Veyis Karakoç

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.

Hemoglobin binding from human blood hemolysate with poly(glycidyl methacrylate) beads.

Metal-chelating affinity beads have attracted increasing interest in recent years for protein purification. In this study, iminodiacetic acid (IDA) was covalently attached to the poly(glycidyl methacrylate) [PGMA] beads (1.6 μm in diameter). Cu(2+) ions were chelated via IDA groups on PGMA beads for affinity binding of hemoglobin (Hb) from human blood hemolysate. The PGMA beads were characterized by scanning electron microscopy (SEM). The PGMA-Cu(2+) beads (628 μmol/g) were used in the Hb binding-elution studies. The effects of Hb concentration, pH and temperature on the binding efficiency of PGMA-Cu(2+) beads were performed in a batch system. Non-specific binding of Hb to PGMA beads in the absence of Cu(2+) ions was very low (0.39 mg/g). The maximum Hb binding was 130.3 mg/g. The equilibrium Hb binding increased with increasing temperature. The negative change in Gibbs free energy (ΔG°<0) indicated that the binding of Hb on the PGMA-Cu(2+) beads was a thermodynamically favorable process. The ΔS and ΔH values were 102.2 J/mol K and -2.02 kJ/mol, respectively. Significant amount of the bound Hb (up to 95.8%) was eluted in the elution medium containing 1.0 M NaCl in 1 h. The binding followed Langmuir isotherm model with monolayer binding capacity of 80.3-135.7 mg/g. Consecutive binding-elution experiments showed that the PGMA-Cu(2+) beads can be reused almost without any loss in the Hb binding capacity. To test the efficiency of Hb depletion from blood hemolysate, eluted portion was analyzed by fast protein liquid chromatography. The depletion efficiency for Hb was above 97.5%. This study determined that the PGMA-Cu(2+) beads had a superior binding capacity for Hb compared to the other carriers within this study.

Performance of Protein-A-Based Affinity Membranes for Antibody Purification

The preparation of affinity membranes for application in antibody purification studies is described here. Protein-A-attached poly(hydroxyethyl methacrylate-N-methacryloyl-L-alanine) (PHEMAAL) membranes were produced by a photopolymerization technique and then characterized by swelling tests, surface area measurements, contact angle and scanning electron microscopy (SEM) studies. The water swelling ratio of the PHEMAAL membrane was 133.2%. PHEMAAL membranes have large pores with a size in the range of 5–10 μm. Protein A was covalently attached onto the PHEMAAL membranes via cyanogen bromide (CNBr) activation. Maximum protein A loading was 4.7 mg/g. There was a very low non-specific IgG adsorption onto the PHEMAAL membranes, about 0.38 mg/g. The maximum IgG adsorption on the PHEMAAL–protein A membrane was found to be 9.8 mg/g at pH 7.4 from aqueous solutions. Higher adsorption amount was observed from human plasma (up to 37.3 mg/g). Adsorbed IgG was eluted using 0.1 M glycine-HCl buffer (pH 3.5) with a purity of 93%. PHEMAAL–protein A membrane was used for repetitive adsorption/elution of IgG without noticeable loss in IgG adsorption amount after 10 cycles. The PHEMAAL–protein A membrane showed several advantages, such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–elution cycles.

Chiral ligand-exchange separation and determination of malic acid enantiomers in apple juice by open-tubular capillary electrochromatography

This study describes the application of an open tubular capillary column for chiral ligand-exchange separation and determination of malic acid enantiomers in apple juice by open-tubular capillary electrochromatography (OT-CEC). The open tubular column was prepared by in-situ grafting polymerization of 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl) and followed by L-Histidine (L-His) modification. L-His was used as a chiral ligand-exchange selector and copper (II) as a central ion. The electrochromatographic characterization of the open tubular column was performed with the use of thiourea as an electroosmotic flow (EOF) marker. Factors affecting electrochromatographic enantioseparation of malic acid were also studied. The running buffer conditions for optimum enantioseparation of malic acid were found to be ACN/5.0mM CuSO4, 20.0mM (NH4)2SO4 (60/40%, v/v) adjusted to pH 3.0. The separation and determination of the enantiomers of malic acid in the apple juice solution diluted 10- to 40-folds were successfully achieved.

