This PhD research relates to the design, fabrication, characterization, and optimization of on-chip electrochemical microelectrode arrays (MEAs) for measurement of transient concentrations and gradients, focusing on fast transients and physiological applications. In particular, this work presents the determination of kinetic mechanisms taking place at an active interface (either physiological or non-physiological) in contact with a liquid phase using the MEA device to simultaneously estimate the concentration and gradient of the analyte of interest at the surface of the active interface. The design approach of the MEA device and the corresponding measurement methodology to acquire reliable concentration information is discussed. The ability of the MEA device to measure fast (i.e., in sub-second time scale) transient gradients is demonstrated experimentally using a controllable diffusion-reaction system which mimics the consumption of hydrogen peroxide by a 2D cell culture. The proposed MEA device and measurement methodology meet effectively most of the requirements for physiological applications and as a demonstration of this, two physiological applications are presented. In one application, the MEA device was tailored to measure the hydrogen peroxide uptake rate of human astrocytes and glioblastoma multiforme cells in 2D cell culture as a function of hydrogen peroxide concentration at the cell surface; the results allowed to quantitatively determine the uptake kinetics mechanisms which are well-described by linear and Michaelis-Menten expressions, in agreement with the literature. In the other application, further customization of the MEA device was realized to study the glucose uptake kinetics of human bronchial epithelial and small cell lung cancer cells, these latter with and without DDX5 gene knockdown; the results allowed to distinguish mechanistic differences in the glucose uptake kinetics among the three cell lines. These results were complemented with measurements of glycolytic and respiration rates to obtain a bigger picture of the glucose metabolism of the three cell lines. Finally, additional applications, both physiological and non-physiological, are proposed for the developed MEA device.

Magistrinio darbo tikslas yra sukurti kompiuterinį amperometrinio biojutiklio su chemiškai modifikuotu elektrodu modelį ir ištirti jo savybes. Modelis papildo egzistuojančius modelius mediatoriaus sluoksniu. Mediatoriaus koncentracija sluoksnyje nėra konstanta, tirpsta biojutiklio veikimo eigoje. Darbe apžvelgiama medžiaga apie amperometrinius biojutiklius, biojutiklio modeliavimo aspektus, mediatoriaus tirpimo priežastis ir veikimo principus. Vėliau suformuluojamas pasirinkto biojutiklio matematinis modelis. Matematiniame modelyje pateikiamos diferencialinės lygtys su dalinėmis išvestinėmis, aprašančios biojutiklyje vykstančias reakcijas ir difuzijas. Pagal matematinį modelį yra sudaromas skaitinis modelis. Pagal žinomus analizinius sprendimus modelis yra ratifikuojamas. Remiantis skaitiniu modeliu buvo sukurta programinė įranga įgyvendinanti diferencialinių lygčių su dalinėmis išvestinėmis sprendimo metodą (išreikštinę baigtinių skirtumų schemą) ir simuliuojanti biojutiklio veikimą. Programinė įranga yra karkasas, leidžiantis nagrinėti sumodeliuoto biojutiklio savybes bei charakteristikas. Buvo ištirtos substrato ir mediatoriaus koncentracijų, mediatoriaus, fermento, difuzijos sluoksnių, bei mediatoriaus sluoksnio difuzijos koeficiento įtakos biojutiklio generuojamos srovės tankiui. Be to gauti dviejų modelių palyginimo rezultatai parodė sumodeliuoto biojutiklio modelio skirtumus ir panaudojimo prasmingumą. / The aim of master thesis is to model amperometric biosensor with chemically modified electrode, introducing into existing model one more additional mediator layer. Mediator concentration inside the layer is changing during the biosensor action. Master thesis content consists of: enzyme kinetics introduction, biosensor modeling peculiarities exploration, chemically modified electrode information, biosensor action aspects description. Following chapter concentrates on mathematical modeling of particular biosensor type. Mathematical model represents differential equations with partial derivatives describing the reactions and diffusion inside biosensor. Next, numerical model is formulated. The explicit method technique was used. Based on numerical model software was made and validated. Using software the biosensor action was simulated in order to investigate biosensor properties and characteristics. In this work several properties were analyzed: impact of substrate and mediator concentrations, size of mediator, enzyme and diffusion layers, mediator layer diffusion coefficient on biosensor response. This model was compared with previous introduced model and it was shown that there is some difference between them and there is a reason to use new model.

