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Background Nanosheets of SnO2 which an n-type semiconductor with a rutile-type crystalline structure are predominantly used as gas sensors. SnO2 nanosheets have a tetragonal crystal structure where growth along the c-axis is suppressed to form a sheet. The major exposed facets of SnO2 nanosheets have {110}, {101} and {211} crystal planes along the a-axis, with the reduced {110} surface having a particularly high surface energy. Identifying peptides that bind to specific crystal planes by using peptide phage-display approach will increase the potential applications of metal oxide nanomaterials by fusing proteins with desirable active sites to peptides that adsorb at high density on the major exposed crystal plane of nanosheets. It may be possible to construct highly sensitive biosensors. Objectives The main objective of the present study is to identify peptides that adsorb preferentially to a SnO2 nanosheet by using peptide-phage display approach. Methods Four milligrams of SnO2 nanosheet were mixed with 1011 plaque-forming units of Ph.D.-12 Phage Display Peptide Library. Phage-bound nanosheet particles were washed 10 times with 1 mL of phosphatebuffered saline containing 0.5% Tween 20. Phages bound to the nanosheet were eluted with three different buffers: (1) high-salt buffer containing 2 M NaCl (pH 7.5); (2) acidic buffer containing 200 mM Gly-HCl (pH 2.2); and (3) high-phosphate-ion buffer containing 500 mM NaH2PO4 (pH 7.5). The eluted phages were subjected to four or five rounds of biopanning. At each round, individual plaques were picked from the plates, and the amino acid sequences of the peptides were identified by DNA sequencing. The identified SnO2-binding peptides labeled with fluorescein isothiocyanate were synthesized. Adsorption isotherms were constructed at peptide concentrations ranging from 0.25 to 2.0 µM with 4mg of nanomaterials. Results We were determined the sequences of 11 clones with the high-salt buffer, 7 with the high-phosphateion buffers, and 6 with the acidic buffer and three peptides (SnO2BPn1, 2, and 3), two peptides (SnO2BPa1 and SnO2BPa2), and one peptide (SnO2BPp1) concentrated under each condition were selected respectively. All six selected peptides contained at least one histidine residue. In addition, the His-Asn-Leu (HNL) sequence was found in two of the peptides (SnO2BPa1 and SnO2BPa2). We constructed adsorption isotherms for the six selected peptides using 4mg of nanosheets. All six peptides were well adsorbed on the SnO2 nanosheet. The adsorption isotherms for SnO2 material with different structure revealed that SnO2BPn1, -2, and -3, and SnO2BPp1, preferentially bound to the spherical SnO2 nanoparticles. SnO2BPa2 preferentially bound to the SnO2 nanosheet, and SnO2BPa1 bound equally to both materials. This result suggested that SnO2BPa2 bound to a specific crystal plane of the nanosheet. The major exposed facet of the SnO2 crystal was the {110} plane, suggesting that SnO2BPa2 likely adsorbed on the {110} plane. SnO2BPn1, SnO2BPn2, SnO2BPn3, SnO2BPa1, and SnO2BPp1 also bound to the other metal oxides, in particular to ZrO2. At pH 7.5, peptides with a negative charge at pH 7.5 (pI 8.5-12) can bind to ZrO2 and SnO2, if the binding is mediated by electrostatic interactions. Thus, it is likely that these five peptides bind to metal oxides via electrostatic interactions. In contrast, SnO2BPa2 had a structurally specific affinity, binding more with the SnO2 nanosheet than with the spherical SnO2 nanoparticles or other metal oxides. Conclusion We identified six peptides that adsorbed on a SnO2 nanosheet. Five of the selected peptides bound preferentially to spherical SnO2 nanoparticles rather than to the SnO2 nanosheet. Whereas, SnO2BPa2 exhibited specifically binding to the SnO2 nanosheet. Our results suggest that crystal plane recognition and material recognition by these peptides are mediated via different, independent mechanisms.

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.

