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Une augmentation de la génération des dérivés réactifs de l’oxygène (DRO), tels que le peroxyde d’hydrogène (H2O2), joue un rôle clé dans la pathophysiologie des maladies cardiovasculaires (MCV). La croissance et la prolifération excessives des cellules musculaires lisses vasculaires (CMLV) ont été suggérées comme étant les mécanismes à la base de la dysfonction vasculaire. Une implication potentielle du facteur de transcription Early growth response protein-1 (Egr-1) dans le développement des dommages vasculaires a été proposée. Des études ont démontré que le H2O2 augmente l’expression de l’Egr-1 dans les CMLV. Cependant, les voies de signalisation intracellulaire menant à l’expression de l’Egr-1 en réponse au H2O2 restent à établir. L’objectif de cette étude vise à examiner les différentes voies de signalisation impliquées dans l’expression de l’Egr-1 induite par le H2O2 dans les CMLV. Le H2O2 augmente l’expression de l’Egr-1 en fonction du temps et de la dose dans les CMLV A10. Le blocage pharmacologique des tyrosines kinases insulin-like growth factor-1 receptor (IGF-1R) et c-Src, par AG1024 et PP2 respectivement, atténue l’expression de l’Egr-1 induite par le H2O2, alors que l’AG1478, un inhibiteur de l’epidermal growth factor receptor (EGFR), et le PP3, l’analogue inactif du PP2, n’ont aucun effet sur l’expression de l’Egr-1. Le blocage pharmacologique de l’extracellular signal-regulated kinase 1/2 (ERK1/2), par UO126, et de la protéine kinase C (PKC), par rottlerin et rö-31-8220, diminue l’expression de l’Egr-1 induite par le H2O2. En résumé, nos résultats suggèrent que le H2O2 déclenche l’expression de l’Egr-1 via l’IGF-1R, la kinase c-Src, l’ERK1/2 et la PKC dans les CMLV. / Increased generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), plays a key role in the pathophysiology of cardiovascular diseases (CVD). Excessive growth and proliferation of vascular smooth muscle cells (VSMCs) has been suggested as an important contributor of vascular dysfunction. A potential involvement of early growth response protein-1 (Egr-1), a zinc-finger transcription factor, in the development of vascular injury has been proposed. Recent studies have shown that H2O2 increases Egr-1 expression in VSMCs. However, signaling events leading to H2O2-induced Egr-1 expression are not fully understood. Therefore, this study aims to examine the signaling pathways implicated in H2O2-induced Egr-1 expression in VSMC. H2O2 increased Egr-1 expression in a time and dose-dependent fashion in A10 VSMC. Pharmacological blockade of tyrosine kinases insulin-like growth factor-1 receptor (IGF-1R) and c-Src, by AG1024 and PP2 respectively, attenuated H2O2-induced Egr-1 expression, while AG1478, an epidermal growth factor receptor (EGFR) inhibitor, and PP3, the inactive analogue of PP2, have no effect on Egr-1 expression. Pharmacological blockade of extracellular signal-regulated kinase 1/2 (ERK1/2), by UO126, and proteine kinase C (PKC), by rottlerin and rö-31-8220, decreased H2O2-induced Egr-1 expression. In summary, our results suggest that H2O2 triggers Egr-1 expression through IGF-1R, c-Src, ERK1/2 and PKC in VSMC.

>Magister Scientiae - MSc / The motivation to determine H2O2 lies in the fact that this chemical species plays a crucial role in diverse fields of practise such as cosmetic, food, diagnostic, pharmaceutical, clinical and environmental protection industries. Several methods such as chromatography, colorimetry, titrimetry and spectrophotometry have been developed for its detection. However, these methods are known to manifest underlying disadvantages such as high cost, time consuming, instability and complicated immobilization procedures. In this present study an enzyme-less electrochemical sensor based on Ag-Fe2O3/POM/RGO nanocomposite (POM stands for polyoxometalate and RGO stands for reduced graphene oxide) was successfully synthesised via a hydrothermal method and a photochemical reduction method for the detection of hydrogen peroxide (H2O2).

