A novel electrochemical biosensing method with double-layered polymer brush modified electrode.

We developed a novel electrochemical biosensor electrode that has a potential to reduce background noise for which we constructed an original conductive substrate modified with a double-layered polymer brush structure that is water impermeable and can control biomolecules adsorption/desorption. In this study, a hydrophobic poly(tert-butyl methacrylate) brush layer was prepared on a gold electrode, and then, the tert-butyl group near the outermost surface was dissociated by the acid treatment to obtain a hydrophilic carboxy group, thereby fabricating a conductive substrate with the double-layered polymer brush structure. Formation of the double-layered polymer brush structure was indicated by surface wettability and optical analyses. The potential difference and hydrogen ion concentration, which is a typical parameter of the surrounding environment, were linearly correlated with the gold electrode having a double-layered polymer brush structure with carboxyl groups. However, there was no correlation on gold electrodes with self-assembled monolayers presenting carboxy groups. It is considered that the pH responsiveness of the carboxy groups on the outermost surface could be exhibited remarkably because the charge state in the vicinity of the surface became constant due to the hydrophobic polymer brush layer having a certain thickness. The target DNA could be captured more efficiently at the probe DNA-immobilized electrode with the double-layered polymer brush structure than when using COOH-SAM. This is the first report of the application of the double-layered polymer brush structure for the electrochemical biosensing, and it will be an excellent surface modification method to reduce background noise.

Cellular Flocculation Driven by Concentrated Polymer Brush-Modified Cellulose Nanofibers with Different Surface Charges.

This study examined the effect of the surface charge of concentrated polymer brush (CPB)-grafted cellulose nanofibers (CNFs) on HepG2 cell flocculation. Four polyelectrolytes, poly(p-styrenesulfonic acid sodium salt) (PSSNa), poly(acrylic acid) (PAA), poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), and poly([(2-methacryloyloxy)ethyl]trimethylammonium chloride) (PMTAC), were grafted onto the CNF surface via surface-initiated atom transfer radical polymerization to form CNF-CPBs. The floc size of HepG2 cells depended on the surface charge of CNF-CPBs, where the anionic CNF-PSSNa formed larger flocs than CNF-PAA; due to the electrostatic repulsive forces, CNF-CPBs with a lower ζ-potential yielded smaller floc sizes. Contrastingly, the cytotoxic cationic CNF-PDMAEMA and CNF-PMTAC limited the floc size growth. Thus, appropriate electrostatic interactions are essential for floc formation and improved cell function in three-dimensional (3D) cell culture systems. Interestingly, while developing a novel 3D cell culture system, we reveal that colloidal flocculation theory is the driving mechanism behind this unique phenomenon.

Cellular Flocculation Using Concentrated Polymer Brush-Modified Cellulose Nanofibers with Different Fiber Lengths.

In this study, concentrated polymer brush-modified cellulose nanofibers (CNFs) with different fiber lengths were used for the flocculation of cells for systematically studying the mechanism of this unique cellular flocculation based on colloidal flocculation theory. Concentrated poly(p-styrenesulfonic acid sodium salt) brush-grafted CNF (CNF-PSSNa) with different fiber lengths were cultured with three different cell types to examine their influence on floc (cell clusters formed by cellular flocculation) characteristics. The floc size and survival rate could be controlled by modifying the CNF-PSSNa fiber lengths. The three cell types showed the same flocculation tendency after culture, indicating the applicability of the method in different cell lines. After 2 weeks of culture, CNF-PSSNa increased the specific expression of hepatocytes compared to the two-dimensional cell culture. Thus, owing to its wide applicability, high cell viability, and ability to control cell size and improve cell function, this technology could be used as a new three-dimensional cell culture method.

Concentrated polymer brush-modified cellulose nanofibers promote chondrogenic differentiation of human mesenchymal stem cells by controlling self-assembly.

In order to develop new three-dimensional (3D) cell culture systems for articular cartilage regeneration, concentrated poly(styrene sulfonate sodium salt) brush-modified cellulose nanofibers were employed as building blocks for the self-assembly of human mesenchymal stem cells (hMSCs). Unique 3D cellular structures, such as giant spheres and sheets, were formed by controlling hMSC self-assembly.

