Eduardo E. Smolko

Immobilized metal ion affinity hollow-fibre membranes obtained by the direct grafting technique

Abstract An immobilized metal ion affinity hollow fibre was prepared by radiation-induced direct grafting of glycidylmethacrylate (GMA) on hydrophilized polyethylene membranes. The epoxy group was converted into an iminodiacetate by iminodiacetic acid treatment. The effect of the radiation dose, salt inhibitors, methanol and GMA concentration, on the grafting degree was studied. The degree of grafting was closely related to the GMA concentration. Salt inhibitors failed in the production of a differential effect on the grafting and homopolymerization processes. Between 30 and 35% of methanol, there is a maximum yield, and no grafting was obtained with a methanol concentration above 50%. Water flux dropped exponentially with the increase in the grafting degree. Scanning-electron microscopy showed that the graft branches are formed on the pores. The pectinesterase adsorption capacity of the membranes was of the same order as of the commercial chelating soft gels.

Hydrogels for immobilization of bacteria used in the treatment of metal-contaminated wastes

Abstract Polymeric matrices prepared by gamma irradiation of 2-hydroxyethyl methacrylate and 2-hydroxyethyl acrylate at −78°C in the presence of water and glycerol and poly(vinyl alcohol) membranes were examined as carriers for immobilization of bacterial cells in experiments of metal decontamination. Bacterial strains capable of growing in the presence of heavy metals were selected from soil and water from the Rio de la Plata coasts in Argentina and cultured in the hydrophilic membranes with the aim of bioremediation of the standard contaminated solutions. The results obtained indicate that removal from free bacteria was more efficient for Pb(II) and Cd(II) than for Cr(III) and Cu(II). It was ascertained that all metals could be immobilized in the polymer matrices to some extent. The Cr(III) ion concentration in bacteria immobilized on the acrylic hydrogel was approximately double of that found in the poly(vinyl alcohol) membrane.

Radiation grafting of different monomers onto PP foils irradiated with a 25 MeV proton beam

Abstract The radiation induced graft polymerisation is a well known method to obtain new materials. Until recently only conventional radiation sources, such as 60Co and electron beams were used. Moreover, part of the damage induced in polymers by heavy ions can produce active sites (peroxides and hydroperoxides) useful to initiate grafting reactions. In this way, the damage induced in polypropylene (PP) by a 25 MeV proton beam was used to initiate grafting reactions by the peroxide method. So, polymer surface modification was obtained by grafting different monomers onto PP films. The present work shows the grafting yield of acrylic acid, methyl methacrylate and styrene as a function of proton fluency and dose. The experimental points obtained by grafting PP foils were fitted according to the equations deduced supposing the target theory

Protein Adsorption onto Tentacle Cation-Exchange Hollow-Fiber Membranes

A sulfonic group (up to 200 μmol/mL) membrane was incorporated to epoxy‐activated microporous hollow fibers to obtain high‐capacity convective ion exchangers. The pure water flux through the membrane decreased exponentially with sulfonic group density and protein binding capacity increased accordingly. At sulfonic group density of 70 μmol/mL, the membrane lysozyme maximum binding capacity was 84 ± 9 mg/mL in comparison with its theoretical monolayer maximum binding capacity of 20 mg/mL, thus evidencing tentacle formation. After a cycle of adsorption in a 30 mM sodium phosphate buffer, pH 7.0, adsorbed lysozyme could be quantitatively recovered following elution with 0.5 M NaCl in the same buffer. Dynamic capacity for lysozyme was 67% of maximum binding, and this value did not change at space velocities ranging from 10 to 40 min−1 as shown by the superimposition of the corresponding breakthrough curves. A cartridge assembled with 21 fibers showed a dynamic‐to‐static capacity ratio for lysozyme of 0.60 with 1 mg/mL pure lysozyme solution, and 0.42 with a particulate feed composed of 1 mg/mL lysozyme and 0.1 mg/mL yeast.

Oriented immobilization of proteins on grafted porous polymers

Abstract The modification of polymers by radiation grafting has been utilized for several decades. The penetrability of gamma rays allows to modify the internal surfaces of porous materials retaining its mechanical properties. In recent years applications of these materials to obtain chromatographic supports and biocatalysts have been reported. In this work, we described the grafting of glycidyl methacrylate (GMA) onto a macroporous polysulfone polymer. Reproducible amount of grafting, from 10% to 60% was obtained by choosing favourable monomer concentration and gamma radiation doses from 6 kGy up. Afterwards, iminodiacetic acid (IDA) and amino phenyl arsine oxide (PAO) were covalently attached to the grafted polyGMA, in correspondence with the grafting degree. Later on, a recombinant histidin-patch thioredoxin protein (HP-rTrx) was immobilized onto this surface by two different ways, involving specific protein orientations. The first one involves an IDA–Ni 2+ complex and three HP-rTrx’s histidines and the other one involves a co-ordination site between PAO and two proximal HP-rTrx’s cysteines, which corresponds to the active site of the enzyme. Specific polyclonal antibodies recognize HP-rTrx on the polymer. Proper orientation of the protein was confirmed by HP-rTrx activity measurements. The described procedure allows the successful oriented immobilization of a protein onto a macroporous polysulfone material.

