We designed glycopolymers carrying sialyl oligosaccharides by “post-click” chemistry and evaluated the interaction with the influenza virus. The glycopolymer structures were synthesized in a well-controlled manner by reversible addition–fragmentation chain transfer polymerization and the Huisgen reaction. Acrylamide-type monomers were copolymerized to give hydrophilicity to the polymer backbones, and the hydrophilicity enabled the successful introduction of the oligosaccharides into the polymer backbones. The glycopolymers with different sugar densities and polymer lengths were designed for the interaction with hemagglutinin on the virus surface. The synthesized glycopolymers showed the specific molecular recognition against different types of influenza viruses depending on the sugar units (6′- or 3′-sialyllactose). The sugar density and the polymer length of the glycopolymers affected the interaction with the influenza virus. Inhibitory activity of the glycopolymer against virus infection was demonstrated.

•Polymerization and phosphorylation of HEMA and PEGMA block copolymers.•Short phosphate segment in polymers shows high affinity to hydroxyapatite.•PEG brushes grafted on the surface inhibit protein adsorption and bacterial adhesion.Four types of phosphorylated 2-hydroxyethyl methacrylate and poly(ethylene glycol) methyl ether methacrylate (PEGMA) block copolymers were synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization and post-phosphorylation. These polymers were composed of different phosphate segments and similar PEG brushes. Polymers with defined phosphate segments were investigated to determine the optimal bonding affinity to hydroxyapatite (HAp). Polymers containing short phosphate segments (as low as 23 mer) were capable of immobilizing on HAp surfaces in situ in a short coating time with considerable durability. After surface modification, the dense PEG brushes altered the interfacial properties of HAp. The protein adsorption on the polymer-grafted HAp was drastically reduced compared with the bare HAp. Furthermore, the presence of the PEG brushes on the HAp surface resulted in bacterial inhibition. The polymer with the shortest phosphate segment (23 mer) showed superior inhibition ability.Download high-res image (158KB)Download full-size image

In this study, macroporous materials, called glycomonoliths, were produced from saccharide-containing monomers, and used for affinity bioseparation of proteins in a continuous-flow system. The porous structure formation of the glycomonoliths involved polymerization-induced phase separation of the polyacrylamide unit. The pore size could be controlled between several hundred nanometers and several micrometers by changing the alcohol used as the porogenic solvent during the preparation of the monolith. The glycomonolith pores allowed for the permeation of solutions through the monoliths, which meant that they could be used in a continuous-flow system. The adsorption capacities of the glycomonoliths for the saccharide-binding protein (concanavalin A) were larger than that of a glycopolymer-grafted material because of the higher saccharide densities in the monoliths than the grafted material. When concanavalin A was eluted from the glycomonolith, the concentration of concanavalin A in the effluent was up to 11 times higher than that in the feed solution. The adsorption of concanavalin A to the glycomonolith was specific, even in the presence of other proteins.

The catalytic activities of basic and thermoresponsive microgel particles for the Knoevenagel condensation reaction in water at ambient temperature were evaluated. The gel particles (GPs) were incorporated with primary-amino, tertiary-amino, imidazole or pyridyl groups. In the Knoevenagel condensation between benzaldehyde and ethyl cyanoacetate, the GPs that had no basic group did not exhibit any catalytic activity. The GPs that had primary-amino, tertiary-amino and imidazole groups exhibited catalytic activity. The catalytic activity of the basic GPs depended on their base strength. The tertiary-aminated microgel particles had quadruple and double the activities of tertiary-aminated silica gel and trimethylamine, respectively. The higher catalytic activity may be attributable to the local enrichment effect of ethyl cyanoacetate. In terms of gel geometry, the activity of the tertiary amines in the microgel particles was higher than that in the bulk gel. The geometry of the microgel particles allowed the rapid uptake of the substrates. Because the base strength of the tertiary amines in the microgel particles decreased with increasing temperature, the catalysts and residual active methylene substrates were separated by dialysis at 80 °C. The recovered microgel particle catalysts were recyclable for an additional Knoevenagel condensation.

Glycosaminoglycans (GAGs) are polysaccharides found in living systems that have key biological roles and function as polyelectrolytes owing to their large number of sulfate groups. There have been many reports describing the syntheses of GAGs and the development of GAG mimetics and analogs. The preparation of such GAG mimics has utilized versatile methods ranging from total syntheses to synthetic polymer chemistry approaches. The core of GAG mimetic production is the fusion of complex chemical structures with polymeric properties. Multivalent interactions of the saccharides with specific biological targets, such as proteins, are an essential function of GAGs and other multivalent saccharides. In this review, methods for generating GAGs from glycopolymers are presented and research reports describing the functional characterization of the synthesized GAGs are outlined.

