Electrochemical quartz crystal impedance study of redox hydrogel mediators for amperometric enzyme electrodes

Quartz crystal impedance around the resonant frequency at 10 MHz of a composite quartz crystal resonator has been studied simultaneously with cyclic voltammetry. A modified quartz crystal with a redox hydrogel (poly(allylamine)-ferrocene cross-linked with glucose oxidase) and immersed in liquid electrolyte was used. Impedance parameters (R(f) and X(L)((f))) of the surface redox gel film were obtained by fitting the resonator transfer function |V(o)/V(i)| vs ω to a BVD equivalent circuit and analyzed with the multiple nonpiezoelectric layer model of Martin. Two limiting hydrogel layers of the same composition were studied while oxidizing and reducing the ferrocene/ferricenium moieties attached to the swollen polymer backbone: thin and thick redox hydrogel films. For the thin films, the Sauerbrey approximation was valid. The mass/thickness and film viscosity changes that resulted from the anion and water exchange were evaluated while redox switching the polymer on the assumption of negligible storage modulus G' and a density of 1. For thick gel layers, on the other hand, the penetration depth of the acoustic wave was far less than the film thickness, and a liquid-like behavior was apparent. Film storage modulus and film loss modulus were simultaneously evaluated with the cyclic voltammetry.     

Alginate hydrogel as a promising scaffold for dental-derived stem cells: an in vitro study

The objectives of this study were to: (1) develop an injectable and biodegradable scaffold based on oxidized alginate microbeads encapsulating periodontal ligament (PDLSCs) and gingival mesenchymal stem cells (GMSCs); and (2) investigate the stem cell viability, and osteogenic differentiation of the stem cells in vitro. Stem cells were encapsulated using alginate hydrogel. The stem cell viability, proliferation and differentiation to adipogenic and osteogenic tissues were studied. To investigate the expression of both adipogenesis and ontogenesis related genes, the RNA was extracted and RT-PCR was performed. The degradation behavior of hydrogel based on oxidized sodium alginate with different degrees of oxidation was studied in PBS at 37?°C as a function of time by monitoring the changes in weight loss. The swelling kinetics of alginate hydrogel was also investigated. The results showed that alginate is a promising candidate as a non-toxic scaffold for PDLSCs and GMSCs. It also has the ability to direct the differentiation of these stem cells to osteogenic and adipogenic tissues as compared to the control group in vitro. The encapsulated stem cells remained viable in vitro and both osteo-differentiated and adipo-differentiated after 4?weeks of culturing in the induction media. It was found that the degradation profile and swelling kinetics of alginate hydrogel strongly depends on the degree of oxidation showing its tunable chemistry and degradation rate. These findings demonstrate for the first time that immobilization of PDLSCs and GMSCs in the alginate microspheres provides a promising strategy for bone tissue engineering.     

Study on the concentration of an enzyme immobilized by Langmuir-Blodgett films

Urease is an enzyme which decomposes urea into NH₃ and CO₂. We can produce a urea sensor by immobilizing urease on the pH sensor, and the Langmuir-Blodgett (LB) method has been expected to be useful as one of the immobilizing methods. We have measured for the first time the amount of urease adsorbed onto the LB film and shown that the relationship between the amount of adsorbed urease and the concentration in the solution can be expressed by an equation similar to the Langmuir adsorption isotherm.     

Native protein proteolysis in an immobilized enzyme reactor as a function of temperature

Trypsin concentration and the unmasking of cleavage sites in proteins play important roles in the stoichiometry of peptide production and the number of limit peptides generated during proteolysis. The hypothesis explored in this work was that native proteins could be digested and identified without disulfide reduction by (i) enhancing the unmasking of cleavage sites through elevated reaction temperatures and (ii) increasing trypsin concentration by use of an immobilized enzyme reactor (IMER). Transferrin was chosen as a model protein for these studies on the basis of its resistance to trypsin digestion. Results from this study showed greater than 70% sequence coverage in the peptides identified when nonreduced transferrin was digested at 60 °C. Large numbers of missed cleavages were observed from specific regions in proteins. Proteolysis appeared to start at a small number of high frequency cleavage sites in the cases of both reduced and nonreduced transferrin. Although approximately the same number of peptides were obtained from both structural forms of transferrin, the location of high frequency cleavage sites and the peptides produced were very different. Results from this study suggest that the location of initial cleavage sites along with the path of subsequent digestion depends strongly on the type of treatment used to open protein structures up for proteolysis.     