Efficient Removal of Bilirubin from Human Serum by Monosize Dye Affinity Beads

Cibacron Blue F3GA (CB) was covalently attached onto poly(glycidyl methacrylate) (PGMA) monosize beads for removal of bilirubin from hyperbilirubinemia human serum. PGMA beads were produced by dispersion polymerization (1.6 μm in diameter). CB loading was 1.73 mmol/g. Bilirubin adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of bilirubin was low (0.4 mg/g polymer). CB attachment onto the PGMA beads significantly increased the bilirubin adsorption (241.5 mg/g) from aqueous solutions. The maximum bilirubin adsorption was observed at pH 6.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption amount of bilirubin decreased drastically. The equilibrium adsorption of bilirubin significantly increased with increasing temperature. Much higher adsorption values up to 332 mg bilirubin/g were achieved in the case of the PGMA/CB beads from human plasma.

Metal-immobilized magnetic nanoparticles for cytochrome C purification from rat liver.

Cu2+‐immobilized magnetic poly(hydroxyethylmethacrylate‐N‐methacryloyl‐(l)‐histidinemethylester) (mPHEMAH) nanoparticles were prepared by surfactant‐free emulsion polymerization for cytochrome C (cyt C) purification from rat liver. Elemental analysis, atomic force microscopy, zeta sizer, and vibrating sample magnetometer were used to characterize mPHEMAH nanoparticles. In addition to these characterization steps, surface area, average particle size, and size distribution of mPHEMAH nanoparticles were determined. Quantity of immobilized Cu2+ was measured using atomic absorption spectrophotometry. N‐Methacryloyl‐(l)‐histidinemethylester and Cu2+ content of mPHEMAH nanoparticles were 0.18 mmol/g polymer and 0.11 mmol/g polymer, respectively. Specific surface area of Cu2+‐immobilized mPHEMAH nanoparticles was 1180 m2/g. Effect of initial cyt C concentration, pH, temperature, and ionic strength on cyt C adsorption onto Cu2+‐immobilized mPHEMAH nanoparticles was investigated. Maximum cyt C adsorption capacity of Cu2+‐immobilized mPHEMAH nanoparticles was 311.9 mg/g polymer. Maximum adsorption was obtained at pH 8.0 and 4 °C. Cu2+‐immobilized mPHEMAH nanoparticles were used ten times with 4.1% decrease in adsorption capacity. In the last stage, Cu2+‐immobilized mPHEMAH nanoparticles were used to purify cyt C from rat liver tissue, and the purity of desorbed fractions was controlled by SDS‐PAGE.

Dye-attached magnetic poly(hydroxyethyl methacrylate) nanospheres for albumin depletion from human plasma

Abstract The selective binding of albumin on dye-affinity nanospheres was combined with magnetic properties as an alternative approach for albumin depletion from human plasma. Magnetic poly(hydroxyethyl methacrylate) (mPHEMA) nanospheres were synthesized using mini-emulsion polymerization method in the presence of magnetite powder. The specific surface area of the mPHEMA nanospheres was found to be 1302 m2/g. Subsequent to Cibacron Blue F3GA (CB) immobilization onto mPHEMA nanospheres, a serial characterization processing was implemented. The quantity of immobilized CB was calculated as 800 μmol/g. Ultimately, albumin adsorption performance of the CB-attached mPHEMA nanospheres from both aqueous dissolving medium and human plasma were explored.

Characterization and cellular interaction of fluorescent-labeled PHEMA nanoparticles

Abstract The aim of this study is to label the cells with polymeric nanoparticles properly and efficiently. For this purpose, acridine orange (AO)-loaded poly(2-hydroxyethyl methacrylate) (PHEMA) nanoparticles were synthesized by miniemulsion polymerization. PHEMA nanoparticles were characterized by zeta sizer, zeta potential, atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), fluorescence microscopy, Fourier transform infrared spectrophotometer (FTIR), and elemental analysis. In addition, the toxicity of the nanoparticles were investigated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.