated to internalise by constitutive endocytosis, by incubating them at 37°C for 1 hour. They were then analysed using identical confocal microscopy conditions (after internalisation).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Inorganic Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Indole Compounds (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

L'invention concerne des colorants à base de cyanine sensibles au pH ainsi que des procédés d'utilisation de ces colorants comme réactifs fluorescents.
PCT/US2000/015682 1999-06-09 2000-06-08 Colorants a base de cyanine sensibles au ph utilises comme reactifs fluorescents WO2000075237A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU57284/00A AU5728400A (en) 1999-06-09 2000-06-08 Ph sensitive cyanine dyes as reactive fluorescent reagents
CA002375740A CA2375740A1 (fr) 1999-06-09 2000-06-08 Colorants a base de cyanine sensibles au ph utilises comme reactifs fluorescents
EP00942696A EP1212375A2 (fr) 1999-06-09 2000-06-08 Colorants a base de cyanine sensibles au ph utilises comme reactifs fluorescents
JP2001502512A JP2003501540A (ja) 1999-06-09 2000-06-08 反応性蛍光試薬としてのｐＨ官能性シアニン染料

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13829799P 1999-06-09 1999-06-09
US60/138,297 1999-06-09

Publications (3)

Publication Number Publication Date
WO2000075237A2 true WO2000075237A2 (fr) 2000-12-14
WO2000075237A3 WO2000075237A3 (fr) 2002-04-11
WO2000075237A9 WO2000075237A9 (fr) 2002-05-10

Family

ID=22481396

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/015682 WO2000075237A2 (fr) 1999-06-09 2000-06-08 Colorants a base de cyanine sensibles au ph utilises comme reactifs fluorescents

Country Status (5)

Country Link
EP (1) EP1212375A2 (fr)
JP (1) JP2003501540A (fr)
AU (1) AU5728400A (fr)
CA (1) CA2375740A1 (fr)
WO (1) WO2000075237A2 (fr)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6348596B1 (en) 1998-01-23 2002-02-19 Pe Corporation (Ny) Non-fluorescent asymmetric cyanine dye compounds useful for quenching reporter dyes
EP1262536A2 (fr) * 2001-05-31 2002-12-04 Nisshinbo Industries, Inc. Composé de carbodiimide contenant un groupe fluorescent et procédé de fabrication du composé
WO2005014723A1 (fr) 2003-08-12 2005-02-17 Giuseppe Caputo Composes de type cyanine avec bras alkynyle de liaison
JP2005536623A (ja) * 2002-05-10 2005-12-02 カーネギー・メロン・ユニヴァーシティ 官能基を含有するキラルインドール中間体およびそれらの蛍光シアニン染料
WO2007059779A2 (fr) * 2005-11-28 2007-05-31 Dako Denmark A/S Nouveaux composés colorants
WO2007076240A2 (fr) * 2005-12-16 2007-07-05 General Electric Company Compositions et procedes de dosage d'absorption de lipoproteines
CN100349869C (zh) * 2004-12-10 2007-11-21 华东理工大学 含端炔基的菁染料及其合成方法
US8051947B2 (en) 2009-03-12 2011-11-08 E.I. Du Pont De Nemours And Company Energy absorbing thermoplastic elastomer
US8465985B2 (en) 2007-03-01 2013-06-18 The University Of Tokyo Fluorescent probe
US8507677B2 (en) 2007-02-16 2013-08-13 The University Of Tokyo Fluorescent probe
US8614302B2 (en) * 2000-09-29 2013-12-24 Life Technologies Corporation Modified carbocyanine dyes and their conjugates
US8889884B1 (en) 2011-07-14 2014-11-18 Pierce Biotechnology, Inc. Phosphine derivatives of fluorescent compounds
US8999653B2 (en) 2008-07-31 2015-04-07 Cis-Bio International Method for detecting membrane protein internalization
CN104610773A (zh) * 2014-02-08 2015-05-13 南京博炫生物科技有限公司 一种荧光染料化合物及其制备方法和应用
US9063127B1 (en) 2013-12-11 2015-06-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells
US9249307B2 (en) 2011-08-16 2016-02-02 Pierce Biotechnology, Inc. Benzocyanine compounds
US9676787B2 (en) 2012-08-28 2017-06-13 Pierce Biotechnology, Inc. Benzopyrylium compounds
US9751868B2 (en) 2012-02-28 2017-09-05 Pierce Biotechnology, Inc. Benzocyanine compounds
US10000467B2 (en) 2012-03-02 2018-06-19 Pierce Biotechnology, Inc. Cyanine compounds
US10351551B2 (en) 2010-12-21 2019-07-16 Pierce Biotechnology, Inc. Fluorescent compounds
US10557851B2 (en) 2012-03-27 2020-02-11 Ventana Medical Systems, Inc. Signaling conjugates and methods of use
US10928405B2 (en) 2013-12-11 2021-02-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells
WO2022129336A1 (fr) 2020-12-17 2022-06-23 Bracco Imaging Spa Colorants de cyanine sensibles au ph et leurs conjugués