A capillary electrophoretic reactor was used to analyze the dissociation kinetics of an enzyme–inhibitor complex in a homogeneous solution without immobilization. The complex consisting of trypsin (Try) and aprotinin (Apr) was used as the model. Capillary electrophoresis provided a reaction field for Try–Apr complex to dissociate through the steady removal of free Try and Apr from the Try–Apr zone. By analyzing the dependence of peak height of Try–Apr on separation time, the dissociation rate kdH was obtained as 2.73 × 10−4 s−1 (298 K) at pH 2.46. The dependence of kdH on the proton concentration (pH = 2.09–3.12) revealed a first-order dependence of kdH on [H+]; kdH = kd + k1[H+], where kd is the spontaneous dissociation rate and was 5.65 × 10−5 s−1, and k1 is the second-order rate constant and was 5.07 × 10−2 M−1 s−1. From the kd value, the half-life of the Try–Apr complex at physiological pH was determined as 3.4 h. The presence of the proton-assisted dissociation can be explained by the protonation of -COO– of the Asp residue in Try, which breaks the salt bridge with the –NH3+ group of Lys in Apr.

The interaction between biomolecules and inorganic materials has gained much interest for the development of sensors and highly controlled materials in scale and composition. For this purpose, 4F2,...

The grating substrate covered with a metal layer, a plasmonic chip, and a bispecific antibody can play a key role in the sensitive detection of a marker protein with an immunosensor, because of the provision of an enhanced fluorescence signal and the preparation of a sensor surface densely modified with capture antibody, respectively. In this study, one of the tumor markers, a soluble epidermal growth factor receptor (sEGFR), was selected as the target to be detected. The ZnO- and silver-coated plasmonic chip with precise regularity and the appropriate duty ratio in the periodic structure further enhanced the fluorescence intensity. As for sensor surface modification with capture antibody, a bispecific antibody (anti-sEGFR and anti-ZnO antibody), the concentrated bispecific antibody solution was found to nonlinearly form a surface densely immobilized with antibody, because the binding process of a bispecific antibody to the ZnO surface can be a competitive process with adsorption of phosphate. As a result, the interface on the plasmonic chip provided a 300× enhanced fluorescence signal compared with that on a ZnO-coated glass slide, and therefore sEGFR was found to be quantitatively detected in a wide concentration range from 10 nM to 700 fM on our plasmonic surface.

The construction of antibody fragments has the potential to reduce the high cost of therapeutic antibody production, but the structures of these fragments, with monovalency and the lack of an Fc region, can lead to reduced function. Multimerization is one strategy for recovering function that also yields better tumor-to-blood ratios than IgGs or monomeric antibody fragments because of rapid tumor uptake and clearance. Here, we constructed single-chain variable fragment (scFv) multimers by modifying the linker length and domain order of humanized anti-(epidermal growth factor receptor) IgG 528 (h528) and tested their ability to inhibit tumor growth. h528 scFv multimers, expressed using a bacterial expression system, were successfully fractionated and inhibited cancer growth in a multimerization-dependent manner, whereas the h528 scFv monomer showed no inhibition. h528 scFv trimers with variable heavy-light domain order and no linkers showed the highest in vitro and in vivo antitumor effects, which were comparable with those of the approved anti-(epidermal growth factor receptor) therapeutic IgG Cetuximab and Panitumumab. The trimers were also structurally stable in vitro and in vivo, which may be attributable to a strong interaction between the variable heavy and variable light domains of h528 Fv. Thus, h528 scFv multimers, especially trimers, are attractive as the next generation of anti-(epidermal growth factor receptor) therapeutic IgG and offer the possibility of low-cost production.

Cellulosomes, which are assemblies of cellulases with various catalytic functions on a giant scaffoldin protein with a carbohydrate-binding module (CBM), efficiently degrade solid cellulosic biomass by means of synergistically coupled hydrolysis reactions. In this study, we constructed hybrid nanocellulosomes from the biotinylated catalytic domains (CDs) of two catalytically divergent cellulases (an endoglucanase and a processive endoglucanase) and biotinylated CBMs by clustering the domains and modules on streptavidin-conjugated nanoparticles. Nanocellulosomes constructed by separately clustering each type of CD with multiple CBMs on nanoparticles showed 5-fold enhancement in cellulase degradation activity relative to that of the corresponding free CDs, and mixtures of the two types of nanocellulosomes gradually and synergistically enhanced cellulase degradation activity as the CBM valency increased (finally, 2.5 times). Clustering the two types of CD together on the same nanoparticle resulted in a great...

