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<p>The transport of ammonia by various tissues throughout the body is of fundamental importance for nitrogen excretion in invertebrates, yet sites and mechanisms of ammonia transport are not presently well understood. In this thesis a novel ammonium-selective microelectrode was developed using the ionophore TD19C6, which is approximately 3800-fold more selective for NH₄⁺ than Na⁺ compared with the 100-fold difference of nonactin used in previous microelectrodes. We investigated the accuracy of the ammonium microelectrode in solutions simulating <em>Drosophila</em> haemolymph (25 mM K⁺) and secreted fluid (120 mM K⁺). In haemolymph-like solutions, ammonium could be measured down to about 1 mM, with an error of 0.5 mM, while in secreted fluid-like conditions ammonium could be determined to within 0.3 mM down to a level of 1 mM NH₄⁺ in the presence of 100 to 140 mM K⁺. These results suggested that the ammonium microelectrode could be used to measure ammonium in the presence of physiological levels of potassium, unlike previous studies. We also quantified ammonium secretion by the Malpighian (renal) tubules of larvae. Ammonium concentrations of secreted fluid were consistently equivalent to or above ammonium concentrations of bathing salines. With a lumen-positive transepithelial potential, these results suggested an active secretory mechanism for ammonia transport. Under conditions of low K⁺ concentrations, the ability of the tubules to concentrate ammonium in secreted fluid was significantly enhanced, indicating some level of competition between NH₄⁺ and K⁺ for common transporters. The new ammonium-selective microelectrode is sufficiently sensitive to detect ammonium at the picomol level.</p> / Master of Science (MSc)

Yes / Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

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Micro- and nano-electrodes have distinct advantages over large electrodes, including their decreased iR drop and enhanced mass transport due to radial diffusion characteristics which leads to the ready establishment of a steady state (or near steady-state) signal without convection. This enhanced mass transport also leads to increased current densities and signal to noise ratios. However, there is a need for fabrication techniques which reproducibly give micro- and nano-electrodes of controlled size and shape. The optimisation of systematic arrays on the nano-scale, open up possibilities for developing highly sensitive electrode devices, for use in physical chemistry and the determination of fast electrode kinetics and rates of reaction, as well as to provide highly sensitive electroanalytical devices, able to detect very low concentrations of substrates. This thesis first presents work involving the fabrication and characterisation on silicon substrates of square platinum microelectrodes. There is already an established theory for the behaviour of microdisc electrodes however, it is easier to make microsquares reproducibly using pixellated photomasks. The voltammetric and ac impedance characteristics of these electrodes in background electrolyte and in the presence of ferri/ferrocyanide redox couple are presented and the response is theoretically analysed. A combination of computer simulation, theory and experimentation show that these electrodes have increased current densities (14% greater) compared with a microdisc of equivalent radius and an alternative theoretical expression is presented to calculate the limiting current of microsquares at all dimensions. This thesis then discusses the development and optimisation of novel nano-band cavity array electrodes (CaviArE), using standard photo-microlithographic techniques. The resulting architecture encloses a Platinum nanoband of 50 nm width within each array element that is positioned half way up the vertical edges of shallow square cavities (depressions), with a total depth of 1050 nm. The width of the square cavity and the separation of the array elements can be controlled and systematically altered, with great accuracy. The CaviArE devices are shown to give quantitative pseudo-steady-state responses characteristic of multiple nanobands, whilst passing overall currents consistent with a macroelectrode. The array has a much enhanced signal-tonoise ratio compared with an equivalent microsquare array, as it has 0.167% of the area and is therefore markedly less affected by non-Faradaic currents, while it passes comparable Faradaic currents. At high sweep rates the response is also virtually unaffected by solution stirring. The impedammetric characteristics presented show different diffusional regimes at high, medium and low frequencies, associated with diffusion within individual square cavities, outside of the cavity and finally across the whole array as the diffusional fields of the neighbouring array elements overlap. Justification and fitting of equivalent circuits to these frequency regions provide details about the charge transfer, capacitance and diffusional processes occurring. The results show that these systems are highly sensitive to surface transfer effects and a rate constant for ferricyanide of 1.99 cm s-1 was observed, suggesting fast kinetic processes can be detected. Together, these characteristics make nanoband electrode arrays, with this architecture, of real interest for sensitive electroanalytical applications, and development of devices for industrial application is currently being undertaken.