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009107769A1 (fr) * 2008-02-29 2009-09-03 国立大学法人東京大学 Réactif pour la mesure d'oxygène réactif
JP2010184866A (ja) * 2009-02-10 2010-08-26 Nagoya Univ 腸管粘膜蛍光観察用検査試薬
EP3333574A4 (fr) * 2015-08-05 2018-06-20 Konica Minolta, Inc. Nanoparticules à colorant accumulé sensibles à l'environnement, et procédé d'analyse d'environnement intracellulaire
JP7423888B2 (ja) * 2019-11-22 2024-01-30 株式会社同仁化学研究所 脂質二分子膜染色用色素及びそれを用いた脂質二分子膜の染色方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4435492A (en) * 1978-10-27 1984-03-06 Hitachi, Ltd. Complex type electrophotographic plate and electrophotographic method using the same
US4524128A (en) * 1983-10-24 1985-06-18 Eastman Kodak Company Spectrally sensitized silver halide photothermographic material
US5268486A (en) * 1986-04-18 1993-12-07 Carnegie-Mellon Unversity Method for labeling and detecting materials employing arylsulfonate cyanine dyes
DE4341618A1 (de) * 1993-12-07 1995-06-08 Univ Schiller Jena Proton-Carrier-Farbstoffe für den Nahen Infrarot-Spektralbereich
EP0747700A2 (fr) * 1995-06-07 1996-12-11 Carnegie-Mellon University Complexe avec un grand décalage de stokes pour marquage fluorescent formé par couplage de cyanine et autres fluorochromes capables de transfert d'énergie résonance

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6048982A (en) * 1986-04-18 2000-04-11 Carnegie Mellon University Cyanine dyes as labeling reagents for detection of biological and other materials by luminescence methods

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4435492A (en) * 1978-10-27 1984-03-06 Hitachi, Ltd. Complex type electrophotographic plate and electrophotographic method using the same
US4524128A (en) * 1983-10-24 1985-06-18 Eastman Kodak Company Spectrally sensitized silver halide photothermographic material
US5268486A (en) * 1986-04-18 1993-12-07 Carnegie-Mellon Unversity Method for labeling and detecting materials employing arylsulfonate cyanine dyes
DE4341618A1 (de) * 1993-12-07 1995-06-08 Univ Schiller Jena Proton-Carrier-Farbstoffe für den Nahen Infrarot-Spektralbereich
EP0747700A2 (fr) * 1995-06-07 1996-12-11 Carnegie-Mellon University Complexe avec un grand décalage de stokes pour marquage fluorescent formé par couplage de cyanine et autres fluorochromes capables de transfert d'énergie résonance

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MUJUMDAR R B ET AL: "CYANINE DYE LABELING REAGENTS CONTAINING ISOTHIOCYANATE GROUPS" CYTOMETRY,US,ALAN LISS, NEW YORK, vol. 10, no. 1, 1989, pages 11-19, XP000034880 ISSN: 0196-4763 cited in the application *
MUJUMDAR R B ET AL: "CYANINE DYE LABELING REAGENTS: SULFOINDOCYANINE SUCCINIMIDYL ESTERS" BIOCONJUGATE CHEMISTRY,US,AMERICAN CHEMICAL SOCIETY, WASHINGTON, vol. 4, no. 2, 1 March 1993 (1993-03-01), pages 105-111, XP000654181 ISSN: 1043-1802 cited in the application *

