The interest in biosensors can be attributed to the first described enzyme containing sensor used to detect levels of glucose in 1962. Although research into biosensors was initially slow to pick up, the field has become increasingly popular and research has been widespread for the last 20 years. The continuing research into biosensors is crucial as this will improve current devices to become smaller, faster and more economical and yield new biosensors. This thesis is mostly concerned with the development of an integral component of a biosensor, the redox mediator. Complexes of ruthenium incorporating electron rich sulfur-donor ligands such as naphthalenedithiol should possess interesting redox qualities which could be used to produce better mediators. A second area covered in this thesis is in the structural studies of a series of iridium and rhodium complexes. Two classes of ruthenium complexes with sulfur-donor ligands have been prepared. The first class incorporates the ruthenium bis-bipyridine moiety while the second contains pentamethylcyclopentadienyl ruthenium. Most complexes synthesised exhibited reversible oxidation waves in the region of -0.1 to 0.2 V vs. Ag/Ag⁺. Their possible use as redox mediators was hindered by several factors, particularly difficulties in purification. They also exhibited oxygen sensitivity and low stability when in solution.The second area covered in this thesis is to further understand the bonding of the ligands used in the above study. A series of pentamethylcyclopentadienyl iridium and rhodium complexes were synthesised with three different dithiolato ligands. Two of the three ligands studies produced structures that included more than one metal centre leading to straining of the ligand. In order to study the monomeric form, the clusters were opened with a neutral phosphine ligand. Both the Ir…S and Rh…S bond lengths of all the complexes were within expected parameters.

An impedance bioanalyzer system comprising an in-vitro biotransducer, instrumentation and control software for the measurement of urea, potentially in blood dialysate, has been developed. The biotransducer comprises of a microlithographically fabricated interdigitated microsensor electrode (IME) onto which was cast a biorecognition layer conferred with the specificity of the enzyme urease. Urease hydrolysis of urea produces NH4+, HC03- and OH- ions that decrease the device's impedance. The temporal rate of change (kinetic) and the extent of change (equilibrium) of ion concentration were measured as the sensor's response. Five formats: [i) unPEGylated urease-containing poly(hydroxyethylmethacrylate) [p(HEMA)] hydrogel, ii) PEGylated urease-containing p(HEMA) hydrogel, iii) via glutaraldehyde crosslinking in the presence of albumin, iv) the direct covalent immobilization of urease to the IME, and v) solution borne urease]. Michaelis-Menten parameters KM, ZMAX and kcat revealed the following rank: PEGylated urease-Gel >> Free Urease > unPEGylated urease-Gel = BSA in Glutaraldehyde > covalently immobilized urease. The unPEGylated-urease sensor provided a higher enzyrne- substrate binding rate and catalysis rate than PEGylated and thus provided a faster impedimetric response to various molar concentrations of urea. Long-term stability (one month) of the PEGylated-urease hydrogel was favorable. A dedicated three-element array impedimetric instrument, the 3EIC BioAnalyzer was designed and produced. A pair of demodulating logarithmic amplifiers (AD8302) was used to calculate the change in phase and amplitude corresponding to the impedimetric response to a 4.0 kHz, 50 mVPP sine wave from a function generator (MAX038). A graphic user interface (GUI), programmed in LabVIEW 7.0 established instrument control, data acquisition via a USB-48A-30A16 μDAQ and graphical data presentation of temporal impedimetric responses.