La primera parte de esta Tesis de Maestría consiste en entender las propiedades catalíticas y de carácter no enzimático de nanopartículas magnéticas (NPM) de óxido de hierro sintetizadas por el método de descomposición térmica en la creación de sensores que permiten detectar analitos tales como el peróxido de hidrógeno (H2O2) y ácido cítrico. La creación del sensor de H2O2 es llevado a cabo mediante la deposición capa por capa [Layer-by-Layer (LbL)] de las NPM suspendidas y funcionalizadas en medio acuoso con la amina cuaternaria y a su vez surfactante Bromuro de cetiltrimetilamonio (Cetyltrimethylammonium bromide-CTAB) y con el polímero poli-aniónicosodio 4-estireno-sulfonato (sodium 4-styrenesulfonate PSS) vía interacción electrostática sobre el sustrato conductor óxido de estaño dopado con flúor (Fluorine doped Tin Oxide-FTO). La sensibilidad del sensor es estudiada en base a su dependencia con las concentraciones aplicadas de H2O2 y ácido cítrico utilizando técnicas electroquímicas como Voltametría Cíclica (VC) y Cronoamperometría. Por otro lado, andamios (scaffolds) que actúen como nexo entre los tejidos conectivos dañados en la matriz ósea es un tema actual de gran impacto. La componente inorgánica principal de los huesos Hidroxiapatita (HAp) en estado macroscópico o bulk ha sido elegida ya desde unos 30 años atrás como un candidato que mimetiza las propiedades de los tejidos óseos, más al parecer el desarrollo de la Nanotecnología, como fuente de nuevas y maravillosas propiedades no solo mejora la viabilidad de estas nano partículas en los análisis biológicos sino que al incorporar vía funcionalización otros nanomateriales como Fe3O4 (magnetita) y su forma oxidada γ-Fe2O3 (maghemita) a la HAp ayuda rotundamente a la proliferación y degeneración de células osteoformadoras u osteoblastos. La segunda parte de este trabajo reside en la comprensión de la aplicación mencionada previamente en el párrafo anterior debido a que es primordial comprender cómo se lleva a cabo el proceso de ligación o funcionalización de una estructura sólida (HAp) a una nanopartícula, tema de gran interés y relevancia en la física-química de superficies, pues en la mayoría de casos son moléculas o ligandos las que se adhieren a través de enlaces covalentes o no covalentes a la superficie de las nanopartículas mediante transferencia de fase discutida posteriormente. Inmediatamente, el análisis citotóxico, e.d., la viabilidad que adquiera este nuevo nanomaterial en las células óseas de SAOS-2 (Sarcoma osteogénico) será evaluado vía el protocolo de cristal violeta, viabilidad celular y análisis ROS, así como el efecto sobre la morfología de las células durante el tiempo de exposición de esta nueva línea de nanomateriales. / --- This Master thesis has two main parts, the first one resides in the understanding of the catalytic properties and non-enzymatic nature of magnetic nanoparticles in order to create sensors that detect analytes such as peroxide hydrogen (H2O2) and citric acid. The creation of H2O2 sensor is carried out by Layer-by-layer (LbL) deposition of the magnetic nanoparticles suspended and functionalized in aqueous medium with the quaternary amine and surfactant cetyltrimethylammonium bromide (cetyltrimethylammonium bromide- CTAB) sodium 4-styrenesulfonate PSS via electrostatic interaction on the conductive substrate fluorine doped tin Oxide-FTO. The sensitivity of the sensor is studied based on their dependence on the applied concentrations of H2O2 using electrochemical techniques such as cyclic voltammetry (CV) and chronoamperometry. On the other hand, scaffolds, who act as a link between the connective tissue damaged in bone matrix is a current issue of great impact and importance. The main inorganic component of bone hydroxyapatite (HAp) on macroscopic or bulk state has been chosen around 30 years ago as the perfect candidate that mimics the properties of bone tissues. Additionally, as part of a new source of wonderful properties nanohydroxyapatite