Characterization of a macroporous epoxy-polymer based resin for the ion-exchange chromatography of therapeutic proteins.

This study investigated the adsorption capacity and mass transfer properties of a novel macroporous epoxy-polymer-based anion-exchanger, MPR Q, for the efficient separation of therapeutic proteins. MPR Q resin was prepared by phase separation based on spinodal decomposition followed by dextran grafting and ligand conjugation. Under static conditions, MPR Q exhibited a binding capacity of 49.8 mg-IgG/cm3-resin at pH 10, whereas the fastest adsorption was observed among the anion-exchanger resins tested. Inverse size-exclusion chromatography (iSEC) experiments revealed that the apparent pore diameter of MPR Q was approximately 90 nm, which was sufficiently large for the penetration of human IgG and bovine IgM. Moreover, the reduced height equivalent to a theoretical plate, h, of human IgG, determined using the linear gradient elution method was 65.8 and was not significantly changed in the range of linear velocities from 20.37 to 50.93 cm/min. The dynamic binding capacity at 10% breakthrough of MPR Q, determined by frontal analysis, exhibited a capacity of 43.8 mg/cm3 at 5.09 cm/min and 58% of DBC10% was maintained even though the linear velocity was increased to 50.93 cm/min. Furthermore, a resolution for separation of IgG and BSA by MPR Q was 1.06 at 5.09 cm/min, while it was higher than that for the conventional resin at all linear velocities from 5.09 cm/min to 50.93 cm/min. Thus, it was suggested that the MPR Q developed in this study is a promising resin that can efficiently separate large biomacromolecules such as human IgG at higher velocities.

Nonbiofouling Coatings Using Bottlebrushes with Concentrated Polymer Brush Architecture.

Concentrated polymer brushes (CPBs) are known to suppress biofouling phenomena, such as protein adsorption and cell adhesion. However, a cumbersome process is needed for their synthesis. Here, we report a simple and versatile method for fabricating nonbiofouling coatings that uses well-defined bottlebrushes instead of CPBs. First, a macroinitiator, poly[2-(2-bromoisobutyryloxy)ethyl methacrylate] (PBIEM), was synthesized by reversible addition-fragmentation chain transfer polymerization. Then, poly[poly(ethylene glycol) methyl ether methacrylate] was grafted from PBIEM through atom transfer radical polymerization to form well-defined bottlebrushes. By controlling the graft chain length, two types of bottlebrushes could be prepared, namely those with a semi-dilute polymer brush (SDPB) structure or a CPB structure on the surface of the outermost layer. Crosslinked films of the bottlebrushes were prepared on silicon wafers by spin-coating and subsequent radical coupling. Importantly, the CPB-type bottlebrush films showed significantly better nonbiofouling characteristics than those of the SDPB-type bottlebrush films.

Well-defined monolith morphology regulates cell adhesion and its functions.

Hydrophilic epoxy resin-based monoliths were employed as cell culture substrates. The monoliths were made of a porous material with a bicontinuous structure that consisted of a porous channel and a resin skeleton. Monolith disks were prepared with a skinless surface through polymerization-induced spinodal decomposition-type phase separation. The pore sizes, which were well controlled by the polymerization temperature, ranged from 70 to 380 nm. The quantity of protein adsorbed per unit area and the early-stage adhesion of HepG2 cells on the monolith substrates were independent of pore size, meaning they were not affected by surface topology. Long-term cell adhesion, as indicated by adherent cell number and shape, as well as liver-specific gene expression were significantly affected by pore size. In terms of cell shape, number, and gene expression, pores of approximately 200 nm were most suitable for HepG2 cell growth. These results highlight the importance of monolith morphology for use as a cell culture substrate. The well-controlled morphology demonstrated in this work indicates monoliths are capable of supporting growth for various types of cells in a range of applications.

Facile Fabrication of Concentrated Polymer Brushes with Complex Patterning by Photocontrolled Organocatalyzed Living Radical Polymerization.

