Fatma Nese Kok

Construction of an acetylcholinesterase-choline oxidase biosensor for aldicarb determination

In this study, acetylcholinesterase and choline oxidase were co-immobilized on poly(2-hydroxyethyl methacrylate) membranes and the change in oxygen consumption upon aldicarb introduction was measured. Immobilization of the enzymes was achieved either by entrapment or by surface attachment via a hybrid immobilization method including epichlorohydrin and Cibacron Blue F36A activation. Immobilized enzymes had a long-storage stability (only 15% activity decrease in 2 months in wet storage and no activity loss in dry storage). Aldicarb detection studies showed that a linear working range of 10-500 and 10-250 ppb aldicarb could be achieved by entrapped and surface immobilized enzymes, respectively. Enzymes immobilized on membrane surfaces responded to aldicarb presence more quickly than entrapped enzymes. Aldicarb concentrations as low as 23 and 12 ppb could be detected by entrapped and surface immobilized enzymes, respectively, in 25 min.

Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications.

Nanofibrous double-layer matrices were prepared by electrospinning technique with the bottom layer formed from PCL (poly-ε-caprolactone)/PLLA (poly-l-lactic acid) nanofibers and the upper layer from PCL/Gelatin nanofibers. Bottom layer was designed to give mechanical strength to the system, whereas upper layer containing gelatin was optimized to improve the cell adhesion. Gelatin microspheres were incorporated in the middle of two layers for controlled growth factor delivery. Successful fabrication of the blend nanofibers were shown by spectroscopy. Scanning electron microscopy results demonstrated that bead-free nanofibers with uniform morphology could be obtained by 10% w/v concentrations of PCL/PLLA and PCL/Gelatin solutions. Microspheres prepared by 15% gelatin concentration and cross-linked with 7.5% glutaraldehyde solution were chosen after in vitro release studies for the incorporation to the double-layer matrices. The optimized conditions were used to prepare fibroblast growth factor-2 (FGF-2) loaded microspheres. Preliminary cell culture studies showed that the FGF-2 could be actively loaded into the microspheres and enhanced the cell attachment and proliferation. The complete system had a slow degradation rate in saline (18% weight loss in 2 months) and it could meanwhile preserve its integrity. This sandwich system prevented microsphere leakage from the scaffold, and the hydrophilic and bioactive nature of the fibers at the upper layer promoted cell attachment to the surface. PLLA/PCL layer, on the other hand, improved the mechanical properties of the system and enabled better handling.

Effect of double growth factor release on cartilage tissue engineering

The effects of double release of insulin‐like growth factor I (IGF‐I) and growth factor β1 (TGF–β1) from nanoparticles on the growth of bone marrow mesenchymal stem cells and their differentiation into cartilage cells were studied on PLGA scaffolds. The release was achieved by using nanoparticles of poly(lactic acid‐co‐glycolic acid) (PLGA) and poly(N‐isopropylacrylamide) (PNIPAM) carrying IGF‐I and TGF–β1, respectively. On tissue culture polystyrene (TCPS), TGF‐β1 released from PNIPAM nanoparticles was found to have a significant effect on proliferation, while IGF‐I encouraged differentiation, as shown by collagen type II deposition. The study was then conducted on macroporous (pore size 200–400 µm) PLGA scaffolds. It was observed that the combination of IGF‐I and TGF‐β1 yielded better results in terms of collagen type II and aggrecan expression than GF‐free and single GF‐containing applications. It thus appears that gradual release of a combination of growth factors from nanoparticles could make a significant contribution to the quality of the engineered cartilage tissue. Copyright

Immobilization of acetylcholinesterase and choline oxidase in/on pHEMA membrane for biosensor construction

In this study, acetylcholinesterase (AChE) and choline oxidase (ChO) were co-immobilized on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes with the aim of using them in biosensor construction. pHEMA membranes were prepared with the addition of different salts in different HEMA : aqueous solution ratios and characterized in terms of porosity, thickness, permeability, and mechanical properties. Membranes prepared in the presence of SnCl4 were found to be superior in terms of porosity and permeability and were chosen as the immobilization matrix. Immobilization of the enzymes was achieved both by entrapment and surface attachment via epichlorohydrin (Epi) and Cibacron Blue F36A (CB) activation. The effect of immobilization on enzyme activity was evaluated by the comparison of Km and Vmax values for the free and immobilized bi-enzyme systems. The increase in Km was negligible (1.08-fold) for the bi-enzyme system upon immobilization on surface but was 2.12-fold upon entrapment. Specific activity of the free enzyme system was found to be 0.306 mVs-1 μg-1 ChO while it was 0.069 (4.43-fold decrease) for entrapped and 0.198 (1.54 fold decrease) for CB-Epi immobilized enzymes. The performance of immobilized enzymes in different buffer types, pH, and temperature conditions were evaluated. The best enzyme activity was obtained at pH 9.0. Activity of the enzymes was found to increase with increasing temperature (in the range 25-40°C).