The advancement of technologies that interface with electrically excitable tissues, such as the cortex and muscle, has the potential to lend greater mobility to the disabled, and facilitate the study of the central and peripheral nervous systems. Myoelectric interfaces are currently limited in their signal fidelity, spatial resolution, and interfacial area. Such interfaces are either implanted in muscle or applied to the surface of the muscle or skin. Thus far, the former technology has been limited in its applications due to the stiffness (several orders of magnitude greater than muscle) of its substrates, such as silicon and polyimide, whereas the latter technology suffers from poor spatial resolution and signal quality due to the physical separation between the electrodes and the signal source. We have developed a stretchable microneedle electrode array (sMEA) that can function while stretching and flexing with muscle tissue, thereby enabling multi-site muscle stimulation and electromyography (EMG) measurement across a large interfacial area. The scope of this research encompassed: (i) the development of a stretchable and flexible array of penetrating electrodes for the purposes of stimulating and measuring the electrical activity of excitable tissue, (ii) the characterization of the electrical, mechanical, and biocompatibility properties of this electrode array, (iii) the measurement of regional electrical activity of muscle via the electrode array, (iv) the study of the effect of spatially distributed stimulation of muscle on the fatigue and ripple of muscle contractions, and (v) the assessment of the extent to which the stretch response of electrically stimulated muscle behaves in a physiological manner.

An overarching goal of the Gerhardt laboratory is the development of an implantable neural device that allows for long-term glutamate recordings in the hippocampus. Proper L-glutamate regulation is essential for hippocampal function, while glutamate dysregulation is implicated in many neurodegenerative diseases. Direct evidence for subregional glutamate regulation is lacking in previous in vivo studies because of limitations in the spatio-temporal resolution of conventional experimental techniques. We used novel enzyme-coated microelectrode arrays (MEAs) for rapid measurements (2Hz) of extracellular glutamate in urethane-anesthetized rats. Potassium-evoked glutamate release was highest in the cornu ammonis 1 (CA1) subregion and lowest in the cornu ammonis 3 (CA3). In the dentate gyrus (DG), evoked-glutamate release was diminished at a higher potassium concentration but demonstrated faster release kinetics. These studies are the first to show subregion specific regulation of glutamate release in the hippocampus. To allow for in vivo glutamate measurements in awake rats, we have adapted our MEAs for chronic use. Resting glutamate measurements were obtained up to six days post-implantation but recordings were unreliable at later time points. To determine the cause(s) for recording failure, a detailed investigation of MEA surface characteristics was conducted. Scanning electron microscopy and atomic force microscopy showed that PT sites have unique surface chemistry, a microwell geometry and nanometer-sized features, all of which appear to be favorable for high sensitivity recordings. Accordingly, studies were initiated to improve enzyme coatings using a computer-controlled microprinting system (Microfab Technologies, Plano, TX). Preliminary testing showed that microprinting allowed greater control over the coating process and produced MEAs that met our performance criteria. Our final studies investigated the effects of chronic MEA implantation. Immunohistochemical analysis showed that the MEA produced minimal damage in the hippocampus at all time points from 1 day to 6 months. Additionally, tissue attachment to the MEA surface was minimal. Taken together with previous electrophysiology data supporting that MEAs are functional up to six months, these studies established that our chronic MEAs technology is capable of maintaining a brain-device interface that is both functional and biocompatible for extended periods of time.

Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 mM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min, consisting of increased spiking and enhanced, coordinated bursting. This was followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Network activity loss generally preceded morphological changes. 20% cell swelling was correlated with 50% activity loss. Cultures pretreated with the GABAA receptor antagonists bicuculline (40 mM) and picrotoxin (1 mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 mM). However, recovery was not complete and slow deterioration of network activity continued over 6 hrs. Removal of zinc by early medium changes showed irreversible, catastrophic network failure to develop in a concentration-dependent time window between 50% and 90% activity loss. Investigation of entry routes suggested the L-type but not N-type calcium channels to be the main entry pathway for zinc. Data are presented implicating the chloride channel to be an additional entry route.

