Molecular Probes, Inc.

Life Technologies Corporation

Yu-Zhong Zhang

Richard P Haugland

John Joseph Naleway

Stephen T Yue

Bruce Branchaud

Lai-Qiang Ying

Nels A Olson

Zhijian Huang

Karen D Larison

Zhenjun Diwu

Hee Chol Kang

Ewald Alois Terpetschnig

Dieter H Klaubert

Yexin Wu

Ram Sabnis

Richard P Haughland

Mohammad N Malekzadeh

Courtenay R Kemper

Biotin derivatives, methods of using the biotin derivatives and kits comprising the biotin derivatives.

Biotin derivatives

Lipophilic fluorescent substances can be used to detect surface defects in materials having hydrophilic (e.g., inorganic) coatings. Use of the described methods makes surface defects appear fluorescent, while the remaining surfaces are not labeled. The disclosed methods are inexpensive, rapid, and easy alternatives to existing approaches.

Methods for detecting defects in inorganic-coated polymer surfaces

The invention relates to fluorescent dyes that are substituted or unsubstituted derivatives of 1-(isoindolyl)methylene-isoindole that are bound through both isoindole nitrogens to a boron difluoride moiety, forming a fluorescent dibenzopyrrometheneboron difluoride compound ##STR1## that is further substituted by bathochromic substituents that are aryl or heteroaryl moieties further substituted by an additional aryl or heteroaryl, that is itself optionally further substituted by an additional aryl or heteroaryl. These aryl and heteroaryl groups are separated by a covalent bond, or by an ethenyl, butadienyl or hexatrienyl linkage. The dyes of the invention are particularly useful as labels for carriers, particularly polymeric microparticles. The resulting microparticles have a long-wavelength fluorescence emission, and possess utility for tracing flow in biological systems, particularly in tracing blood flow.

Long wavelength dyes for infrared tracing

Dipyrrometheneboron difluoride dyes possessing a covalently attached basic amine moiety are described that have utility for staining acidic organelles. Samples comprising isolated acidic organelles, or a cell or cells containing acidic organelles, are stained by preparing an aqueous labeling solution of the dye and incubating the sample in the labeling solution for a time sufficient to produce fluorescent labeled acidic organelles.

Stains for acidic organelles

The invention describes novel fluorescently labeled microspheres, where the microspheres possess at least one internal fluorescent spherical zone. The invention also describes the method of preparing the novel microspheres, the method of calibrating microscopy instrumentation using the novel microspheres, the method of using the novel microspheres as distinct labels for combinatorial analysis and the use of the labeled microspheres as tagging agents and tracers.

Microspheres with fluorescent spherical zones

The present invention describes a method of staining mitochondria, and analyzing mitochondrial function, using a class of fluorescent substituted 3',6'-diaminoxanthenes and their reduced analogs 3',6'-diaminodihydroxanthenes, which are oxidized to the fluorescent form of the dye in situ. In their oxidized form, the dyes selectively localize within mitochondria. The dyes of the invention are substituted by an alkylating group that allows their retention in mitochondria even after cell death, fixation, and permeabilization.

Xanthylium dyes that are well retained in mitochondria

The dyes of the present invention are fluorescent substituted 3',6'-diaminoxanthenes and their reduced analogs 3',6'-diaminodihydroxanthenes, which are oxidized to the fluorescent form of the dye in situ. In their oxidized form, the dyes selectively localize within mitochondria. The dyes of the present invention include an alkylating group that is covalently attached that allows their retention in mitochondria even after cell death, fixation, and permeabilization.

Activity of enzymes and enzyme conjugates is detected using novel substrates made from a class of fluorophores, generally including quinazolinones (quinazolones), benzimidazoles, benzothiazoles, benzoxazoles, quinolines, indolines, and phenanthridines, having the general formula: ##STR1## where carbon atoms of --C.sup.1 .dbd.C.sup.2 -- are joined to complete a first 5- or 6-membered aromatic ring which optionally contains one of the hetero atoms N, O or S, and carbon atoms of --C.sup.4 --N.dbd.C.sup.3 -- are joined to complete a second 5- or 6-membered aromatic ring that contains a nitrogen between C.sup.3 and C.sup.4 and optionally contains an additional hetero atom N, O or S, where the first and second aromatic rings may be joined by a 5- or 6-membered bridging ring that contains at least the C.sup.2 from the first aromatic ring and the C.sup.3 from the second aromatic ring, where each of the first and second aromatic rings may be fused to at least one additional aromatic ring that may contain at least one of the hetero atoms N, O or S, and where each of said aromatic rings may be further modified by substitution of any hydrogens on an aromatic carbon by substituents that are halogen, nitro, cyano, aryl, lower alkyl (1-4 carbons), perfluoroalkyl (1-4 carbons), or alkoxy (1-4 carbons), or any combination thereof; and the fluorophore is covalently linked to a blocking group through an oxygen --O-- at C.sub.1 ; such that removal of the blocking group by enzyme activity yields a precipitate.

Enzymatic analysis using substrates that yield fluorescent precipitates

The subject invention provides a method for analyzing the metabolic activity in cells by improving the retention of a detectable reporter molecule only in intact cells where a particular enzyme is present. In particular, improved retention results from a two part process involving conjugation of haloalkyl-substituted derivatives of a reporter molecule with intracellular cysteine-containing peptides while unblocking the reporter molecule. The method for analyzing metabolic activity of cells involves the use of a substrate having the form

Fluorescent haloalkyl derivatives of reporter molecules well retained in

Novel fluorescent precipitating substrates made from a class of fluorophores, generally including quinazolinones (quinazolones), benzimidazoles, benzothiazoles, benzoxazoles, quinolines, indolines, and phenanthridines, having the general formula: ##STR1## where carbon atoms of --C.sup.1 .dbd.C.sup.2 -- are further joined so as to complete a first 5- or 6-membered aromatic ring which may contain at least one of the hetero atoms N, O or S,

The claimed invention relates to a substrate for evaluating glycosidic enzymes comprising a resorufin derivative of the general formula: ##STR1## wherein Gly is a carbohydrate bonded to resorufin by a glycosidic linkage; where at least one of substituents R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.8, and R.sub.9 is a lipophilic residue of the formula --L(CH.sub.2).sub.n CH.sub.3, where n is greater than 3 and less than 22, and where L is a methylene --CH.sub.2 --, an amide --NHCO--, a sulfonamide --NHSO.sub.2 --, a carboxamide --CONH--, a carboxylate ester --COO--, a urethane --NHCOO--, a urea --NHCONH--, or a thiourea --NHCSNH--; and

Long wavelength lipophilic fluorogenic glycosidase substrates

The claimed invention relates to a substrate for evaluating glycosidic enzymes comprising a fluorescein derivative of the general formula: ##STR1## wherein GlyX is a carbohydrate bonded to fluorescein by a glycosidic linkage;

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Eugene, OR, United States of America

Yu-Zhong Zhang