Huseyin Bekir Yildiz

Redox regulation of antioxidant enzymes: post-translational modulation of catalase and glutathione peroxidase activity by resveratrol in diabetic rat liver

Resveratrol is a strong antioxidant that exhibits blood glucose-lowering effects, which might contribute to its usefulness in preventing complications associated with diabetes. The present study aimed to investigate resveratrol effects on catalase (CAT) and glutathione peroxidase (GPx) gene and protein expression, their phosphorylation states and activities in rat liver of STZ-induced diabetes. Diabetes increased the levels of total protein phosphorylation and p-CAT, while mRNA expression, protein levels, and activity were reduced. Although diabetes induced transcriptional repression over GPx, it did not affect the protein levels and activity. When resveratrol was administered to diabetic rats, an increase in activity was associated with an increase in p-GPx levels. Decrease in Sirtuin1 (SIRT1) and nuclear factor erythroid 2-related factor (Nrf2) and increase in nuclear factor kappa B (NFκB) gene expression in diabetes were associated with a decrease in CAT and GPx mRNA expression. A possible compensatory mechanism for reduced gene expression of antioxidant enzymes is proved to be nuclear translocation of redox-sensitive Nrf2 and NFκB in diabetes which is confirmed by the increase in nuclear and decrease in cytoplasmic protein levels of Nrf2 and NFκB. Taken together, these findings revealed that an increase in the oxidized state in diabetes intricately modified the cellular phosphorylation status and regulation of antioxidant enzymes. Gene regulation of antioxidant enzymes was accompanied by nuclear translocation of Nrf2 and NFκB. Resveratrol administration also activated a coordinated cytoprotective response against diabetes-induced changes in liver tissues.

Enzyme immobilization in biosensor constructions: self-assembled monolayers of calixarenes containing thiols

Herein, an amperometric glucose oxidase (GOx) biosensor is presented using calixarenes as an immobilization matrix of the biomolecule. Firstly, thiol-containing calixarenes (Calix-SH) were synthesized, then self-assembled monolayers (SAMs) of Calix-SH on a gold surface were formed and hydroxyl groups of Calix-SH were activated using 1,1′-carbonyldiimidazole (CDI) chemistry. To test the usability of Calix-SH modified surfaces as a biosensor, glucose oxidase was used as a model biological component. After optimization of preparation and working conditions, our results indicate that the Calix-SH/GOx biosensor has a linear range in the range 0.1–1.0 mM (LOD: 0.015 mM) for glucose with a 25 s response time. Finally, the application of the biosensor was examined to detect glucose in real samples. The glucose amounts were calculated as 19.460 ± 0.521 and 31.647 ± 2.125 mM in coke and fizzy drink (with orange), respectively. To confirm the reliability of the Calix-SH/GOx biosensor, the calculated glucose concentrations which were analyzed by the Calix-SH/GOx biosensor were compared to conventional spectrophotometric glucose kits. The glucose amounts in coke and fizzy drink were calculated as 18.509 ± 0.732 mM and 31.579 ± 4.466 mM, respectively.

Synthesis of an amine-functionalized naphthalene-containing conducting polymer as a matrix for biomolecule immobilization

N-functionalized dithienopyrroles (DTP-NH2) were synthesized and electropolymerized onto a graphite electrode as a novel conducting polymer matrix for biomolecule immobilization. 1H-NMR and 13C-NMR were utilized to investigate the characteristics of the monomer. After that, glucose oxidase (GOx) was immobilized onto the amino-functionalised matrix by means of glutaraldehyde. The surface morphologies of both DTP-NH2 and DTP-NH2–GOx were visualised by using SEM and fluorescence microscopy. The chronoamperometric signals of the electrochemical DTP-NH2–GOx biosensors were measured by monitoring the O2 consumption during an enzymatic reaction in the presence of glucose at −0.7 V. After the optimization of the pH and scan number of the polymer deposition in batch mode, the DTP-NH2–GOx biosensor was also tested in Flow Injection Analysis (FIA) mode. The DTP-NH2–GOx biosensors had a very good linearity between 0.05 and 1.0 mM, and between 0.1 and 2.5 mM for glucose in batch and FIA modes, respectively. Finally, it was applied for glucose analysis in real samples where commercial glucose kits were used as the reference method to verify the data obtained with the proposed biosensor.

