Hirokazu Seto

Polymer-modified gold nanoparticles via RAFT polymerization: A detailed study for a biosensing application

Glycopolymers of polyacrylamide derivatives with mannose were prepared via the living radical polymerization of a reversible addition–fragmentation chain transfer reagent. The polymers obtained showed narrow polydispersities. The polymer terminal group was reduced to a thiol, and the resulting polymers were mixed with gold nanoparticles to prepare glycopolymer-substituted gold nanoparticles. The mannose density was adjusted by varying the copolymer preparation and the glycopolymer–polyacrylamide mixture. The colloidal stability of the polymer-coated gold nanoparticles is dependent on the mannose density. Polymer-coated nanoparticles with low mannose densities showed better colloidal stabilities. The molecular recognition abilities of the polymer were investigated using UV-vis spectroscopy. The polymer-coated nanoparticles showed strong protein recognition abilities because of multivalent binding effects. Polymers with high mannose densities showed stronger recognition abilities. The molecular recognition abilities of the glycopolymer–polyacrylamide mixed nanoparticles are dependent on the mannose density. An immunochromatographic assay was performed using the polymer-coated nanoparticles. The color was detected from the gold nanoparticles in the nanoparticle systems with strong molecular recognition and good colloid stability.

Selective Protein Separation Using Siliceous Materials with a Trimethoxysilane-Containing Glycopolymer

A copolymer with α-D-mannose (Man) and trimethoxysilane (TMS) units was synthesized for immobilization on siliceous matrices such as a sensor cell and membrane. Immobilization of the trimethoxysilane-containing copolymer on the matrices was readily performed by incubation at high heat. The recognition of lectin by poly(Man-r-TMS) was evaluated by measurement with a quartz crystal microbalance (QCM) and adsorption on an affinity membrane, QCM results showed that the mannose-binding protein, concanavalin A, was specifically bound on a poly(Man-r-TMS)-immobilized cell with a higher binding constant than bovine serum albumin. The amount of concanavalin A adsorbed during permeation through a poly(Man-r-TMS)-immobilized membrane was higher than that through an unmodified membrane. Moreover, the concanavalin A adsorbed onto the poly(Man-r-TMS)-immobilized membrane was recoverable by permeation of a mannose derivative at high concentration.

Surface modification of siliceous materials using maleimidation and various functional polymers synthesized by reversible addition-fragmentation chain transfer polymerization.

A novel surface modification method was investigated. The surface of siliceous materials was modified using polystyrene, poly(acrylic acid), poly(N-isopropylacrylamide), and poly(p-acrylamidophenyl-α-mannoside) synthesized by reversible addition-fragmentation chain transfer polymerization. Thiol-terminated polymers were obtained by reduction of the thiocarbonate group using sodium borohydride. The polymers were immobilized on the surface via the thiol-ene click reaction, known as the Michael addition reaction. Immobilization of the polymers on the maleimidated surface was confirmed by X-ray photoelectron spectroscopy, infrared spectroscopy, and contact angle measurements. The polymer-immobilized surfaces were observed by atomic force microscopy, and the thickness of the polymer layers was determined by ellipsometry. The thickness of the polymer immobilized by the maleimide-thiol reaction was less than that formed by spin coating, except for polystyrene. Moreover, the polymer-immobilized surfaces were relatively smooth with a roughness of less than 1 nm. The amounts of amine, maleimide, and polymer immobilized on the surface were determined by quartz crystal microbalance measurements. The area occupied by the amine-containing silane coupling reagent was significantly less than the theoretical value, suggesting that a multilayer of the silane coupling reagent was formed on the surface. The polymer with low molecular weight had the tendency to efficiently immobilize on the maleimidated surface. When poly(p-acrylamidophenyl-α-mannoside)-immobilized surfaces were used as a platform for protein microarrays, strong interactions were detected with the mannose-binding lectin concanavalin A. The specificity of poly(p-acrylamidophenyl-α-mannoside)-immobilized surfaces for concanavalin A was compared with poly-l-lysine-coated surfaces. The poly-l-lysine-coated surfaces nonspecifically adsorbed both concanavalin A and bovine serum albumin, while the poly(p-acrylamidophenyl-α-mannoside)-immobilized surface preferentially adsorbed concanavalin A. Moreover, the poly(p-acrylamidophenyl-α-mannoside)-immobilized surface was applied to micropatterning with photolithography. When the micropattern was formed on the poly(p-acrylamidophenyl-α-mannoside)-spin-coated surface by irradiation with ultraviolet light, the pattern of the masking design was not observed on the surface adsorbed with fluorophore-labeled concanavalin A using a fluorescent microscope because of elution of poly(p-acrylamidophenyl-α-mannoside) from the surface. In contrast, fluorophore-labeled concanavalin A was only adsorbed on the shaded region of the poly(p-acrylamidophenyl-α-mannoside)-immobilized surface, resulting in a distinctive fluorescent pattern. The surface modification method using maleimidation and reversible addition-fragmentation chain transfer polymerization can be used for preparing platforms for microarrays and micropatterning of proteins.