Cited By (45)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6348596B1 (en) 1998-01-23 2002-02-19 Pe Corporation (Ny) Non-fluorescent asymmetric cyanine dye compounds useful for quenching reporter dyes
AU2003301687B2 (en) * 1999-06-09 2010-05-13 Amersham Biosciences Uk Limited Chiral indole intermediates and their fluorescent cyanine dyes containing functional groups
US8614302B2 (en) * 2000-09-29 2013-12-24 Life Technologies Corporation Modified carbocyanine dyes and their conjugates
EP1439214A3 (fr) * 2001-05-31 2006-07-05 Nisshinbo Industries, Inc. Composé de carbodiimide contenant un groupe fluorescent et procédé de fabrication du composé
US6825195B2 (en) 2001-05-31 2004-11-30 Nisshinbo Industries, Inc. Fluorescent group-containing carbodiimide compound and process for producing the compound
EP1262536A3 (fr) * 2001-05-31 2003-03-26 Nisshinbo Industries, Inc. Composé de carbodiimide contenant un groupe fluorescent et procédé de fabrication du composé
EP1262536A2 (fr) * 2001-05-31 2002-12-04 Nisshinbo Industries, Inc. Composé de carbodiimide contenant un groupe fluorescent et procédé de fabrication du composé
JP2005536623A (ja) * 2002-05-10 2005-12-02 カーネギー・メロン・ユニヴァーシティ 官能基を含有するキラルインドール中間体およびそれらの蛍光シアニン染料
JP2010202876A (ja) * 2002-05-10 2010-09-16 Carnegie Mellon Univ 官能基を含有するキラルインドール中間体およびそれらの蛍光シアニン染料
US8044203B2 (en) 2002-05-10 2011-10-25 Carnegie Mellon University Chiral indole intermediates and their fluorescent cyanine dyes containing functional groups
JP4749718B2 (ja) * 2002-05-10 2011-08-17 カーネギー・メロン・ユニヴァーシティ 官能基を含有するキラルインドール中間体およびそれらの蛍光シアニン染料
US7615646B2 (en) 2002-05-10 2009-11-10 Carnegie Mellon University Chiral indole intermediates and their fluorescent cyanine dyes containing functional groups
WO2005014723A1 (fr) 2003-08-12 2005-02-17 Giuseppe Caputo Composes de type cyanine avec bras alkynyle de liaison
US7892297B2 (en) 2003-08-12 2011-02-22 Cyanine Technologies S.p.A. Cyanin-type compounds having an alkynyl linker arm
CN100349869C (zh) * 2004-12-10 2007-11-21 华东理工大学 含端炔基的菁染料及其合成方法
WO2007059779A3 (fr) * 2005-11-28 2008-06-19 Dako Denmark As Nouveaux composés colorants
US7972789B2 (en) 2005-11-28 2011-07-05 Dako Denmark A/S Dye compounds
WO2007059779A2 (fr) * 2005-11-28 2007-05-31 Dako Denmark A/S Nouveaux composés colorants
US7846735B2 (en) 2005-12-16 2010-12-07 General Electric Company Compositions and methods for lipoprotein uptake assays
WO2007076240A3 (fr) * 2005-12-16 2007-09-13 Gen Electric Compositions et procedes de dosage d'absorption de lipoproteines
WO2007076240A2 (fr) * 2005-12-16 2007-07-05 General Electric Company Compositions et procedes de dosage d'absorption de lipoproteines
US8507677B2 (en) 2007-02-16 2013-08-13 The University Of Tokyo Fluorescent probe
US8465985B2 (en) 2007-03-01 2013-06-18 The University Of Tokyo Fluorescent probe
US8999653B2 (en) 2008-07-31 2015-04-07 Cis-Bio International Method for detecting membrane protein internalization
US8051947B2 (en) 2009-03-12 2011-11-08 E.I. Du Pont De Nemours And Company Energy absorbing thermoplastic elastomer
US11053222B2 (en) 2010-12-21 2021-07-06 Pierce Biotechnology, Inc. Fluorescent compounds
US10351551B2 (en) 2010-12-21 2019-07-16 Pierce Biotechnology, Inc. Fluorescent compounds
US8889884B1 (en) 2011-07-14 2014-11-18 Pierce Biotechnology, Inc. Phosphine derivatives of fluorescent compounds
US9365598B2 (en) 2011-07-14 2016-06-14 Pierce Biotechnology, Inc. Phosphine derivatives of fluorescent compounds
US10125120B2 (en) 2011-08-16 2018-11-13 Pierce Biotechnology, Inc. Benzocyanine compounds
US10730857B2 (en) 2011-08-16 2020-08-04 Pierce Biotechnology, Inc. Benzocyanine compounds
US9249307B2 (en) 2011-08-16 2016-02-02 Pierce Biotechnology, Inc. Benzocyanine compounds
US10526317B2 (en) 2012-02-28 2020-01-07 Pierce Biotechnology, Inc. Benzocyanine compounds
US9751868B2 (en) 2012-02-28 2017-09-05 Pierce Biotechnology, Inc. Benzocyanine compounds
US10000467B2 (en) 2012-03-02 2018-06-19 Pierce Biotechnology, Inc. Cyanine compounds
US10696653B2 (en) 2012-03-02 2020-06-30 Pierce Biotechnology, Inc. Cyanine compounds
US10557851B2 (en) 2012-03-27 2020-02-11 Ventana Medical Systems, Inc. Signaling conjugates and methods of use
US11906523B2 (en) 2012-03-27 2024-02-20 Ventana Medical Systems, Inc. Signaling conjugates and methods of use
US10174045B2 (en) 2012-08-28 2019-01-08 Pierce Biotechnology, Inc. Benzopyrylium compounds
US9676787B2 (en) 2012-08-28 2017-06-13 Pierce Biotechnology, Inc. Benzopyrylium compounds
US9709556B2 (en) 2013-12-11 2017-07-18 Analiza, Inc. Devices and methods for determining and/or isolating circulating cancer cells
US9063127B1 (en) 2013-12-11 2015-06-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells
US10928405B2 (en) 2013-12-11 2021-02-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells
CN104610773A (zh) * 2014-02-08 2015-05-13 南京博炫生物科技有限公司 一种荧光染料化合物及其制备方法和应用
WO2022129336A1 (fr) 2020-12-17 2022-06-23 Bracco Imaging Spa Colorants de cyanine sensibles au ph et leurs conjugués