Nesta tese foram desenvolvidos sensores amperométricos baseados em porfirinas supramoleculares e hidróxido de níquel nanoestruturado e estabilizado na fase alfa, bem como novos materiais baseados em hidróxidos mistos de níquel e cobalto nanoetruturados e estabilizados na fase alfa. Também, um analisador FIA amperométrico, foi desenvolvido em colaboração com o Laboratório de Instrumentação Analítica do IQ-USP, para a determinação e quantificação de SO2 livre em amostras reais, como vinhos, águas de coco e sucos de frutas. Os sensores amperométricos baseados em porfirinas supramoleculares foram empregados na determinação de SO2 livre, presente em sucos de frutas e águas de coco, utilizando um sistema FIA amperométrico. O sistema FIA amperométrico foi constituído de um injetor manual, uma cela amperométrica em fluxo integrada a uma unidade de difusão de gases e um fluxo de uma solução de ácido sulfúrico e de uma solução de eletrólito. Possíveis interferentes como ácido ascórbico, catecol, glicose e benzoato de sódio foram avaliados antes de iniciar as análises das amostras reais. Os resultados das análises foram comparados com o método oficial Monier-Williams. Os níveis de SO2 livre encontrados nas amostras estavam de acordo com o que é preconizado pela Legislação Brasileira. Os eletrodos de FTO, modificados com hidróxido de níquel estabilizado na fase alfa, foram utilizados como sensores amperométricos para a determinação de glicose, usando um sistema FIA amperométrico. Estes sensores apresentaram excelentes respostas lineares em concentrações inferiores a 100 µM. Ainda estes sensores têm apresentado uma sensibilidade específica muito elevada para a glicose, provavelmente associada com a sua natureza nanoestruturada, e consequêntemente sua área superficial aumentada. No entanto o eletrodo modificado apresentou uma tendência de mudar de fase, da fase meta-estável alfa para a fase termodinamicamente estável beta, com o aumento da concentração de glicose. Novos materiais baseados em hidróxidos mistos de níquel e cobalto estabilizados na fase alfa também foram desenvolvidos. A partir de técnicas como difratometria de raio-x e Microbalança Eletroquímica de Cristal de Quartzo (MECQ) foi possível determinar a fase polimórfica dos materiais. Técnicas como AFM e MEV demonstraram que estes materiais são formados por nanopartículas com tamanhos de 5 nm. Estes nanomateriais apresentaram uma maior quantidade de carga específica, em comparação com o hidróxido de níquel puro. E por último um analisador FIA amperométrico automatizado foi desenvolvido para a realização de análises de SO2 livre em amostras líquidas. Este analisador é composto por reservatórios de soluções, sistema de amostragem, sistema de propulsão das soluções, cela FIA amperométrica integrada a uma unidade de difusão de gases, um sensor amperométrico, além de placas controladoras, mini-potenciostato e um software para controlar o equipamento. Uma elevada reprodutibilidade foi alcançada para análises sequenciais de uma amostra, mas quando a amostra foi trocada a reprodutibilidade foi muito baixa, sendo necessário mais desenvolvimento do sistema FIA automatizado nesta área. / New nanomaterials based on mixed nickel and cobalt hydroxides stabilized in the alpha phase, as well as amperometric sensors based on supramolecular porphyrins and stabilized alpha nickel hydroxide were developed in this thesis. An amperometric FIA sulfite analyzer was also developed in colaboration with of Group of Analytical Instrumentation of IQ-USP, for determination of free SO2 present in liquid samples such as wine, coconut water and fruit juices. The amperometric FIA system is constituted by manual injector, amperometric FIA cell with integrated gas diffusion unit and amperometric sensor based on supramolecular porphyrins, whereas sulfuric acid and electrolyte were used as reactive and carrier solution. Some species such as ascorbic acid, cathecol, glucose and sodium benzoate were evaluated as possible interferents before starting the analyses of real samples. The results were compared with those obtained with the official Monier-Williams method. The concentrations of free SO2 found in fruit juices and coconut water were in accordance with Brazilian law. The amperometric FIA sensors for determination of glucose were based on FTO electrodes modified stabilized alpha nickel hydroxide, which presented excellent linear responses at concentrations below 100 µM. These sensors have shown a very high specific sensitivity for glucose probably associated with their nanostructured nature, and consequent enhanced surface area. Nevertheless, the electrode material was shown to have an increasing tendency to change from alpha to the beta phase as function of the glucose concentration. New materials based on mixed nickel and cobalt hydroxides, stabilized in the alpha phase was prepared and characterized by techniques such as X-ray diffraction, thermogravimetric analysis and Electrochemical Quartz Crystal Microbalance to determine their polymorphic phase. The nanostructured nature of the mixed nickel and cobalt hydroxide materials was confirmed by microscopy techniques such as AFM and SEM. These new materials showed a high specific charge, in fact higher than of nanostructured stabilized alpha nickel hydroxide. An amperometric FIA sulfite analyzer system for liquid samples consisting of solution reservoirs, a sampling system, a solution propulsion system, an amperometric FIA cell with integrated gas diffusion unit, an amperometric sensor based on supramolecular porphyrins, in addition to controller boards, a mini-potentiostat and control software, was developed and tested for determination of free SO2. High reproducibility was achieved for sequential analyses of a sample but the reproducibility after exchange of samples was relatively low, needing further developments in the sampling system.

