On Going to a New Era of Microgel Exhibiting Volume Phase Transition.

The discovery of phenomena of volume phase transition has had a great impact not only on bulk gels but also on the world of microgels. In particular, research on poly(N-isopropylacrylamide) (PNIPAM) microgels, whose transition temperature is close to body temperature, has made remarkable progress in almost 35 years. This review presents some breakthrough findings in microgels that exhibit volume phase transitions and outlines recent works on the synthesis, structural analysis, and research direction of microgels.

Micro hydrogels: preparation, properties, and applications.

A micro hydro-gel is a submicron- or micron-sized network polymer particle that is insoluble in water but highly swellable. This review presents the following topics: preparation, properties, and applications of micro hydrogels. First, two types of preparation methods for micro hydrogels are presented: (i) particle-forming polymerization and (ii) molecular assembly of polymer chains dissolved in water. Next, the characteristic properties of micro hydrogels are discussed. Finally, the applications of micro hydrogels are reviewed, with special emphasis on environmentally sensitive optical/photonic, biological/biomedical, and chemical applications. This review is not comprehensive, but is rather a "mini-review" primarily focused on results obtained in the author's laboratories.

Enhancement of sensitivity of SPR protein microarray using a novel 3D protein immobilization.

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.

Thermosensitive pickering emulsion stabilized by poly(N-isopropylacrylamide)-carrying particles.

Poly(N-isopropylacrylamide) (PNIPAM)-carrying particles were characterized as thermosensitive Pickering emulsifiers. Emulsions were prepared from various oils, such as heptane, hexadecane, trichloroethylene, and toluene, with PNIPAM-carrying particles. PNIPAM-carrying particles preferentially formed oil-in-water (O/W)-type emulsions with a variety of oils. All the emulsions stabilized by PNIPAM-carrying particles were stable for more than 3 months as long as they were stored at room temperature. However, when the emulsions were heated from room temperature to 40 degrees C, at which point the PNIPAM layer caused a coil-to-globule transition, phase separation occurred. Thus, by using thermosensitive PNIPAM-carrying particles as emulsifiers, the stability of the Pickering emulsions could be controlled by a slight change in temperature.

Development of novel magnetic nano-carriers for high-performance affinity purification.

We developed novel magnetic nano-carriers around 180 nm in diameter for affinity purification. Prepared magnetic nano-carriers possessed uniform core/shell/shell nano-structure composed of 40 nm magnetite particles/poly(styrene-co-glycidyl methacrylate (GMA))/polyGMA, which was constructed by admicellar polymerization. By utilizing relatively large 40 nm magnetite particles with large magnetization, the magnetic nano-carriers could show good response to permanent magnet. Thanks to uniform polymer shell with high physical/chemical stability, the magnetic nano-carriers could disperse in a wide range of organic solvent without disruption of core/shell structure and could immobilize various kinds of drugs. We examined affinity purification using our prepared magnetic nano-carriers with anti-cancer agent methotrexate (MTX) as ligand. Our magnetic nano-carriers showed higher performance compared to commercially available magnetic beads in terms of purification efficiency of target including extent of non-specific binding protein.

Impact of initiators in preparing magnetic polymer particles by miniemulsion polymerization.

Sub-micron sized polystyrene particles containing magnetite more than 30 wt.% were prepared by miniemulsion polymerization with commercially available ferricolloid. The effects of some water-soluble initiators and/or oil-soluble initiators on the particles characteristics, such as the size, morphology, magnetic properties and colloidal stability, were studied. The size of monomer droplets/polymer particles increased from 60 to 300 nm during polymerization, keeping magnetic in core when potassium persulfate (KPS) or ammonium persulfate (APS) was used as the sole initiator. These particles were easily separated from the medium within short time scale in external magnetic field, while such characteristics were controlled by the amount of persulfate used for the polymerization. In contrast, when 2,2'-azobis isobutyronitrile (AIBN) was used as the initiator, the size of droplets/particles was retained to be 90 nm at the most and magnetite nanoparticles located at the surface of polystyrene particles, which were so colloidally stable that they were not separated in external magnetic field. The above-mentioned effect of initiators on particle size in persulfate system was likely originated from the decrease of pH value and the increase of ionic strength, which induced the fusion of droplets/particles containing magnetite. Mixed-initiators system resulted in intermediate characteristics, compared with each initiator system. The location of magnetite in the particle seems to depend on where initiation/polymerization occurred in each initiator system.

Hybrid microgels with reversibly changeable multiple brilliant color.