Photocontrolled surface-initiated reversible complexation mediated polymerization (photo-SI-RCMP) was successfully applied to fabricate concentrated polymer brushes with complex patterning structures. Positive-type patterned polymer brushes were obtained by photo-SI-RCMP under visible light (550(±50) nm) using photomasks. A particularly interesting finding was that negative-type patterned polymer brushes were also obtainable in a facile manner. A nonspecial UV light (250-385 nm) enabled the preparation of pre-patterned initiator surfaces in a remarkably short time (1 min), leading to negative-type patterned polymer brushes. Based on this unique selectivity between visible and UV light, the combination of two patterning techniques enabled the preparation of complex patterned brushes, including diblock copolymers, binary polymers, and functional binary polymers, without multistep immobilization of one or more initiators on the surfaces.

USAXS analysis of concentration-dependent self-assembling of polymer-brush-modified nanoparticles in ionic liquid: [I] concentrated-brush regime.

Using ultra-small angle X-ray scattering (USAXS), we analyzed the higher-order structures of nanoparticles with a concentrated brush of an ionic liquid (IL)-type polymer (concentrated-polymer-brush-modified silica particle; PSiP) in an IL and the structure of the swollen shell layer of PSiP. Homogeneous mixtures of PSiP and IL were successfully prepared by the solvent-casting method involving the slow evaporation of a volatile solvent, which enabled a systematic study over an exceptionally wide range of compositions. Different diffraction patterns as a function of PSiP concentration were observed in the USAXS images of the mixtures. At suitably low PSiP concentrations, the USAXS intensity profile was analyzed using the Percus-Yevick model by matching the contrast between the shell layer and IL, and the swollen structure of the shell and "effective diameter" of the PSiP were evaluated. This result confirms that under sufficiently low pressures below and near the liquid/crystal-threshold concentration, the studied PSiP can be well described using the "hard sphere" model in colloidal science. Above the threshold concentration, the PSiP forms higher-order structures. The analysis of diffraction patterns revealed structural changes from disorder to random hexagonal-closed-packing and then face-centered-cubic as the PSiP concentration increased. These results are discussed in terms of thermodynamically stable "hard" and/or "semi-soft" colloidal crystals, wherein the swollen layer of the concentrated polymer brush and its structure play an important role.

Characterization of Initial Cell Adhesion on Charged Polymer Substrates in Serum-Containing and Serum-Free Media.

Charged substrates are expected to promote cell adhesion via electrostatic interaction, but it remains unclear how cells adhere to these substrates. Here, initial cell adhesion (<30 min) was re-examined on charged substrates in serum-containing and serum-free media to distinguish among various cell adhesion mechanisms (i.e., electrostatic interaction, hydrophobic interaction, and biological interaction). Cationic and anionic methacrylate copolymers were coated on nonionic nontissue culture-treated polystyrene to create charged substrates. Cells adhered similarly on cationic, anionic, and nonionic substrates in serum-free medium via integrin-independent mechanisms, but their adhesion forces differed (anionic > cationic > nonionic substrates), indicating that cell adhesion is not mediated solely by the cells' negative charge. In serum-containing medium, the cells adhered minimally on anionic and nonionic substrates, but they adhered abundantly on cationic substrates via both integrin-dependent and -independent mechanisms. These results suggest that neither electrostatic force nor protein adsorption is accountable for cell adhesion. Conclusively, the observed phenomena revealed a gap in the generally accepted understanding of cell adhesion mechanisms on charged polymeric substrates. A reanalysis of their mechanisms is necessary.

Strategy for the Improvement of the Mechanical Properties of Cellulose Nanofiber-Reinforced High-Density Polyethylene Nanocomposites Using Diblock Copolymer Dispersants.

Cellulose nanofibers (CNFs) hold great potential as sustainable reinforcement fillers with excellent mechanical, thermal, and chemical properties. However, in polyolefin nanocomposite materials, the rational control of dispersion and the improvement of interfacial strength remain challenging. Herein we propose the tuning of the interface between CNF and high-density polyethylene by the design of polymer dispersants on the basis of surface free energy and the glass transition temperature. The former is related to the wettability against the polymer matrix and is therefore critical to the dispersion of CNF whereas the latter is related to the interfacial strength between CNF and HDPE. As a result of this investigation, we discovered a suitable dispersant for CNFs, poly(dicyclopentenyloxyethyl methacrylate)-block-poly(2-hydroxyethyl methacrylate), which played a pivotal role in achieving both a uniform dispersion of CNF and greatly improved mechanical properties, including a 4-fold increase of the Young's modulus over that of neat HDPE with 10 wt % CNF loading.

