The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Martin A Philbert - One of the best experts on this subject based on the ideXlab platform.
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a real time ratiometric method for the determination of molecular oxygen inside living cells using sol gel based spherical optical nanosensors with applications to rat c6 glioma
Analytical Chemistry, 2001Co-Authors: Hao Xu, Raoul Kopelman, Jonathan W Aylott, Terry J Miller, Martin A PhilbertAbstract:The first sol−gel-based, ratiometric, optical nanosensors, or sol−gel probes encapsulated by biologically localized embedding (PEBBLEs), are made and demonstrated here to enable reliable, real-time measurements of subcellular molecular oxygen. Sensors were made using a modified Stober method, with poly(ethylene glycol) as a steric stabilizer. The radii of these spherical PEBBLE sensors range from about 50 to 300 nm. These sensors incorporate an oxygen-sensitive Fluorescent Indicator, Ru(II)−tris(4,7-diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+), and an oxygen-insensitive Fluorescent dye, Oregon Green 488-dextran, as a reference for the purpose of ratiometric intensity measurements. The PEBBLE sensors have excellent reversibility, dynamic range, and stability to leaching and photobleaching. The small size and inert matrix of these sensors allow them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. Applications of sol−gel PEBBLEs insert...
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optical nanosensors for chemical analysis inside single living cells 2 sensors for ph and calcium and the intracellular application of pebble sensors
Analytical Chemistry, 1999Co-Authors: Heather A Clark, Raoul Kopelman, Ron Tjalkens, Martin A PhilbertAbstract:Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective Fluorescent Indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of Fluorescent Indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB. Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.
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optical nanosensors for chemical analysis inside single living cells 2 sensors for ph and calcium and the intracellular application of pebble sensors
Analytical Chemistry, 1999Co-Authors: Heather A Clark, Raoul Kopelman, Ron Tjalkens, Martin A PhilbertAbstract:Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective Fluorescent Indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of Fluorescent Indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Spec...
Alan S. Verkman - One of the best experts on this subject based on the ideXlab platform.
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k waves in brain cortex visualized using a long wavelength k sensing Fluorescent Indicator
Nature Methods, 2005Co-Authors: Prashant Padmawar, Orin Bloch, Geoffrey T Manley, Alan S. VerkmanAbstract:K + waves in brain cortex visualized using a long-wavelength K + -sensing Fluorescent Indicator
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k waves in brain cortex visualized using a long wavelength k sensing Fluorescent Indicator
Nature Methods, 2005Co-Authors: Prashant Padmawar, Orin Bloch, Geoffrey T Manley, Xiaoming Yao, Alan S. VerkmanAbstract:We synthesized a water-soluble, long-wavelength K(+) sensor, TAC-Red, consisting of triazacryptand coupled to 3,6-bis(dimethylamino)xanthylium, whose fluorescence increased 14-fold at 0-50 mM K(+) with K(+)-to-Na(+) selectivity >30. We visualized K(+) waves in TAC-Red-stained brain cortex in mice during spreading depression, with velocity 4.4 +/- 0.5 mm/min, and K(+) release and reuptake half-times (t(1/2)) of 12 +/- 2 and 32 +/- 4 s, respectively. Aquaporin-4 (AQP4) deletion slowed K(+) reuptake about twofold, suggesting AQP4-dependent K(+) uptake by astroglia.
Raoul Kopelman - One of the best experts on this subject based on the ideXlab platform.
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a real time ratiometric method for the determination of molecular oxygen inside living cells using sol gel based spherical optical nanosensors with applications to rat c6 glioma
Analytical Chemistry, 2001Co-Authors: Hao Xu, Raoul Kopelman, Jonathan W Aylott, Terry J Miller, Martin A PhilbertAbstract:The first sol−gel-based, ratiometric, optical nanosensors, or sol−gel probes encapsulated by biologically localized embedding (PEBBLEs), are made and demonstrated here to enable reliable, real-time measurements of subcellular molecular oxygen. Sensors were made using a modified Stober method, with poly(ethylene glycol) as a steric stabilizer. The radii of these spherical PEBBLE sensors range from about 50 to 300 nm. These sensors incorporate an oxygen-sensitive Fluorescent Indicator, Ru(II)−tris(4,7-diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+), and an oxygen-insensitive Fluorescent dye, Oregon Green 488-dextran, as a reference for the purpose of ratiometric intensity measurements. The PEBBLE sensors have excellent reversibility, dynamic range, and stability to leaching and photobleaching. The small size and inert matrix of these sensors allow them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. Applications of sol−gel PEBBLEs insert...
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optical nanosensors for chemical analysis inside single living cells 2 sensors for ph and calcium and the intracellular application of pebble sensors
Analytical Chemistry, 1999Co-Authors: Heather A Clark, Raoul Kopelman, Ron Tjalkens, Martin A PhilbertAbstract:Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective Fluorescent Indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of Fluorescent Indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB. Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.
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optical nanosensors for chemical analysis inside single living cells 2 sensors for ph and calcium and the intracellular application of pebble sensors
Analytical Chemistry, 1999Co-Authors: Heather A Clark, Raoul Kopelman, Ron Tjalkens, Martin A PhilbertAbstract:Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective Fluorescent Indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of Fluorescent Indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Spec...