We report reversibly color changeable hybrid microgels that tune multiple brilliant colors due to interparticle interactions of SPR using several structured nanoparticles. The interparticle interactions were brought out using the thermosensitive swelling/deswelling property of microgel. We employ N-isopropylacrylamide (NIPAM) and glycidyl methacrylate (GMA) copolymerized microgels (NG microgels) as templates for in situ synthesis of Au nanoparticles. The seed Au nanoparticles could be stably grown by successive reduction of Au and Ag in the microgels. Interestingly, the hybrid microgels were able to exhibit multiple brilliant colors by attaching Au/Ag multiple core/shell bimetallic nanoparticles in the microgels, and the color change reversibility of each hybrid microgel was accomplished by adjusting the nanoparticles' sizes. Obtained microgels shown in this study will find important applications such as in biomedical and electronic devices.

Affinity identification of delta-opioid receptors using latex nanoparticles.

Three types of latex nanoparticles carrying naltrindole (NTI) derivatives were synthesized as probes for the affinity isolation of their binding proteins including the delta-opioid receptor. The effect of the attachment of NTI to different positions on the linker was investigated. Only latex nanoparticles in which the NTI derivative was linked through the phenol group were useful for isolating the recombinant delta-opioid receptor solubilized from CHO cell membrane. These latex nanoparticles could be a useful tool for investigations of the pharmacological activity of NTI.

Gold nanoparticle localization at the core surface by using thermosensitive core-shell particles as a template.

We report novel thermosensitive hybrid core-shell particles via in situ gold nanoparticle formation using thermosensitive core-shell particles as a template. This method for the in situ synthesis of gold nanoparticles with microgel interiors offers the advantage of eliminating or significantly reducing particle aggregation. In addition, by using thermosensitive microgel structures in which the shell has thermosensitive and gel properties in water--whereas the core itself is a water-insoluble polymer--we were able to synthesize the gold nanoparticles only at the surface of the core, which had reactive sites to bind metal ions. After the gold nanoparticles were synthesized, electroless gold plating was carried out to control the thickness of the gold nanoshells. The dispersions of the obtained hybrid particles were characterized by dynamic light scattering and UV-vis absorption spectroscopy, and the dried particles were also observed by electron microscopy. Adaptation of the technique shown here will create a number of applications as optical, electronic, and biomedical functional materials.

Modification of gold nanoparticle composite nanostructures using thermosensitive core-shell particles as a template.

We report the formation of novel thermosensitive hybrid core-shell particles via in situ synthesis of gold nanoparticles using thermosensitive core-shell particles as a template. The template core-shell particles, with cores composed mainly of poly(glycidyl methacrylate) (GMA) and shells composed mainly of poly(N-isopropylacrylamide) (PNIPAM), were synthesized in aqueous medium, and functional groups such as thiol groups were incorporated into each particle. We found that these particles containing thiol groups were effective for the in situ synthesis of gold nanoparticles in long-term storage. The obtained hybrid particles exhibited a reversible color change from red to purple, which originated from the surface plasmon resonance of gold nanoparticles and which was temperature-dependent in the range of 25-40 degrees C. In addition to their thermosensitive property, the hybrid particles exhibited the unique characteristic of uniform distribution on a solid substrate. The particles obtained by this approach have potential thermosensitive applications such as in sensors and photonic or electronic devices.

Colored thin films prepared from hydrogel microspheres.

Ordered 2-D structures composed of poly(N-isopropylacrylamide) (PNIPAM) microgel particles that had regularity on a sub-micrometer length scale were prepared. By using sterically stabilized PNIPAM microgel particles as components, the ordered array was formed by a self-assembly process. The particle array was prepared by depositing a droplet of the microgel dispersion on a substrate. Atomic force microscopy observation of the resulting thin films revealed that they comprised a monolayer particle array. The periodic structure of the array produced iridescent colors due to optical diffraction. Since a homogeneous particle array can be prepared simply by drying the dispersion, this particle dispersion may be considered as a new ink whose color is generated from the microstructure in the films produced.

Development of polymer latex particles for selective cleavage of mismatched DNA and their application for DNA diagnosis.

We developed functional polymer latex particles that can catch and cleave mismatched DNA selectively and propose a new mismatch detection system using the functional particles. The aimed particles possess two functional units composed of mismatch binding protein (MutS) and an anthraquinone derivative (AQ), a light-activated agent that photocleaves dsDNA. Use of the functional particles made it possible to discriminate complementary and mismatched DNAs and photocleave mismatched DNA selectively. The efficiency of photocleavage of mismatched DNA by the functional particles increased with UV irradiation time. It was also found that the functional particles were reusable and had dissociation constants (K(d)) of 1000 and 68.5 nM for G/C homoduplex and G/T heteroduplex, respectively. Using the functional particles and a dsDNA-binding fluorescent dye, SYBR-Gold, we could construct the system for detection of mismatched DNA that was 40 base pairs. The functional particles prepared in this study will be an absolutely new tool for mismatch detection in DNA diagnosis.

