Nitsa Rosenzweig

Quantum Dot FRET-Based Probes in Thin Films Grown in Microfluidic Channels

This paper describes the development of new fluorescence resonance energy transfer (FRET)-based quantum dot probes for proteolytic activity. The CdSe/ZnS quantum dots are incorporated into a thin polymeric film, which is prepared by layer-by-layer deposition of alternately charged polyelectrolytes. The quantum dots, which serve as fluorescent donors, are separated from rhodamine acceptor molecules, which are covalently attached to the film surface by a varying number of polyelectrolyte layers. When excited with visible light, the emission color of the polyelectrolyte multilayer film appears orange due to FRET between the quantum dots and molecular acceptors. The emission color changes to green when the rhodamine molecules are removed from the surface by enzymatic cleavage. The new probe design enables the use of quantum dots in bioassays, in this study for real-time monitoring of trypsin activity, while alleviating concerns about their potential toxicity. Application of these quantum dot FRET-based probes in microfluidic channels enables bioanalysis of volume-limited samples and single-cell studies in an in vivo-like environment.

Characterization of aggregation and protein expression of bovine corneal endothelial cells as microcarrier cultures in a rotating-wall vessel.

Rotating-wall vessels are beneficial to tissue engineering in that the reconstituted tissue formed in these low-shear bioreactors undergoes extensive three-dimensional growth and differentiation. In the present study, bovine corneal endothelial (BCE) cells were grown in a high-aspect rotating-wall vessel (HARV) attached to collagen-coated Cytodex-3 beads as a representative monolayer culture to investigate factors during HARV cultivation which affect three-dimensional growth and protein expression. A collagen type I substratum in T-flask control cultures increased cell density of BCE cells at confluence by 40% and altered the expression of select proteins (43, 50 and 210 kDa). The low-shear environment in the HARV facilitated cell bridging between microcarrier beads to form aggregates containing upwards of 23 beads each, but it did not promote multilayer growth. A kinetic model of microcarrier aggregation was developed which indicates that the rate of aggregation between a single bead and an aggregate was nearly 10 times faster than between two aggregate and 60 times faster than between two single beads. These differences reflect changes in collision frequency and cell bridge formation. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls perhaps due to hypoxia, fluid flow or distortion of cell shape. In addition to the insight that this work has provided into rotating-wall vessels, it could be useful in modeling aggregation in other cell systems, propagating human corneal endothelial cells for eye surgery and examining the response of endothelial cells to reduced shear.

Dynamic analytical chemistry: A kinetic study of the labeling of normal and age fractionated human erythrocytes with monobromobimane

Abstract The application of digital fluorescence imaging microscopy for the analysis of the thiol content and labeling kinetics of normal and age fractionated erythrocytes on a single cell basis is described. Monobromobimane (mBBr), a thiol selective dye, reacts primarily with glutathione, the major thiol compound in erythrocytes. The fluorescence intensity of thiol bound mBBr molecules is about 14-fold higher than the fluorescence intensity of unbound mBBr molecules. The labeling process is monitored in-situ using a slow scan high performance charge coupled device (CCD) camera. A 4-fold cell-to-cell variation is found in the maximum fluorescence intensity of mBBr labeled cells within a large normal population of erythrocytes. This observation is in agreement with previous results obtained for the same cellular system using capillary electrophoresis and laser excited fluorescence detection. In addition, a 2-fold variation is observed in the rate of labeling of the cells with mBBr. Quantitative kinetic measurements of the labeling of age fractionated erythrocytes with mBBr show that the cellular variation in the labeling rate and in the cellular thiol content are related to the cell age.