Depuis l'apparition du terme "biosensor" à travers un article de Lyons et Clark en 1962, les biocapteurs ont connu un véritable essor tant au niveau académique qu'industriel. Le principal objectif de ce travail de thèse était de créer une surface permettant l'immobilisation spécifique par liaison covalente de simple ou double brin d'ADN puis d'étudier les interactions pouvant exister entre une protéine donnée et l'ADN. Pour préparer la surface à cette immobilisation, nous avons opéré une réduction électrochimique de sel d'aryldiazoniums. Ce type de modification nous a permis de fixer de manière covalente sur la surface conductrice des fonctions de type Ar-SO2Cl. Par l'utilisation de la QCM et de l'AFM, nous avons pu par la suite détailler les mécanismes de fonctionnement de protéines (HsRad51 et Transposase) en interaction avec l'ADN simple ou double brin fixé, que ce soit d'un point de vue cinétique ou bien structural. / Since the emergence of the term "biosensor" through an article of Clark and Lyons in 1962, such devices have experienced a tremendous activity both in the academic and industries. The main objective of this thesis work was to create a surface allowing the specific immobilization of single or double DNA strand by covalent bonding and then study the interactions that may exist between a given protein and DNA. To functionalize the surface, we firstly investigated the electrochemical reduction of aryldiazoniums salt. This kind of methodology has allowed us to covalently graft Ar-SO2Cl functions over the conductive surface which can further react with DNA to immobilize it. By using the QCM and AFM methodologies, we are able to kinetically or structurally detail the intimate mechanisms of interactions between two proteins (HsRad51 and Transposase) and single or double strand DNAs.

Les Kinases cycline-dépendantes (CDK / cyclines) jouent un rôle majeur dans la régulation de la progression du cycle cellulaire et la prolifération des cellules cancéreuses, et constituent ainsi des cibles d'intérêt pour le développement de stratégies de diagnostic et thérapeutiques anticancéreuse. L'objectif de cette étude a consisté à développer une famille de biosenseurs fluorescents pour mesurer l'activité des CDK/ cycline in vitro, in cellulo et in vivo.Nous avons conçu et développé un biosenseur polypeptidique sensible à l'environnement comprenant une séquence substrat des CDKs, qui est marquée avec une sonde fluorescente sensible à l'environnement à proximité du site de phosphorylation, et un domaine de liaison phospho-amino acide, qui se lie à la séquence du substrat lorsqu'il est phosphorylé, ce qui modifie l'environnement de la sonde fluorescente et conduit par conséquent à l'augmentation de la fluorescence. Plusieurs variants de ce premier biosenseur CDKACT ont été développés. Les biosenseurs ont d'abord été caractérisés in vitro en utilisant plusieurs complexes CDK / cycline recombinants et avec des CDK / cyclines endogènes à partir d'extraits cellulaires, induisant des changements dynamiques de l'intensité de fluorescence, qui ont été mesurés en temps réel. Nous avons caractérisé la spécificité de ces biosenseurs pour les kinases CDK/ cycline par rapport à d'autres kinases (Plk1, Plk3, CIV, PKA, MAPK). En outre ces biosenseurs permettent de mesurer des différences dans l'activité des CDK/Cyclines entre différentes lignées cellulaires saines et cancéreuses. Enfin, nous avons mis en place les conditions pour internaliser ces biosenseurs dans des cellules vivantes grâce à des formulations de peptides pénétrants, afin de mesurer l'activité des CDK / cycline en temps réel. L'imagerie time-lapse et la quantification ratiométrique de fluorescence de la sonde sensible à l'environnement par rapport à une sonde fluorescente standard a permis de suivre l'activité des CDK/ cycline au cours du cycle cellulaire de cellules en division, des cellules ne se divisant pas et des cellules traitées avec des inhibiteurs des CDK / cycline. Les biosenseurs ont également été utilisé pour établir des conditions nécessaires à réaliser un criblage haut débit et des essais d'imagerie in vivo dans des modèles de souris comportant des xénogreffes. / Cyclin-dependent kinases (CDK/Cyclins) play central roles in regulation of cell cycle progression and proliferation of cancer cells, thereby constituting attractive targets for development of cancer diagnostics and therapeutics. The objective of this study consisted in developing a family of fluorescent biosensors to probe CDK/Cyclin activity in vitro, in cellulo and in vivo. To this aim, we designed and engineered an environmentally sensitive polypeptide sensor consisting of a CDK substrate sequence labelled with an environmentally-sensitive dye proximal to the phosphorylation site, and a phospho-amino acid binding domain, which binds the substrate sequence when it is phosphorylated, thereby altering the environment of the fluorescent probe and consequently leading to fluorescence enhancement. Several variants of this first CDKACT biosensor were further engineered. The biosensors were first characterized in vitro using several recombinant CDK/Cyclin complexes and endogenous CDK/Cyclins from cell extracts, inducing dynamic changes in fluorescence intensity, which were measured in real-time. We further characterized the specificity of these biosensors for CDK/Cyclin kinases as opposed to other kinases (Plk1, Plk3, CIV, PKA, MAPK). We further applied CDK biosensors to measure CDK/Cyclin kinase activity between different healthy and cancer cell lines. Finally, we established conditions to deliver the biosensors into living cells thanks to cell-penetrating peptide formulations, to monitor CDK/Cyclin activity in real time. Time-lapse imaging and ratiometric quantification of fluorescence of the environmentally sensitive probe over that of a fluorescent standard allowed to monitor CDK/Cyclin activity throughout the cell cycle of dividing cells, non-dividing cells and cells treated with CDK/Cyclin inhibitors. The biosensors were further applied to establish conditions for a high throughput screen and an in vivo imaging assay using xenografted mouse models.