We present electric field-based in-vitro control of intracellular protein nanowires, so-called microtubules, using a pair of metallic glass (Pd 42.5 Cu 30 Ni 7.5 P 20 ) microwire electrodes. We can reversibly control the spatio-temporal localization, orientation, as well as guided translocation of microtubules suspended in a buffer solution. Using freestanding wire electrodes to localize microtubules allows for an assembly of nanowires in a 0 or 1-dimensional environment, with the potential of producing more complex geometries. This is of particular interest where bio-mimetic applications require control beyond planar devices.

Microtubules play an important role in intraneuronal communication. A barrier to microtubule mediated neuronal transport can initiate dementia. The nanoscale modeling of these biological events is expected to be useful for healthcare applications and auxiliary biosensor based diagnostics. Here, we report a model system by using an in vitro bio-motility assay to predict events related to dementia at excess metal ion concentration. We demonstrate how metal ions (calcium, iron, zinc) at pathophysiologically relevant concentrations have the ability to affect the gliding motion of microtubules on kinesin. The addition of the neurotransmitter (acetyl choline) has no apparent effect on MT gliding. Therefore, the reagents that can stabilize the bio-motility system have potential to regulate dementia.

Background Biological applications of nanoparticles are rapidly increasing, which introduces new possibilities to improve the efficacy of radiotherapy. Here, we synthesized titanium peroxide nanoparticles (TiOxNPs) and investigated their efficacy as novel agents that can potently enhance the effects of radiation in the treatment of pancreatic cancer. Methods TiOxNPs and polyacrylic acid-modified TiOxNPs (PAA-TiOxNPs) were synthesized from anatase-type titanium dioxide nanoparticles (TiO2NPs). The size and morphology of the PAA-TiOxNPs was evaluated using transmission electron microscopy and dynamic light scattering. The crystalline structures of the TiO2NPs and PAA-TiOxNPs with and without X-ray irradiation were analyzed using X-ray absorption. The ability of TiOxNPs and PAA-TiOxNPs to produce reactive oxygen species in response to X-ray irradiation was evaluated in a cell-free system and confirmed by flow cytometric analysis in vitro. DNA damage after X-ray exposure with or without PAA-TiOxNPs was assessed by immunohistochemical analysis of γ-H2AX foci formation in vitro and in vivo. Cytotoxicity was evaluated by a colony forming assay in vitro. Xenografts were prepared using human pancreatic cancer MIAPaCa-2 cells and used to evaluate the inhibition of tumor growth caused by X-ray exposure, PAA-TiOxNPs, and the combination of the two. Results The core structures of the PAA-TiOxNPs were found to be of the anatase type. The TiOxNPs and PAA-TiOxNPs showed a distinct ability to produce hydroxyl radicals in response to X-ray irradiation in a dose- and concentration-dependent manner, whereas the TiO2NPs did not. At the highest concentration of TiOxNPs, the amount of hydroxyl radicals increased by >8.5-fold following treatment with 30 Gy of radiation. The absorption of PAA-TiOxNPs enhanced DNA damage and resulted in higher cytotoxicity in response to X-ray irradiation in vitro. The combination of the PAA-TiOxNPs and X-ray irradiation induced significantly stronger tumor growth inhibition compared to treatment with either PAA-TiOxNPs or X-ray alone (p

Production of various combinations of bispecific variable domain of heavy chain of heavy chain-only antibody (VHH) constructs to evaluate their therapeutic potential usually requires several gene-engineering steps. Here, we present an alternative method of creating bispecific VHH constructs in vivo through protein trans-splicing (PTS) reaction; this method may reduce the number of gene manipulation steps required. As a proof-of-concept, we constructed a bispecific antibody (bsAb) containing an anti-epidermal growth factor receptor VHH and anti-green fluorescent protein VHH, and we evaluated and confirmed its bispecificity. We also tested antibody labeling by fluorescent protein tagging using the PTS reaction. Compared with the conventional gene construction method, bsAb construction via PTS is a promising alternative approach for generating multiple bsAb combinations.