has captured the attention by incorporating inside its structure other nanomaterials such as Fe3O4 (magnetite) and its oxidized form γ-Fe2O3 (maghemite) which strongly support the proliferation and degeneration of bone-forming cells or osteoblasts. However the rol that nanoparticles play in this processes has not been explained yet. For that, the second part of this Master thesis explained at first how to carry out the process of functionalization of a solid structure (HAp) to a nanoparticle, a subject of great interest and relevance in the physical-chemical surfaces, because in most cases are molecules or ligands which are attached by covalent or noncovalent bounds to the surface of the nanoparticles linked by phase transfer discussed in the preliminary concepts section later on. As a complement for the characterization studies, cytotoxic analysis, i.e., feasibility to acquire this new nanomaterial in SAOS-2 bone cells will be evaluated using crystal violet protocol; besides cell viability, analysis ROS and the effect on cells morphology during time exposure of this new line of nanomaterials will be studied. Keywords: non-enzymatic sensor, magnetic nanoparticles, H2O2, hydroxyapatite, SAOS-2 cells, tissue regeneration. / Tesis

La NADPH oxydase, Nox4, appartient à la famille des Nox qui génèrent les espèces radicalaires de l'oxygène, ROS, en transférant un électron à l'oxygène moléculaire. Malgré sa large distribution dans les tissus, Nox4 est encore mal comprise. Contrairement aux autres Nox, Nox4 est unique par son activité constitutive et sa capacité à former H2O2. Les ROS sont des espèces bactéricides dans les phagocytes et des outils de signalisation dans les cellules non phagocytaires en étant associés à de nombreuses pathologies inflammatoires et du vieillissement. Une étude de la structure en lien avec la fonction de Nox4 permettra de mettre l'accent sur un mécanisme de fonctionnement et sur de nouvelles cibles thérapeutiques. 5 nouveaux anticorps monoclonaux ont été générés contre une construction recombinante tronquée (AA: 206-578) de Nox4. La spécificité de 3 anticorps monoclonaux (8E9, 5F9, 6B11) a été confirmée par western blot dans les cellules HEK293 transfectées et le cortex de rein humain. L'anticorps 8E9 est le seul à permettre un marquage des cellules TRex-Nox4 sans perméabilisation par FACS. L'immunofluorescence confocale a montré que Nox4 est localisée dans la zone périnucléaire et le réticulum endoplasmique. La microscopie TIRF a confirmé sa présence dans la membrane plasmique. Un phénomène intéressant est que 5F9 ne détecte pas Nox4 à la membrane plasmique. L'épitope de 8E9 reconnaît une région sur la dernière boucle E extracellulaire de Nox4 (222H-E241), tandis que les anticorps monoclonaux, 6B11 et 5F9 marquent respectivement les régions 6B11 (389S-P416) et 5F9 (392D-F398). Par ailleurs, seuls 5F9 et 6B11 inhibent l'activité de Nox4, ce qui suggère que les deux régions marquées par ces ACm sont impliquées dans le transfert d'électrons. Une étude ciblée sur la boucle E de Nox4 a permis de montrer que le changement de 2 cystéines modifie la nature des ROS générés par Nox4 avec la production de O2- au lieu de H2O2. O2- est mis en évidence par la formation de peroxynitrite en présence de NO. Par ailleurs l'ACm 8E9 diminue la production de H2O2 dans les cellules COS7 qui expriment Nox4 à la membrane plasmique alors que celle de O2- est augmentée. Des constructions recombinantes de Nox4 (native ou tronquée) ont été générées par induction bactérienne, E.Coli, et par un système de transcription/traduction (RTS). Les protéines correspondantes, solubles, ont été produites à grande échelle et l'activité diaphorase mesurée; cette activité est constitutive. L'étude de la topologie membranaire de Nox4 et p22phox a été abordée en préparant des protéines de fusion avec l'ubiquitine marquée à la GFP. Cette méthode, TDUFA, particulièrement originale, devrait permettre d'appréhender la topologie de l'hétérodimère Nox4/p22phox, actif.