Modification of the interface properties on hydroxyapatite and tooth enamel surfaces was investigated to fabricate bacterial resistance in situ. A series of copolymers containing pendants of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and ethylene glycol methacrylate phosphate (Phosmer) were polymerized by conventional free radical polymerization and changing the feed ratio of monomers. The copolymers were immobilized on hydroxyapatite and tooth enamel via the affinity of phosphate groups to hydroxyapatite to form the stable and durable polymer brushes on the surfaces. The amounts of polymer immobilized depended on the phosphate group ratio in the copolymers. Surface modification altered the interfacial properties of hydroxyapatite and inhibited bacterial adhesion. Copolymers containing 40–60% PEGMA segments showed a significant inhibitory effect on bacterial adhesion of S. epidermidis both in the presence and absence of plaque model biomacromolecules.Keywords: antibacterial; copolymers; hydroxyapatite; surface modification

The specific binding and uptake of protein molecules to individual hydrogel nanoparticles is measured with real-time single-nanoparticle surface plasmon resonance imaging (SPRI) microscopy. Nanoparticles that adsorb onto chemically modified gold thin films interact with traveling surface plasmon polaritons and create individual point diffraction patterns in the SPRI microscopy differential reflectivity images. The intensity of each point diffraction pattern depends on the integrated refractive index of the nanoparticle; an increase in this single nanoparticle point diffraction intensity (Δ%RNP) is observed for nanoparticles that bind proteins. SPRI adsorption measurements can be used to measure an average increase in Δ%RNP that can be correlated with bulk dynamic light scattering measurements. Moreover, the distribution of Δ%RNP values observed for individual nanoparticles can be used to learn more about the nature of the protein–nanoparticle interaction. As a first example, the binding of the lectin Concanavalin A to 180 nm N-Isopropylacrylamide hydrogel nanoparticles that incorporate a small percentage of mannose sugar monomer units is characterized.

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.

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.Keywords: bacteria; glycopolymer; label-free biosensor; metal mesh device; protein

We report the effect of physical properties, such as flexibility and polymer density, of nanogel particles (NPs) on the association/dissociation rates constant (kon and koff) and equilibrium constants (Kd) of multipoint protein recognition process. NPs having different flexibilities and densities at 25 °C were synthesized by tuning cross-linking degrees and the volume phase transition (VPT) temperature. Rate constants were quantified by analyzing time course of protein binding process on NPs monitored by a quartz crystal microbalance (QCM). Both kon and koff of swollen phase NPs increased with decreasing cross-linking degree, whereas cross-linking degree did not affect kon and koff of the collapsed phase NPs, indicating that polymer density of NPs governs kon and koff. The results also suggest that the mechanical flexibility of NPs, defined as the Young’s modulus, does not always have crucial roles in the multipoint molecular recognition process. On the other hand, Kd was independent of the cross-linking degree and depended only on the phase of NPs, indicating that molecular-scale flexibility, such as side-chain and segmental-mode mobility, as well as the conformation change, of polymer chains assist the formation of stable binding sites in NPs. Our results reveal the rationale for designing NPs having desired affinity and binding kinetics to target molecules.

The glycopolymers for glycosaminoglycan mimic were synthesized, and the inhibitory effects of Alzheimer’s 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.

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.

Herein we report that an aqueous solution of temperature-responsive micro- and nanogel particles (GPs) consisting of N-isopropylacrylamide (NIPAm) and N-[3-(dimethylamino)propyl]methacrylamide (DMAPM) reversibly absorbs and desorbs CO2 via a phase transition induced by cooling and heating cycles (30–75 °C). Below the phase-transition temperature, most of the amines in the swollen GPs are capable of forming ion pairs with absorbed bicarbonate ions. However, above the phase-transition temperature, shrinkage of the GPs lowers the pKa and the number of amine groups exposed to water, thereby resulting in almost complete desorption of CO2. The GPs can reversibly absorb more than the DMAPM monomer and polymer without NIPAm, which indicates the importance of the temperature-responsive phase transition of polymers in determining the degree of absorption. The results show the potential of temperature-responsive polymer solutions as absorbents to sequester CO2 at a low energy cost.

