Sinan Akgöl

Hydrolysis of sucrose by invertase immobilized onto novel magnetic polyvinylalcohol microspheres

Abstract The magnetic polyvinylalcohol (PVAL) microspheres were prepared by crosslinking glutaraldehyde. 1,1′-Carbonyldiimidazole (CDI), a carbonylating agent was used for the activation of hydroxyl groups of polyvinylalcohol, and invertase immobilized onto the magnetic PVAL microspheres by covalent bonding through the amino group. The retained activity of the immobilized invertase was 74%. Kinetic parameters were determined for immobilized invertase, as well as for the free enzyme. The K m values for immobilized invertase (55 mM sucrose) were higher than that of the free enzyme (24 mM sucrose), whereas V max values were smaller for the immobilized invertase. The optimum operational temperature was 5°C higher for immobilized enzyme than that of the free enzyme. The operational inactivation rate constant ( k opi ) of the immobilized invertase at 35°C with 200 mM sucrose was 5.83×10 −5 min −1 . Thermal and storage stabilities were found to increase with immobilization.

A Novel Magnetic Adsorbent for Immunoglobulin‐G Purification in a Magnetically Stabilized Fluidized Bed

A novel magnetic poly(ethylene glycol dimethacrylate‐N‐methacryloly‐l‐histidinemethylester) [m‐poly(EGDMA‐(MAH)] support was prepared for purification of immunoglobulin G (IgG) in a magnetically stabilized fluidized bed by suspension polymerization. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 μmol MAH/g polymer. Magnetic poly(EGDMA‐MAH) beads were used in the separation of immunoglobulin‐G (IgG) from aqueous solutions and/or human plasma in a magnetically stabilized fluidized bed system. IgG adsorption capacity of the beads decreased with an increase in the flow rate. The maximum IgG adsorption was observed at pH 6.0 for MES buffer. IgG adsorption onto the m‐poly(EGDMA) was negligible. Higher adsorption values (up to 262 mg/g) were obtained in which the m‐poly(EGDMA‐MAH) sorbents were used from aqueous solutions. Higher amounts of IgG were adsorbed from human plasma (up to 320 mg/g) with a purity of 87%. IgG molecules could be repeatedly adsorbed and desorbed with these sorbents without noticeable loss in their IgG adsorption capacity.

Phenylalanine containing hydrophobic nanospheres for antibody purification

In this study, novel hydrophobic nanospheres with an average size of 158 nm utilizing N‐methacryloyl‐(L)‐phenylalanine methyl ester (MAPA) as a hydrophobic monomer were produced by surfactant free emulsion polymerization of 2‐hydroxyethyl methacrylate (HEMA) and MAPA conducted in an aqueous dispersion medium. MAPA was synthesized using methacryloyl chloride and L‐phenylalanine methyl ester. Specific surface area of the nonporous nanospheres was found to be 1874 m2/g. Poly(HEMA–MAPA) nanospheres were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Average particle size, size distribution, and surface charge measurements were also performed. Elemental analysis of MAPA for nitrogen was estimated as 0.42 mmol/g polymer. Then, poly(HEMA–MAPA) nanospheres were used in the adsorption of immunoglobulin G (IgG) in batch system. Higher adsorption values (780 mg/g) were obtained when the poly (HEMA–MAPA) nanospheres were used from both aqueous solutions and human plasma. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It was observed that IgG could be repeatedly adsorbed and desorbed without significant loss in adsorption amount. These findings show considerable promise for this material as a hydrophobic support in industrial processes.

High capacity binding of antibodies by poly(hydroxyethyl methacrylate) nanoparticles.

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 150 nm in diameter and with a poly-dispersity index of 1.171 were produced by a surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 1779 m(2)/g. Reactive imidazole containing 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was used as a pseudo-specific ligand. IMEO was attached covalently onto the nanoparticles. PHEMA-IMEO nanoparticles were used for the affinity binding of immunoglobulin-G (IgG) from human plasma. To evaluate the degree of IMEO loading, the PHEMA nanoparticles were subjected to Si analysis by using flame atomizer atomic absorption spectrometer and it was estimated as 64.5 mg/g of polymer. The nanoparticles were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). IgG binding onto the PHEMA nanoparticles was found to be 5.2 mg/g. Much higher binding values (up to 843 mg/g) were obtained for the PHEMA-IMEO nanoparticles. IgG could be repeatedly bound and eluted on PHEMA-IMEO nanoparticles without noticeable loss in the IgG binding capacity.

Glucose oxidase and catalase adsorption onto Cibacron Blue F3GA-attached microporous polyamide hollow-fibres

Abstract The aim of this study was to explore in detail the performance of polyamide hollow fibers to which Cibacron Blue F3GA was attached for adsorption of proteins. Model proteins were glucose oxidase, as a flavo-enzyme and contains two tightly bound flavine adenine dinucleotide cofactor, and catalase as a heme-containing metallo-enzyme. The hollow fiber structure was characterized by scanning electron microscopy. These dye-carrying hollow-fibers (35.8 μmol/g) were used in the glucose oxidase and catalase adsorption–elution studies. The non-specific adsorption values were 1.25 mg/g for glucose oxidase and 1.97 mg/g for catalase. Cibacron Blue F3GA attachment increased the adsorption capacity up to 248 mg/g. Langmuir adsorption model was found to be applicable in interpreting glucose oxidase and catalase adsorption by Cibacron Blue F3GA-attached hollow fibers. Significant amount of the adsorbed proteins (up to 97%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0. In order to determine the effects of adsorption and elution conditions on possible conformational changes of studied protein structures, fluorescence spectrophotometry was employed. It was concluded that polyamide dye-affinity hollow-fibers can be applied for glucose oxidase and catalase adsorption without causing any significant conformational changes. Repeated adsorption/elution processes showed that these dye-attached hollow-fibers are suitable for glucose oxidase and catalase separation.