Signal amplified two-dimensional photonic crystal biosensor immobilized with glyco-nanoparticles

A two-dimensional, glycopolymer-immobilized, photonic crystal (PhC) biosensor was developed for the detection of proteins. Glycopolymers with different conformations, homopolymers and sugar-incorporating nanoparticles were immobilized on the PhC using intermediate succinimide-containing polymers and proteins. The surface modification was analyzed in detail, and the sugar-protein interaction was detected by monitoring changes in the reflection intensity that was expressed by the two-dimensional PhC. The surface modifications were performed successfully, and specific interactions were detected between the glycopolymers and the proteins. Stronger bonds were present between the glycopolymers and the target proteins than between the glycopolymers and the monovalent sugar, because of a clustering effect. The sugar-incorporating nanoparticles showed a larger binding capacity compared with the homopolymers, and low protein concentrations (with a detection limit of 6.0 ng mL-1) were detected using the sugar-incorporating nanoparticle-immobilized PhC. The detection limit of the developed biosensor was lower than that of surface plasmon resonance sensor (1.43 μg mL-1). The results of this study indicated the potential of the developed biosensor for the detection of a variety of biomolecules.

Biotinylation of Silicon and Nickel Surfaces and Detection of Streptavidin as Biosensor

The availability of metal mesh device sensors has been investigated using surface-modified nickel mesh. Biotin was immobilized on the sensor surfaces consisting of silicon and nickel via a thiol-ene click reaction, known as the Michael addition reaction. Biotinylation on the maleimidated surface was confirmed by X-ray photoelectron spectroscopy. The binding of streptavidin to the biotinylated surfaces was evaluated using a quartz crystal microbalance and a metal mesh device sensor, with both techniques providing similar binding constant value. The recognition ability of the biotin immobilized using the thiol-maleimide method for streptavidin was comparable to that of biotin immobilized via several other methods. The adsorption of a biotin conjugate onto the streptavidin-immobilized surface via the biotin-streptavidin-biotin sandwich method was evaluated using a fluorescent microarray, with the results demonstrating that the biological activity of the streptavidin remained.

Metal mesh device sensor immobilized with a trimethoxysilane-containing glycopolymer for label-free detection of proteins and bacteria

Biosensors for the detection of proteins and bacteria have been developed using glycopolymer-immobilized metal mesh devices. The trimethoxysilane-containing glycopolymer was immobilized onto a metal mesh device using the silane coupling reaction. The surface shape and transmittance properties of the original metal mesh device were maintained following the immobilization of the glycopolymer. The mannose-binding protein (concanavalin A) could be detected at concentrations in the range of 10(-9) to 10(-6) mol L(-1) using the glycopolymer-immobilized metal mesh device sensor, whereas another protein (bovine serum albumin) was not detected. A detection limit of 1 ng mm(-2) was achieved for the amount of adsorbed concanavalin A. The glycopolymer-immobilized metal mesh device sensor could also detect bacteria as well as protein. The mannose-binding strain of Escherichia coli was specifically detected by the glycopolymer-immobilized metal mesh device sensor. The glycopolymer-immobilized metal mesh device could therefore be used as a label-free biosensor showing high levels of selectivity and sensitivity toward proteins and bacteria.

