Emrah Kilinc

Horseradish peroxidase immobilized electrode for phenothiazine analysis

A horseradish peroxidase (HRP) immobilized carbon composite electrode has been developed for the amperometric study of phenothiazine analogues. Flow injection analysis and batch experiments have been realized in acetate buffer in the presence of hydrogen peroxide. Cyclic voltammetry and amperometry using a thin-layer flow cell (dual configuration, serial mode) have permitted one to suggest the mechanisms governing the biosensor signal at −0.1 V (vs. Ag/Ag+), in the presence of hydrogen peroxide, by addition of a phenothiazine derivative. It was inferred that electron transfer mediation by the phenothiazine occcurred at HRP/graphite adsorbed sites, in addition to peroxidation by dispersed HRP with substrate recycling at the graphite array-like structure of the biosensor. Thanks to these processes, high sensitivities were achieved especially in batch configurations, with amperometric detection capabilities down to 10−8 M in acetate buffer pH 4.7. Application of the biosensor to the determination of phenothiazines in drug formulations were realized.

Determination of the hydroxyl radical by its adduct formation with phenol and liquid chromatography/electrochemical detection.

An HPLC-ECD method is described for the indirect determination of the hydroxyl (OH) radical. Fentons reaction is used to produce OH, which simultaneously attacks phenols (phenol or pyrocatechol) to form the adducts, pyrocatechol or pyrogallic acid. Thus, [OH] quantification is based on the separation and detection of pyrogallic acid and/or pyrocatechol by an isocratic HPLC-ECD method. The quantification of OH is also performed alternatively by a chronoamperometric detection in an electrochemical cell, where simultaneously formed Fe(III) (Fentons reaction) combines [Fe(II)(CN)(6)](4-) to produce the Prussian blue (PB) molecules (Fe(4)(III)[Fe(II)(CN)(6)](3)). Newly formed PB molecules are then immediately converted to colorless Everitts salt (K(4)Fe(4)(II)[Fe(II)(CN)(6)](3)) with the reduction of the high-spin Fe(III) to Fe(II) at the surface of a glassy carbon electrode at +0.150V (versus Ag/AgCl). The calculated concentration of OH during incubation (0.626ppm) can be detected with negative errors by the HPLC-ECD (0.595 and 0.615ppm with the errors -5.2 and -1.8%, respectively) and by the chronoamperometric method (0.552 and 0.607ppm with the errors -11.8 and -3.0%, respectively). For the comparison of the two sets of data, HPLC-ECD method is much more promising.

Buttermilk Based Cobalt Phthalocyanine Dispersed Ferricyanide Mediated Amperometric Biosensor for the Determination of Xanthine

A tissue-based amperometric biosensor for xanthine determination has been developed. The biosensor uses a carbon paste electrode (CPE) which contains buttermilk (BTM) as source of xanthine oxidase (XOD) (EC.1.1.3.2.2.) and cobalt phthalocyanine (CoPc) as mediator. The system is also mediated with ferricyanide in solution to obtain a double-mediated biosensor. The effect of various variables upon the response was studied. Linearity was observed over the concentration range of 1–15 mM xanthine. The optimum operational pH range for the electrode is 8.0–9.0. Reproducibility was studied and a relative standard deviation (rsd) of 10.26 % was found. After 60 days the normalized response of the BTM-modified electrode was 60 % of the first day.

Online electrochemical monitoring of nitric oxide during photodynamic therapy

Photodynamic therapy (PDT), as a novel treatment modality, is based on the use of a photosensitizing agent with an excitation light source for the treatment of various malignancies. Its effect is mediated through reactive oxygen species and nitric oxide (NO), which are shown to be present in apoptosis. Individual differences among patients and even in different areas of the same tumor in one patient may cause a major problem with PDT: dose calculation during application of the light. An electrochemical sensor is proposed for online monitoring of NO generation as a solution of this problem. 5-Aminolevulinic acid (ALA) was administered as the photosensitizer in rat cerebellum. An amperometric sensor, selective to NO, was designed and tested both in vitro and in vivo during PDT. ALA-mediated PDT resulted in rapid generation of NO, starting as early as the application of light on the tissue. Simultaneous amperometric recordings have been carried out for 5 min during PDT. The progressive increase in NO concentration peaked at 1.10 min and then the response current began to decrease until it reached a plateau at around 70% of its peak value. This study, for the first time, electrochemically demonstrates the generation of NO during PDT. Rapid and stable responses obtained by the experimental setup confirmed that this method could be used as an online monitoring system for PDT-mediated apoptosis.