A novel architecture based on a conducting polymer and calixarene derivative: its synthesis and biosensor construction

In this study, a novel amperometric glucose biosensor based on a selenium comprising conducting polymer and calixarene was developed. Firstly, poly(2-(2-octyldodecyl)-4,7-di(selenoph-2-yl)-2H-benzo[d][1,2,3]triazole), poly((SBTz)) was electrodeposited onto a graphite electrode by an electropolymerization technique. Then, a newly synthesized calixarene and gold nanoparticle (AuNP) mixture was used for the improvement of biosensor characteristics. GOx, as a model enzyme was immobilized on the modified electrode surface. The constructed surface serves as a sufficient immobilization platform for the detection of glucose. Calixarenes and their derivatives may be a favouring agent for enzyme immobilization due to their specific configurations. Moreover, through the covalent binding between the carboxylic groups of the calixarenes and amino groups of the biomolecule, effective enzyme immobilization can be achieved while protecting the well-ordered structure of the enzyme molecule. Amperometric detection was carried out following oxygen consumption at −0.7 V vs. the Ag reference electrode in phosphate buffer (50 mM, pH 6.5). The proposed biosensor showed a linear amperometric response for glucose within a concentration range of 0.005 to 0.5 mM (LOD: 0.004 mM). Kappm and sensitivity were calculated as 0.025 mM and 102 μA mM−1 cm−2, respectively. Scanning Electron Microscopy (SEM) was used to investigate the surface morphologies of successive modifications. Finally, the constructed biosensor was tested successfully to detect glucose in beverage samples.

L-Dopa synthesis catalyzed by tyrosinase immobilized in poly(ethyleneoxide) conducting polymers.

1-3,4-Dihydroxy phenylalanine called as l-Dopa is a precursor of dopamine and an important neural message transmitter and it has been a preferred drug for the treatment of Parkinsons disease. In this study, with regards to the synthesis of L-Dopa two types of biosensors were designed by immobilizing tyrosinase on conducting polymers: thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinylbenzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemically and tyrosinase immobilized by entrapment during electropolymerization. L-Tyrosine was used as the substrate for L-Dopa synthesis. The kinetic parameters of the designed biosensors, maximum reaction rate of the enzyme (Vmax) and Michaelis Menten constant (Km) were determined. Vmax were found as 0.007 μmol/(minelectrode) for PEO-co-PPy matrix and 0.012 μmol/(minelectrode) for CP-co-PPy matrix. Km values were determined as 3.4 and 9.2 mM for PEO-co-PPy and CP-co-PPy matrices, respectively. Optimum temperature and pH, operational and shelf life stabilities of immobilized enzyme were also examined.

New Amperometric Cholesterol Biosensors Using Poly(ethyleneoxide) Conducting Polymers

Accumulation of cholesterol in human blood can cause several health problems such as heart disease, coronary artery disease, arteriosclerosis, hypertension, cerebral thrombosis, etc. Therefore, simple and fast cholesterol determination in blood is clinically important. In this study, two types of amperometric cholesterol biosensors were designed by physically entrapping cholesterol oxidase in conducting polymers; thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemically and cholesterol oxidase was immobilized by entrapment during electropolymerization. The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H2O2 at +0.7 V in the absence of a mediator. Kinetic parameters, such as Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. Km values were found as 1.47 and 5.16 mM for PEO-co-PPy and CP-co-PPy enzyme electrodes, respectively. By using these Km values, it can be observed that ChOx immobilized in PEO-co-PPy shows higher affinity towards the substrate.