Surface Engineering of Cellulose Nanofiber by Adsorption of Diblock Copolymer Dispersant for Green Nanocomposite Materials.

An effective approach for the dispersion of hydrophilic cellulose nanofiber (CNF) in hydrophobic high-density polyethylene (HDPE) is presented using adsorption of a diblock copolymer dispersant. The dispersant consists of both resin compatible poly(lauryl methacrylate) (PLMA) and cellulose interactive poly(2-hydroxyethyl methacrylate) blocks. The PLMA-adsorbed CNFs are characterized by FT-IR and contact angle measurement, revealing successful hydrophobization. X-ray CT imaging shows there are apparently less CNF aggregates in the nanocomposites if adding amount of the dispersant was enough. The good dispersion results in a high mechanical reinforcement, corresponding to 140% higher Young's modulus and 84% higher tensile strength than the neat HDPE. This approach is broadly applicable and allows for easy manufacturing process for strong and lightweight CNF-reinforced nanocomposite materials.

Aligned 1-D nanorods of a π-gelator exhibit molecular orientation and excitation energy transport different from entangled fiber networks.

Linear π-gelators self-assemble into entangled fibers in which the molecules are arranged perpendicular to the fiber long axis. However, orientation of gelator molecules in a direction parallel to the long axes of the one-dimensional (1-D) structures remains challenging. Herein we demonstrate that, at the air-water interface, an oligo(p-phenylenevinylene)-derived π-gelator forms aligned nanorods of 340 ± 120 nm length and 34 ± 5 nm width, in which the gelator molecules are reoriented parallel to the long axis of the rods. The orientation change of the molecules results in distinct excited-state properties upon local photoexcitation, as evidenced by near-field scanning optical microscopy. A detailed understanding of the mechanism by which excitation energy migrates through these 1-D molecular assemblies might help in the design of supramolecular structures with improved charge-transport properties.

Conformational interchange of a carbohydrate by mechanical compression at the air-water interface.

Methyl xylopyranoside containing three 4-(pyrene-1-yl)benzoyl groups (PyXy) undergoes conformational interchange within a Langmuir monolayer upon mechanical compression. This xylose-type molecular machine PyXy was immobilized within two different matrix lipids, methyl stearate and methyl 2,3,4-tri-O-stearoyl-ß-D-xylopyranoside, which respectively form rigid and soft monolayers. Structural properties of the monolayer were characterized by assessing the compressibility, compression modulus, and ideal limiting molecular area of PyXy, all of which were estimated from the π-A isotherm measurements. Only the rigid monolayer exhibited a transition to the condensed phase with a limiting molecular area of PyXy smaller than that of the cross-sectional area of the xylopyranose ring in its C1 chair conformation. This suggests conformational interchange of PyXy from the most stable (4)C1 (C1) form to the metastable (1)C4 (1C) form. Surface-reflective fluorescence spectroscopy of the monolayer was applied to detect excimer emission resulting from the face-to-face dimerization of pyrenes attached at the O-2 and O-4 positions of xylose. Fluorescence intensity of the excimer increased abruptly in the condensed region only when the rigid monolayer was applied. These results indicate that the rigidity of the matrix monolayer is a critical aspect of the precise manipulation of molecular machines at interfaces. Consequently, this study demonstrates that including a molecular machine into a rigid lipid matrix is a promising means for the preparation of a novel nanoassembly with dynamic functionalities variable depending on a mechanical stimulus.

Light-harvesting nanorods based on pheophorbide-appending cellulose.

In contrast to the success in artificial DNA- and peptide-based nanostructures, the ability of polysaccharides to self-assemble into one-, two-, and three-dimensional nanostructures are limited. Here, we describe a strategy for designing and fabricating nanorods using a regioselectively functionalized cellulose derivative at the air-water interface in a stepwise manner. A semisynthetic chlorophyll derivative, pyro-pheophorbide a, was partially introduced into the C-6 position of the cellulose backbone for the design of materials with specific optical properties. Remarkably, controlled formation of cellulose nanorods can be achieved, producing light-harvesting nanorods that display a larger bathochromic shift than their solution counterparts. The results presented here demonstrate that the self-assembly of functionalized polysaccharides on surfaces could lead the nanostructures mimicking the naturally occurring chloroplasts.

