A highly photoreflective and heat-insulating alumina film composed of stacked mesoporous layers in hierarchical structure.

An alumina film with highly photoreflective and heat-insulating properties can be simply synthesized using a sol of fibrous boehmite with an additive. The entangled fibers bring about mesopores among them and form stacked 2D nonwoven-like nanosheets. The porosity and the layered structure of alumina accompanying the heat resistivity provide the upper properties that are usually difficult to realize simultaneously.

Electrochemical enzymatic biosensor with long-term stability using hybrid mesoporous membrane.

An acetylcholinesterase-immobilized sensor unit was successfully prepared by encapsulating the enzyme within hybrid mesoporous silica membranes (F127-MST). Through a novel combination with tetracyanoquinodimethane, both acetylcholine and organophosphorus pesticides were successfully detected with high sensitivity. Furthermore, we manufactured the working prototype of an enzyme sensor with this sensor unit for detecting dichlorvos, aldicarb and parathion. At present, the detection limit in this working prototype either equaled or surpassed that of others. Also, we have the advantage of increased stability of the enzyme against the outer environment by encapsulation of the enzymes into a silica nanospace. Consequently, acetylcholinesterase immobilized in F127-MST is a practical sensor with high sensitivity, reusability, and storage stability.

Preparation of plate-like mesoporous material from layered silicate RUB-15.

A plate-like mesoporous material was formed from the lamellar structure of layered silicate RUB-15. RUB-15 was synthesized by a hydrothermal method, as reported previously. TMA (tetramethylammonium) ions exist in the interlayer of RUB-15 were exchanged with C16 TMA (hexadecyl-trimethyl-ammonium) ions, and TEOS (tetraethylorthosilicate) was then intercalated in between the layers. After steaming, the obtained powder was calcined and characterized by XRD, N2 gas adsorption, and scanning electron microscopy (SEM). The XRD patterns and N2 adsorption-desorption isotherms of the finally obtained powders indicated the presence of mesopores in the sample. The morphology of powders was plate-like which originates from the structure of the starting material. Cross-sectional FE-SEM images of the final obtained powders revealed existence of mesopores between the layers. The morphology of the final obtained mesoporous materials was affected by their remaining layered structure due to the starting material RUB-15.

An enzyme-encapsulated microreactor for efficient theanine synthesis.

A flow-type microreactor containing glutaminase-mesoporous silica composites with 10.6 nm pore diameter (TMPS10.6) was developed for the continuous synthesis of theanine, a unique amino acid. High enzymatic activity was exhibited by the local control of the reaction temperature.

Heterogeneous nucleation of protein crystals on fluorinated layered silicate.

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface.

Increasing particle size of a synthetic smectite for polymer nanocomposites using a supercritical hydrothermal treatment.

We have studied the effect of a supercritical hydrothermal process on the structural and surface morphological properties of synthetic smectite clay, stevensite (ST), in terms of the particle size, in order to enhance the functionality of the synthetic smectites as an inorganic filler for transparent clay/polymer nanocomposites. The ST aqueous suspensions were treated in a flow reactor system at 673 K and 25 MPa. The structural characterizations revealed that the ST retained a layered structure composed of polymeric sheets of SiO(4) tetrahedra after the treatment. The treated ST possessed a particle size of 71 nm, approximately twice that of the original ST (36 nm) for the 0.1 wt.% suspension using an operation condition at a flow rate of 0.085 g s(-1). SEM observation revealed that an enlarged particle was formed from cohesive aggregates, suggesting that the increase in size of the particles was caused by the cohesion of the microcrystallites or primary particles of ST. The treated ST was subsequently used to prepare nanocomposites with carboxymethylcellulose sodium salt (CMC Na) to evaluate the effect of the supercritical treatment. The treated ST nanocomposite films retained their transparency which is very similar to the original ST nanocomposite films. Furthermore, the nanocomposite films, which had a high CMC Na ratio ranging from 40 to 90 wt.%, showed improved oxygen barrier properties when compared with those of original ST. The tortuous model revealed that this improvement was mainly due to the increase of the particle size. Consequently, the supercritical treatment successfully brought about the growth of the ST particles, leading to the development of functional synthetic clays for clay/polymer nanocomposites.