Novel carbonyl-group-containing dextran synthesis by pyranose-2-oxidase and dextransucrase.

The carbonyl polysaccharide, keto-dextran, was synthesized by the regioselective oxidation of sucrose and by the subsequent transfer reaction of the oxidized sucrose. The regioselective oxidation of sucrose was performed by bioconversion with pyranose-2-oxidase (EC 1.1.3.10). After 24h, the conversion percentage of sucrose into keto-sucrose was 100% as determined by a colorimetric method with dinitrophenylhydrazine. Converted keto-sucrose was polymerized to keto-dextran by dextransucrase (EC 2.4.1.5). Polymerization of keto-dextran was confirmed by the increase in molecular weight and amount of keto-dextran produced. The amount of keto-dextran produced decreased to 80% of the amount of dextran produced owing to the substrate recognition of DSase. From a Lineweaver-Burk reciprocal plot, the Michaelis constants for sucrose and keto-sucrose were 4.6 mmol L(-1) and 14.0 mmol L(-1), respectively. The keto-dextran had a carbonyl group in all glucose units.

Affinity Separation of Lectins Using Porous Membranes Immobilized with Glycopolymer Brushes Containing Mannose or N-Acetyl-d-Glucosamine

Porous membranes with glycopolymer brushes were prepared as biomaterials for affinity separation. Glycopolymer brushes contained acrylic acid and D-mannose or N-acetyl-D-glucosamine, and were formed on substrates by surface-initiated atom transfer radical polymerization. The presence of glycopolymer brush was confirmed by X-ray photoelectron spectroscopy, contact angle, and ellipsometry measurements. The interaction between lectin and the glycopolymer immobilized on glass slides was confirmed using fluorescent-labeled proteins. Glycopolymer-immobilized surfaces exhibited specific adsorption of the corresponding lectin, compared with bovine serum albumin. Lectins were continuously rejected by the glycopolymer-immobilized membranes. When the protein solution was permeated through the glycopolymer-immobilized membrane, bovine serum albumin was not adsorbed on the membrane surface. In contrast, concanavalin A and wheat germ agglutinin were rejected by membranes incorporating D-mannose or N-acetyl-D-glucosamine, respectively. The amounts of adsorbed concanavalin A and wheat germ agglutinin was increased five- and two-fold that of adsorbed bovine serum albumin, respectively.

Syntheses of sulfated glycopolymers and analyses of their BACE-1 inhibitory activity.

The glycopolymers for glycosaminoglycan mimic were synthesized, and the inhibitory effects of Alzheimers β-secretase (BACE-1) were examined. The regio-selective sulfation was conducted on N-acetyl glucosamine (GlcNAc), and the acrylamide derivatives were synthesized with the consequent sulfated GlcNAc. The glycopolymers were synthesized with acrylamide using radical initiator. The glycopolymer with sulfated GlcNAc showed the strong inhibitory effect on BACE-1, and the inhibitory effects were dependent on the sulfation positions. Especially, glycopolymers carrying 3,4,6-O-sulfo-GlcNAc showed the strong inhibitory effect. The docking simulation suggested that glycopolymers bind to the active site of BACE-1.

Dynamic rejection of colloidal particles with generating dextran by enzymatic reaction

Dextransucrase forms a complex with dextran during an enzymatic reaction with sucrose. Using its enzymatic character, we performed a continuous and dynamic rejection of colloidal particles by generating dextran with dextransucrase immobilized in an inorganic porous membrane. Inorganic membranes having 1.9 and 3.0 U/g of immobilized dextransucrase, and 4.1 and 9.4 mg/g of generated dextran, respectively, had constant rejection percentages for 55 and 100 nm colloidal particles in permeating solutions. On the other hand, permeating sucrose solutions containing colloidal particles through a dextran-immobilized membrane dynamically increased the rejection percentages of the colloidal particles owing to dextran generation via enzymatic reaction. The dynamic increase was due to the gradually generating dextran dynamically occupying the membrane pore with its steric volume.