Preparation and characterisation of immobilised metal ion hollow-fibre polysulphone membranes. Their application in high-speed pectic enzyme fractionation

Abstract Chelating hollow-fibre membranes were prepared from epoxy-activated polysulphone microfiltration fibres by introducing iminodiacetic acid (IDA) groups in the presence of dimethyl sulphoxide. Fibres with 160, 350 and 620 μmol epoxy groups/ml provided ligand densities of 69, 134 and 203 μmol IDA/ml and pure water fluxes of 7.8, 5.8 and 0.42 cm/min, respectively. However, lysozyme capacity was close to 4 μmol/ml for all fibres. Adsorption isotherms for lysozyme and pectinesterase did not fit Langmuir-type curves and the existence of two types of ligand (A and B) with different accessibility to proteins was assumed. For pectinesterase, maximum capacities of 5100 and 2900 U/ml and dissociation constants of 25 and 316 U/ml were found, respectively, for ligands A and B. For lysozyme, maximum capacities were 2.9 and 0.9 μmol/ml and dissociation constants 5.0 and 102 μM, respectively, for said ligands. A cartridge assembled with IDA hollow fibres had a dynamic capacity for pectinesterase of 7509 U/ml. Productivity of this cartridge for pectic enzyme fractionation was 750 pectinesterase U/ml min, far higher than that obtained with a chelating soft gel (81 pectinesterase U/ml min).

Chromatographic characterization of immobilized metal ion hollow-fiber affinity membranes obtained by direct grafting

Abstract Iminodiacetic acid was immobilized onto membranes with different grafting degrees by reaction in phosphate buffer or water/dimethyl sulfoxide. Membranes subjected to conversion in water/dimethyl sulfoxide underwent greater conversion than those modified in phosphate buffer, despite their grafting degree. Copper saturation capacity increased consistently with the grafting degree and histidine saturation capacity was approximately half than that of copper. When working with proteins, membrane behavior was related to the molecular weight of the protein tested. Accessible sites for lysozyme decreased with the increase in the grafting degree and the rise in the conversion of epoxy groups in iminodiacetic groups in water/dimethyl sulfoxide while they remained practically unchanged when the conversion step was performed in phosphate buffer. When working with hemoglobin, this effect was the same but at lower capacities. For hollow fibres with 60 and 75% grafting, capacities were the same despite the conv...

Sera radiosterilization : studies and applications

Abstract The application of ionizing radiation for sterilization of animal serum increased rapidly over the last few years, due to the fact that most radiation-resistant living organisms (fungi, bacteria, viruses, etc.) can be readily inactivated without damage to the serum. The quality of sera sterilized by ionizing radiation has been investigated. Radiosterilization was carried out in the frozen state at doses between 25 and 50 kGy. The source of gamma radiation was 60 Co. To demonstrate that serum proteins did not suffer alterations during the process of irradiation, SDS-PAGE, electrofocusing and non equilibrium pH gradient electrophoresis of the sera were carried out before and after irradiation. The biological efficiency of the irradiated sera was demonstrated by growth curves of several cell lines in cell cultures supplemented with it. The results demonstrated that radiosterilization is a simple and effective method for sera.

Ampicillin release from swellable controlled system

Abstract Ampicillin was incorporated to HEMA Hydrogel produced by radio-induced polymerization. Gamma radiation from 60 Co was used at low temperature with doses ranging up to 25 kGy. The following studies were performed: (a) In vitro ampicillin release kinetics, through dilution test and by biological activity measurements in Micrococcus luteus . (b) in vivo release kinetics and comparison with a traditional commercial preparation. Results show that released ampicillin represented as the accumulated amount by unit area is proportional to the square root of time and that it inhibited Micrococcus luteus growth in agar for 24 h. Drug release from the new formulation, as measured by instantaneous concentration in dog blood and compared with the drug release from a commercial tablet showed improved bioavailability, resulting in an average blood availability about twice the traditional one.

Cryo-gamma radiation inactivation of bovine herpesvirus type-1

Abstract The radioresistance of bovine herpesvirus-1 (BHV-1), commonly known as infectious bovine rhinotracheitis virus (IBRV), suspended in free serum Glasgow-MEM medium and frozen at −78°C was studied. The number of surviving virus at a given dose of gamma-radiation was determined by a plaque assay system. D 10 values were calculated before and after removal of cell debris. The D 10 values obtained were 4.72 kGy and 7.31 kGy before and after removal of cell debris, respectively. Our results indicate that the inactivated viral particles could be used for vaccine preparation or diagnostic reagents.