Effect of Additives on Freeze-Drying and Storage of Yarrowia lipolytica Lipase

The extracellular lipase of Yarrowia lipolytica presents numerous potentialities for biotechnological applications. This work describes the development and storage of powders obtained from supernatants containing Y. lipolytica lipase by freeze-drying as downstream process that is important in obtaining a stable lipase powder with high enzymatic activity. Lipase was produced by Y. lipolytica U6 mutant strain in 20-L bioreactor. Non-concentrated cell-free culture supernatant samples were supplemented with different concentrations (0.5?C1?%) of maltodextrin and glycerol as additives to freeze-drying. Effects of additives, temperature, pH, and storage time on lipase powders were determined. After addition of additives, freeze-drying yield increased 3.5-fold compared to supernatant without additive. Maltodextrin with 0.5?% concentration gave the best protection of lipase during dehydration treatment and its freeze-drying yield (77?%) is better than other formulations. Lipase powders were stored at 4 and 25?°C for 46?weeks without loss of lipase activity. A common impediment to the production of commercial enzyme is their low-stability aqueous solutions. The present study shows that freeze-dried lipase powders of Y. lipolytica have good stability for storage and various applications.     

Effect of sodium tetraborate (borax) on the thermal properties of frozen aqueous sugar and polyol solutions

The effect of sodium tetraborate (Na(2)B(4)O(7), borax) on the thermal property of frozen aqueous sugar and polyol solutions was studied through thermal analysis. Addition of borax raised the thermal transition temperature (glass transition temperature of maximally freeze-concentrated solutes; T(g)') of frozen sucrose solutions depending on the borax/sucrose concentration ratios. Changes in the T(g)' of frozen mono- and disaccharide solutions suggested various forms of complexes, including those of a borate ion and two saccharide molecules. Borax exerted the maximum effect to raise the oligosaccharide and dextran T(g)'s at borax/saccharide molar ratios of approximately 1-2 (maltose and maltooligosaccharides), 2 (dextran 1060), 5 (dextran 4900), and 10 (dextran 10200). Further addition of borax lowered T(g)'s of the saccharide solutions. Borax also raised T(g) and T(g)' temperatures of frozen aqueous glycerol solutions. The decreased solute mobility in frozen solutions by the borate-polyol complexes suggested higher collapse temperature in the freeze-drying process and improved stability of biological systems in frozen solutions.     

Performance of a Faradaic impedimetric immunosensor for blood group antigen A

The performance is described of a label-free Faradaic impedimetric immunosensor based on immobilized monoclonal IgM antibodies to blood group antigen A (anti–A) for blood typing. Anti–A was directly immobilized onto gold electrodes modified with an amine-reactive self assembled monolayer of dithiobis(succinimidylundecanoate). The alteration of the interfacial features of the electrodes due to different modification or recognition steps was probed by Faradaic impedance spectroscopy and cyclic voltammetry in the presence of a hexacyanoferrate(II)/(III) redox couple. Various optimization studies were undertaken with respect to the construction and potential use of the immunosensors as a diagnostic tool for blood typing.     

Paper-based device for rapid typing of secondary human blood groups

We report the use of bioactive paper for typing of secondary human blood groups. Our recent work on using bioactive paper for human blood typing has led to the discovery of a new method for identifying haemagglutination of red blood cells. The primary human blood groups, i.e., ABO and RhD groups, have been successfully typed with this method. Clinically, however, many secondary blood groups can also cause fatal blood transfusion accidents, despite the fact that the haemagglutination reactions of secondary blood groups are generally weaker than those of the primary blood groups. We describe the design of a user-friendly sensor for rapid typing of secondary blood groups using bioactive paper. We also present mechanistic insights into interactions between secondary blood group antibodies and red blood cells obtained using confocal microscopy. Haemagglutination patterns under different conditions are revealed for optimization of the assay conditions.     