High-performance fluorescent particles prepared via miniemulsion polymerization.

Highly fluorescent polymer particles were prepared with Eu beta-diketonates complex as a fluorophore by miniemulsion polymerization technique. Eu beta-diketonates complex has a long decay time, a large Stokes shift, and very narrow emission bands in comparison with other organic fluorescent compounds. Aqueous miniemulsion was prepared by mixing monomer, crosslinker, hydrophobe, and Eu beta-diketonates complex and then putting the mixture into an aqueous solution of surfactant, followed by ultrasonication. An aqueous solution of initiator was added to the miniemulsion to obtain fluorescent polymer particles, which were monodispersed without aggregation. Particle size was decreased to deca-nano scale by increasing the amount of surfactant. Fluorescent intensity was increased by using Eu beta-complex coordinated with additional ligand. Further fluorescence quantum yields and fluorescent properties in the presence of DNA were investigated to the confirm superiority of Eu beta-diketonates complexes in polymer particles.

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique.

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K-ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin-SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K-ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.

Selective ligand purification using high-performance affinity beads.

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.

Self-assembly of poly(N-isopropylacrylamide)-carrying microspheres into two-dimensional colloidal arrays.

Droplets containing polymer particles were deposited on a substrate. Poly(N-isopropylacrylamide) (PNIPAM) hydrogel and particles with PNIPAM graft chains on the surface self-assembled into a two-dimensional (2-D) superlattice when their dilute dispersions were dried on substrates. The capillary force between the particles induced ordered array formation during water evaporation. The presence of a PNIPAM layer on the particle surface gave the particles steric stability during ordered array formation. By grafting PNIPAM chains on particle surfaces by living radical polymerization, we successfully controlled the structural patterns of the colloidal arrays. These, controllable, 2-D colloidal arrays were generated on various substrates upon air-drying.

Design and synthesis of a solid-supported FR225659 derivative for its receptor screening.

[structure: see text] We describe the design and synthesis of latex particles attached to an FR225659 derivative to identify its receptor proteins. Two key building blocks were prepared by two-step degradation of FR225659 under basic conditions. The designed ligand showed an acceptable level of biological activity to make it of potential value for use in affinity-supported receptor identification. Affinity purification of FR225659-binding proteins using the latex nanoparticles provided three candidate receptor peptides for the biological activity.

Flow-stress-induced discrimination of a K-ras point mutation by sandwiched polymer microsphere-enhanced surface plasmon resonance.

The highly sensitive detection of a K-ras point mutation with the aid of DNA-carrying microspheres as a flow-stress receptor is proposed at the surface of a surface plasmon resonance (SPR) biosensor. Single-stranded DNAs were immobilized onto epoxy-group-derivatized gold surfaces and the hybridization of DNA targets was monitored. The subsequent interaction with DNA-carrying micospheres enhanced the SPR response. The increase of flow rate during the event of dissociation changed the amount of detachment of the DNA-carrying microspheres for the mismatched pair. In addition, the viscosity was changed by addition of glycerol to the buffer. The increase of shear stress from the flow resulted in detachment of DNA-carrying microspheres hybridized with the mismatched sequence and increased the ability to discriminate a point mutation. This is a new method which not only increases the lower detection limit of evanescent wave-based biosensors, but also the ability to discriminate a point mutation which is a critical factor for ultrasensitive DNA detection in flow devices.

Temperature-sensitive hairy particles prepared by living radical graft polymerization.

Four types of temperature-sensitive hairy particles were prepared by living radical graft polymerization using a photoiniferter. The hairs were poly(N-isopropylacrylamide) (N), poly(N-isopropylacrylamide)ran-poly(acrylic acid) (NA), and diblock copolymers composed of N and NA. The block copolymer was attached to the particle in different modes, that is, one has a N-block inner and a NA-block outer but the other has the inverse arrangement. The acrylic acid content in NA was adjusted to be only 1%, but NA had a higher transition temperature by 5 degrees C than N in a neutral aqueous solution. The sequence of blocks attached onto the particle was the key factor to control the temperature responsiveness of the particle. The hairy particles exhibited a two-step transition with increasing temperature under certain conditions. The hairy particle also responded to the pH and ionic strength. Some unique behaviors of the hairy particles were studied in detail in terms of electrophoretic mobility and adsorption of dye molecules as well as swelling/deswelling.