Fatma Ayhan

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.

Sterilization of Sutures by Low Temperature Argon Plasma

Nondegradable and biodegradable, mono- and multifilament, natural and synthetic sutures were sterilized by treating in an argon gas plasma glow-discharge system at different glow-discharge powers (10, 20 and 40 watt) and exposure times (5, 15 and 30 min). All of the sutures (except plain and chromic catguts) were successfully sterilized. Plain and chromic catgut could not be sterilized even at very high discharge power and exposure time. A five watt discharge and 5 min of exposure time were considered optimal conditions for argon plasma sterilization without changing the desired suture mechanical properties (extensibility, knot-pull breaking and tensile strengths).

Surface Modification and Covalent Coupling of Concanavalin A onto Poly(EGDMA/HEMA) Microbeads for Cell Affinity Applications

The surfaces of nonporous and porous Poly(EGDMA/HEMA) microbeads were modified by the reaction of sodium periodate, hexamethylenediamine and glutaraldehyde. Covalent immobilization of Concanavalin A (Con A) was optimized for maximum loading at various Con A concentrations, pH, temperatures, and ionic strengths. The optimal Con A immobilized concentration was determined as 0.37 and 0.75 mg/g microbeads for the nonporous and porous microbeads, respectively. Covalent coupling was achieved at 37 C, with 0.5 mg/mL of Con A at pH 7.4 and ionic strength of 0.01. The specific activity of Con A immobilized microbeads to carbohydrate structure was tested for affinity for myeloma HeLa cells. The data indicates that Con A may play a role in myeloma cell adhesion.

Water based PHEMA hydrogels for controlled drug delivery

Abstract Objective: In the scope of presented work, synthesis of water based acrylate hydrogels, characterization, and their usage in controlled drug release systems were aimed to investigate. Methods: Synthesis of acrylate based hydrogels that have different properties was carried out by free radical photopolymerization using photoinitiator. Because of its high biocompatibility, 2-hydroxyethyl metacrylate (HEMA) was used as monomer. Then drug release experiments were performed in pH 7.4 and 1.2 buffer solutions with certain ionic strength while the dynamic swelling behaviors were also determined. In the last part of the work, drug activities of synthesized drug-loaded hydrogels were tested in mediums containing Staphylococcus aureus and Pseudomonas aeruginosa bacteria cultures. Results: ATR-FTIR spectrums of all synthesized hydrogels were analyzed. The characteristic O-H, C-H, C=O, C-O tension vibrations bands were observed in the spectrums of the hydrogels. The rate of drug release in acidic pH 1.2 for two types of hydrogels was observed to be much faster than at pH 7.4. It was determined that hydrogel swelling ratio decrease with increasing monomer ratio. All drug loaded hydrogels were effective to inhibit the growth of both two bacterial strains. Conclusion: Hydrogels synthesized were found to be suitable for the controlled drug delivery applications.

Homocysteine-BSA Affinity Based Biosensor Design

Objective: In this research, the detection and Reusability of Homocysteine by piezoelectric based Homocysteine Bioaffinity sensor using Quartz Crystal Microbalance (QCM) was aimed. Methods: The surface of silver sensor was subjected to a series of pretreatments. Surface activation process was done with the interaction of silver crystals with NaOH, acetone and methanol, respectively at definite times. Then, systeamine, glutaraldehyde attachments and BSA immobilization were performed to surfaces. BSA concentration was assayed within a range and the highest coupling BSA amount of 0.1 mg/mL was chosen as the most convenient concentration. Results: In the scope of BSA-Homocysteine affinity, the calibration graph of Homocysteine was formed after surface modification steps and the range of 0.5-2 μmol was estimated to give the best determination coefficient. Reusability of quartz crystals were tested by the cleavage of BSA-Homocysteine affinity in acidic and basic conditions. The NaOH solutions of 10-6, 10-3, 10-1, 1, 10 ve 100 mM (pH 8-9) were used to cleave the affinity and it was observed that no basic medium trials were convenient to reuse. Assays performed to break BSA-Homocysteine affinity in acidic condition were done with 0.01, 1, 10, 100 mM Glisin-HCl solutions of pH 2.5, 3.5, 5.5 and 6.5. The crystal reusability of 12 was reached with Glisin-HCl solution of 0.01, mM in pH 5.5. Conclusion: The highest reusability value of 30 repeat was reached when Glisin-HCl concentration is 1mM with pH 5.5 achieved to cleave BSA-Homocysteine affinity experiments. The experiments carried out in 10 mM Glisin-HCl acidic medium concentration gave oppurtunity to four reuse of silver crystal.

In Vitro Evaluation of 3T3 and MDBK Cells Attachment and Proliferation on Collagen and Fibronectin Immobilized Nonwoven Polylactide Matrices

Fibroblast-like, 3T3 (Mouse Swiss Albino) and epithelial-like MDBK (Madine Darby Bovine Kidney) cells were selected as model cell lines and the adhesion of these cells on nonwoven poly DL-lactide (PDLLA) matrices were investigated. PDLLA was synthesized by ring-opening polymerization of DL-lactide. Nonwoven PDLLA matrices were prepared by an extrusion/winding process. Surface modification of these matrices was achieved by glow-discharge treatment, which was followed by glutaraldehyde incorporation. Biologically modification was performed by immobilization of collagen and/or fibronectin for cell attachment and proliferation. Adhesion values of fibroblast-like and epithelial-like cells after incubation for 2 h was 90 and 85% of initial cells, respectively. The 3T3 cells grown on PDLLA, after 11 days of incubation, was 9 105 cells while the control group was 1.2 106 cells. MDBK cells grown on the same polymeric matrices were determined to be 1.5 106 cells and 1.8 106cells for the control group. These results indicate that biodegradable nonwoven PDLLA matrices can be used for adhesion and proliferation of primary cell cultures and that implants of polymer-cells composites can be used in wound healing. An important advantage of these matrices is that a second surgical operation is not necessary to remove the implant due to the biodegradation of polymer in tissue.