Controlled release of aldicarb from carboxymethyl cellulose microspheres: in vitro and field applications

Aldicarb is a carbamate pesticide that is widely used throughout the world in the protection of crops (eg cotton, nuts, potatoes, onion, tobacco, sugar beet and sugar cane). In Turkey, especially in the Cukurova region, it is used for the control of the cotton white fly (Bemisia tabaci) which attacks cotton plants cultivated in this region. Aldicarb contamination in surface and ground water is a serious problem in several countries, partly due to its high water solubility. It is also highly toxic to mammals. In order to overcome these problems, microspheres of aldicarb were prepared using carboxymethyl cellulose (CMC) as the biodegradable support material cross-linked with aluminium chloride. A strong hysteresis behaviour was observed upon drying and reswelling. Encapsulation efficiency was in the range 12-23% and aldicarb contents of 5.7-10.3 mg per 100 mg of microspheres was achieved. In vitro release was distinctly Fickian, and Higuchi constants were very close to 0.5. Release in pots revealed that only one sample had a release capability for more than four weeks. In the cotton plot much longer durations of release (more than seven weeks) were observed while a commercial granular formulation released its content immediately. It was thus possible to construct a controlled pesticide release system that prolonged the bioavailability to about eight weeks. # 1999 Society of Chemical Industry The control of pests often requires periodic applica- tion of pesticide to the crop using conventional formulations, eg powders, granules, or concentrated emulsions. These lead to significant levels of environ- mental pollution due to the application of extensive quantities of pesticide required to prolong effective- ness. Losses can occur due to wash-out, evaporation, surface run-off and dispersion to unintended regions, 1-3 which also has undesirable economical consequences. Such disadvantages can be overcome by providing low levels of the pesticide over the desired period using a bioactive material release system based on the diffusion of the bioactive compound through a matrix or membrane. 4-6

Immobilization of laccase on polymer grafted polytetrafluoroethylene membranes for biosensor construction

In this study, Trametes versicolor laccase was immobilized on polytetrafluoroethylene (PTFE) membranes using two different techniques, entrapment to gelatin and covalent immobilization to the surface. For surface immobilization, functional groups were formed on PTFE surface by radiofrequency (RF) plasma treatment followed by polymer grafting. Two different polymers, polyacrylamide (pAAm) and polyacrylic acid (pAAc) were tried. For polyacrylamide grafted PTFE, a two-step polymerization process was used. The membranes were first treated with hydrogen plasma and pAAm grafted PTFE (pAAm-g-PTFE) was then formed by argon plasma treatment. To produce pAAc grafted PTFE (pAAc-g-PTFE), the surface was first treated with argon plasma and AAc was then attached to the surface by heat treatment (70°C, 6h). For both cases, an optimized carbodiimide coupling reaction was used for laccase immobilization. Enzyme activity was measured by an oxygen electrode using guaiacol as substrate. All three biosensing membranes were characterized and compared in terms of optimum working conditions, storage stability and reusability. Our study concluded that although a higher activity was obtained by gelatin entrapped laccase, its mechanical instability and poor storage life makes the gelatin biosensor unattractive for multiple usages and for field measurements. pAAc-g-PTFE biosensor was found to be more stable and highly reusable (ca. 50 times) when compared with the other two biosensors. In addition, its sensitivity was suitable for field applications. Therefore, the pAAc-g-PTFE biosensor could be proposed as an alternative on-site detection tool for phenolic compound monitoring.

Magnesium substituted hydroxyapatite formation on (Ti,Mg)N coatings produced by cathodic arc PVD technique

In this study, formation of magnesium substituted hydroxyapatite (Ca10-xMgx(PO4)6(OH)2) on (Ti,Mg)N and TiN coating surfaces were investigated. The (Ti1-x,Mgx)N (x=0.064) coatings were deposited on titanium substrates by using cathodic arc physical vapor deposition technique. TiN coated grade 2 titanium substrates were used as reference to understand the role of magnesium on hydroxyapatite (HA) formation. The HA formation experiments was carried out in simulated body fluids (SBF) with three different concentrations (1X SBF, 5X SBF and 5X SBF without magnesium ions) at 37 °C. The coatings and hydroxyapatite films formed were characterized by scanning electron microscope (SEM), X-ray diffraction (XRD) and FTIR Spectroscopy techniques. The energy dispersive X-ray spectroscopy (EDS) analyses and XRD investigations of the coatings indicated that magnesium was incorporated in the TiN structure rather than forming a separate phase. The comparison between the TiN and (Ti, Mg)N coatings showed that the presence of magnesium in TiN structure facilitated magnesium substituted HA formation on the surface. The (Ti,Mg)N coatings can potentially be used to accelerate the HA formation in vivo conditions without any prior hydroxyapatite coating procedure.