<div> <p>Fluorescent proteins and the biosensors created with them have been used extensively to monitor chemical species inside and outside of the cell. They have been used to increase our knowledge of cellular function in normal and diseased states. Fluorescent biosensors are advantageous because they can be genetically encoded, do not require exogenous reagents, and can be quantitative. Fluorescent biosensors are also able to measure analytes with high spatial and temporal resolutions, enabling measurements at the scale of physiological events. In this thesis efforts have made to increase the available fluorescent biosensor tools for imaging cellular events. This work includes creation of new sensors for two molecules not yet detectable via fluorescent protein biosensor, acetylcholine and adenosine diphosphate. Efforts were also made to improve the current available biosensors for adenosine triphosphate and cellular redox, to make them more compatible with multiplex and deep tissue imaging. Here I present my work to design, characterize and utilize these fluorescent biosensors.</p> </div> <br>

No description available.

The cyclic nucleotides cAMP and cGMP are two ubiquitous important second messengers, which regulate diverse physiological responses from vision and memory to blood pressure and thrombus formation. They act in cells via cAMP- and cGMP-dependent protein kinases (PKA and GK), cyclic nucleotide-gated channels and Epac. Although the concept of cyclic nucleotide signalling is well developed based on classical biochemical studies, these techniques have not allowed to analyze cAMP and cGMP in live cells with high temporal and spatial resolution. In the present study fluorescence resonance energy transfer was used to develop a technique for visualization of cAMP and cGMP in live cells and in vitro by means of fluorescent biosensors. Ligand-induced conformational change in a single nucleotide-binding domain flanked with green fluorescent protein mutants was used for dynamic, highly sensitive measurements of cAMP and cGMP. Such biosensors retained binding properties and chemical specificity of unmodified domains, allowing to image cyclic nucleotides in a physiologically relevant range of concentrations. To develop cAMP-sensors, binding domains of PKA, Epac and cAMP-gated HCN-channel were used. cGMP-sensors were based on single domains of GK and phosphodiesterases (PDEs). Sensors based on Epac were used to analyze spatio-temporal dynamics of cAMP in neurons and macrophages, demonstrating that cAMP-gradients travel with a high speed (~ 40 &#956;m/s) throughout the entire cytosol. To understand the mechanisms of cAMP-compartmentation, kinetics properties of phosphodi-esterase (PDE2) were, next, analyzed in aldosterone producing cells. PDE2 is able to rapidly hydrolyze extensive amounts of cAMP, so that the speed of cAMP-hydrolysis is much faster than that of its synthesis, which might serve as a basis of compartmentation. cAMP-sensors were also used to develop a clinically relevant diagnostic method for reliable detection of &#946;1-adrenergic receptor autoantibodies in cardiac myopathy patients, which has allowed to significantly increase the sensitivity of previously developed diagnostic approaches. Conformational change in a single binding domain of GK