Detection of hetero-proteins-mesoporous silica assembly by BRET.

The assembly of a hetero-protein (Renilla reniformis luciferase (Rluc) and a green fluorescence protein (sGFP)) encapsulated in folded-sheet mesoporous material with 7.1 nm pore diameter (FSM7.1), which was used for studying protein-protein interactions in pores of mesoporous silica, has been confirmed by the detection of bioluminescence resonance energy transfers (BRET).

Enhancement in thermal stability and resistance to denaturants of lipase encapsulated in mesoporous silica with alkyltrimethylammonium (CTAB).

We assembled a highly durable conjugate with both a high-density accumulation and a regular array of lipase, by encapsulating it in mesoporous silica (FSM) with alkyltrimethylammonium (CTAB) chains on the surface. The activity for hydrolyzing esters of the lipase immobilized in mesoporous silica was linearly related to the concentration of lipase, whereas that of non-immobilized lipase showed saturation due to self-aggregation at a high concentration. The lipase conjugate also had increased resistance to heating when stayed in the silica coupling with CTAB. In addition, encapsulating the enzyme with FSM coupled CTAB caused the lipase to remain stable even in the presence of urea and trypsin, suggesting that the encapsulation prevented dissociation and denaturing. This conjugate had much higher activity and much higher stability for hydrolyzing esters when compared to the native lipase. These results show that FSM provides suitable support for the immobilization and dispersion of proteins in mesopores with disintegration of the aggregates.

Importance of the support material in thin palladium composite membranes for steady hydrogen permeation at elevated temperatures.

Hydrogen permeation performance of palladium membranes supported on porous alpha-alumina and yttria-stabilized zirconia (YSZ) was studied at 300-850 degrees C. The hydrogen permeation flux across the palladium-alpha-alumina membrane decreased markedly during permeation tests conducted at >600 degrees C. The SEM and XPS studies of the post-test membrane revealed the presence of aluminium in the palladium layer. Such migration of aluminium was not observed by heating the palladium-alpha-alumina membrane under an argon atmosphere, indicating that hydrogen is responsible for this phenomenon. Hydrogen-induced strong metal-support interaction might be related to this considerable loss of the hydrogen flux. Reduction of alumina to Al(0) by active hydrogen at the membrane-support interface and subsequent migration of Al(0) into the palladium layer represents the most plausible mechanism for the aluminium diffusion. Actually, Al(0) that migrated into the palladium membrane layer generated less hydrogen-permeable palladium-aluminium alloy or inter-metallic compound phase. In contrast, no such strong interaction was found between the YSZ support and the palladium membrane. This composite membrane exhibited a steady permeation of hydrogen at 650 degrees C for 336 h. Having a remarkably high reduction potential, Y(III) is unlikely to be reduced to Y(0), although Zr(IV) has a comparable reduction potential to that of Al(III). A binary phase diagram shows a liquid alloy phase present for the Pd/Al couple at temperatures greater than 615 degrees C (eutectic point), while an inter-metallic compound or liquid alloy phase in the Pd-Zr binary system is not apparent at temperatures less than 750 degrees C. Consequently, inter-diffusion of zirconium with palladium did not occur during operations at 650 degrees C.

High-throughput protein refolding screening method using zeolite.

We established a 96-well-plate-based refolding screening system using zeolite. In this system, protein denatured and solubilized with 6 M guanidine hydrochloride is adsorbed onto zeolite placed in a 96-well plate. The refolding conditions can be tested by incubating the samples with refolding buffers under various conditions of pH, salts, and additives. In this study, we chose green fluorescent protein as the model protein. Green fluorescent protein was expressed as inclusion bodies, and we tested the effects of four pH conditions and six additives on its refolding. The results demonstrate that green fluorescent protein was more efficiently refolded with zeolite than with the conventional dilution method.

Refolding of lactate dehydrogenase by zeolite beta.