Alteration of PTFE Surface to Increase Its Blood Compatibility

The aim of this study is to increase the blood compatibility of polytetrafluoroethylene (PTFE), one of the preferred materials for soft-tissue application, by a two-step procedure: first, the surface was activated by hydrogen plasma followed by acrylamide attachment and, secondly, hirudin, a potent antithrombogenic protein from leeches, was immobilized to the surface. Plasma treatment conditions were optimized and different surfaces were characterized by water contact angle measurements, ATR–FT-IR and X-ray photoelectron spectroscopy (XPS). It was seen that the contact angle of the PTFE decreased from 126° to 55° in optimum conditions. Acrylamide (25% (w/v) in ethanol/acetone (50%, v/v)) was grafted to the surface by the help of argon plasma treatment (1 min, 50 W, 13 Pa). The water contact angle was further decreased to 33° with acrylamide grafting and amide groups, which were subsequently used in protein immobilization, and could be detected both by ATR–FT-IR and XPS analysis. In the second part, hirudin was attached to these amide groups on PTFE surface by an optimized EDC/NHS activation procedure. Then a thrombogenicity test was done to detect hirudin activity. The results showed that there is a significant decrease in the clot formation compared with the untreated PTFE samples and ca. 0.3–0.4 ATU/cm2 (22–29 ng/cm2) of hirudin was enough to prevent the clot formation. A preliminary study showed that the hirudin immobilized membranes keep their antithrombogenic activity for at least 40 days in 37°C in PBS (0.1 M, pH 7.4). As a result, the blood compatibility of PTFE surfaces was ameliorated by plasma-induced monomer grafting and hirudin immobilization, and an alternative material was obtained to be used in medical applications such as vascular grafts, catheters, etc.

Biodegradation of aldicarb in a packed-bed reactor by immobilized Methylosinus

Abstract Carboxymethylcellulose microspheres cross-linked via aluminum ions were used as a support material for immobilization of Methylosinus isolated from soil contaminated with aldicarb. The degradation capacity of immobilized bacteria, different parameters such as substrate concentration (50–800 ppm), flow rate (10–60 ml h −1 ), and continuous contact with reaction medium (flow rate, 20 ml h −1 and concentration, 100 ppm) that affect aldicarb degradation were investigated in a packed-bed reactor. Increases in the flow rate decreased the conversion of aldicarb into its metabolites. On the other hand, increasing the substrate concentration up to 400 ppm led to an increase in the amount of aldicarb converted (max 16%). Beyond this, the proportion of aldicarb that converted was decreased, reaching approximately 7% at 800 ppm. The apparent kinetic parameters, K ′ m and V max , were determined to be as 310.11 ppm and 2.29 × 10 −2 ppm s −1 , respectively. Operation of the bioreactor in the recycled mode was much more efficient, degrading 50% of the aldicarb in 24 h and 100% in four days.

Microfluidic device on a nonwoven fabric: A potential biosensor for lactate detection

In the present study, a novel, wearable textile based microfluidic device was developed that provides a non-invasive, rapid, semi-quantitative detection of the lactate level in simulated sweat solution. The potential application was envisioned to be a biosensor that can monitor an athlete’s physical status during exercise. A photolithography technique was used for the fabrication of hydrophilic micro channels and reservoirs surrounded by hydrophobic barriers made from SU-8 negative photoresist. The reservoirs were functionalized by co-immobilization of lactate oxidase (LOX) and horseradish peroxidase (POX) enzymes. LOX uses L-(+)-Lactic acid as substrate and produces H2O2 which is a POX substrate. Then, POX oxidases H2O2 in the presence of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and results in color formation. The studies showed that excess amount of analyte presence resulted in analyte inhibition. It was also shown that analyte pH and temperature were effective on the color formation. For effective results, analyte pH and temperature should be ≥5℃ and 25–30℃, respectively. Lower pH and higher temperature values resulted in a decrease in the enzyme activity. The textile based biosensor system could make a semi-quantitative visual detection to differentiate between the normal (<5 mM) and high (≥5 mM) lactate level: while a high lactate level led to a denser purple color formation, normal levels led to a light purple formation and a green color started to be observed.