Peptide aptamers in label-free protein detection: 1. Characterization of the immobilized scaffold

Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplify the production of microarrays. This paper describes the generation and assaying characteristics of a scaffold protein adlayer. Orientational control of the scaffold protein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with a surface cysteine residue, which leads to the presentation of the scaffold recognition surface to solution. Operational stability of the system is excellent, with only a minor decrease in detection sensitivity over time (less than 1% h-1). We use this system to establish a surface plasmon resonance assay offering a limit of detection of 1 nM (150 ng mL-1) and determine the affinity constant of interaction of STM for a cognate antibody to be KD = 1.47 +/- 0.23 nM. Thus, we have established a solid foundation for the future creation of highly multiplexed peptide aptamer microarrays that will be compatible with a broad range of label-free detection technologies.     

Enzyme inhibitor screening by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust.     

An efficient green-emitting luminescent material: Tb3+-activated monoclinic Gd2O3

Monoclinic gadolinium oxide activated with Tb³⁺ is an efficient green-emitting phosphor, even for low Tb³⁺ concentrations. Under X-ray excitation its efficiency is about 50% of that of Gd₂O₂S-Tb. The low amount of blue ⁵D₃ emission from Gd₂O₃-Tb is ascribed to the low-energy position of the 4f-5d band (~ 280 nm). The intensity ratio of the Gd³⁺ excitation lines in the photoluminescence excitation spectrum provides information on the location of the quenching sites (probably Tb⁴⁺).     

Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: horseradish peroxidase immobilized on magnetic beads

Horseradish peroxidase was immobilized on the magnetic poly(glycidylmethacrylate-co-methylmethacrylate) (poly(GMA-MMA)), via covalent bonding and used for the treatment of phenolic wastewater in continuous systems. For this purposes, horseradish peroxidase (HRP) was covalently immobilized onto magnetic poly(GMA-MMA) beds using glutaraldehyde (GA) as a coupling agent. The maximum HRP immobilization capacity of the magnetic poly(GMA-MMA)-GA beads was 3.35 mg g(-1). The immobilized HRP retained 79% of the activity of the free HRP used for immobilization. The immobilized HRP was used for the removal of phenol and p-chlorophenol via polymerization of dissolved phenols in the presence of hydrogen peroxide (H(2)O(2)). The effect of pH and temperature on the phenol oxidation rate was investigated. The results were compared with the free HRP, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP. The optimum pH value for free and immobilized HRP was observed at pH 7.0. The optimum temperature for phenols oxidation with immobilized HRP was between 25 and 35 degrees C and the immobilized HRP has more resistance to temperature inactivation than that of the free form. Finally, the immobilized HRP was operated in a magnetically stabilized fluidized bed reactor, and phenols were successfully removed in the enzyme reactor.     

Reduced-graphene-oxide-and-strontium-titanate-based double-layered composite: an efficient microwave-absorbing material

Microwave-absorbing materials based on reduced graphene oxide (r-GO)/strontium titanate were prepared by embedding in epoxy matrix. R-GO and strontium titanate were synthesized and characterized before composite fabrication. Microstructures of the constituent elements were studied by scanning electron microscopy and X-ray diffraction (XRD). Microwave absorption capabilities of the composite absorbers were investigated using a Vector Network Analyser in the range 8–12 GHz. A maximum reflection loss of ?7.5 and ?16.4 dB was obtained at 9.3 and 12.08 GHz, respectively, for 2% (w/w) r-GO-loaded epoxy composites. A maximum attenuation of ?12.8 dB at 9.3 GHz was obtained for the strontium titanate/epoxy composite. However, double-layer composite with r-GO/strontium titanate/epoxy composition showed the maximum reflection loss of ?15.1 dB at 9.47 GHz and ?9.65 dB at 12.3 GHz. All the results are discussed in terms of complex permeability and permittivity. The study revealed that intrinsic conductivity and polarization of the r-GO particles and dielectric polarization of the strontium titanate within epoxy matrix contribute to the microwave absorption.     