Rheological behaviour of irradiated wound dressing poly(vinyl pyrrolidone) hydrogels

The use of hydrogels as biomaterials has increased lately. Poly(vinyl pyrrolidone) (PVP) is an example of polymer hydrogels applied for the synthesis of hydrogel to be used in different biomedical applications. This paper describes a study on rheological properties of PVP hydrogels obtained by gamma radiation techniques. PVP hydrogels were obtained by gamma radiation of PVP water solutions with different radiation doses. It was studied the influence of additives such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO) and glycerol on the rheological behaviour of the gel. The rheological behaviour of hydrogel samples was characterized by measuring the shear storage modulus (G′) under dynamic shear loading. Besides this, sterility and cytotoxicity tests were performed. The study on rheological behaviour of hydrogels showed that G′ of PVP gels change according to the additive used. Glycerol increases the fluidity of the gel. The influence of PEG depends on the amount and on its molecular mass. The increase on PEG amount and molecular mass cause a decrease of G′ and an increase in the crosslinking density of PVP hydrogel network. The use of high molecular weight PEO allows the increase of the elasticity of the PVP gels.     

Surface properties of chitosan composites with poly(<Emphasis Type="Italic">N</Emphasis>-vinylpyrrolidone) and montmorillonite

Surface properties of composites containing chitosan (Ch) with poly(N-vinylpyrrolidone) (PVP) and montmorillonite (MMT) additives were investigated by contact angle measurements, thermogravimetric analysis (TGA), atomic force microscopy (AFM) and tensile tests. Composites were obtained by drop casting suspensions of montmorillonite (1 wt % relative to chitosan) and polymer (1 wt %) in 0.1 mol/dm³ acetic acid. Contact angle measurements for diiodomethane (D) and glycerol (G) on the surfaces of the chitosan films, PVP and their composite films were made; surface free energies were also calculated. It was found that the PVP/MMT or Ch/PVP/MMT blend surface is enriched with a high surface energy component, i.e., polyvinylpyrrolidone. The roughness of chitosan composites increases after the addition of montmorillonite; this may indicate an increase in the heterogeneity of this composition in comparison to other compositions. The TGA thermograms and mass loss percentages at different decomposition temperatures showed that the thermal stability of the binary composite slightly increases upon the addition of polyvinylpyrrolidone. The mechanical properties such as tensile strength and Young modulus depend on the composition and varied non-uniformly.     

Association between branched poly(ethyleneimine) and sodium dodecyl sulfate in the presence of neutral polymers

In the present paper, the effect of different neutral polymers on the self-assemblies of hyperbranched poly(ethyleneimine) (PEI) and sodium dodecyl sulfate (SDS) has been investigated at different ionization degrees of the polyelectrolyte molecules. The investigated uncharged polymers were poly(ethyleneoxide), poly(vinylpyrrolidone) and dextran samples of different molecular mass. Dynamic light scattering and electrophoretic mobility measurements demonstrate that the high molecular mass PEO or PVP molecules adsorb considerably onto the surface of the PEI/SDS nanoparticles. At appropriate concentrations of PVP or PEO, sterically stabilized colloidal dispersions of the polyelectrolyte/surfactant nanoparticles with hydrophobic core and hydrophilic corona can be prepared. These dispersions have considerable kinetic stability at high ionic strengths where the accelerated coagulation of the PEI/SDS nanoparticles results in precipitation in the absence of the neutral polymers. In contrast, the addition of dextran does not affect considerably the kinetic stability of PEI/SDS mixtures because of its low adsorption affinity towards the surface of the polyelectrolyte/surfactant nanoparticles.     

一种苯并咪唑黄原酸酯的合成及其摩擦学性能

由于操作条件及环境保护的要求日益苛刻,环境友好型润滑油添加剂越来越受到人们的关注,润滑油添加剂向着低灰份、多功能、绿色环保方向发展。本文设计合成了三个环保型含氮硫化合物,采用质谱,红外光谱,元素分析对其结构进行了表征。考察了该类添加剂在菜籽油中的溶解性;采用热重分析对其热稳定性进行了评价;并在四球摩擦磨损试验机上考察了所合成的含氮硫化合物在菜籽油中的摩擦学性能。结果表明,该类添加剂在菜籽油中的溶解度都能达到2%,最低热分解温度为129℃,最高达532℃;在菜籽油中的极压值最高达1186N,是菜籽油的2倍,是ZDDP的1.3倍,抗磨性能最好的是B3,极压值最好的是B2。     