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Fluorescent fiber optic calcium sensor for physiological measurements
Analytical Chemistry, 1996Co-Authors: Michael R Shortreed, Raoul Kopelman, Michael A Kuhn, Brian HoylandAbstract:A new optical sensor based on covalent immobilization of a newly synthesized calcium-selective, long-wavelength, Fluorescent Indicator has been constructed, with a response dynamic range optimal for physiological measurements. Immobilization occurs via photoinitiated copolymerization of the Indicator with acrylamide on the distal end of a silanized 125 μm diameter multimode optical fiber. The working lifetime of this sensor is limited only by photobleaching of the Indicator. Due to the inherent hydrophilic nature of the acrylamide polymer, the response time of this new sensor is governed by simple aqueous diffusion of the ionic calcium. This results in sensor response times fast enough to monitor some concentration fluctuations at physiological rates. The ability to monitor calcium concentration fluctuations in a high background level of magnesium is also demonstrated with a calculated selectivity of 10-4.5.
Prashant Padmawar - One of the best experts on this subject based on the ideXlab platform.
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k waves in brain cortex visualized using a long wavelength k sensing Fluorescent Indicator
Nature Methods, 2005Co-Authors: Prashant Padmawar, Orin Bloch, Geoffrey T Manley, Alan S. VerkmanAbstract:K + waves in brain cortex visualized using a long-wavelength K + -sensing Fluorescent Indicator
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k waves in brain cortex visualized using a long wavelength k sensing Fluorescent Indicator
Nature Methods, 2005Co-Authors: Prashant Padmawar, Orin Bloch, Geoffrey T Manley, Xiaoming Yao, Alan S. VerkmanAbstract:We synthesized a water-soluble, long-wavelength K(+) sensor, TAC-Red, consisting of triazacryptand coupled to 3,6-bis(dimethylamino)xanthylium, whose fluorescence increased 14-fold at 0-50 mM K(+) with K(+)-to-Na(+) selectivity >30. We visualized K(+) waves in TAC-Red-stained brain cortex in mice during spreading depression, with velocity 4.4 +/- 0.5 mm/min, and K(+) release and reuptake half-times (t(1/2)) of 12 +/- 2 and 32 +/- 4 s, respectively. Aquaporin-4 (AQP4) deletion slowed K(+) reuptake about twofold, suggesting AQP4-dependent K(+) uptake by astroglia.
Kazuhiko Nakatani - One of the best experts on this subject based on the ideXlab platform.
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structure activity studies on the Fluorescent Indicator in a displacement assay for the screening of small molecules binding to rna
Chemistry: A European Journal, 2012Co-Authors: Shiori Umemoto, Jinhua Zhang, Masaki Hagihara, Asako Murata, Yasue Harada, Takeo Fukuzumi, Takahiro Wazaki, Shinichi Sasaoka, Kazuhiko NakataniAbstract:A series of xanthone and thioxanthone derivatives with aminoalkoxy substituents were synthesized as Fluorescent Indicators for a displacement assay in the study of small-molecule-RNA interactions. The RNA-binding properties of these molecules were investigated in terms of the improved binding selectivity to the loop region in the RNA secondary structure relative to 2,7-bis(2-aminoethoxy)xanthone (X2S) by fluorimetric titration and displacement assay. An 11-mer double-stranded RNA and a hairpin RNA mimicking the stem loop IIB of Rev response element (RRE) RNA of HIV-1 mRNA were used. The X2S derivatives with longer aminoalkyl substituents showed a higher affinity to the double-stranded RNA than the parent molecule. Introduction of a methyl group on the aminoethoxy moiety of X2S effectively modulated the selectivity to the RNA secondary structure. Methyl group substitution at the C1' position suppressed the binding to the loop regions. Substitution with two methyl groups on the amino nitrogen atom resulted in reducing the affinity to the double-stranded region by a factor of 40%. The effect of methyl substitution on the amino nitrogen atom was also observed for a thioxanthone derivative. Titration experiments, however, suggested that thioxanthone derivatives showed a more prominent tendency of multiple binding to RNA than xanthone derivatives. The selectivity index calculated from the affinity to the double-stranded and loop regions suggested that the N,N-dimethyl derivative of X2S would be suitable for the screening of small molecules binding to RRE.
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Fluorescent Indicator displacement assay for ligand rna interactions
Journal of the American Chemical Society, 2010Co-Authors: Jinhua Zhang, Shiori Umemoto, Kazuhiko NakataniAbstract:Since the discovery of the RNA functions modulating gene expression, functional RNA has become an attractive drug target. Ligand−RNA interactions have been studied by covalent labeling of either the ligand or RNA with Fluorescent dyes or immobilization on the sensing devices. However, it is desirable to investigate the interactions without any chemical modifications in order to keep the native secondary and tertiary structures. Displacement assays satisfy this criterion because neither covalent labeling nor immobilization is necessary for the assay. We here report a Fluorescent Indicator displacement assay for the investigation of a ligand−RNA interaction that exploits a novel Fluorescent Indicator, a 2,7-disubstituted 9H-xanthen-9-one derivative. The assay was found to be applicable for screening new ligands binding to RRE RNA from chemical library.