Akifumi Kawamura

Synthesis of glucose-responsive bioconjugated gel particles using surfactant-free emulsion polymerization.

Bioconjugated gel particles that have complexes composed of lectin concanavalin A (ConA) and 2-glucosyloxyethyl methacrylate (GEMA) were synthesized by the surfactant-free emulsion copolymerization of N,N-diethylaminoethyl methacrylate (DEAEMA), poly(ethylene glycol) dimethacrylate (PEGDMA), GEMA, and modified-ConA with polymerizable groups. The resultant gel particles having GEMA-ConA complexes (GEMA-ConA gel particles) were colloidally stable in a phosphate buffer solution and had a diameter of approximately 750nm. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) measurements implied that GEMA-ConA gel particles have core-shell structures consisting of a hydrophobic core of DEAEMA and a hydrophilic shell of GEMA and PEGDMA containing ConA. GEMA-ConA gel particles underwent a change in size in response to glucose in a phosphate buffer solution. The swelling ratio of GEMA-ConA gel particles gradually increased with an increase in the glucose concentration. On the other hand, the swelling ratio of GEMA-ConA gel particles remained unchanged in a phosphate buffer solution containing galactose. The glucose-responsive swelling of GEMA-ConA gel particles was induced by the dissociation of GEMA-ConA complexes acting as reversible cross-links, because free glucose behaved as an inhibitor of GEMA-ConA complexes. These results indicate that GEMA-ConA gel particles can recognize glucose selectively and undergo changes in size in response to the glucose concentration. The smart functions of glucose-responsive gel particles can provide tools for constructing self-regulated drug delivery systems and sensor systems useful for treating diabetes.

Preparation of Molecule-Responsive Microsized Hydrogels via Photopolymerization for Smart Microchannel Microvalves

Microdevices designed for practical environmental pollution monitoring need to detect specific pollutants such as dioxins. Bisphenol A (BPA) has been widely used as a monomer for the synthesis of polycarbonate and epoxy resins. However, the recent discovery of its high potential ability to disrupt human endocrine systems has made the development of smart systems and microdevices for its detection and removal necessary. Molecule-responsive microsized hydrogels with β-cycrodextrin (β-CD) as ligands are prepared by photopolymerization using a fluorescence microscope. The molecule-responsive micro-hydrogels show ultra-quick shrinkage in response to target BPA. Furthermore, the flow rate of a microchannel is autonomously regulated by the molecule-responsive shrinking of their hydrogels as smart microvalves.

Development of protein-recognition SPR devices by combination of SI-ATRP with biomolecular imprinting using protein ligands

Abstract Molecularly imprinted polymer brush layers and gel layers with both a lectin (ConA) and an antibody-IgG as biomolecular ligands for a target protein were formed on surface plasmon resonance (SPR) sensor chips via surface-initiated atom transfer radical polymerization (SIATRP) without and with a crosslinker, respectively. While the IgG-imprinted brush layers chip had almost the same affinity constant for target IgG as the nonimprinted brush layer chip, the affinity constant of the IgG-imprinted gel layer chip was approximately twice than that of the nonimprinted gel layer chip. These indicate that chemical crosslinks are very important factor to create distinct molecular recognition sites by molecular imprinting. Thus, biomolecular imprinting that uses biomolecular ligands and crosslinkers enables us to design polymer layer chips with distinct molecular recognition sites with a strong affinity for a target biomolecule. The molecularly imprinted gel layers chips with lectin and antibody ligands are promising candidates for fabricating SPR sensor systems to monitor target biomolecules such as proteins.

Effect of Enzyme Modification by Well-Defined Multi-Armed Poly(Ethylene Glycol) Synthesized Using Polyamidoamine Dendron

Egg white lysozyme was chemically modified by PEGylated PAMAM 1st, 2nd and 3rd generation dendrons, which had 2, 4 and 8 PEG arms, respectively. The number of PEG chains introduced to the lysozyme molecule drastically increased with an increase in dendron generation, although the number of PEGylated PAMAM dendrons introduced decreased due to steric repulsion. The lytic activity of lysozyme to Micrococcus luteus cells was effectively inhibited by conjugating PEGylated PAMAM dendron to the lysozyme, indicating steric stabilization of PEG chains at the surface of lysozyme molecule. In addition, the enzymatic reaction of the lysozyme with oligosaccharide substrate was apparently accelerated by a substrate condensation effect due to the multi-armed structure of PEG.

Mechanical and responsive properties of temperature-responsive gels prepared via atom transfer radical polymerization

Temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm) gels were prepared via atom transfer radical polymerization (ATRP), and their mechanical and responsive properties were investigated from the viewpoint of their network homogeneity. When the PNIPAAm gels were prepared by ATRP and free radical polymerization (FRP) at a low temperature, no difference was observed in the mechanical properties. However, the PNIPAAm gels prepared via ATRP at room temperature showed a higher elastic modulus and greater temperature-responsive change than the gels prepared via FRP. The temperature-responsive shrinkage rate of the PNIPAAm gel prepared via ATRP was slower than that of the gel prepared via FRP. Dynamic light scattering (DLS) measurements revealed that these results can be attributed to the more homogeneous network structure in the PNIPAAm gel prepared via ATRP than in the gel prepared via FRP.

