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The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trio-ctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thioi group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FRET signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 [micro]g/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRET-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency.

In this paper, we describe the synthesis and characterization of analytical properties of fluorescence-based zinc ion-sensing glass slides and their application in monitoring zinc ion release from beta pancreatic cells in cell cultures. To fabricate the sensors, the zinc ion indicator ZnAF-2 {6-[N-[N',N'-bis(2-pyridinylmethyl)-2-aminoethyl]amino3', 6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xan-thene]-3-one} was modified to include a sufficiently long linking aliphatic chain with a terminal carboxyl functional group. The recently synthesized ZnAF-2 zinc ion indicator provided high zinc ion selectivity in physiological solutions containing millimolar levels of calcium and other possible interfering cations. The carboxyl-modified ZnAF-2 was conjugated to the activated surface of glass slides, which then served as zinc ion sensors. It was possible to grow pancreatic cells directly on the zinc-sensing glass slide or on a membrane placed on these glass slides. The sensors were used to monitor zinc ion release events from glucose-stimulated pancreatic cells. The study showed that the zinc ion sensors responded effectively to the release of zinc ions from pancreatic cells at the nanomolar level with high selectivity and rapid subsecond response time.

This paper describes the development of novel particle-based fluorescence resonance energy transfer (FRET) sensors to quantify the concentration and monitor the binding affinity of carbohydrates and glycoproteins to lectins, which are carbohydrate binding proteins. The sensing approach is based on FRET between fluorescein (donor)-labeled lectin molecules, adsorbed on the surface of micrometric polymeric beads, and polymeric dextran molecules labeled with Texas Red (acceptor). The FRET efficiency of the donor-acceptor pair decreases in the presence of carbohydrates or glycoproteins that compete with the Texas Red-labeled dextran molecules on the lectinic binding sites. The inhibitory effect is concentration and time dependent. The sensing technique enables the discrimination between carbohydrates and glycoproteins based on their binding affinity to the FRET sensing particles as well as quantitative analysis of carbohydrates and glycoproteins in aqueous samples. In the future, the newly developed sensors could enable screening glycoprotein-based drugs for their binding affinity toward selective receptors.

Phagocytosis of bioparticles such as bacteria and viruses by macrophages is a critical component of the immune response against infections. In this paper we describe the synthesis of submicrometer fluorescent particles with pH sensing capability. The particles are used to measure the pH and to monitor the effect of chloroquine, an antimalarial drug, on the pH in the lysosome, the cellular organelle involved in the phagocytosis process. The synthesis of the pH sensing particles is realized by the covalent attachment of amine reactive forms of Oregon Green (pH sensitive dye) and Texas Red (pH insensitive dye) to the surface of amino-modified submicrometer polystyrene particles. The particles are absorbed by J774 Murine Macrophages through phagocytosis and directed to lysosomes. Despite the high lysosomal levels of digestive enzymes and acidity, the absorbed particles remain stable for 12 h in the cells when they are stored in a PBS buffer solution at pH 7.4. The pH dynamic range of the sensing particles is between pH 4.5 and 7 with a sensitivity of 0.1 pH units. Exposure of the cells to chloroquine increases the lysosomal pH from 4.8 to 6.5. The effect is concentration-dependent

The development of new fluorescence resonance energy transfer (FRET)-based quantum dot probes for proteolytic activity is described. The newly developed quantum dot FRET-based probes could be applied in microfluidic channels to facilitate bioanalysis of volume-limited samples and single-cell studies in an in vivo-like environment.

Intracellular oxygen concentration is of primary importance in determining numerous physiological and pathological processes in biological systems. This paper describes the development and application of micrometersized oxygen-sensitive fluorescence lipobeads for intracellular measurements of molecular oxygen in J774 murine macrophages. `A ruthenium diimine complex [Ru(bpy-pyr)[(bpy).sub.2]][Cl.sub.2] (bpy = 2,2'-bipyridine, bpy-pyr = 4-(1'-pyrenyl)-2,2'-bipyridine) is used as the oxygen indicator. The indicator exhibits high chemical and photostability and high sensitivity to oxygen. The indicator molecules are immobilized in a phospholipid membrane that coats polystyrene microparticles. The fluorescence of the lipobeads is effectively quenched by molecular oxygen. The fluorescence intensity of the oxygen-sensitive lipobeads is 3 times higher in a nitrogenated solution than in an oxygenated solution. The lipobeads are internalized by murine macrophages through phagocytosis. They maintain their spectral properties for 24 h in living cells when the cells are stored in phosphate-buffered saline at pH 7.4. The photostability, reversibility, and effect of hypoxia, hyperoxia, and oxidative stress on the intracellular level of oxygen in J774 murine macrophages are described.

This paper describes the synthesis and characterization of micrometric phospholipid-coated polystyrene particles, named lipobeads, with pH-sensing capability and their application for intracellular pH measurements in murine macrophages. The phospholipids used to coat the particles are labeled with fluorescein (a pH-sensitive dye) and tetramethylrhodamine (a pH-insensitive dye), which serves as a referencing fluorophore for increased accuracy of the pH measurements. The synthesis of the pH-sensing lipobeads is realized by the covalent attachment of the fluorescent phospholipids to the surface of carboxylated polystyrene particles. The pH dynamic range of the sensing particles is between 5.5 and 7.0 with a sensitivity of 0.1 pH unit. The excitation light intensity is reduced to minimize photobleaching of the fluorescein--phospholipid conjugates. The fluorescent lipobeads are used to measure the pH in single macrophages. The lipobeads are ingested by the macrophages and directed to lysosomes, which are the cellular organelles involved in the phagocytosis process. Despite the high lysosomal levels of digestive enzymes and acidity, the absorbed particles remain stable for over 6 h in the cells when they are stored in a phosphate-buffered saline solution at pH 7.4.

The development of Rhodamine-labeled peptide-coated CdSe/ZnS quantum dots FRET-based protease sensors and their application for the measurement of extracellular matrix metalloproteinases (MMPs) activity in normal and cancerous breast cells are described. The synthesis enabled to discriminate between normal and cancerous cell cultures.

The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trio- ctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thiol group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FREF signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 µg/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRFT-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency. [ABSTRACT FROM AUTHOR]

Describes the development and application of micrometer-sized oxygen-sensitive fluorescence lipobeads for intracellular measurements of molecular oxygen in J774 murine macrophages. Use of a ruthenium diimine complex as the oxygen indicator; Internalization of the lipobeads by murine macrophages; Effect of hypoxia, hyperoxia and oxidative stress on the intracellular level of oxygen in J774 murine macrophages.

Describes the synthesis of submicrometer fluorescent particles with pH sensing capability. Utilization of the particles to measure the pH and to monitor the effect of chloroquine, an antimalarial drug, on the pH in the lysosome; Absorption of the murine macrophages through phagocytosis; Stability of the absorbed particles in the cells.