We used zeolite beta as an adsorbing matrix to refold recombinant lactate dehydrogenase (LDH) protein collected as an insoluble aggregate from a bacterial expression system. The adsorption isotherm revealed that 1 g of zeolite adsorbed 200 mg of denatured LDH solubilized with a buffer containing 6 M of guanidine hydrochloride. The pH of the buffer had little effect on the adsorption, but this property was abolished by preincubation of the zeolite with polyethylene glycol (PEG) in a weight ratio of 1:10. These data suggest that the adsorption of LDH depends on the hydrophobicity of the zeolite surface, and that the adsorption of PEG to zeolite is sufficient to release LDH from its surface. LDH was thus released by refolding buffer containing PEG and arginine, and soluble LDH was obtained in its active enzymatic form. The addition of arginine dramatically increased the yield of LDH in a dose-dependent manner. The overall refolding efficiency was optimized to 35%.

Amperometric determination of choline with enzyme immobilized in a hybrid mesoporous membrane.

Choline sensor is successfully prepared by using immobilized enzyme, i.e., choline oxidase (ChOx) within a hybrid mesoporous membrane with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide, which is the co-product of the enzymatic choline oxidation. The determination range and the response time are 5.0-800 microM and approximately 2 min, respectively. The sensor is very stable compared to the native enzyme sensor and 85% of the initial response was maintained even after storage for 80 days. These results indicate that ChOx is successfully immobilized and well stabilized, and at the same time, enzyme reaction proceeds efficiently. Such ability of hybrid mesoporous membrane F127M suggests great promise for effective immobilization of enzyme useful for electrochemical biosensors.

Use of zeolite to refold a disulfide-bonded protein.

Zeolites are microporous crystalline aluminosilicates with a highly ordered structure. Using zeolite beta as an adsorbent, denatured/reduced hen egg lysozyme was refolded to the active form at high concentrations. The denatured/reduced lysozyme was adsorbed onto the zeolite and the protein was refolded by desorbing it into refolding buffer, consisting of redox reagents, guanidine hydrochloride, polyethylene glycol, and L-arginine. This zeolite refolding method could be highly effective for various kinds of proteins, refolding them with high efficiency even when they contain disulfide bonds.

Preparation of a microporous layered organic-inorganic hybrid nanocomposite using p-aminotrimethoxysilane and a crystalline layered silicate, ilerite.

The silylation of ilerite with p-aminophenyltrimethoxysilane (denoted as APhS) was conducted in order to attempt preparation of a layered organic-inorganic hybrid nanocomposite having interlayer microporosity, in which the APhS molecule bridges between the silicate layers using two heterofunctional groups containing an amino group and a methoxy one. The APhS molecules were successfully condensed and immobilized in the interlayer of the protonated ilerite. Chemical analysis and (29)Si solid-state NMR results indicated that the interlayer space was occupied by preferential condensation between the APhS molecules. Subsequent HCl treatment could remove the excess amounts of the oligomeric species of the APhS molecules. The removal brought about vacant spaces in the interlayer supported by the bridging of the residual APhS molecules. Furthermore, the immobilized APhS molecules had a significant influence on the surface properties of the resultant nanocomposite, which showed high toluene adsorptivity. Consequently, we found a means to construct a microporous, layered nanocomposite by the bridging of the organic spaces with two heterofunctional groups.

Selective adsorption of bacterial cells onto zeolites.

Zeolites adsorb microbial cells on their surfaces and selective adsorption for specific microorganisms was seen with certain zeolites. Tests for the adsorption ability of zeolites were conducted using various established microbial cell lines. Specific cell lines were shown to selectively absorb to certain zeolites, species to species. In order to understand the selectivity of adsorption, we tested adsorption under various pH conditions and determined the zeta-potentials of zeolites and cells. The adsorption of some cell lines depended on the pH, and some microorganisms were preferentially adsorbed at acidic pH. The values of zeta-potentials were used for calculating the electric double layer interaction energy between zeolites and microbial cells. There was a correlation between the experimental adsorption results and the interaction energy. Moreover, we evaluated the surface hydrophobicity of bacterial cells by using the microbial adherence to hydrocarbon (MATH) assay. In addition, we also applied this method for zeolites to quantify relative surface hydrophobicity. As a result, we found a correlation between the adsorption results and the hydrophobicity of bacterial cells and zeolites. These results suggested that adsorption could be explained mainly by electric double layer interactions and hydrophobic interactions. Finally, by using the zeolites Na-BEA and H-Y, we succeeded in clearly separating three representative microbes from a mixture of Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Zeolites could adsorb each of the bacterial cell species with high selectivity even from a mixed suspension. Zeolites can therefore be used as effective carrier materials to provide an easy, rapid and accurate method for cell separation.