Production of various combinations of bispecific variable domain of heavy chain of heavy chain-only antibody (VHH) constructs to evaluate their therapeutic potential usually requires several gene-engineering steps. Here, we present an alternative method of creating bispecific VHH constructs in vivo through protein trans-splicing (PTS) reaction; this method may reduce the number of gene manipulation steps required. As a proof-of-concept, we constructed a bispecific antibody (bsAb) containing an anti-epidermal growth factor receptor VHH and anti-green fluorescent protein VHH, and we evaluated and confirmed its bispecificity. We also tested antibody labeling by fluorescent protein tagging using the PTS reaction. Compared with the conventional gene construction method, bsAb construction via PTS is a promising alternative approach for generating multiple bsAb combinations.

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

We have realized chemical recovery of a phenolic compound, 2-(hydroxy-benzyl)-4-methyl-phenol (BMP), from organosolv lignin without forming char in a mixture of supercritical water and p-cresol (Okuda, K.; et al. J. Phys.: Condens. Matter2004, 16, S1325). In this paper, we evaluated the reaction rate constants of the depolymerization process of organosolv lignin using Monte Carlo simulation. We also evaluated the formation and decomposition rate of BMP based on the proposed reaction scheme. The obtained reaction rate constants gave insight into the mechanism why the mixture of supercritical water and p-cresol suppressed the formation of char. They also suggested the reaction path for the formation of BMP. In addition, these reaction rate constants enable the design and optimization of the chemical recovery processes to realize the highest yield.

We report that several tryptophan-rich peptides exhibit an affinity for a hydrophobic ionic liquid (IL) (1-ethyl-3-methylimidazolium bis-trifluoromethanesulfonyl imide), and that green fluorescent protein (GFP) fused to a peptides, “SSSWWSWWWW” (SW1) or “SWWWWSWWWW” (SW2), containing serine (S) and tryptophan (W) at the C terminus localized at the IL/water interface. While GFPs without W-rich peptide distributed only in water phase, SW1- and SW2-GFPs were accumulated at the interface. The localization of SW1-GFP showed biphasic behavior, and most distinctive localization was observed at 7.1 μM. The localization of SW2-GFP presumably occurred at largely lower concentration (≤ 0.5 μM) than that of SW1-GFP, which difference was due to the higher hydrophobicity of SW2 peptide.

We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR × anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity-determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single-chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti-EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.

This review reports fabrication methods for ordered metallic nanostructures such as nanowires and nanoparticles based on deoxyribonucleic acid (DNA) templates. The phosphate groups in DNA are negatively charged; consequently, the DNA conformation may mineralize metals, e.g., palladium (Pd) at a relatively high metal concentration. We successfully form unique spherically shaped moss-like hybrid Pd nanoparticles using the small compacted globular state of DNA by controlling the reductive reaction. Pd can absorb hydrogen to become PdHx, and hydrogen storage increases the electrical resistance and volume of Pd materials. Hence, the use of this material is attracting growing interest as a reliable, cheap, ultracompact, and safe hydrogen sensor. Pd–DNA hybrid nanoparticles can be used as highly sensitive hydrogen sensors, which exhibit a switch response that depends on the volume expansion in a cyclic atmosphere exchange. This paper also shows the fabrications of Pd–carbon nanotube (CNT) hybrid nanostructures.

A plasmonic biosensor chip of silver-coated PMMA grating with a zinc oxide (ZnO) overlayer is fabricated for surface plasmon field-enhanced fluorescence (SPF) detection of Cy5-labeled green fluorescent protein (GFP). A bispecific antibody (anti-GFP x anti-ZnO antibody) prepared in our lab is densely immobilized on the sensor chip for GFP detection. The sensitivity of the plasmonic biosensors is improved due to densely packed antibodies and ZnO-coating that suppresses nonspecific protein adsorption and fluorescent quenching. With the ZnO-coated plasmonic chip, Cy5-labeled GFP of 10 pM can be detected through SPF. This sensitivity is 100 higher compared with the normal fluorescent detection on a ZnO-coated glass slide.

