Development of Fe3O4/Ag core/shell-based multifunctional immunomagnetic nanoparticles for isolation and detection of CD34+ stem cells.

Fe3O4/Ag core/shell nanoparticles functionalized with the free amino (NH2) functional groups (Fe3O4/Ag-NH2) were conjugated with fluorescent electron coupled dye (ECD)-antiCD34 antibody using the 1-ethyl-3-(3'-dimethyl-aminopropyl) carbodiimide (EDC) catalyst (ECD - Electron Coupled Dye or R Phycoerythrin-Texas Red is a fluorescent organic dye attached to the antibody). The characteristic fluorescence of ECD in the antibody was investigated and was used as a good indicator for estimating the percentage of the antibodies that were successfully conjugated with the nanoparticles. The conjugation efficiency was found to increase depending on the VNP:VAB ratio, where VNP and VAB are the volumes of the nanoparticle solution (concentration of 50 ppm) and the as-purchased antibody solution, respectively. The conjugation efficiency rapidly increased from approximately 18% to approximately 70% when VNP:VAB was increased from 2:1 to 100:1, and it gradually reached the saturated state at an efficiency of 95%, as the VNP:VAB was equal to 300:1. The bioactivity of the abovementioned conjugation product (denoted by Fe3O4/Ag-antiCD34) was evaluated in an experiment for the collection of stem cells from bone marrow samples.

Thermodynamics and kinetics of the pressure unfolding of phosphoglycerate kinase.

Due to the relationship between compressibility and volume fluctuations, high-pressure studies provide vital insight into protein dynamics and function. Most high-pressure experiments were performed on small and fast folding proteins or model peptides. Here we show that a detailed kinetic study is necessary to extract reliable information from the high-pressure-induced structural conversion of large, slowly folding proteins. The pressure-jump unfolding kinetics of yeast phosphoglycerate kinase was recorded at pressures between 50 and 150 MPa. The time dependence of the conformational state of the protein was followed by tryptophan fluorescence measurements from 30 s to 2 h. The observed changes were described by a three-state model, and the volume change and the activation volume as well as the midpoint pressure of the transitions between the folded, intermediate, and unfolded states were determined. An interesting feature of the pressure unfolding of phosphoglycerate kinase was that the unfolding process speeds up with increasing pressure, which is the consequence of negative activation volumes for the folded --> intermediate, intermediate --> unfolded, and unfolded --> intermediate transitions.