The ensemble of hetero-proteins in inorganic nanochannels.

The assembly and proper alignment of two heterofluorescent proteins (sGFP and DsRed) in the mesoporous channels of ethanol-treated FSM6.2 (a folded-sheet mesoporous material with a pore diameter of 6.2 nm) was confirmed using a fluorescence resonance energy transfer (FRET) technique. The sGFP-DsRed-FSM6.2 conjugate showed a large decrease in the emission of donor (sGFP) fluorescence, indicating that the conjugate functions as an energy transfer system through the combination of the two heteroproteins, due to the successful encapsulation of the sGFP-DsRed pairs in the mesopores. Fluorescence spectral analysis demonstrated that the proteins were highly dispersed and homogeneously encapsulated in the mesopores of FSM6.2, even at high concentration, although they spontaneously aggregated and showed a red shift in solution at the concentration corresponding to that in the conjugate. Furthermore, an increase in the amount of sGFP and DsRed adsorbed to the pores of FSM6.2 led to a decrease in the distance between these proteins, resulting in enhancement of FRET efficiency.

Use of layer silicate for protein crystallization: effects of Micromica and chlorite powders in hanging drops.

Two kinds of layer silicate powder, Micromica and chlorite, were used to aid protein crystallization by the addition to hanging drops. Using appropriate crystallization buffers, Micromica powder facilitated crystal growth speed for most proteins tested in this study. Furthermore, the addition of Micromica powder to hanging drops allowed the successful crystallization of lysozyme, catalase, concanavalin A, and trypsin even at low protein concentrations and under buffer conditions that otherwise would not generate protein crystals. Except for threonine synthase and apoferritin, the presence of chlorite delayed crystallization but induced the formation of large crystals. X-ray analysis of thaumatin crystals generated by our novel procedure gave better quality data than did that of crystals obtained by a conventional hanging drop method. Our results suggest that the speed of crystal growth and the quality of the corresponding X-ray data may be inversely related, at least for the formation of thaumatin crystals. The effect of Micromica and chlorite powders and the application of layer silicate powder for protein crystallization are discussed.

Formation of cage-like hollow spherical silica via a mesoporous structure by calcination of lysozyme-silica hybrid particles.

Calcination of lysozyme-silica hybrid hollow particles gives novel cage-like hollow spherical silicas with differently patterned through-holes on their shell structure.

Optical nanosensor design with uniform pore geometry and large particle morphology.

Appropriate design of nanosensors for optically selective, sensitive sensing systems is needed for naked-eye detection of pollutants for environmental cleanup of toxic heavy-metal ions. Mesostructured materials with two- or three-dimensional (2D or 3D) geometries and large particle morphologies show promise as probe carriers, and can therefore be used to reproducibly fabricate uniformly packed nanosensors. This is the first report on the effects of significant key properties of the mesostructured carriers, such as morphology, geometry, and pore shape, on the functionality of optical nanosensor designs. Such mesostructured sensors with superior physical characteristics can be used as components in sensing systems with excellent stability and sensitivity, and with rapid detection response. The nanosensor design can enhance the selectivity even at low concentrations of the pollutant target ions (nanomolar level). Among the nanosensors developed here, the large pore-surface grains of highly ordered 3D monoliths (HOM) exhibited a high adsorption capability of the Pyrogallol Red probe and high accessibility to analyte ion transport, leading to possible naked-eye detection of Sb(III) ions at concentrations as low as 10(-9) mol dm(-3) and at a wide detection range of 0.5 ppb to 3 ppm. A key finding in our study was that our mesostructured nanosensor designs retained highly efficient sensitivity without a significant increase in kinetic hindrance, despite the slight decrease of the specific activity of the electron acceptor/donor strength of the probe functional group after several regeneration/reuse cycles. The results, in general, indicate that large-scale reversibility of optical nanosensors is feasible in such metal-ion sensing systems.