The assembly of metal oxide nanoparticles (NPs) on a biomolecular template by a one-pot hydrothermal synthesis method is achieved for the first time. Magnetite (Fe3O4) nanoneedles (length: ~100 nm; width: ~10 nm) were assembled on cyclic-diphenylalanine (cFF) nanorods (length: 2–10 μm; width: 200 nm). The Fe3O4 nanoneedles and cFF nanorods were simultaneously synthesized from FeSO4 and l-phenylalanine by hydrothermal synthesis (220 °C and 22 MPa), respectively. The samples were analyzed by powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (IR), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Experimental results indicate that Fe3O4 nanoneedles were assembled on cFF nanorods during the hydrothermal reaction. The composite contained 3.3 wt% Fe3O4 nanoneedles without any loss of the original magnetic properties of Fe3O4.

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.

Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self-assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO-binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO-binding peptide, 125 spot-synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO-binding sites were found as 6-mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles.

We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy.

The conjugation of polyethylene glycol (PEGylation) with downsized compact antibodies is an effective method for overcoming the problem of rapid elimination of the compact antibodies from the body. We integrated site-specific PEGylation with the refolding of a single-chain Fv (scFv) of humanized monoclonal antibody 528 with affinity for the epidermal growth factor receptor, to prepare active PEGylated scFv from insoluble aggregates produced in an Escherichia coli expression system. The insertion of a cysteine residue at the C-terminus of scFv to serve as the conjugation site for PEG led to the formation of highly multimeric scFv during the refolding process; however, PEGylation after refolding drastically dispersed the multimer into monomeric active scFv fragments. Further, the PEGylation of partially refolded scFv during the refolding process improved the PEGylation efficiency and suppressed the formation of highly multimeric scFv; consequently, monomeric active scFv fragments were obtained directly from the insoluble aggregates in E. coli. We show that in vitro refolding of PEGylated scFv should be useful for improving downstream processing performance in the production of clinically useful small antibodies from insoluble fractions.

A novel method that enables antibody fragments to be immobilized on a sensor substrate with a high binding capability using molecular recognition has been developed. Using genetic engineering, we fabricated bispecific recombinant antibody fragments, which consist of two kinds of antibody fragments: a gold antibody fragment and a target molecule antibody fragment. Surface plasmon resonance (SPR) analysis indicated that these gold-binding bispecific antibody fragments bind directly to the gold substrate with high affinity (K(D) approximately 10(-9) M). About 70% of the bispecific antibody fragments immobilized on the gold substrate retained their target protein-binding efficiency. The Sips isotherm was used to assess the heterogeneity in antibody affinity for the bispecific antibody fragments. The results showed that the immobilized bispecific antibody fragments exhibited an increased homogeneity of affinity (K(D)) to target molecules when compared with monospecific antibody fragments immobilized by conventional methods. The use of bispecific antibody fragments to directly immobilize antibody fragments on a solid-phase substrate offers a useful platform for immunosensor applications.

Antibodies and their fragments are attractive binding proteins because their high binding strength is generated by several hypervariable loop regions, and because high-quality libraries can be prepared from the vast gene clusters expressed by mammalian lymphocytes. Recent explorations of new genome sequences and protein structures have revealed various small, nonantibody scaffold proteins. Accurate structural descriptions of protein-protein interactions based on X-ray and NMR analyses allow us to generate binding proteins by using grafting and library techniques. Here, we review approaches for generating binding proteins from small scaffold proteins on the basis of tertiary structural information. Identification of binding sites from visualized tertiary structures supports the transfer of function by peptide grafting. The local library approach is advantageous as a go-between technique for grafted foreign peptide sequences and small scaffold proteins. The identification of binding sites also supports the construction of efficient libraries with a low probability of denatured variants, and, in combination with the design for library diversity, opens the way to increasing library density and randomized sequence lengths without decreasing density. Detailed tertiary structural analyses of protein-protein complexes allow accurate description of epitope locations to enable the design of and screening for multispecific, high-affinity proteins recognizing multiple epitopes in target molecules.