Recent and future trends in blood group typing

Blood group typing is the process of testing red blood cells to determine which antigens are present and which are absent. It is standard practice to test for A, B, and D (Rh) antigens and to perform tests for other antigens in selected cases. ABO blood group typing is confirmed by reverse grouping that detects expected isoagglutinins. Unexpected antibodies can be demonstrated by antibody screening tests. For transfusion, donor units compatible with the patient are selected. Prior to transfusion, a crossmatch is performed as a final check for incompatibility. This article describes the recent and future methods of blood group typing and testing of serological compatibility. In addition, methods for blood bank automation are presented.     

Synthesis of nanosized silver colloids by microwave dielectric heating

Silver nanosized crystallites have been synthesized in aqueous and polyols viz., ethylene glycol and glycerol, using a microwave technique. Dispersions of colloidal silver have been prepared by the reduction of silver nitrate both in the presence and absence of stabilizer poly(vinylpyrolidone) (PVP). It was observed that PVP is capable of complexing and stabilizing Ag nanoparticles formed through the reduction of Ag⁺ ions in water and ethylene glycol. In the case of ethylene glycol, it has been shown that the use of PVP leads to particles with a high degree of stability. The colloids are stable in glycerol for months even in the absence of stabilizer.     

Fabrication and cytocompatibility evaluation for blood‐compatible polyethersulfone membrane modified by a synthesized poly (vinyl pyrrolidone)‐block‐poly (acrylate‐graft‐poly(methyl methacrylate))‐block‐poly‐(vinyl pyrrolidone)

Polyethersulfone (PES) membrane, one of the most important polymeric materials because of its good chemical resistance, thermal stability, mechanical, and film‐forming properties, has already been used in hemodialysis, tissue engineering, and artificial organs. In order to improve the blood compatibility of PES membrane, many amphiphilic block copolymers have been synthesized and used as additives for surface modification. The object of this study is to develop a hydrophilic PES membrane by blending a comblike amphiphilic block copolymer poly (vinyl pyrrolidone)‐block‐poly [acrylate‐graft‐poly (methyl methacrylate)]‐block‐poly‐(vinyl pyrrolidone) [PVP‐b‐P (AE‐g‐PMMA)‐b‐PVP] synthesized by RAFT polymerization. The cytocompatibility performance of PVP‐b‐P (AE‐g‐PMMA)‐b‐PVP modified PES membrane was evaluated, which showed better cytocompatibility compared with that of pristine PES membrane. Endothelial cells cultured on the modified membranes present improved growth in terms of scanning electron microscope observation, MTT assay, and confocal laser scanning microscope observation. These results indicate that the modified membrane has great potential application in blood‐contact fields such as hemodialysis and bio‐artificial liver supports. Copyright © 2015 John Wiley & Sons, Ltd.     

Towards dynamic coating of glass microchip chambers for amplifying DNA via the polymerase chain reaction

As microchip technology evolves to allow for the integration of more complex processes, particularly the polymerase chain reaction (PCR), it will become necessary to define simple approaches for minimizing the effects of surfaces on the chemistry/processes to be performed. We have explored alternatives to silanization of the glass surface with the use of additives that either dynamically coat or adsorb to the glass surface. Polyethylene glycol, polyvinylpyrrolidone (PVP), and hydroxyethylcellulose (HEC) have been explored as potential dynamic coatings and epoxy (poly)dimethylacrylamide (EPDMA) evaluated as an adsorbed coating. By carrying out analysis of the PCR products generated under different conditions via microchip electrophoresis, we demonstrate that these coating agents adequately passivate the glass surface in a manner that prevents interference with the subsequent PCR process. While several of the agents tested allowed for PCR amplification of DNA in glass, the EPDMA was clearly superior with respect to ease of preparation. However, more efficient PCR (larger mass of amplified product) could be obtained by silanizing the glass surface.     

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.     