Persistent organic pollutants (POPs) are organic compounds of anthropogenic origin that have been linked to the development of cancer, neurobehavioural impairment, and immune system biochemical alterations. These chemicals have various industrial applications as well as acting as pesticides. Dioxins and furans are some of these compounds that are unintentionally produced in combustion and industrial processes. By definition these compounds have 4 common qualities: they are highly toxic, they are resistant to environmental degradation, they are introduced into the air and water where they travel long distances, and they accumulate in fatty tissues. Photochemical degradation is a method that has been extensively researched in the last few decades. In the aqueous phase it has already been shown to be able to degrade a number of refractory organics, such as dioxins and furans. The ultimate products of this process tend to be carbon dioxide, water, and mineral anions. Air phase work has been also gaining attention in recent decades as a possible alternative to incineration methods in air pollution control. The advantages of photochemical degradation processes are that they can be initiated at low temperatures, are relatively low cost compared to incineration processes, environmentally benign, and have the potential for quick and complete degradation of organic compounds. The main aim of the research is to investigate the photochemical degradation potential of PCDD/ PCDFs in gaseous air streams as a potential air pollution control technology. In order to do this, the photodegradation reaction kinetics were determined for chlorobenzene as a suitable surrogate for PCDD/PCDFs. Three different photodegradation schemes were employed: direct photolysis, UV/O3, and UV/H2O2. In addition, ozonolysis reaction rates were also determined to evaluate the effects of on the overall photodegradation rates for the UV/O3 process. Factors such as humidity levels and temperature were investigated to determine their effects on degradation rates. Temperature and humidity were not greatly influential on the degradation rates of direct photolysis. The degradation rate of chlorobenzene at a temperature of 100°C and high humidity was noticeably reduced, but unchanged at the 10% RH and 60% RH levels for all temperatures. Ozonolysis of chlorobenzene was negligible at 30°C for all humidity levels. Ozonolysis reactions at the 60°C and 100°C levels were higher than direct photolysis rates and in the 100°C case exceeded the UV/O3 degradation rates. Ozone coupled with UV experiments proved to be the most destructive at the low temperature of 30°C and molar ratio of 10:1 ozone to chlorobenzene. There was a clear and positive relationship between the amount of ozone present in the reactor and the degradation rate. At lower ozone to chlorobenzene molar ratios the degradation rates were not much higher than those for direct photolysis of ozone. The 5:1 molar ratio saw a significant increase in degradation rates over the photolysis rates. The fastest degradation rate was achieved for the 10:1 molar ratio and high humidity, which was over 10 times the rate of direct photolysis. In addition, humidity had a noticeably significant positive effect in these reactions. The effect of temperature on the UV/ozone reaction scheme was determined for the 5:1 ozone to chlorobenzene ratio. Temperature had an interesting effect on the degradation rates at higher temperatures. As the reactor temperature increased, the degradation rates from ozonolysis and UV/O3 began to converge at 60°C, ultimately leading to the ozonolysis reaction being faster than the UV/O3. Exploratory experiments for the H2O2 scheme were performed. H2O2 had a positive influence on the degradation rate of chlorobenzene and was about 26% higher than the direct photolysis rates. However for similar conditions, the UV/O3 process had higher degradation rates as was expected from the difference in absorption values between ozone and hydrogen peroxide.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)- benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells and OC2 human oral cancer cells was unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended MDCK and OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 0.1-20 £gM increased [Ca2+]i in a concentration-dependent manner in MDCK cells, however in OC2 cells, m-3M3FBS at concentrations between 10-60 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signals were reduced partly by removing extracellular Ca2+ in the two cell types. m-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, m-3M3FBS pretreatment abolished the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, cyclopiazonic acid or BHQ partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. Collectively, in MDCK and OC2 cells, m-3M3FBS induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels and other unidentified Ca2+ channels. Additionally, 5-100 £gM of m-3M3FBS killed cells in a concentration-dependent manner in OC2 cells. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 £gM) induced apoptosis in a Ca2+-independent manner. We were also interested in knowing whether BAPTA suppressed cell death during oxidative stress in MDCK cells. BAPTA loading altered tBHP (tert-butyl hydroperoxide) and H2O2-induced cell death in a concentration-dependent manner. This suggests that the cell death induced by tBHP and H2O2 appears to be Ca2+-dependent in MDCK cells. The tBHP and H2O2-induced cell death was not suppressed by 2 £gM U73122 (PLC inhibitor), 50 £gM zVAD-fmk (caspase inhibitor), 2 £gM cyclosporin A (a potent inhibitor of the MPTP), 20 £gM PD98059 (ERK inhibitor) or 2 £gM SP600125 (JNK inhibitor). This suggests that the tBHP and H2O2-induced MDCK cells death was not via the PLC, MPTP, caspase, ERK or JNK pathways. Propidium iodide staining, caspase-3 activity assay and cell morphology data suggest that tBHP and H2O2-induced cell death was necrosis, not via apoptosis, and the cell death appears to be caspase-independent and Ca2+-dependent. The effect of the antidepressant paroxetine on [Ca2+]i in OC2 human oral cancer cells is unclear. This study also explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1000 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine¡Vinduced [Ca2+]i rise. Inhibition of PLC with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 £gM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with BAPTA. Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine also induced cell death in a Ca2+-independent manner.

<p>The kinetics for radiation induced dissolution of spent nuclear fuel is a key issue in the safety assessment of a future deep repository. Spent nuclear fuel mainly consists of UO₂ and therefore the release of radionuclides (fission products and actinides) is assumed to be governed by the oxidation and subsequent dissolution of the UO₂ matrix. The process is influenced by the dose rate in the surrounding groundwater (a function of fuel age and burn up) and on the groundwater composition. In this licentiate thesis the effects of HCO₃- (a strong complexing agent for UO2²⁺) and ionic strength on the kinetics of UO₂ oxidation and dissolution of oxidized UO₂ have been studied experimentally.</p><p>The experiments were performed using aqueous UO₂particle suspensions where the oxidant concentration was monitored as a function of reaction time. These reaction systems frequently display first order kinetics. Second order rate constants were obtained by varying the solid UO₂surface area to solution volume ratio and plotting the resulting pseudo first order rate constants against the surface area to solution volume ratio. The oxidants used were H₂O₂(the most important oxidant under deep repository conditions), MnO₄- and IrCl₆^2-. The kinetics was studied as a function of HCO₃- concentration and ionic strength (using NaCl and Na₂SO₄as electrolytes).</p><p>The rate constant for the reaction between H₂O₂ and UO₂ was found to increase linearly with the HCO3- concentration in the range 0-1 mM. Above 1 mM the rate constant is independent of the HCO3- concentration. The HCO₃- concentration independent rate constant is interpreted as being the true rate constant for oxidation of UO₂ by H₂O₂ [(4.4 ± 0.3) x 10-6 m min-1] while the HCO3- concentration dependent rate constant is used to estimate the rate constant for HCO₃- facilitated dissolution of UO₂2+ (oxidized UO₂) [(8.8 ± 0.5) x 10-3 m min-1]. From experiments performed in suspensions free from HCO₃- the rate constant for dissolution of UO₂2+ was also determined [(7 ± 1) x 10^-8mol m^-2 s^-1]. These rate constants are of significant importance for simulation of spent nuclear fuel dissolution.</p><p>The rate constant for the oxidation of UO₂ by H₂O₂ (the HCO₃- concentration independent rate constant) was found to be independent of ionic strength. However, the rate constant for dissolution of oxidized UO₂ displayed ionic strength dependence, namely it increases with increasing ionic strength.