Recent advances in molecular evolution technology enabled us to identify peptides and antibodies with affinity for inorganic materials. In the field of nanotechnology, the use of the functional peptides and antibodies should aid the construction of interface molecules designed to spontaneously link different nanomaterials; however, few material-binding antibodies, which have much higher affinity than short peptides, have been identified. Here, we generated high affinity antibodies from material-binding peptides by integrating peptide-grafting and phage-display techniques. A material-binding peptide sequence was first grafted into an appropriate loop of the complementarity determining region (CDR) of a camel-type single variable antibody fragment to create a low affinity material-binding antibody. Application of a combinatorial library approach to another CDR loop in the low affinity antibody then clearly and steadily promoted affinity for a specific material surface. Thermodynamic analysis demonstrated that the enthalpy synergistic effect from grafted and selected CDR loops drastically increased the affinity for material surface, indicating the potential of antibody scaffold for creating high affinity small interface units. We show the availability of the construction of antibodies by integrating graft and evolution technology for various inorganic materials and the potential of high affinity material-binding antibodies in biointerface applications.

In this study, we report the synthesis of uniform and narrowly size-distributed ZnO nanoparticles with sizes of approximately 3 nm; the nanoparticles were prepared by means of organic-ligand-assisted hydrothermal conditions with various organic modifiers. The results obtained herein revealed that among the various functional groups tested (alcohols, aldehydes, carboxylic acids, and amines), only hexanol effectively controlled the nucleation and crystal growth of spherical ZnO nanoparticles. The use of hexanol also caused the surface of the ZnO particles to change from hydrophilic to hydrophobic, which would enhance the dispersion of these particles in polymer matrices, paints, cosmetics, and other organic application media.

We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 (hEx3-Db) for cancer immunotherapy. Bacterial expression can be used to express small recombinant antibodies on a large scale; however, their overexpression often results in the formation of insoluble aggregates, and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography. Here, we propose a novel method for preparing refined, functional, tag-free bi-specific diabodies from IgG-like bi-specific antibodies (BsAbs) in a mammalian expression system. We created an IgG-like BsAb in which bi-specific diabodies were fused to the human Fc region via a designed human rhinovirus 3C (HRV3C) protease recognition site. The BsAb was purified by protein A affinity chromatography, and the refined tag-free hEx3-Db was efficiently produced from the Fc fusion format by protease digestion. The tag-free hEx3-Db from the Fc fusion format showed a greater inhibition of cancer growth than affinity-tagged hEx3-Db prepared directly from Chinese hamster ovary cells. We also applied our novel method to another small recombinant antibody fragment, hEx3 single-chain diabody (hEx3-scDb), and demonstrated the versatility and advantages of our proposed method compared with papain digestion of hEx3-scDb. This approach may be used for industrial-scale production of functional tag-free small therapeutic antibodies.

We prepared palladium–polyelectrolyte hybrid nanoparticles by using a metallization of polyelectrolyte. In this study, we selected polyacrylic acid (PAA) as a polyelectrolyte and reduced the palladium ions on the PAA by using ascorbic acids in order to form a unique spherically shaped mosslike hybrid nanoparticle. Palladium (Pd) can absorb hydrogen to become PdHx, and the storage of hydrogen increases the electrical resistance and volume of Pd materials. The use of this material is attracting growing interest as a reliable, cheap, ultracompact, and safe hydrogen sensor for use in fuel cells. We showed the utilization of the Pd–PAA hybrid nanoparticles as a highly sensitive hydrogen sensor that exhibited a switch response depending on volume expansion in a cyclic atmosphere exchange.

The interface molecules designed to exhibit molecular recognitions between different species have become attractive tools for the bottom-up fabrication and hybridization of nanostructured units. Here, we focus on antibodies with high binding ability and specificity to construct a novel biomolecule interface for recognizing an inorganic material. Careful selection from a phage-displayed library of variable region heavy and light Fv chains of human antibodies using enzyme-linked immunosorbent assay and surface plasmon resonance assay resulted in the identification of an antibody fragment, A14P-b2, with high affinity (KD = 1.7 nm) and specificity for gold materials. Our results indicated the potential usefulness of human antibody libraries and the effectiveness of the antibody framework for recognizing bulk material surfaces. Construction of bivalent and bispecific antibodies on the A14P-b2 platform with high affinity by means of fusion technology enabled the functionalization of gold nanoparticles and allowed selective protein accumulation on gold spots patterned on a silicon substrate. This type of antibody engineering is potentially applicable to bio-inspired materials and nanobiosensing.