EFFECT OF MELATONIN COTREATMENT AGAINST KAINIC ACID ON COENZYME Q10, LIPID PEROXIDATION AND TRX mRNA IN RAT HIPPOCAMPUS

Oxidative stress is a likely molecular mechanism in the neurotoxicity of kainic acid (KA), an excitotoxic substance. The aim of this report was to assess the effect of melatonin co-treatment against KA by measuring the levels of Coenzyme Q10 (CoQ 10), lipid peroxidation (LPO), and Thioredoxin (Trx) mRNA in the rat hippocampus. The male rats were divided into three groups as saline, KA treatment (15 mg/kg), and KA plus melatonin (20 mg/kg). The levels of LPO and CoQ10 were determined by high pressure liquid chromatography (HPLC) consisting of fluorescence and electro-chemical detectors, respectively. The expression of the Trx gene was quantified using reverse transcription followed by real-time polymerase chain reaction (RT-PCR). The results show that the level of LPO increased although the level of CoQ10 decreased both in homogenates and mitochondria in KA-treated rats However, melatonin co-treatment attenuated the level of LPO and partially restored the level of CoQ10. Melatonin co-treatment against KA did not affect the regulation of Trx. Finally, in the context of the decreased LPO and the increased CoQ10, the results suggest that melatonin may be protective against central nervous system pathologies involving excitotoxicity or where oxidative damage may contribute to mitochondrial dysfunction.

Microelectrode for in vivo real-time detection of NO.

Nitric oxide (NO) is gaining importance with its diverse spectrum of clinic effects. However, there is still a need for an ideal sensor to monitor its concentration in tissue. An ideal sensor should not interfere with the ongoing physiological process, while making fast, reliable, and repeatable measurements. We have designed a microelectrode for electrochemical NO measurement from tissue with relatively low interference and reliable results upon calibration. Details of electrode preparation and calibration procedure are explained along with an experiment to monitor effects of photodynamic therapy.

Comparison of electrochemical detection of acetylcholine-induced nitric oxide release (NO) and contractile force measurement of rabbit isolated carotid artery endothelium

Since the identification of nitric oxide (NO) as an endothelial-derived relaxing factor, it became very important to quantify NO in biological models eventhough it is present in very low concentrations with a very short half-life. The use of electrochemistry as an alternative detection method is quite promising and electrochemical probes are now being developed to detect NO. This paper consists of an amperometric, bi-polymer modified, platinum-iridium microelectrode (Pt 90%-Ir 10% alloy, multistranded, total diameter 130 microm) design and its application for NO detection in acetylcholine (Ach) introduced, rabbit isolated carotid artery endothelium model. In a pH range of 3.0-10.0. pH 3.0 was found to be the optimum pH. As the pH values increased up to 10.0, the response current decreased as the oxidation of NO is catalyzed by H(+) in the acidic media. Temperature effect was checked at 25 degrees C (room temperature), 30 and 40 degrees C. An increasing trend was observed in sensor response with the increasing temperature. Most common biological interferences as ascorbic acid, uric acid and glucose were eliminated via bi-polymer coatings of four layers of Nafion and a layer of 50 mM o-phenylenediamine (OPD). When S/N ratio was accepted as 3, limit of detection was calculated as 15 nM. NO release from carotid artery endothelium was also determined by measuring response force in thermostatic isolated organel baths. Obtained force responses (mg) were compared with the electrochemical (nA) sensor responses.

Interactions between verapamil and digoxin in Langendorff-perfused rat hearts: the role of inhibition of P-glycoprotein in the heart.

P-glycoprotein (P-gp) is expressed in tumour cells as well as normal tissues including heart. Modulation of P-gp transport in vivo may lead to increased drug penetrance to tissues with resulting increases in toxicity. We aimed to investigate the effects of P-gp on the isolated heart by digoxin infusion in the absence and presence of verapamil. The study was performed in Langendorff isolated perfused rat hearts. After a 20 min. stabilisation period with Tyrode Buffer, digoxin (125 μg/5 mL) was infused for 10 min. in the control group (n = 7). The same dose of digoxin was infused during perfusion with verapamil (1 nm) containing Tyrode Buffer (n = 8) in the study group. Outflow concentration and cardiac parameters of digoxin were measured at frequent intervals for 40 min. AUEC((0-40 min)) for left ventricular developed pressure was significantly increased in the presence of verapamil (4260 ± 39.37 mmHg min versus 4607 ± 98.09 mmHg min; 95% CI -587.7 to -105.8; p = 0.0083). The significant increases in left ventricular developed pressure were at 20, 25, 30, 35 and 40 min. AUC((0-40 min)) value for outflow digoxin concentration-time curve was significantly lower in the presence of verapamil. Verapamil increased the positive inotropic effect of digoxin, probably through the inhibition of P-gp, which effluxes digoxin out of cardiac cells.

Multielectrode array for simultaneous recording of glucose, oxygen and electrocorticography from cerebral cortex in experimental focal epilepsy

Delineating epileptic tissue before and during surgery is still a major problem. Electroencephalography (EEG), electrocorticography (ECoG), and magnetoencephalography (MEG) evaluations may not always correlate with outcome. Metabolic mapping, i.e. positron emission tomography (PET) or single photon emission tomography (SPECT), is not practical or not sensitive--both spatial and temporal--enough for use in neurosurgery. Amperometric electrochemical electrodes for recording oxygen and glucose from nervous tissue are developed and tested in rat experimental focal penicillin epilepsy model. With a three-by-three array of glucose electrodes, epileptic focus is mapped. Simultaneous recordings of ECoG, oxygen and glucose levels performed. During seizure, extracellular glucose level showed a biphasic response pattern while oxygen level decreased slightly. It has been concluded that such a combined subdural grid recording might help before and during surgery. This method can be used in ischemia and such experimental metabolic studies.