Toru Hoshi

Dual aptamer-immobilized surfaces for improved affinity through multiple target binding in potentiometric thrombin biosensing

We developed a label-free and reagent-less potentiometric biosensor with improved affinity for thrombin. Two different oligomeric DNA aptamers that can recognize different epitopes in thrombin were introduced in parallel or serial manners on the sensing surface to capture the target via multiple contacts as found in many biological systems. The spacer and linker in the aptamer probes were optimized for exerting the best performance in molecular recognition. To gain the specificity of the sensor to the target, an antifouling molecule, sulfobeaine-3-undecanethiol (SB), was introduced on the sensor to form a self-assembled monolayer (SAM). Surface characterization revealed that the aptamer probe density was comparable to the distance of the two epitopes in thrombin, while the backfilling SB SAM was tightly aligned on the surface to resist nonspecific adsorption. The apparent binding parameters were obtained by thrombin sensing in potentiometry using the 1:1 Langmuir adsorption model, showing the improved dissociation constants (Kd) with the limit of detection of 5.5 nM on the dual aptamer-immobilized surfaces compared with single aptamer-immobilized ones. A fine control of spacer and linker length in the aptamer ligand was essential to realize the multivalent binding of thrombin on the sensor surface. The findings reported herein are effective for improving the sensitivity of potentiometric biosensor in an affordable way towards detection of tiny amount of biomolecules.

Control of surface modification uniformity inside small-diameter polyethylene/poly(vinyl acetate) composite tubing prepared with supercritical carbon dioxide

A small-diameter polymer composite tubing comprising polyethylene (PE) and poly(vinyl acetate) (PVAc) with a biocompatible surface was developed for application to medical devices. In this study, the following modification process was applied to the narrow PE tubing (inside diameter: 300 μm, outside diameter: 600 μm, length: 5.0 m). The polymer composite (PE/PVAc) was prepared by supercritical impregnation of a vinyl acetate monomer and initiator into PE tubing, followed by in situ radical polymerization within the polymer matrix. Infrared imaging measurement of a cross-section confirmed that PVAc was uniformly generated in the PE tubing. After the acetyl groups on the surface were hydrolyzed, a phospholipid polymer with both phosphorylcholine groups and silane coupling groups was immobilized onto the hydroxyl groups on the surface. These reactions were performed over the entire surface of the PE tubing with a high-aspect-ratio uniformly. This surface possessing the phospholipid polymer shows high hydrophilicity and a remarkable ability to suppress protein adsorption. This novel surface modification by molecular composite formation using supercritical CO2 can modify areas such as the lumenal surface of very narrow PE tubing which are difficult to modify in conventional modification methods. This new procedure could have applications for the preparation of new polymeric materials, including biomaterials.

Preparation of temperature-responsive, cationized, poly(ε-caprolactone)-based, cross-linked materials by a macromonomer design and positive charge control on the surface

AbstractIn this study, a convenient method to synthesize cationic macromonomers containing branched poly(ε-caprolactone) (PCL) was developed, and stable materials were derived by photo-cross-linking reactions. In fact, a bromomethyl-terminated modification was carried out at the hydroxyl end groups of the starting PCL; then, the terminal groups reacted with 2,2′-dimethylaminoethyl methacrylate to afford the objective macromonomers, which had N,N′-dimethylmethacrylamino groups at the chain ends. The resulting PCL-based materials were cross-linked by UV light irradiation and were stable against exposure to organic solvents and heating above the softening points. The surface properties of the cationic, PCL, cross-linked membrane were evaluated by measuring the zeta potentials and performing anionic dye adsorption tests using Acid Red 87. As expected, the cationic, PCL, cross-linked membrane surfaces showed a positive charge and greater dye adsorption than the naked PCL, which depended on the cationic contents and temperature. Over the softening point, the positive charge steeply increased. The morphologies of adhered human mesenchymal stem cells on the PCL materials with lower cationic contents were preliminarily observed and shown to be well dispersed. The PCL-based materials in this study could enhance cell interaction and be useful for scaffold or mechanobiology studies.This study demonstrated convenient preparation methods for the introduction of cationic and cross-linkable moieties into 2-branched and 4-branched PCL and their corresponding stable materials. The cationic content and the ratio of 2-branched and 4-branched monomers could be simultaneously controlled by incorporating non-cationic macromonomers. Zeta potential measurements proved that the cationic charge could be controlled by changing the temperatures. Human MSC adhesion was observed on the PCL materials with different cationic contents and lower contents of cationic contents seem to be preferable. Consequently, such materials are promising for biomaterials research.