Although a number of procedures to create synthetic polymer nanoparticles (NPs) with an intrinsic affinity to target biomacromolecules have been published, little has been reported on strategies to control the binding kinetics of target recognition. Here, we report an enzyme-mimic strategy to control binding/dissociation rate constants of NPs, which bind proteins through multipoint interactions, by taking advantage of the temperature-responsive coil–globule phase transition of poly-N-isopropylacrylamide (PNIPAm)-based NPs. PNIPAm NPs with a “flexible” random-coil conformation had a faster binding rate than NPs with a “rigid” globule conformation; however, the dissociation rate constant remained unchanged, resulting in stronger affinity. The dissociation rate of the “flexible” NPs was decelerated by the “induced-fit”-type conformation change of polymers around the coil–globule phase transition temperature, resulting in the formation of the most stable NP–protein complexes. These results provide a guide for designing plastic antibodies with tailor-made binding kinetics and equilibrium constants.

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.Keywords: glycopolymer; lectin; membrane; QCM; silane coupling reagent; surface modification;

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.Keywords: maleimide; microarray; RAFT polymerization; surface modification; thiol;

This review addresses the design and synthesis of synthetic glycopolymers. Glycopolymers with pendant saccharides exhibit high affinities for proteins owing to their multivalency. Glycopolymers have molecular recognition abilities and amphiphilicity and can be applied as biomaterials and in bioassays. Most synthetic glycopolymers are prepared from polymerizable saccharide derivatives, and the rest are prepared by saccharide addition to a polymer backbone. Because sugar-recognizing proteins have precise structures, the living polymerization of glycopolymers is important in the fabrication of well-defined multiple ligands of saccharides. Glycopolymers with narrow polydispersity are useful for determining the interactions between saccharides and proteins. The living polymerization of glycopolymers provides hybrid materials by the terminal modification of the polymers. The glycopolymers of block polymers, star polymers and polymer brushes have been investigated for use in novel biomaterials.

The inhibitory effect of disaccharides and their polyvalent compounds toward aggregation of Aβ(1-40) was investigated. Polyvalent trehalose, maltose and lactose were synthesized, and their inhibitory effects were investigated together with those of the corresponding monomeric disaccharides. The inhibitory effects of the monomeric disaccharides were weak, but were amplified by multivalency. Trehalose and polyvalent trehalose induced formation of aggregates with specific pseudo-spherical morphology, which did not occur in the presence of the other disaccharides or in the absence of additives. Addition of polyvalent trehalose neutralized the cytotoxicity of Aβ, while monomeric trehalose induced weak cytotoxicity (toward HeLa cells). The specific inhibitory effects of trehalose and polyvalent trehalose on Aβ aggregation are discussed in the terms of the conformation of those molecules.

Interactions between proteins and biomaterial surfaces correlate with many important phenomena in biological systems. Such interactions have been used to develop various artificial biomaterials and applications, in which regulation of non-specific protein adsorption has been achieved with bioinert properties. In this research, we investigated the protein adsorption behavior of polymer brushes of dendrimer self-assembled monolayers (SAMs) with other generations. The surface adsorption properties of proteins with different pI values were examined on gold substrates modified with poly(amidoamine) dendrimer SAMs. The amount of fibrinogen adsorption was greater than that of lysozyme, potentially because of the surface electric charge. However, as the generations increased, protein adsorption decreased regardless of the surface charge, suggesting that protein adsorption was also affected by density of terminal group.Graphical abstractResearch highlights▶ Dendrimer SAM represented bioinert surface for protein adsorption using SPR analysis. ▶ Some correlations between amount of protein adsorption and pI of protein were found. ▶ As generation increased, the amount of proteins adsorption was decreased. ▶ Protein adsorption was affected by density of terminal group.

In this study, macroporous materials, called glycomonoliths, were produced from saccharide-containing monomers, and used for affinity bioseparation of proteins in a continuous-flow system. The porous structure formation of the glycomonoliths involved polymerization-induced phase separation of the polyacrylamide unit. The pore size could be controlled between several hundred nanometers and several micrometers by changing the alcohol used as the porogenic solvent during the preparation of the monolith. The glycomonolith pores allowed for the permeation of solutions through the monoliths, which meant that they could be used in a continuous-flow system. The adsorption capacities of the glycomonoliths for the saccharide-binding protein (concanavalin A) were larger than that of a glycopolymer-grafted material because of the higher saccharide densities in the monoliths than the grafted material. When concanavalin A was eluted from the glycomonolith, the concentration of concanavalin A in the effluent was up to 11 times higher than that in the feed solution. The adsorption of concanavalin A to the glycomonolith was specific, even in the presence of other proteins.

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.