Recently, we selected the antibody fragment with high affinity for the biopolymer film of polyhydroxybutyrate (PHB) from human antibody fragment libraries. In this study, we functionalized CdSe quantum dot (QD) nanoparticles by orderly conjugating the anti-PHB antibody fragments to perform spontaneous and selective stacking of inorganic particles on PHB-coated plates in neutral solutions at room temperature. Surface plasmon resonance analysis showed that the orderly clustering of anti-PHB antibody fragment on QD particles led to no dissociation of QD on PHB-coated plates, indicating the availability of avidity effect. The strong spontaneous immobilization using biomolecular recognition enabled stepwise stacking of inorganic particles on PHB-coated plates only by mixing operation in neutral solutions at room temperature. We show the potential of recombinant anti-material antibody fragments for the bottom-up stacking procedures for hybrid assembly.

We report the utility of in vitro refolding in the preparation of monomorphous hEx3 bispecific diabodies with epidermal growth factor receptor and CD3 retargeting from insoluble aggregates in Escherichia coli. Appropriate interaction between cognate variable heavy and light chains led to the formation of functional hEx3 heterodimers in a diabody format rather than inactive homodimers. The refolded hEx3 was found to exhibit almost the equivalent activity to the hEx3 and single-chain hEx3 (hEx3-scDb) prepared in a mammalian secretion system. We suggest that the preparation of hEx3 from bacterial insoluble material by means of in vitro refolding would be useful for industrial-scale production of the diabody for its potential use in clinical studies.

Synthesis and in situ surface modification of copper manganese oxide nanoparticles have been carried out under supercritical water conditions. Flow type and batch type reactors were used in this study. Effect of different reactors on the phase formation was studied in detail. The addition of organic modifier into the reaction system has significant influence on the particle size, morphology and the dispersion of nanocrystal. The particle size was greatly reduced and single nanocrystal particles were formed by the in situ surface modification of nanoparticles with organic reagents during the synthesis. The surface modified particles showed a very high surface area when compared with unmodified particles. The particles are dispersed in a binary solvent system where organic-modified particles are dispersed in the upper organic solvent medium. In contrast, the particles without surface modification are settled down in the water phase.

The aim of the study was to gain insight into the role of the each unit of lignin in the formation of products. Glycerol, guaiacol, the mixture of glycerol and guaiacol (Gly&Gua), and guaiacylglycerol-β-guaiacyl ether (GGGE) were used as lignin model compounds to study fragmentation of lignin in an excess of water and p -cresol at 400 °C. The products have been analyzed employing gas chromatography (GC)–mass spectrometer (MS) and gas chromatography-frame ionization detector for qualitative and quantitative analysis. GC–MS analysis indicated that phenol, o -cresol, methyl-anisole, di-methyl-phenol, ethyl-methyl-phenol, 2-(hydroxy-benzyl)-4-methyl-phenol (BMP) and 2-(2-hydroxy-5-methyl-benzyl)-4-methyl-phenol were formed. The products were similar to the products by the fragmentation of lignin. The products, except o -cresol, were primarily derived from glycerol and/or guaiacol. We also found that the amount of BMP derived from GGGE, which has glycerol unit and guaiacol unit in its structure, is much more than that derived from Gly&Gua. The increase of the BMP yield by reaction with GGGE compared with (glycerol and/or guaiacol) indicates that guaiacylglycerol unit with linkage of Gly&Gua plays an important role in the formation of BMP and also it is suggested that the BMP formation from GGGE has pathways other than that from Gly&Gua.

Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials.

Continuous production of highly crystalline ZnO nanorods by supercritical hydrothermal synthesis was reported in this article. Zinc nitrate aqueous solution was pressurized to 30 MPa at room temperature and rapidly heated to 673 K by mixing with supercritical water and then fed into a tubular reactor. Residence time is about 10 s. Production of ZnO nanorod particles with uniform particle size distribution showed a strong ultraviolet light emission at room temperature. This article also reported in-situ surface modification of ZnO nanorods with organic reagents by the supercritical hydrothermal synthesis.