Calixarene modified montmorillonite: a novel design for biosensing applications†

Here we report the synthesis, characterization and application of calixarene (Calix) modified montmorillonite (Mt) as a platform for bio-applications such as biomolecule immobilization and biosensing technologies. This modification enhanced the biomolecule immobilization capability of Mt. Initially, amino-functionalised calixarenes (Calix-NH2) were synthesized and used as a modifier. X-ray diffraction, Fourier transform infrared spectroscopy, zeta potential and thermal gravimetric analysis were performed to verify the modification of the clay minerals. For the biosensor construction, Calix-NH2 modified Mt (Calix-NH2/Mt), bovine serum albumin (BSA), glutaraldehyde (GA) and pyranose oxidase were immobilized on the surface of a glassy carbon electrode which was then referred to as a Calix-NH2/Mt/PyOx biosensor. After optimization of the enzyme amount and pH, analytical characteristics were investigated in detail.

Construction of ferrocene modified conducting polymer based amperometric urea biosensor

Herein, an electrochemical urea sensing bio-electrode is reported that has been constructed by firstly electropolymerizing 4-(2,5-Di(thiophen-2-yl)-1H-pyrrol-1-yl)aniline monomer (SNS-Aniline) on Pencil Graphite Electrode (PGE), then modifying the polymer coated electrode surface with di-amino-Ferrocene (DAFc) as the mediator, and lastly Urease enzyme through glutaraldehyde crosslinking. The effect of pH, temperature, polymer thickness, and applied potential on the electrode current response was investigated besides performing storage and operational stability experiments with the interference studies. The resulting urea biosensors amperometric response was linear in the range of 0.1-8.5mM with the sensitivity of 0.54μA/mM, detection limit of 12μM, and short response time of 2s. The designed bio-electrode was tested with real human blood and urine samples where it showed excellent analytical performance with insignificant interference.

Electrospun Polyvinyl Borate/Poly(Methyl Methacrylate) (PVB/PMMA) Blend Nanofibers

The aim of this study was to prepare polyvinyl borate (PVB)/poly(methyl methacrylate) (PMMA) blend nanofibers by electrospinning process. Polyvinyl borate was synthesized by the condensation reaction of polyvinyl alcohol and boric acid. FTIR analyses showed that boron atoms were found to be integrated into the polymer network. Blending PMMA with PVB decreased the fiber diameter and enhanced the surface roughness of PVB/PMMA blend nanofiber mats. The water wetting property of the nanofiber mats was influenced by the surface roughness. The blend composition with the highest polyvinyl borate content was found to be suitable for thermally stable nanofiber formation.

Novel Photoelectrochemical Biosensors for Cholesterol Biosensing by Photonic “Wiring” of Cholesterol Oxidase

In this study, a novel approach for constructing different very sensitive and efficient photoelectrochemical biosensors called as P(SNS-NH2)/ChOx/[Ru(bpy)3]2+ and ChOx/[Ru(bpy)3]2+, were fabricated by bonding ChOx covalently to P(SNS-NH2) modified electrode and bare thioaniline modififed gold slide, respectively, using gluteraldehyde and tethering the N-hydroxysuccinimidyl ester functionalized Ru(II)-trisbipryridine to the ChOx enzyme. In the presence of different concentrations of cholesterol, the photocurrents were obtained by irradiating of the photoelectrochemical cell containing P(SNS-NH2)/ChOx/[Ru(bpy)3]2+ or ChOx/[Ru(bpy)3]2+ electrode as the anode under air. The bipyridine complex [Ru(bpy)3]2+ was used to activate photoinduced electron-transfer reaction and it acted as a redox mediator to activate the bioelectrocatalytic functions of ChOx. Therefore, it was shown the photonic electron-transfer wiring of ChOx with the electrode. ChOx/[Ru(bpy)3]2+ and P(SNS-NH2)/ChOx/[Ru(bpy)3)2+ biosensors showed a very good linearity between 0.05–0.9 mM and 0.00625–0.6 mM for cholesterol respective. LOD values for P(SNS-NH2)/ChOx/[Ru(bpy)3)2+ and ChOx/[Ru(bpy)3)2+ electrodes were obtained as 9.86 × 10−5 mM and 3.48 × 10−4 mM cholesterol respectively according to S/N = 3 ratios. Kinetic parameters, such as Km and Imax operational and storage stabilities, effects of pH and temperature were determined for both enzyme electrodes.