Spider silk as an active scaffold in the assembly of gold nanoparticles and application of the gold-silk bioconjugate in vapor sensing

Spider silk is being viewed with interest by materials scientists due to its excellent resilience and mechanical properties. In this paper we show that spider silk is an excellent scaffold for the one-step synthesis and assembly of gold nanoparticles. Formation of a gold nanoparticle-spider-silk bioconjugate material is accomplished by simple reaction of the fibers with aqueous chloroauric acid. The gold nanoparticles thus formed are strongly bound to the spider-silk fiber surface enabling study of the electrical properties of the nanobioconjugate. Using the well-known contraction/expansion behavior of the fibers in solvents of varying polarity, we show that exposure of the gold nanoparticle-spider silk bioconjugate to vapors of methanol and chloroform leads to changes in electrical transport through the nanoparticles and thus, the possibility of developing a vapor sensor. The bioconjugate shows excellent response time and cycling efficiency to methanol vapors. The activation energy of electron transport from one gold nanoparticle to another in the nanobiocojugate was determined from temperature-dependent electron-transport measurements to be approximately 1.7 eV.     

Mesoflowers: A new class of highly efficient surface-enhanced Raman active and infrared-absorbing materials

A method for the synthesis of a new class of anisotropic mesostructured gold material, which we call “mesoflowers”, is demonstrated. The mesoflowers, unsymmetrical at the single particle level, resemble several natural objects and are made up of a large number of stems with unusual pentagonal symmetry. The mesostructured material has a high degree of structural purity with star-shaped, nano-structured stems. The mesoflowers were obtained in high yield, without any contaminating structures and their size could be tuned from nano- to meso-dimensions. The dependence of various properties of the mesoflowers on their conditions of formation was studied. The near-infrared-infrared (NIR-IR) absorption exhibited by the mesoflowers has been used for the development of infrared filters. Using a prototypical device, we demonstrated the utility of the gold mesoflowers in reducing the temperature rise in an enclosure exposed to daylight in peak summer. These structures showed a high degree of surface-enhanced Raman scattering (SERS) activity compared to spherical analogues. SERS-based imaging of a single mesoflower is demonstrated. The high SERS activity and NIR-IR absorption property open up a number of exciting applications in diverse areas. Electronic Supplementary Material  Supplementary material is available in the online version of this article at http://dx.doi.org/ and is accessible free of charge.     

The New Morris Method: an efficient second-order screening method

The New Morris Method was proposed by Campolongo and Braddock [Reliab. Engng Syst. Saf. 64 (1999) 1] as an extension of the Morris Method [Technometrics 33 (1991) 161] to include estimation of two-factor interaction effects. An undetected programming error prevented Campolongo and Braddock from appreciating the efficacy of the method. Testing on an analytic function reveals that the method is more powerful and efficient than previously thought.     

Nanoscaled redox active protein adsorption on Au-dot arrays: An electrochemical scanning probe microscopic investigation for application in nano-biodevices

Highly dense and uniform protein dot arrays on Au-nanodots using size controllable method were fabricated on indium tin oxide (ITO) substrate in order to develop an electrochemical nanobiochip. Cysteine modified azurin was directly immobilized on the fabricated Au-nanodots without any linker materials. Atomic force microscopy was used for characterizing Au-dots formed on ITO substrate. Electrochemical scanning tunneling microscopy (ECSTM) revealed the monolayer formation with an in situ cyclic voltammetry to observe redox behaviour of both bare Au-dots and protein immobilized Au-dots. I–V characteristics were obtained on both bare Au-dots and protein immobilized Au-dots structured on ITO conductive electrodes.     