Also Published As

Publication number Publication date
WO2000075237A3 (fr) 2002-04-11
EP1212375A2 (fr) 2002-06-12
CA2375740A1 (fr) 2000-12-14
WO2000075237A9 (fr) 2002-05-10
JP2003501540A (ja) 2003-01-14
AU5728400A (en) 2000-12-28

Similar Documents

Publication Publication Date Title
EP1212375A2 (fr) Colorants a base de cyanine sensibles au ph utilises comme reactifs fluorescents
US5852191A (en) Rigidized monomethine cyanines
EP2596065B1 (fr) Colorants luminescents avec pont intramoléculaire hydrosoluble et leurs conjugués biologiques
US6545164B1 (en) Fluorochromes labelling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer
US5569766A (en) Method for labeling and detecting materials employing arylsulfonate cyanine dyes
US5627027A (en) Cyanine dyes as labeling reagents for detection of biological and other materials by luminescence methods
US5569587A (en) Method for labeling and detecting materials employing luminescent arysulfonate cyanine dyes
US6004536A (en) Lipophilic cyanine dyes with enchanced aqueous solubilty
CA2314188C (fr) Colorants de trimethine cyanine rigidifiee
US7767829B2 (en) Water-soluble fluoro-substituted cyanine dyes as reactive fluorescence labelling reagents
JP2001524969A (ja) 複合糖質蛍光標識試薬
US10316136B2 (en) Polyfluoreno[4,5-cde]oxepine conjugates and their use in methods of analyte detection
US5986093A (en) Monomethine cyanines rigidized by a two-carbon chain
EP0747448B1 (fr) Colorants cyanines monométhiniques rigidifiés
EP1394219A1 (fr) Colorants à base de cyanine sensibles au pH utilisés comme réactifs fluorescents
EP1810998B1 (fr) Colorant Cyanine Fluorescent
US20030211454A1 (en) Detection reagent

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK 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 MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2000942696

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 57284/00

Country of ref document: AU

ENP Entry into the national phase in:

Ref country code: CA

Ref document number: 2375740

Kind code of ref document: A

Format of ref document f/p: F

Ref country code: JP

Ref document number: 2001 502512

Kind code of ref document: A

Format of ref document f/p: F

Ref document number: 2375740

Country of ref document: CA

AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK 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 MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

COP Corrected version of pamphlet

Free format text: PAGES 1/8-8/8, DRAWINGS, REPLACED BY NEW PAGES 1/7-7/7; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

WWP Wipo information: published in national office

Ref document number: 2000942696

Country of ref document: EP

WWR Wipo information: refused in national office

Ref document number: 2000942696

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2000942696

Country of ref document: EP