Nesta tese foram desenvolvidos sensores amperométricos baseados em porfirinas supramoleculares e hidróxido de níquel nanoestruturado e estabilizado na fase alfa, bem como novos materiais baseados em hidróxidos mistos de níquel e cobalto nanoetruturados e estabilizados na fase alfa. Também, um analisador FIA amperométrico, foi desenvolvido em colaboração com o Laboratório de Instrumentação Analítica do IQ-USP, para a determinação e quantificação de SO2 livre em amostras reais, como vinhos, águas de coco e sucos de frutas. Os sensores amperométricos baseados em porfirinas supramoleculares foram empregados na determinação de SO2 livre, presente em sucos de frutas e águas de coco, utilizando um sistema FIA amperométrico. O sistema FIA amperométrico foi constituído de um injetor manual, uma cela amperométrica em fluxo integrada a uma unidade de difusão de gases e um fluxo de uma solução de ácido sulfúrico e de uma solução de eletrólito. Possíveis interferentes como ácido ascórbico, catecol, glicose e benzoato de sódio foram avaliados antes de iniciar as análises das amostras reais. Os resultados das análises foram comparados com o método oficial Monier-Williams. Os níveis de SO2 livre encontrados nas amostras estavam de acordo com o que é preconizado pela Legislação Brasileira. Os eletrodos de FTO, modificados com hidróxido de níquel estabilizado na fase alfa, foram utilizados como sensores amperométricos para a determinação de glicose, usando um sistema FIA amperométrico. Estes sensores apresentaram excelentes respostas lineares em concentrações inferiores a 100 µM. Ainda estes sensores têm apresentado uma sensibilidade específica muito elevada para a glicose, provavelmente associada com a sua natureza nanoestruturada, e consequêntemente sua área superficial aumentada. No entanto o eletrodo modificado apresentou uma tendência de mudar de fase, da fase meta-estável alfa para a fase termodinamicamente estável beta, com o aumento da concentração de glicose. Novos materiais baseados em hidróxidos mistos de níquel e cobalto estabilizados na fase alfa também foram desenvolvidos. A partir de técnicas como difratometria de raio-x e Microbalança Eletroquímica de Cristal de Quartzo (MECQ) foi possível determinar a fase polimórfica dos materiais. Técnicas como AFM e MEV demonstraram que estes materiais são formados por nanopartículas com tamanhos de 5 nm. Estes nanomateriais apresentaram uma maior quantidade de carga específica, em comparação com o hidróxido de níquel puro. E por último um analisador FIA amperométrico automatizado foi desenvolvido para a realização de análises de SO2 livre em amostras líquidas. Este analisador é composto por reservatórios de soluções, sistema de amostragem, sistema de propulsão das soluções, cela FIA amperométrica integrada a uma unidade de difusão de gases, um sensor amperométrico, além de placas controladoras, mini-potenciostato e um software para controlar o equipamento. Uma elevada reprodutibilidade foi alcançada para análises sequenciais de uma amostra, mas quando a amostra foi trocada a reprodutibilidade foi muito baixa, sendo necessário mais desenvolvimento do sistema FIA automatizado nesta área. / New nanomaterials based on mixed nickel and cobalt hydroxides stabilized in the alpha phase, as well as amperometric sensors based on supramolecular porphyrins and stabilized alpha nickel hydroxide were developed in this thesis. An amperometric FIA sulfite analyzer was also developed in colaboration with of Group of Analytical Instrumentation of IQ-USP, for determination of free SO2 present in liquid samples such as wine, coconut water and fruit juices. The amperometric FIA system is constituted by manual injector, amperometric FIA cell with integrated gas diffusion unit and amperometric sensor based on supramolecular porphyrins, whereas sulfuric acid and electrolyte were used as reactive and carrier solution. Some species such as ascorbic acid, cathecol, glucose and sodium benzoate were evaluated as possible interferents before starting the analyses of real samples. The results were compared with those obtained with the official Monier-Williams method. The concentrations of free SO2 found in fruit juices and coconut water were in accordance with Brazilian law. The