Immobilization of aptamer-based molecular beacons onto optically-encoded micro-sized beads.

This paper presents a method for the novel immobilization of aptamer-based molecular beacons (apta-beacons) onto optically-encoded micro-sized beads (apta-beacon beads). To immobilize apta-beacons onto flourescently-encoded micro-sized beads, core-shell type beads containing a fluorescent dye-encoded core and apta beacon-coupled shell were prepared. The fluorescent dye-encoded beads were prepared from TentaGel resins by coupling RITC to the amino groups of the core region, after partial protection of amino groups with Fmoc-OSu in a diffusion-controlled manner. After deprotection of the Fmoc-amino groups, FITC-coupled molecular beacons (MBs) were immobilized to the beads together with a quencher by covelent bonding. Briefly, aspartic acid (Asp) was coupled to the shell part of the beads. Then, the quencher was coupled to the N-terminal amino group of Asp and the MBs were coupled to the side chain carboxyl group. In a model study, thrombin was directly detected using this apta-beacon bead method. The thrombin-bound apta-beacon beads were easily recognized by the appearance of fluorescence without any further labeling step.

Enhanced cell permeability of kojic acid-phenylalanine amide with metal complex.

Kojic acid-phenylalanine amide (KA-F-NH(2)), which showed an excellent tyrosinase inhibitory activity, did not inhibit melanogenesis in melanocyte due to its low cell permeability. To enhance its cell permeability by increasing lipophilicity, we prepared metal coordination compounds of KA-F-NH(2) and characterized them by FT-IR and ICP analysis. The metal complex of KA-F-NH(2) inhibited mushroom tyrosinase activity as much as KA-F-NH(2) and reduced melanin contents in melanocyte efficiently.

Multilayer fluorescence optically encoded beads for protein detection.

An easy preparation method of multilayer fluorescence optically encoded beads for protein detection is presented. The beads, which consist of multicolored layers, are made from amino polyethylene glycol grafted polystyrene (PS-g-PEG) beads by using several fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine via controlling diffusion of an Fmoc-protecting group after HCl solution swelling. A biotin, glutathione S-transferase (GST) antibody, and an RNA aptamer that specifically recognize streptavidin, GST antigen, and hepatitis C virus (HCV) helicase are introduced to the optically encoded beads and monitored for their binding activity to the target molecules. After binding, the ligands are identified easily by their color codes.

Surface-grafted polystyrene beads with comb-like poly(ethylene glycol) chains: preparation and biological application.

We prepared surface-grafted polystyrene (PS) beads with comb-like poly(ethylene glycol) (PEG) chains. To accomplish this, conventional gel-type PS beads (35-75 microm) were treated with ozone gas to introduce hydroperoxide groups onto the surface. Using these hydroperoxide groups, poly(methyl methacrylate) (PMMA, Mn= 22,000-25,000) was grafted onto the surface of the PS beads. The ester groups of the grafted PMMA were reduced to hydroxyl groups with lithium aluminum hydride (LAH). After adding ethylene oxide (EO) to the hydroxyl groups, we obtained the PS-sg-PEG beads, which had a rugged surface and a diameter of 80-150 microm. We could obtain several kinds of the PS-sg-PEG beads by controlling the chain lengths of the grafted PMMA and the molecular weights of the PEG chains. The grafted PEG layer was about 30-50 microm thick, which was verified from the cross-sectioned views of the fluorescamine-labeled beads. These fluorescence images proved that the beads possessed a pellicular structure. Furthermore, we found that the surface-grafted PEG chains had the characteristic property of reducing non-specific protein adsorption on the beads.