Production of hollow-type spherical bacterial cellulose as a controlled release device by newly designed floating cultivation

We developed a novel cultivating system for hollow-type spherical bacterial cellulose (HSBC) gel production without any molds or template. It consisted of floating aqueous medium droplet containing Gluconacetobacter xylinus (G. xylinus) at the boundary of two non-mixed silicone oil layers. The fibrils of bacterial cellulose (BC) were produced at the interface of water and oil phases. Fibril layers effectively thickened layer-by-layer and eventually formed a shell structure. The size of the HSBC gel can be controlled by the volume of dropped cell suspension. For cell suspensions of 50 μL and 10 μL, HSBC gels of approximately 4.0 mm and 2.5 mm were obtained, respectively. The shell of the HSBC gel is the gelatinous membrane formed by well-organized fibril networks; they comprised type-I crystal structure of cellulose. Additionally, we studied release profile of the fluorescein isothiocyanate-dextran (FITC-Dex) and observed that it released rapidly from the HSBC gels compared to from the BC gels obtained by the static culture method. The release behavior from HSBC gel agreed satisfactorily with Higuchi model. Therefore, the shell of HSBC gel is surely a thin gelatinous membrane of BC, and would be useful as a drug release device.

Surface Modification of SiO2 Microchannels with Biocompatible Polymer Using Supercritical Carbon Dioxide

The surface of 500-mm-long microchannels in SiO2 microchips was modified using supercritical CO2 (scCO2) and a biocompatible polymer was coated on it to confer biocompatibility to the SiO2 surface. In this method, the SiO2 surface of a microchannel was coated with poly(ethylene glycol monomethacrylate) (PEGMA) as the biocompatible polymer using allyltriethoxysilane (ATES) as the anchor material in scCO2 as the reactive medium. Results were compared with those using the conventional wet method. The surface of a microchannel could not be modified by the wet method owing to the surface tension and viscosity of the liquid, but it was modified uniformly by the scCO2 method probably owing to the near-zero surface tension, low viscosity, and high diffusivity of scCO2. The effect of the surface modification by the scCO2 method to prevent the adsorption of protein was as high as that of the modification by the wet method. Modified microchips can be used in biochemical and medical analyses.

New Nanocomposite Biomaterials Controlling Surface and Bulk Properties using Supercritical Carbon Dioxide

The molecular composite composed of polyethylene (PE) and poly(vinyl acetate) (PVAc) prepared using supercritical carbon dioxide (scCO 2 ) and its surface modification for biocompatible surface demonstrated the creation of novel polymer biomaterials. In this study, this modification process was applied to the PE narrow tube (inside diameter: 300μm, outside diameter: 600μm, length: 5m). It was confirmed that PVAc was uniformly generated in PE tube by infrared imaging measurement of cross-section. After the acetyl group on the surface was hydrolyzed, phospholipid polymer was immobilized to the hydroxyl group on the surface of the tube. The phospholipid polymer immobilized surface showed a drastic reduction in protein adsorption. The surface of the minute and slender tube can be effectively modified using the feature of carbon dioxide whose surface tension is near zero. The modification technology by scCO 2 is a promising for creation of variously-shaped new biomaterials.