ABSTRACT
We have discovered a novel protein immobilization method, i.e., a "Three-Dimensional Nanostructured Protein Hydrogel" (3-D NPH), which is composed of protein-reactive polymer hybrid nanoparticles to detect protein-protein interactions. The 3-D NPH can be easily prepared by spotting a protein/reactive polymer mixture on a substrate. The resulting 3-D NPH is characterized by large amounts of immobilized proteins and a novel porous structure.The 3-D NPH technology was applied to immobilize streptavidin (SA) onto Au-coated surface for surface plasmon resonance imaging (SPRi). By using 3-D NPH method, it was possible to improve the sensitivity of protein-protein interactions drastically comparing to the conventional protein immobilization method.
Subject(s)
Protein Array Analysis/methods , Gold , Hydrogels , Immobilized Proteins , Molecular Biology/methods , Nanoparticles , Polymers , Protein Interaction Mapping/methods , Streptavidin , Surface Plasmon Resonance/methodsABSTRACT
A pod-like form of a hollow nanocarbon "carbon nanopod" with controllable graphene layer orientation and good water-dispersibility has been synthesized with extremely high selectivity and without any impurities.
ABSTRACT
We discovered a novel method to prepare a protein-based hydrogel, that is, a "Three-Dimensional Nanostructured Protein Hydrogel (3D NPH)", which is composed of protein-polymer hybrid nanoparticles. In this study, we propose a novel protein microarray whose 3D NPH spots were prepared by dispensing a small volume of the solution of protein-polymer mixture on a substrate. The dispensed solution had a short time for cross-linking before its drying-up and the resulting 3D NPH had loosely cross-linked, thin spongy structure. Therefore, the reaction ratio between ligands and analytes was drastically improved in this system compared with the large volume system for Surface Plasmon Resonance (SPR) protein microarray.
Subject(s)
Biosensing Techniques/methods , Imaging, Three-Dimensional/methods , Immobilized Proteins/chemistry , Protein Array Analysis/methods , Animals , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/pharmacology , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Hydrogels/chemistry , Ligands , Mice , Microscopy, Electron, Transmission/methods , Polymers/chemistry , Streptavidin/chemistry , Surface Plasmon ResonanceABSTRACT
A microfluidic chip carrying three reaction chambers was designed and constructed to examine sequential multiple enzymatic reactions. The synthesis of oligosaccharides in living cells is carried out in the Golgi apparatus where multiple enzymes such as glycosidase and glycosyltransferases act on a variety of substrates to generate glycoconjugates that include glycolipids and glycoproteins. The regulatory mechanism of the process however remains unknown. A microchip-based analysis platform may provide a valuable tool with which to address the issue by mimicking the Golgi function. We thus examined 3 sequential glycosyltransfer reactions on a chip, and succeeded in the synthesis of a tetrasaccharide using immobilized enzymes. Also, the kinetic parameters for a recently identified glycosyltransferase, proteoglycan GalT-I, were obtained for the first time.