Detecting trace melamine in solution by SERS using Ag nanoparticle coated poly(styrene-co-acrylic acid) nanospheres as novel active substrates

A systematic study for the preparation of Ag nanoparticle (Ag-NP) coated poly(styrene-co-acrylic acid) (PSA) composite nanospheres by in situ chemical reduction is reported. The experimental results showed that the reaction temperature and the surface coverage of the -COOH determined the surface coverage and grain size of Ag nanoparticles on the PSA nanospheres. The surface enhanced Raman spectroscopy (SERS) sensitivity was investigated using 4-hydroxythiophenol (4-HBT) as the model probe in the solution of composite nanospheres stabilized by polyvinylpyrrolidone (PSA/Ag-NPs/PVP), with the detection limit of about 1 × 10(-6) M. Potential application of the new SERS substrate was demonstrated with the detection of melamine, and the detection limit was about 1 × 10(-3) M. Chemical noises from PVP and other impurities were observed and attributed mainly to the competitive adsorption of PVP on the surfaces of Ag-NPs. After tetrahydrofuran washing of the PSA/Ag-NPs/PVP substrates that removed the PVP and other residuals, the signal/noise levels of SERS were greatly improved and the detection limit of melamine was determined to be 1 × 10(-7) M. This result indicated that the new PSA/Ag-NPs system is highly effective and can be used as the SERS-active substrate for trace analysis of a variety of drugs and food additives.     

大豆油生物柴油低温流动性能影响的研究

以大豆油为原料采用碱催化酯交换法合成生物柴油,测定了其酸值、游离甘油、总甘油、灰分、黏度和磷含量等指标。实验表明,共存的甲醇、水分和甘油酯对生物柴油的低温流动性能影响极少,饱和脂肪酸甲酯的同时析出对生物柴油低温流动性能起关键作用。考察了5种柴油降凝剂、0号和20号柴油以及乙醇对生物柴油低温流动性能的影响。0号柴油有效地改善生物柴油黏度,但对其低温流动性能影响不大。20号柴油和乙醇能显著降低生物柴油的凝点、倾点、冷滤点和黏度。其中3种降凝剂有效降低生物柴油的凝点和倾点,1种降凝剂能小幅度改善冷滤点,5种降凝剂都能使生物柴油的黏度小幅上升。     

Molecular mechanism of the protective effect of monomer polyvinylpyrrolidone on antioxidants – experimental and computational studies

We previously developed a lutein–polyvinylpyrrolidone (PVP) complex with improved aqueous saturation solubility and stability, though the conjugation mechanism is still unclear. In this paper, experiments with astaxanthin–PVP complex and curcumin–PVP complex were carried out, which indicated that PVP could improve the solubility and stability of astaxanthin and curcumin. We aimed to construct a computational model capable of understanding the protective effect in complexes formed between PVP and antioxidants, through which the binding mode of PVP and antioxidants was investigated with molecular modelling in order to obtain the interactions, binding energy, binding site and surface area between PVP and antioxidants. Solubility enhancement was attributed to the H-bonds between PVP and antioxidants, and the saturation solubility was curcumin > lutein > astaxanthin. Binding energy, binding site and surface area were beneficial for the stability of complex, and the stability enhancement was lutein > astaxanthin > curcumin. The experimental results were in agreement with the computational results. Furthermore, we established a method for the exploration of a similar system with other polymer complexes. Additionally, the proposed PVP model could predict the interactions between PVP and various ligands, such as antioxidants and drugs.     

Drying of a cellulose II gel: effect of physical modification and redispersibility in water

The agglomeration of cellulosic materials upon drying, often called hornification, causes a reduction of water retention, among other undesired effects. It is one of the main issues in industrial cellulose processing, especially with regard to nanocelluloses. As a consequence, high transportation and storage costs arise since nanocelluloses need to remain in aqueous suspensions unless trade-offs in reactivity, redispersibility and surface properties are accepted. In this study, different drying strategies for TENCEL^® gel, a nanostructured gel derived from the Lyocell process consisting of spherical particles, are compared and evaluated. First, freeze-drying with consideration of the influence of freezing temperature and the use of tert-butanol as cryo-protectant, and second, simple oven-drying at 60 °C. Surprisingly, oven-dried xerogels showed higher water retention values and also better colloidal stability than the cryogels. This is in stark contrast to cellulose nanofibrils for which freeze-drying has been shown to be significantly superior to oven drying in terms of redispersibility. For the TENCEL^® gel, oven-drying was thus selected and the influence of additives on the redispersibility of the cellulose II gel was studied by means of the common water retention value, particle size, colloidal stability, appearance of the redispersed gel and viscosity. The addition of the polysaccharides carboxymethyl cellulose or xanthan showed the most promising results with regard to redispersibility. Also sucrose and ammonium bicarbonate provided higher colloidal stabilities than that of the untreated TENCEL^® gel. The redispersibility of the cellulose II xerogels could thus be significantly improved by simple and cost-efficient mixing with additives prior to drying.     