amperometric FIA sensors for determination of glucose were based on FTO electrodes modified stabilized alpha nickel hydroxide, which presented excellent linear responses at concentrations below 100 µM. These sensors have shown a very high specific sensitivity for glucose probably associated with their nanostructured nature, and consequent enhanced surface area. Nevertheless, the electrode material was shown to have an increasing tendency to change from alpha to the beta phase as function of the glucose concentration. New materials based on mixed nickel and cobalt hydroxides, stabilized in the alpha phase was prepared and characterized by techniques such as X-ray diffraction, thermogravimetric analysis and Electrochemical Quartz Crystal Microbalance to determine their polymorphic phase. The nanostructured nature of the mixed nickel and cobalt hydroxide materials was confirmed by microscopy techniques such as AFM and SEM. These new materials showed a high specific charge, in fact higher than of nanostructured stabilized alpha nickel hydroxide. An amperometric FIA sulfite analyzer system for liquid samples consisting of solution reservoirs, a sampling system, a solution propulsion system, an amperometric FIA cell with integrated gas diffusion unit, an amperometric sensor based on supramolecular porphyrins, in addition to controller boards, a mini-potentiostat and control software, was developed and tested for determination of free SO2. High reproducibility was achieved for sequential analyses of a sample but the reproducibility after exchange of samples was relatively low, needing further developments in the sampling system.

Presented Ph.D. Thesis is focused on the use of the boron-doped diamond (BDD) electrodes for voltammetric and amperometric determination of selected nitrophenols: 2-nitrophenol (2NP), 4-nitrophenol (4NP), and 2,4-dinitrophenol (2,4DNP). These compounds are listed as "priority pollutants" by United States Environmental Protection Agency (US EPA) due to their negative impact on living organisms and are mainly used in agriculture as plant growth stimulators. BDD electrodes are used for determination of wide range of electrochemically both reducible and oxidisable organic compounds and have become a popular electrode material thanks to its commercial availability and excellent mechanical and electrochemical properties. A differential pulse voltammetric method was developed for the determination of 2NP, 4NP and 2,4DNP at a BDD film electrode using electrochemical reduction and of 4NP and 2,4DNP using electrochemical oxidation. The method was successfully applied for the direct determination of these compounds in drinking and river water in the concentration range from 4×10-7 to 2×10-5 mol.L-1 . To improve the limit of quantification, a preconcentration by solid phase extraction from 100 mL (drinking and river water) and 1000 mL (drinking water) of water samples was used with limit of determination...

Present Ph.D. Thesis is focused on the development of electrochemical methods for determination of anticancer drugs using various types of membranes for their preliminary separation. Furthermore, this Thesis reports the study of transport mechanisms of heavy metals in the presence of phytochelatins across biological membranes. Sodium anthraquinone-2-sulphonate (AQS) was used as a model compound for its similar structure with anthraquinone-based (AQ-based) anticancer drugs (doxo/daunorubicin) and also due to its better availability. All these compounds can be easily electrochemically oxidized and/or reduced. Redox behaviour of AQS was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in a cathodic region on mercury meniscus modified (m-AgSAE) and polished silver solid amalgam (p-AgSAE) electrodes, Obtained results were used for the development of a micro-volume voltammetric cell (MVVC). Its applicability for voltammetric determination of anticancer drugs was verified by using doxorubicin (DX) as a model substance. The second part of this Thesis deals with therapeutic monitoring of anticancer drugs in the blood circulation of the patients. For pilot experiments, a liquid-flow system with dialysis catheter and amperometric detection was used. The flow rate of carrier...