</p><p>The HCO₃- concentration and ionic strength dependence for the anionic oxidants is more complex since also the electron transfer process is expected to be ionic strength dependent. Furthermore, the kinetics for the anionic oxidants is more pH sensitive. For both MnO₄- and IrCl₆2- the rate constant for the reaction with UO₂was found to be diffusion controlled at higher HCO3- concentrations (~0.2 M). Both oxidants also displayed ionic strength dependence even though the HCO₃- independent reaction could not be studied exclusively.</p><p>Based on changes in reaction order from first to zeroth order kinetics (which occurs when the UO₂ surface is completely oxidized) in HCO₃- deficient systems the oxidation site density of the UO₂ powder was determined. H₂O₂and IrCl₆2- were used in these experiments giving similar results [(2.1 ± 0.1) x 10-4 and (2.7 ± 0.5) x 10^-4 mol m^-2, respectively].</p>

The significantly increased copper and oxidative stress levels are characteristic hallmarks of cancer cells. These differences provide a unique opportunity for selective targeting of cancer cells. D-penicillamine (D-pen) has been proposed to generate reactive oxygen species (ROS) in presence of copper. Therefore, these studies were aimed at investigating the potential application of a currently marketed copper chelator, D-pen, as a novel cytotoxic anti-cancer agent. D-pen was shown to produce ROS, specifically hydrogen peroxide (H2O2), in the presence of cupric sulfate through a copper catalyzed oxidation reaction. During this process D-pen was converted to D-pen disulfide. The experimental proof of the H2O2 generation was conclusively shown with the aid of a novel High Performance Liquid Chromatography (HPLC) assay. The in-vitro cytotoxicity of D-pen co-incubated with cupric sulfate was examined in human beast cancer (MCF-7 and BT474) and leukemia cells (HL-60, HL-60/VCR, and HL-60/ADR). D-pen was shown to cause concentration dependent cytotoxicity in both leukemia and breast cancer cells. A direct correlation between the detection of intracellular ROS and cytotoxicity was established. The treatment of D-pen plus cupric sulfate resulted in a significant reduction in the intracellular thiol content. D-pen is highly hydrophilic and is rapidly eliminated from the body; therefore to improve the intracellular uptake and to protect the thiol group of D-pen, we carried out the synthesis and the in-vitro characterization of a novel gelatin-D-pen conjugate. It was shown that D-pen alone does not enter cells. Confocal microscopy was employed to exhibit the uptake of the novel gelatin-D-pen conjugate by cancer cells. As the cancer cells in-vitro do not accumulate the same levels of copper as reported for cancer cells in-vivo, cancer cells were pre-treated with cupric sulfate to simulate the elevated copper levels. The cupric sulfate pretreatment resulted in reduced thiol level and significantly increased cellular copper content compared to untreated cells. Whereas both free D-pen and gelatin-D-pen conjugate lacked cytotoxicity in un-treated cells, both agents caused concentration dependent cytotoxicity in cupric sulfate pre-treated leukemia cells. Therefore, it was shown that the administration of D-pen as polymer conjugate would potentially provide cytotoxicity and specificity in the treatment of cancer.

L-glutamate plays a vital role in central nervous system. It is a neurotransmitterassociated with several neurological disorders like Parkinson's disease, epilepsyand stroke. Continuous and fast monitoring of this neurotransmitter has become amajor concern for neuroscientists throughout the world. A simple, sensitive, and reliable L-glutamate microsensor with short responsetime has been developed using ceramic-based microelectrode arrays with platinum recording sites. The electrodes were modified by electrodeposition of Platinum black (Pt-black) to detect hydrogen peroxide (H2O2) which was produced by enzymatic reactions of glutamate oxidase immobilized on the electrode surface. Modification of Pt electrodes with Pt-black has been adoptedbecause the microscale roughness of Pt-black increases the effective surface area of the electrode and promotes efficiency of H2O2 electro-oxidation. The modified Pt recording sites were coated with m-phenylenediamine (mPD) and L-glutamate oxidase (L-GluOx). mPD acts as an barrier for extracellular interferents such as ascorbic acid and dopamine, thus increasing the selectivity of electrode for Glutamate (Glu). This modified microsensor was highly sensitive to H2O2(686.3??156.48 ??AmM-1cm-2), and Glutamate (492.2??112.67 ??AmM-1cm-2) at 700mV versus Ag/AgCl reference. Deposition of Pt nano-particles on recording sites enhanced the sensitivity to H2O2 by 2 times and the sensitivity to glutamate by 1.5 times.