and PDE was, next, used to create novel fluorescent biosensors for cGMP. These sensors demonstrated high spatio-temporal resolution and were applied to analyze rapid dynamics of cGMP production by soluble and particulate guanylyl cyclases as well as to image cGMP in mesangial cells. In summary, highly sensitive biosensors for cAMP and cGMP based on single cyclic nucleotide-binding domains have been developed and used in various biological and clinically relevant applications. / Die zyklischen Nukleotide cAMP and cGMP sind zwei ubiquitäre Botenstoffe, die verschiedene physiologische Prozesse regulieren, vom Sehen und Gedächtnis bis zu Blutdruck und Thrombusbildung. Sie wirken über cAMP- und cGMP-abhängige Kinasen (PKA und GK), Kanäle und Epac. Obgleich die Funktionen von zyklischen Nukleotiden in klassischen biochemischen Studien gut untersucht sind, ermöglichen diese Methoden nicht, cAMP und cGMP in lebenden Zellen mit hoher zeitlicher und räumlicher Auflösung zu analysieren. In dieser Arbeit wurde Fluoreszenzresonanzenergietransfer benutzt, um eine Technik für die Visualisierung von cAMP and cGMP in lebenden Zellen und in vitro zu entwickeln. Ligand-induzierte Konformationsänderung in einer einzelnen, mit Grünfluoreszenzproteinmutanten fusionierten Bindungsdomäne diente als Grundlage für Biosensoren, die dynamische, hochsensitive Messungen von cAMP und cGMP ermöglichen. Bei solchen Sensoren wurden die chemischen und Bindungseigenschaften von unmodifizierten Domänen aufrechterhalten, was die cAMP- und cGMP-Messungen im physiologischen Konzentrationsbereich in lebenden Zellen ermöglicht. Für die Entwicklung der cAMP-Sensoren wurden die Domänen von PKA, Epac und von einem cAMP- gesteuerten HCN-Kanal benutzt. cGMP-Sensoren beruhen sich auf den Bindungsdomänen von GK und Phosphodiesterasen (PDEs). Mit Hilfe der auf Epac-basierten Sensoren wurde die cAMP-Dynamik in Neuronen und Makrophagen zeitlich und räumlich aufgelöst. In diesen Zellen diffundiert cAMP mit hoher Geschwindigkeit (~ 40 &#956;m/s) frei durch das ganze Zytosol. Um die Mechanismen der cAMP-Kompartimentierung besser zu verstehen, wurden die kinetischen Eigenschaften der PDE2 in aldosteronproduzierenden Zellen analysiert. PDE2 ist imstande, große Mengen von cAMP äußerst schnell zu hydrolisieren, so dass die Geschwindigkeit der cAMP-Hydrolyse viel höher ist als von cAMP-Synthese, was eine Grundlage der cAMP-Kompartimentierung sein könnte. cAMP-Sensoren wurden auch benutzt, um eine klinisch relevante diagnostische Methode zu entwickeln, die Autoantikörper gegen &#946;1-adrenergen Rezeptoren bei Herzinsuffizienzpatienten zuverlässig nachweist. Diese Methode hat ermöglicht, die Sensitivität der früher entwickelten Techniken zu verbessern. Konformationsänderung in einzelnen Bindungsdomänen von GK und PDE wurde als nächstes benutzt, um ein Reihe neuer fluoreszierender Biosensoren für cGMP zu entwickeln. Diese Sensoren zeigten hohe räumliche und zeitliche Auslösung und wurden zur Analyse schneller Dynamik von cGMP-Synthese und für cGMP-Imaging in Mesangialzellen angewandt. Zusammenfassend wurden hochsensitive Biosensoren für cAMP und cGMP auf Grund einzelner, mit Grünfluoreszenzproteinmutanten fusionierter Bindungs-domäne entwickelt und in verschiedenen biologischen und klinisch relevanten Applikationen eingesetzt.