O biodiesel (BD) tem sido peça chave para redução das emissões de gases de efeito estufa em diversos países em virtude da sua compatibilidade com motores de ciclodiesel automotivos ou estacionários. Contudo, por ser um meio orgânico e altamente resistivo, o uso de técnicas eletroquímicas para estudar sua corrosividade frente a metais é bastante desafiador. No presente trabalho, foi estudada a corrosividade do BD através do uso de microeletrodos (MES) de níquel, alumínio e cobre, o potencial desempenho de amidas graxas na redução da corrosividade desse meio e, ainda, a viabilidade do uso da técnica Scanning Vibrating Electrode Technique (SVET). Para tal, foram obtidos e caracterizados por voltametria cíclica staircase (VCS) MEs de 25 µm de diâmetro de platina, cobre, níquel e alumínio. Com exceção do ME de Pt, todos os MEs foram caracterizados em meios em que são ativos, sendo os resultados obtidos para os MEs de Ni e de Al não encontrados em buscas nas bases de dados da literatura. Foram realizados ensaios gravimétricos para o Ni, Al e para o Cu em BD, sendo o último utilizado também para o estudo das amidas graxas 1-(1-pirrolidinil)-1-octadecanona e N-[2-hidroxi-1,1-bis(hidroximetil)etil]stearamida como inibidores de corrosão. Foram feitas medidas de espectroscopia de impedância eletroquímica (EIE) com todos os MEs em BD bom (BDB) e degradado (BDDE), sendo também inéditos os resultados referentes aos MEs de Ni e de Al nesses meios. Nos ensaios com SVET foram avaliados, imersos em BDB e BDDE, os pares galvânicos Cu/Zn e Cu/Ni, com e sem polarização potenciostática e, ainda, o cobre puro. Os resultados de VCS evidenciaram que a técnica utilizada para obtenção dos MEs foi exitosa. Os ensaios gravimétricos evidenciaram que a corrosão é maior para o Cu, seguida do Al e do Ni. Ainda, estes ensaios evidenciaram que nenhuma das amidas testadas foi capaz de inibir a corrosão do Cu. Para os MEs de Al e Cu, nos espectros de EIE obtidos em BDB foi possível identificar a presença de duas constantes de tempo (CT), enquanto no BDDE as duas CT foram identificadas para todos os MEs. A primeira CT foi associada a uma resposta conjunta da capacitância do BD e da difusão frente à geometria do ME, sendo a segunda CT associada a resposta da dupla camada. A técnica SVET demonstrou que corrosão ao Zn do par Zn/Cu é maior do que frente a do Ni no par Cu/Ni, conforme esperado. Os resultados mostram a viabilidade de uso da técnica de SVET para avaliar o comportamento de metais em BD. / Biodiesel has been a key to reduce greenhouse gas emissions in several countries because of its compatibility with automotive or stationary cycle motors. However, because it is an organic and highly resistive medium, the use of electrochemical techniques to study its corrosiveness toward metals is quite challenging. In the present work, biodiesel\'s corrosiveness was studied with nickel, aluminum and copper MEs by Electrochemical Impedance Spectroscopy (EIS). Furthermore, it was studied the potential performance of fatty amides as corrosion inhibitor for copper, and the feasibility of using Scanning Vibrating Electrode Technique (SVET) in studies with biodiesel. Therefore, platinum, copper, nickel and aluminum ME\'s (Æ=µm) were obtained and characterized by staircase cyclic voltammetry (VCS). Gravimetric tests were run for nickel, aluminum and copper in pure biodiesel. For biodiesel doped with 1- (1-pyrrolidinyl) -1-octadecanone and N- [2-hydroxy-1,1- bis (hydroxymethyl) ethyl] stearamide, the gravimetric tests were restricted to copper. EIS tests were carried out with all MEs on good (GB) and degraded (DB) biodiesel. From our best knowledge, it is the first time that nickel and aluminum MEs are being reported in studies with biodiesel. For SVET experiments, it was used Cu, Cu/Zn and Cu/Ni samples. The VCS results showed that the technique used to obtain the MEs was successful. The gravimetric tests showed that the biodiesel corrosiveness is higher for copper, followed by aluminum and nickel. Further, these tests showed that none of the amides were able to inhibit copper corrosion in biodiesel. For Cu and Al MEs in GB, the EIS spectra showed two-time constants (TC), while in DB the second TC could be seen for all MEs. The first TC was associated to biodiesel\'s capacitance and a ME\'s geometry response, while the second TC was related to double layer response. Also, from the fitting of the data to an equivalent electric circuit it was possible to determine the charge transfer resistance for each of the systems that showed two TCs. The SVET results showed that the corrosion of Zn in Zn / Cu pair is higher than that of Ni in the Cu / Ni pair, as expected. This fact proved that the SVET is feasible to study the corrosion behavior of metals in biodiesel.

