Voltammetric determination of hafnium with calconcarboxylic acid

The interaction of Hf(IV) with calconcarboxylic acid (1-(2-hydroxy-4-sulfo-1-naphtylazo)-3-naphtolic acid) was investigated by cyclic voltammetry varying various factors (pH, ionic strength, contents of ethanol and scan rate). Optimal conditions of Hf(IV) determination in the presence of calconcarboxylic acid were found: acetic buffer solution pH 2.6, scan rate 0.5 Vs⁻¹. The detection limit of Hf(IV) concentration was 2.46 × 10⁻⁷ mol L⁻¹. The influence of foreign ions on the Hf(IV) determination was studied. It was established that some metals like cadmium, nickel, zinc, copper and titanium could be determined with Hf(IV) simultaneously. The method of voltammetric determination of hafnium was tested on model solutions and used in the determination of Hf(IV) in terbium-base alloy.     

Separation by hydrophobic interaction chromatography and structural determination by mass spectrometry of mannosylated glycoforms of a recombinant transferrin-exendin-4 fusion protein from yeast

Obtaining sufficient amounts of pure glycoprotein variants to characterize their structures is an important goal in both functional biology and the biotechnology industry. We have developed preparative HIC conditions that resolve glycoform variants on the basis of overall carbohydrate content for a recombinant transferrin-exendin-4 fusion protein. The fusion protein was expressed from the yeast Saccharomyces cerevisiae from high density fermentation and is post-translationally modified with mannose sugars through O-glycosidic linkages. Overall hydrophobic behavior appeared to be dominated by the N-terminal 39 amino acids from the exendin-4 and linker peptide sequences as compared to the less hydrophobic behavior of human transferrin alone. In addition, using LC techniques that measure total glycans released from the pure protein combined with new high resolution technologies using mass spectrometry, we have determined the locations and chain lengths of mannose residues on specific peptides derived from tryptic maps of the transferrin-exendin-4 protein. Though the protein is large (80,488 kDa) and contains 78 possible serine and threonine residues as potential sites for sugar addition, mannosylation was observed on only two tryptic peptides located within the first 55 amino acids of the N-terminus. These glycopeptides were highly heterogeneous and contained between 1 and 10 mannose residues scattered among the various serine and threonine sites which were identified by electron transfer dissociation mass spectrometry. Glycan sequences from 1 to 6 linear mannose residues were detected, but mannose chain lengths of 3 or 4 were more common and formed 80% of the total oligosaccharides. This work introduces new technological capabilities for the purification and characterization of glycosylated variants of therapeutic recombinant proteins.     

Reagentless amperometric formaldehyde-selective biosensors based on the recombinant yeast formaldehyde dehydrogenase

Novel formaldehyde-selective amperometric biosensors were developed based on NAD(+)- and glutathione-dependent formaldehyde dehydrogenase isolated from a gene-engineered strain of the methylotrophic yeast Hansenula polymorpha. Electron transfer between the immobilized enzyme and a platinized graphite electrode was established using a number of different low-molecular free-diffusing redox mediators or positively charged cathodic electrodeposition paints modified with Os-bis-N,N-(2,2'-bipyridil)-chloride ([Os(bpy)(2)Cl]) complexes. Among five tested Os-containing redox polymers of different chemical structure and properties, complexes of osmium-modified poly(4-vinylpyridine) with molecular mass of about 60 kDa containing diaminopropyl groups were selected. The positively charged cathodic paint exhibited the best electron-transfer characteristics. Moreover, the polymer layers simultaneously served as a matrix for keeping the negatively charged low-molecular cofactors, glutathione and NAD(+), in the bioactive layer. Additionally, covering the enzyme/polymer layer with a negatively charged Nafion membrane significantly decreased cofactors leakage and simultaneously enhanced the sensor' stability. The developed sensors revealed a high selectivity to formaldehyde (FA) and a low cross-sensitivity to other substances (such as, e.g. butyraldehyde, propionaldehyde, acetaldehyde, methylglyoxal). The maximum current value was 34.2+/-0.72 microA/mm(2) (3.05 mm diameter electrode) and the apparent Michaelis-Menten constant (K(M)(app)) derived from the FA calibration curves was 120+/-5mM with a linear detection range for FA up to 20mM. The best observed sensitivity for reagentless sensor was 1.8 nA microM(-1) (358 Am(-2)M(-1)). The developed sensors had a good operational and storage stability. The laboratory prototype of the sensor was applied for FA testing in some real samples of pharmaceutical (formidron), disinfectant (descoton forte) and industrial product (formalin). A good correlation was revealed between the concentration values measured using the developed FdDH-based sensor, an enzymatic method and standard chemical methods of FA determination.     

Electrodeposition polymers as immobilization matrices in amperometric biosensors: improved polymer synthesis and biosensor fabrication

Electrodeposition polymers can be precipitated on electrode surfaces upon electrochemical-induced modulations of the pH value in the diffusion zone in front of the electrode. The formed polymer films can be used as immobilization matrices in amperometric biosensors. In order to rationally control the thus obtained biosensor properties, it is indispensable to develop strategies for the reproducible synthesis of electrodeposition polymers as well as methods for the non-manual and reproducible sensor fabrication. Based on instrumental developments such as a specifically designed parallel synthesizer with improved stirring and temperature control, an automatic pipetting robot for the preparation of the monomer mixtures and controlled removal of polymerization inhibitors, the reproducible synthesis of libraries of electrodeposition polymers was achieved. Moreover, the polymerization process could be monitored using in-line thermocouples, and it could be shown that the chosen strategies led to reproducible polymerization reactions. By adaptation of an electrochemical robotic system integrating a Au microtiter plate and automatic electrode cleaning by means of a polishing wheel reproducible biosensor fabrication using glucose oxidase as a model enzyme could be demonstrated. These results open the route for the rational development of biosensors and control of the sensor properties by choosing specifically designed electrodeposition polymers.      

Bioelectrochemical detection of L-lactate respiration using genetically modified Hansenula polymorpha yeast cells overexpressing flavocytochrome b2

In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, also known as flavocytochrome b₂, FC b₂) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b₂ the O₂ reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b₂, cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN⁻, lactate respiration could be proven for causing the decrease in the O₂ reduction.     

Inverse problem with a time‐integral condition for a fractional diffusion equation

We find the conditions for the unique solvability of the inverse problem for a time‐fractional diffusion equation with Schwarz‐type distributions in the right‐hand sides. This problem is to find a generalized solution of the Cauchy problem and an unknown space‐dependent part of an equation's right‐hand side under a time‐integral overdetermination condition.     

Welding dynamics in an atomistic model of an amorphous polymer blend with polymer–polymer interface

We consider an atomistic model of thermal welding at the polymer-polymer interface of a polyetherimide/polycarbonate blend, motivated by applications to 3D manufacturing in space. We follow diffusion of semiflexible chains at the interface and analyze strengthening of the samples as a function of the welding time t_w by simulating the strain–stress and shear viscosity curves. The time scales for initial wetting, and for fast and slow diffusion, are revealed. It is shown that each component of the polymer blend has its own characteristic time of slow diffusion at the interface. Analysis of strain–stress demonstrates saturation of the Young's modulus at t_w = 240 ns, while the tensile strength continues to increase. The shear viscosity is found to have a very weak dependence on the welding time for t_w > 60 ns. It is shown that both strain–stress and shear viscosity curves agree with experimental data.