A novel biosensor for specific determination of hydrogen peroxide: catalase enzyme electrode based on dissolved oxygen probe.

A biosensor for the specific determination of hydrogen peroxide was developed using catalase (EC 1.11.1.6) in combination with a dissolved oxygen probe. Catalase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane served as enzyme electrode. The electrode response was maximum when 50 mM phosphate buffer was used at pH 7.0 and at 35 degrees C. The biosensor response depends linearly on hydrogen peroxide concentration between 1.0x10(-5) and 3.0x10(-3) M with a response time of 30 s. The sensor is stable for >3 months so in this period >400 assays can be performed.

Selective separation of human serum albumin with copper(II) chelated poly(hydroxyethyl methacrylate) based nanoparticles.

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 300 nm in diameter and with a polydispersity index of 1.156 were produced by surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 996 m(2)/g. Metal-chelating ligand 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was covalently attached to the PHEMA nanoparticles. IMEO content was 0.97 mmol IEMO/g. The morphology and properties of these nanoparticles were characterized with scanning electron microscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The Cu2+-chelated PHEMA-IMEO nanoparticles were used in the adsorption-elution studies of human serum albumin (HSA) in a batch system. Maximum HSA adsorption amount of the Cu2+ chelated nanoparticles was 680 mg HSA/g. The PHEMA-IMEO-Cu2+ nanoparticles exhibited a quite high adsorption capacity and fast adsorption rate due to their high specific surface area and the absence of internal diffusion resistance.

Silane‐Modified Magnetic Beads: Application to Immunoglobulin G Separation

The magnetic poly(2‐hydroxyethyl methacrylate ethylene glycol dimethacrylate) [m‐poly(HEMA‐EGDMA)] beads (150–250‐μm diameter in spherical form) were prepared by a radical suspension polymerization technique. The pseudo‐specific ligand, reactive imidazole containing 3–(2‐imidazoline‐1‐yl)propyl (triethoxysilane) (IMEO) was selected as a silanization agent. IMEO was covalently immobilized onto the magnetic beads. IMEO‐immobilized m‐poly(HEMA‐EGDMA) beads were used for the affinity adsorption of immunoglobulin‐G (IgG) from aqueous solutions and human plasma. To evaluate the degree of IMEO attachment, the m‐poly(HEMA‐EGDMA) beads were subjected to Si analysis by using flame atomizer atomic absorption spectrometer, and it was estimated as 36.6 mg IMEO/g of polymer. The nonspecific IgG adsorption onto the plain m‐poly(HEMA‐EGDMA) beads was very low (about 0.4 mg/g). Higher adsorption values (up to 55 mg/g) were obtained when the m‐poly(HEMA‐EGDMA)/IMEO beads were used from both aqueous solutions and human plasma. The maximum IgG adsorption on the m‐poly(HEMA‐EGDMA)‐IMEO beads was observed at pH 7.0. The IgG molecules could be repeatedly adsorbed and desorbed with m‐poly(HEMA‐EGDMA)‐IMEO beads without noticeable loss in the IgG adsorption capacity. The adsorption capacity from human plasma in magnetically stabilized fluidized bed decreased drastically from 78.9 to 19.6 mg/g with the increase of the flow rate from 0.2 to 3.5 mL/min.

Immobilized metal affinity beads for ferritin adsorption.

A new metal–chelate adsorbent utilizing N-methacryloyl-(L)-cysteine methyl ester (MAC) was prepared as a metal-chelating ligand. MAC was synthesized by using methacryloyl chloride and L-cysteine methyl ester dihydrochloride. Spherical beads with an average diameter of 150–200 μm were produced by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAC carried out in an aqueous dispersion medium. Then, Fe3+ ions were chelated directly on the beads. Properties such as specific surface area, specific pore volume and ligand occupation were determined. The specific surface area of the beads was found to be 18.9 m2/g. The total pore volume was 2.8 ml/g and represented a porosity over 52%. The average pore size of the poly(HEMA-MAC) beads was 620 nm. Fe3+ -chelated beads were used in the adsorption of ferritin from aqueous solutions. Ferritin adsorption increased with increasing ferritin concentration. The maximum ferritin adsorption capacity of the Fe3+ -chelated poly(HEMA-MAC) beads (Fe3+ loading 0.81 mmol/g) was found to be 3.7 mg/g at pH 4.0 in acetate buffer. The non-specific ferritin adsorption on the poly(HEMA-MAC) beads were 0.4 mg/g. Adsorption behavior of ferritin could be modelled using both the Langmuir and Freundlich isotherms. Adsorption capacity decreased with increasing ionic strength of the binding buffer. Ferritin molecules could be adsorbed and desorbed five times with these adsorbents without noticeable loss in their ferritin adsorption capacity.

The fabrication of nanosensor-based surface plasmon resonance for IgG detection.

Abstract Poly(2-hydroxyethyl methacrylate)/3-(2-imidazoline-1-yl)propyl(triethoxysilane) (PHEMA/IMEO) nanoparticles were attached on surface plasmon resonance (SPR) sensor for the real-time detection of human immunoglobulin G (IgG) in human serum. The PHEMA/IMEO nanoparticles–attached SPR sensor was characterized by Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. IgG detection studies were performed using aqueous IgG solutions at different concentrations. In order to show the selectivity and specificity of the SPR sensor, competitive kinetic analyses were performed using IgG, albumin, hemoglobin in singular and competitive manner. Finally, IgG detection in human serum was carried out.