Preparation of molecularly imprinted hydrogel layer SPR sensor chips with lectin-recognition sites via SI-ATRP

AbstractMolecularly imprinted hydrogel layers with lectin-recognition sites were prepared on surface plasmon resonance (SPR) sensor chips via surface-initiated atom transfer radical polymerization (SI-ATRP) combined with molecular imprinting. The lectin-imprinted hydrogel layer sensor chips showed larger SPR signal change in response to a target lectin than nonimprinted hydrogel layer sensor chips. The larger SPR signal change was attributed to the strong affinity constant of the lectin-imprinted hydrogel layer for the target lectin. These results suggest that molecular recognition sites for the lectin were formed within the hydrogel layers by molecular imprinting. On the other hand, the SPR signal change of the lectin-imprinted hydrogel layer chip in the presence of other lectin was very small. Poly(2-methacryloxyethyl phosphorylcholine) as a main chain of the hydrogel layer inhibited nonspecific adsorption of other lectin. This paper describes that SI-ATRP with biomolecular imprinting is a useful method to design highly sensitive and selective SPR sensor chips with molecular recognition sites for a target lectin.Molecularly imprinted hydrogel layer SPR sensor chips with lectin-recognition sites, which were prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) combined with molecular imprinting, exhibited not only large SPR signal change in response to a target lectin but also inhibited nonspecific protein adsorption.

Reductively Responsive Gel Capsules Prepared Using a Water-Soluble Zwitterionic Block Copolymer Emulsifier

Utilizing the unique solubility of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), which is soluble in only water and alcohol, we synthesized a water-soluble block copolymer emulsifier composed of a hydrophilic PMPC block and an amphiphilic poly[oligo(ethylene glycol) methacrylate] (POEGMA) block via reversible addition-fragmentation chain transfer (RAFT) polymerization. Water-in-oil (W/O) emulsions were successfully formed in the presence of the resulting PMPC- b-POEGMA, which acted as a stabilizer of water droplets in a chloroform continuous phase because the PMPC and POEGMA blocks were distributed to the water and chloroform phases, respectively. Next, the amphiphilic poly[poly(ethylene glycol) methacrylate] (PPEGMA) gel layer, which contained bis(2-methacryloyl)oxyethyl disulfide as a reductively responsive cross-linker, was prepared by inverse miniemulsion periphery RAFT polymerization from the PMPC- b-POEGMA that stabilized the W/O emulsions. The resulting PPEGMA gel capsules were colloidally stable in not only chloroform but also water without additional hydrophilic surface modification. The drug-release behavior from the PPEGMA gel capsules in response to dithiothreitol (DTT), which is a reducing agent, was investigated using fluorescein-conjugated dextran (FITC-Dex) as a model drug. The FITC-Dex release rate from the gel capsules in a phosphate buffer solution (pH 7.4, 20 mM) with DTT was fast compared to that without DTT. The reductively responsive FITC-Dex release is attributed to the cleavage of disulfide bonds that act as cross-links in the PPEGMA gel layer. The fascinating properties of the PPEGMA gel capsules suggest that they can provide a useful platform for designing drug carriers for protein and gene delivery and nanobioreactors.

Biomolecularly stimuli-responsive tetra-poly(ethylene glycol) that undergoes sol–gel transition in response to a target biomolecule

Stimuli-responsive polymers that undergo a sol–gel transition in response to changes in environmental factors such as pH and temperature have attracted considerable attention for biomedical applications such as drug reservoirs for controlled release and scaffolds for tissue engineering. Although numerous stimuli-responsive polymers that undergo a sol–gel transition have been reported, the literature contains few accounts of biomolecularly stimuli-responsive polymers that undergo a sol–gel transition in response to a specific biomolecule. In previous studies, we designed biomolecule-responsive hydrogels that undergo changes in volume in response to a target biomolecule; the strategy involves using biomolecular complexes as dynamic cross-links in the gel networks. In the present study, we designed biotin-conjugated four-armed poly(ethylene glycol) (biotinylated Tetra-PEG) as biomolecular stimuli-responsive sol–gel transition polymers that underwent the phase transition from a sol to a gel state in response to avidin as a target biomolecule. When avidin that forms a biomolecular complex with biotin was added to a buffer solution containing biotinylated Tetra-PEG, the solution transformed to a gel state immediately. However, the addition of a buffer solution with free biotin to the resulting hydrogel induced its dissociation to a sol state. The sol–gel transition of a buffer solution with biotinylated Tetra-PEG was directly affected by polymer concentration and the biotin/avidin molar ratio. The phase diagram of the sol–gel transition of biotinylated Tetra-PEG in a buffer solution as a function of polymer concentration and the biotin/avidin molar ratio is presented.

4.2 – Biosensors

Monitoring biological or biochemical processes is of utmost importance for medical and biological applications. Development of highly sensitive, specific, and cost-effective biosensors is in considerable demand because they lead to highly precise diagnosis and individualized medicine. This chapter provides an overview of widely used biosensors and biosensing techniques such as enzyme-based electrochemical biosensors, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and DNA microarray. These are widely utilized diagnostic tools and methods for exploring biomarkers in medical and biological fields. In addition, important research into the development of biosensors by utilizing surface plasmon resonance sensors, field-effect transistors, and gold nanoparticles is also described in this chapter. This representative research will contribute to the realization of next-generation medical systems such as individualized medicine and ultrasensitive point-of-care detection of biomarkers.