Langmuir nanoarchitectonics: one-touch fabrication of regularly sized nanodisks at the air-water interface.

In this article, we propose a novel methodology for the formation of monodisperse regularly sized disks of several nanometer thickness and with diameters of less than 100 nm using Langmuir monolayers as fabrication media. An amphiphilic triimide, tri-n-dodecylmellitic triimide (1), was spread as a monolayer at the air-water interface with a water-soluble macrocyclic oligoamine, 1,4,7,10-tetraazacyclododecane (cyclen), in the subphase. The imide moieties of 1 act as hydrogen bond acceptors and can interact weakly with the secondary amine moieties of cyclen as hydrogen bond donors. The monolayer behavior of 1 was investigated through π-A isotherm measurements and Brewster angle microscopy (BAM). The presence of cyclen in the subphase significantly shifted isotherms and induced the formation of starfish-like microstructures. Transferred monolayers on solid supports were analyzed by reflection absorption FT-IR (FT-IR-RAS) spectroscopy and atomic force microscopy (AFM). The Langmuir monolayer transferred onto freshly cleaved mica by a surface touching (i.e., Langmuir-Schaefer) method contained disk-shaped objects with a defined height of ca. 3 nm and tunable diameter in the tens of nanometers range. Several structural parameters such as the disk height, molecular aggregation numbers in disk units, and 2D disk density per unit surface area are further discussed on the basis of AFM observations together with aggregate structure estimation and thermodynamic calculations. It should be emphasized that these well-defined structures are produced through simple routine procedures such as solution spreading, mechanical compression, and touching a substrate at the surface. The controlled formation of defined nanostructures through easy macroscopic processes should lead to unique approaches for economical, energy-efficient nanofabrication.

Chiroptical properties of an alternatingly functionalized cellotriose bearing two porphyrin groups.

Right-handedness derived from bisporphyrins attached to a cellotriose backbone at O-6 and O''-6 positions is revealed for the first time. This cellotriose is proposed as a model of alternatingly functionalized cellulosics, which have promising properties for applications in optoelectronics and molecular receptors owing to the chirality and rigid backbone effects.

Natural tubule clay template synthesis of silver nanorods for antibacterial composite coating.

Halloysite is naturally available clay mineral with hollow cylindrical geometry and it is available in thousands of tons. Silver nanorods were synthesized inside the lumen of the halloysite by thermal decomposition of the silver acetate, which was loaded into halloysite from an aqueous solution by vacuum cycling. Images of individual ca. 15 nm diameter silver nanorods and nanoparticles were observed with TEM. The presence of silver inside the tubes was also verified with STEM-EDX elemental mapping. Nanorods had crystalline nature with [111] axis oriented ~68° from the halloysite tubule main axis. The composite of silver nanorods encased in clay tubes with the polymer paint was prepared, and the coating antimicrobial activity combined with tensile strength increase was demonstrated. Coating containing up 5% silver loaded halloysite did not change color after light exposure contrary to the sample prepared with loading with unshelled silver nanoparticles. Halloysite tube templates have a potential for scalable manufacturing of ceramic encapsulated metal nanorods for composite materials.

Preparation of 6-azafulleroid-6-deoxy-2,3-di-O-myristoylcellulose.

6-Azafulleroid-6-deoxy-2,3-di-O-myristoylcellulose (3) was synthesized from 6-azido-6-deoxycellulose (1) by two reaction steps. The myristoylation of compound 1 with myristoyl chloride/pyridine proceeded smoothly to give 6-azido-6-deoxy-2,3-di-O-myristoylcellulose (2) in 97.0% yield. The reaction of compound 2 with fullerene (C(60)) was carried out by microwave heating to afford compound 3 in high yield. It was found from FT-IR, (13)C NMR, UV-vis, differential pulse voltammetry (DPV), SEC analyses that compound 3 was the expected C(60)-containing polymer. Consequently, maximum degree of substitution of C(60) (DS(C60)) of compound 3 was 0.33.