Preparation of polysaccharide-coated nanoparticles by emulsion polymerization of styrene

It is the aim of this paper to describe the preparation of polysaccharide-coated nanoparticles by direct emulsion polymerization of styrene in the presence of native dextran. In spite of the lack of surface-active properties of native dextran, stable latexes with very low amount of coagulate were obtained. Particle size decreased with dextran concentration and molecular weight. The amount of permanently adsorbed dextran was determined by direct titration of the polysaccharide present on the surface of the nanoparticles. A maximum value of 2.5 mg m⁻² was found. Zeta-potential measurements allowed us to estimate the thickness of the hydrophilic layer, which regularly increased with dextran aqueous concentration. The dextran-coated polystyrene nanoparticles were stable in concentrated NaCl solutions and could be redispersed after freeze-drying. The mechanism of chemical modification of dextran was studied by nuclear magnetic resonance and matrix-assisted laser desorption/ionization-time of flight spectrometry studies. Graft copolymers are supposed to be formed.     

Mechanisms of red blood cells agglutination in antibody-treated paper

Recent reports on using bio-active paper and bio-active thread to determine human blood type have shown a tremendous potential of using these low-cost materials to build bio-sensors for blood diagnosis. In this work we focus on understanding the mechanisms of red blood cell agglutination in the antibody-loaded paper. We semi-quantitatively evaluate the percentage of antibody molecules that are adsorbed on cellulose fibres and can potentially immobilize red blood cells on the fibre surface, and the percentage of the molecules that can desorb from the cellulose fibre surface into the blood sample and cause haemagglutination reaction in the bulk of a blood sample. Our results show that 34 to 42% of antibody molecules in the papers treated with commercial blood grouping antibodies can desorb from the fibre surface. When specific antibody molecules are released into the blood sample via desorption, haemagglutination reaction occurs in the blood sample. The reaction bridges the red cells in the blood sample bulk to the layer of red cells immobilized on the fibre surface by the adsorbed antibody molecules. The desorbed antibody also causes agglutinated lumps of red blood cells to form. These lumps cannot pass through the pores of the filter paper. The immobilization and filtration of agglutinated red cells give reproducible identification of positive haemagglutination reaction. Results from this study provide information for designing new bio-active paper-based devices for human blood typing with improved sensitivity and specificity.     

Protection of aluminum hydroxide during lyophilisation as an adjuvant for freeze-dried vaccines

Lyophilisation of an aluminum hydroxide adjuvant containing formulation for use in vaccine development has been investigated. Novel formulation approaches using disaccharides, polysaccharides, polyols and polymers have been investigated as stabilising excipients to prevent aluminum hydroxide adjuvant from aggregation during a freeze–thaw cycle or the lyophilisation process itself. Amorphous character of the lyophilisates was investigated by wide angle X-ray diffraction. Sedimentation assay, particle size analysis and optical microscopy could show that aluminum adjuvant gel character of selected formulations could be maintained within acceptable quality or stayed the same in some cases. In principle, it could be demonstrated that not less than 10% excipient is necessary to yield good results within applied analytics evaluating the excipients’ protecting qualities. Based on results obtained by this study, the formulations containing trehalose at a concentration of 15 or 20%, dextran, PVP K 25, HES 450 and 200, saccharose and sorbitole along with polysorbate 80 and buffer promise to be good candidates for the protection of aluminum adjuvanted vaccines during exposure to coldness stresses as during freezing or lyophilisation.