Nesse trabalho são apresentados resultados sobre o desenvolvimento de microeletrodos por desgaste eletroquímico de microfibras de dimensões da ordem de 100 µm de diferentes materiais (platina, ouro e fibra de carbono) visando à utilização em micro-ambientes. Na grande maioria dos casos esse processo foi realizado utilizando transformadores AC. Arranjo de microeletrodos construídos a partir de técnicas litográficas também foram construídos. A aplicabilidade de microeletrodos de Pt construídos por desgaste eletroquímico foi avaliada para monitorar a concentração \"in-situ\" de ácido ascórbico em diferentes frutas. O sensor foi capaz de avaliar a distribuição espacial da concentração de ácido ascórbico \"in-situ\" em laranjas. A partir desses resultados, mapas para a concentração de ácido ascórbico foram construídos. A correlação entre o estado de maturação e a concentração de ácido ascorbico também foi observada a partir das medições realizadas e concentrações maiores foram encontradas em frutas mais maduras. O arranjo de microeletrodos foi utilizado para avaliar a concentração de iodato em amostra de sal de cozinha utilizado pequenos volumes de amostra (500 µL). Verificada a possibilidade de fabricação de microeletrodos de diferentes geometrias e dimensões, estudos visando ao monitoramento e à detecção de DNA e ácido ascórbico foram efetuados. Superfícies eletródicas modificadas com filmes de óxido de rutênio hexacianoferrato (RuOHCF) foram utilizadas para a detecção amperométrica de 2\'-deoxiguanosina (dG) e ácido ascórbico por análise por injeção em fluxo (FIA). O método para detecção de dG apresentou um resposta linear de 3,8 a 252 µmol L-1, com um limite de detecção de 94 nmol L-1. Aplicações em amostras de DNA também foram realizadas, e os resultados para determinação da dG concordaram com os obtidos pelo método padrão (HPLC). Estudos sobre o processo eletrocatalítico de oxidação da dG em meio ácido em filmes de RuOHCF foram investigados utilizando eletrodo rotativo. Sobre esse mesma superfície modificada, o processo eletrocatalítico permitiu a determinação de ácido ascórbico em pH = 6,9 a 0,0 V vs Ag/AgCl em FIA, com um limite de detecção de 2,2 µmol L-1. Estudos sobre o processo eletrocatalítico de oxidação do ácido ascórbico também foram avaliados com eletrodo rotativo. O método foi aplicado para a determinação de ácido ascórbico em urina, obtendo-se bons valores de recuperação (96 - 104 %). Microeletrodos contendo filmes eletrodepositados de RuOHCF foram utilizados para monitorar o consumo de ácido ascórbico em 3 diferentes tipos de células neuronais (SH-SY5Y, células transfectadas com SOD humana e mutante típica da doença de Esclerose Lateral Amiotrófica (ALS)). Os resultados do consumo de ácido ascórbico para essas células estão de acordo com os níveis de estresse oxidativo induzido pela SOD mutante. Experimentos visando a aplicabilidade de microeletrodos inseridos em células também foram efetuados. / Results on the development of microelectrodes fabricated by electrochemical etching using fibers of different materials (platinum, gold and carbon fiber) and dimensions (starting from 100 µm) for the application in micronenvironments are presented. In almost all cases this procedure was carried out using AC transformer. Array of microelectrodes were also fabricated by using litographic techniques. The applicability of Pt microelectrodes fabricated by electrochemical etching was evaluated in the in-situ monitoring of the ascorbic acid concentration in different fruits. The sensor allowed spatial distribution of ascorbic acid concentration in oranges to be found and concentration maps were constructed. A correlation between the ripening stage and the ascorbic acid concentration was also observed from electrochemical measurements, the ascorbic acid content being higher in mature fruits. Studies on the detection of species involved in the oxidative stress process, such as DNA and ascorbic acid, were also performed. Ruthenium oxide hexacyanoferrate (RuOHCF) modified electrode surfaces were used as amperometric detectors for 2\'-deoxyguanosine (dG) and ascorbic acid determinations in FIA apparatus. The method exhibited a linear response range to 2\'-deoxyguanosine from 3.8 to 252 µmol L-1 dG with detection limit of 94 nmol L-1. Applications in DNA samples were examined, and the results for determination of 2\'-deoxyguanosine were in good agreement with those obtained by HPLC analysis. The dG electrocatalytic oxidation at a RuOHCF glassy carbon (GC) modified electrode was investigated in acid medium by using rotating disc electrode (RDE) voltammetry. On this modified surface, the electrocatalytic process allowed the determination of ascorbic acid to be performed at 0.0 V and pH = 6.9 with a limit of detection of 2.2 µmol L-1 in a flow injection configuration. The usefulness of the method was demonstrated by an addition-recovery experiment with urine samples and the recovered values were in the 96 to 104 % range. Investigations on the mechanism of the electrocatalytic oxidation of ascorbic acid was also investigated at pH = 6.9 by using RDE voltammetry. The RuOHCF carbon fiber modified microelectrode was used to monitor the ascorbate uptake by control SH-SY5Y cells, and cells transfected with wild-type Cu,Zn-Superoxide Dismutase (SOD) or with a mutant SOD (SOD G93A) typical of familial Amyotrophic Lateral Sclerosis (ALS). Data on the rate of ascorbate uptake by these cells were in agreement with the level of oxidative stress induced by the mutant SOD. Attemps to use the microelectrodes inside single cells were also performed