The DNA, which forms a double helical conformation by the highly selective base-pairing rule with the width of 2 nm, is one of the polymers utilized as a template for nanoparticle assembly. Then, holiday junction structure, where two different DNA double strands intersect and replace each complement strand, is an important artificial motif for forming two-dimensional mesh-like DNA morphology. In this study, the two-dimensional sheet with the mesh-like conformation is formed and the growth of the DNA morphology is controlled by changing the temperature in the formation process. The influence of the cooling procedure on the formation process of the two-dimensional DNA sheet is observed by atomic force microscope, and the formation of double strands in the process is traced by absorption spectra. The spectroscopic results demonstrate that the two-dimensional DNA sheet is strongly dependent on the correlation between the applied cooling process and the melting temperature of double helical conformation, and that the growth of the two-dimensional DNA sheet can be manipulated by the cooling procedure. Further, we will show the size control of the DNA sheet by mixing the designed DNA which inhibits the growth of the DNA sheet.

We synthesized surface-modified iron oxide nanoparticles in aqueous phase by heating an aqueous solution of iron sulfate (FeSO 4 ) at 473 K with a small amount of either n -decanoic acid (C 9 H 19 COOH) or n -decylamine (C 10 H 21 NH 2 ), which is not miscible with water at room temperature. Transmission electron microscopy showed that the addition of n -decanoic acid or decylamine changed the shape of the obtained nanoparticles. X-ray diffraction spectra revealed that the synthesized nanoparticles were in α-Fe 2 O 3 or Fe 3 O 4 phase while Fourier transform infrared spectroscopy and thermogravimetry indicated the existence of an organic layer on the surface of the nanoparticles. In the synthetic condition, decreased dielectric constant of water at higher temperature increased the solubility of n -decanoic acid or n -decylamine in water to promote the reaction between the surface of iron oxide nanoparticles and the organic reagents. After the synthesis, the used organic modifiers separated from the aqueous phase at room temperature, which may help the environmentally benign synthesis of surface-modified metal oxide nanoparticles.

We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgG1 (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1-100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced antitumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense antitumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed.

Fine BaTiO3 nanoparticles were prepared by hydrothermal synthesis under supercritical condition (400 °C and 30 MPa) from mixture of barium hydroxide and titanium dioxide as starting precursors. First, conditions for synthesizing BaTiO3 were examined by using batch reactors. High pH condition, pH > 13, is necessary to obtain phase pure BaTiO3. The reason was discussed based on the solubility of titanium dioxide, which that dissolution–recrystallization process is essential for the synthesis of BaTiO3 nanoparticles. Rapid heating of the starting precursors by mixing with high temperature water in a flow reactor is effective to synthesize smaller size and narrower particle size distribution for the BaTiO3 nanoparticles, compared with the case of slow heating with a batch reactor.

In situ surface modification of boehmite (AlOOH) nanoparticles during hydrothermal synthesis in supercritical water was examined by adding CH3(CH2)4CHO and CH3(CH2)5NH2 as modifier reagents to the reactants. Changes in surface properties of the nanoparticles by surface modification was observed by FTIR, dispersion in solvents and TEM analyses, which demonstrated that reagents chemically binded onto the surface of the AlOOH nanoparticles. The results of SEM and TEM pictures show that the surface modification affects crystal growth and reduces the particle size and changes the morphology of the particles.

Titanium oxide (TiO2) nanocyrstals which are perfectly dispersed in organic solvents are synthesized by organic-ligand-assisted supercritical hydrothermal synthesis. The addition of hexaldehyde to the supercritical hydrothermal synthesis of TiO2 leads to the in-situ surface modification, which enables the synthsized TiO2 nanocrystals to be perfectly dispersed in iso-octane because of its hydrophobic nature. Further, the one-pot synthesis of hybrid materials results in the significant reduction of the particles size, probably due to the capping effect of hexaldehyde to suppress the particles growth.

We report a new method to synthesize cobalt (Co) nanoparticles using supercritical hydrothermal synthesis. An important aspect of supercritical hydrothermal synthesis of nano particles is the ability to create a homogeneous reducing atmosphere by introducing H2 gas. Co aqueous solution with formic acid was pressurized to 30 MPa at room temperature and rapidly heated to 400°C by mixing with supercritical water and then fed into a tubular reactor. The size of obtained Co nanoparticles was about 10nm and its particle size distribution was very narrow. The coercivity force at room temperature of the nanopartricles showed about 90 Oe. In this study, another important aspect of supercritical hydrothermal synthesis is reported; that is, in-situ surface modification of Co nanoparticles with carboxylic acids during crystallization.