Novel chitosan hydrogel formed by ethylene glycol chitosan, 1,6-diisocyanatohexan and polyethylene glycol-400 for tissue engineering scaffold: in vitro and in vivo evaluation

Traditional chitosan hydrogels were prepared by chemical or physical crosslinker, and both of the two kinds of hydrogels have their merits and demerits. In this study, researchers attempted to prepare one kind of chitosan hydrogel by slightly crosslinker, which could combine the advantages of the two kinds of hydrogels. In this experiment, the crosslinker was formed by a reaction between the isocyanate group of 1,6-diisocyanatohexan and the hydroxyl group of polyethylene glycol-400 (PEG-400), then the crosslinker reacted with the amidine and the hydroxyl group of ethylene glycol chitosan to form the network structure. Physical properties of the hydrogel were tested by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and biodegradation. Biocompatibility was assessed by cell implantation in vitro and the scaffold was used as a cartilage tissue engineering scaffold to repair a defect in rabbit knee joints in vivo. FTIR results show the formation of a covalent bond during thickening of the ethylene glycol chitosan. SEM and degradation experiments showed that the ethylene glycol chitosan hydrogel is a 3-D, porous, and degradable scaffold. The hydrogel contained 2 % ethylene glycol chitosan and 10 μl crosslinker was selected for the biocompatibility experiment in vitro and in vivo. After chondrocytes were cultured in the ethylene glycol chitosan hydrogel scaffold for 1 week cells exhibited clustered growth and had generated extracellular matrix on the scaffold in vitro. The results in vivo showed that hydrogel-chondrocytes promoted the repair of defect in rabbits. Based on these results, it could be concluded that ethylene glycol chitosan hydrogel is a scaffold with excellent physicochemical properties and it is a promising tissue engineering scaffold.     

ChemTS: an efficient python library for de novo molecular generation

Abstract

Automatic design of organic materials requires black-box optimization in a vast chemical space. In conventional molecular design algorithms, a molecule is built as a combination of predetermined fragments. Recently, deep neural network models such as variational autoencoders and recurrent neural networks (RNNs) are shown to be effective in de novo design of molecules without any predetermined fragments. This paper presents a novel Python library ChemTS that explores the chemical space by combining Monte Carlo tree search and an RNN. In a benchmarking problem of optimizing the octanol-water partition coefficient and synthesizability, our algorithm showed superior efficiency in finding high-scoring molecules. ChemTS is available at https://github.com/tsudalab/ChemTS.     

Finite separation method: an efficient boundary element crack modeling technique

In computational fracture mechanics, great benefits are obtained from the reduced modeling dimension order and the accurate integral formulation of the boundary element method (BEM). However, the direct representation of co-planar surfaces (i.e., cracks) causes a degeneration of the standard displacement BEM formulation which can only be circumvented with special modeling techniques. Aiming to simplify the generalized application of the BEM to fracture mechanics problems, this paper presents a two-dimensional crack modeling approach. The method uses the direct BEM displacement formulation within a single-domain model to efficiently and precisely calculate any mixed mode crack tip stress intensity factor. Details of the application of the method are presented, while its accuracy and reliability are demonstrated through numerous comparisons with benchmark results.     

Shape optimal design of structures: an efficient shape representation concept

This paper describes a shape representation concept that may be used in general shape optimization procedures. It relies on the assumption that the finite element mesh is defined as a convective mesh following automatically the shape changes of a conveniently parameterized body. Suitable parameterization of the body is achieved by combining the design element technique and a convenient design element. The design element is defined as a rational Bézier body. It represents a general‐purpose design element that may serve as the geometrical data provider for the response and sensitivity analysis for virtually any finite element type. Practical implementation of the proposed approach for space frame structures is discussed in detail. The validity of the proposed approach as well as the use of a body‐like design elements are illustrated by three numerical examples. Copyright © 2000 John Wiley & Sons, Ltd.     

Novel precipitated silicas: an active filler of synthetic rubber

Highly dispersed silicas have been obtained at a semi-technical scale by precipitation from sodium metasilicate solutions using appropriately diluted solutions of ammonium hydrogencarbonate or ammonium chloride. The difference, as compared to the generally applied technology of silica precipitation using acids or their anhydrides, has been the involvement of the precipitation reaction in alkaline media. Attempts have been made at changing the silica surface character from hydrophilic to more hydrophobic, using a modifier from the group of silane coupling agents. The alkoxysilanes modifiers contained groups with chemical affinity towards functional groups of butadiene–styrene rubber. Modification of silica surface using silane coupling agents has been found to improve the reinforcement of vulcanizates, by increasing the tensile and tear strength in particular.