Neste trabalho são apresentados resultados relacionados a estudos sobre o comportamento eletroquímico do lactato e do peróxido de hidrogênio em diversos eletrodos em meio de diferentes pHs utilizando voltametria cíclica. Também foi investigado o comportamento eletroquímico do peróxido de hidrogênio em eletrodos modificados com filmes de hexacianoferratos utilizando eletrodos de platina e carbono vítreo. A vantagem do uso do CTAB na modificação da superfície de carbono vítreo com Azul da Prússia foi confirmada no que se refere à melhora da sensibilidade e da estabilidade das medidas. Numa etapa posterior desenvolveu-se um biossensor para a determinação de lactato pelo monitoramento de peróxido de hidrogênio produzido na reação catalisada de lactato com oxigênio na presença da enzima lactato oxidase. Nessa etapa, o trabalho consistiu em imobilizar a enzima lactato oxidase na superfície do eletrodo, previamente, modificado com Azul da Prússia, com o auxílio de Nafion&#174;. Após a construção do biossensor e a otimização das condições de análise (pH, quantidade de enzima, volume da amostra e vazão) para obtenção de maior sinal analítico no desenvolvimento do método para a determinação de lactato por análise em fluxo, averiguou-se a repetibilidade das medidas (Clactato = 0,28 mmol L-1) obtendo-se um desvio padrão de 2,2% para 18 repetições. A freqüência analítica foi estimada em 160 injeções por hora com limite de detecção de 0,84 &#181;mol L-1 e linearidade até 0,28 mmol L-1 de lactato. O biossensor foi aplicado na quantificação de lactato em amostras alimentares (cervejas alcoólicas e não alcoólicas) e biológicas (sangue liofilizado e recém coletado). Por fim, realizaram-se estudos envolvendo a variação da concentração de lactato sanguíneo em função da intensidade de atividade esportiva. Os resultados obtidos pelo método proposto foram comparados com aqueles oriundos do uso de método de referência. / Results on the investigation of the electrochemical behavior of lactate and hydrogen peroxide at various electrodic surfaces at different pHs using cyclic voltammetry are presented. Experiments were also carried out with platinum and glassy carbon electrodes modified with hexacyanoferrate films. The advantage of using CTAB in the electrodeposition step of Prussian Blue films onto glassy carbon surfaces was confirmed taking into account both the stability and sensitivity of measurements. The immobilization of lactate oxidase onto glassy carbon electrodes modified with a Prussian Blue layer using Nafion® was performed to fabricate a biosensor for lactate. The biosensor was used in the development of a FIA amperometric method for the determination of lactate. Under optimal operating conditions (pH = 6.9, E = -0.1 V), the linear response of the method was extended up to 0.28 mmol L-1 lactate with a limit of detection of 0.84 &#181;mol L-1. The repeatability of the method for injections of a 0.28 mmol L-1 lactate solution was 2.2 % (n = 18). The analytical frequency was calculated as 160 injections h-1. The usefulness of the method was demonstrated by determining lactate in beer (alcoholic and nonalcoholic beers) and blood samples (lyophilized and freshly collected). Finally, the influence of physical exercise on the blood lactate level was studied. Results obtained by using the proposed amperometric detector compared well with the reference method.

Nanotubos de Carbono (Carbon Nanotubes - CNTs) são nanoestruturas de carbono na forma de tubos cilíndricos que apresentam excelentes propriedades ópticas, elétricas, térmicas e mecânicas. A imobilização dos CNTs em conjunto com polieletrólitos condutores e naturais na forma de filmes ultrafinos, utilizando a técnica automontagem, apresenta uma nova alternativa para o desenvolvimento de nanocompósitos aplicados em dispositivos sensores e biossensores. Neste trabalho de mestrado foram construídos e estudados filmes automontados contendo CNTs funcionalizados com grupos ácidos carboxílicos e imobilizados com os polieletrólitos polianilina (PANI) e quitosana (QUIT). Também foram fabricados para comparação filmes de PANI e poliestireno sulfonado (PSS). O crescimento dos filmes de PANI/CNT e PANI/PSS revelou um crescimento linear com o número de bicamadas, conforme mostraram as medidas de espectroscopia UV-Vis e voltametria cíclica (CV), enquanto que para o filme de QUIT/CNT, as medidas de microbalança de cristal de quartzo (QCM) mostraram um crescimento exponencial. Análises de espectroscopias Raman e infravermelho com transformada de Fourier (FTIR) indicaram alterações nas bandas características dos filmes, comparadas com seus materiais precursores, indicando interação entre os componentes nas multicamadas dos filmes. Análises morfológicas obtidas por AFM mostraram um aumento da rugosidade com o aumento do número de bicamadas. O mapeamento Raman revelou que os filmes contendo CNTs apresentaram maior heterogeneidade química na superfície do que aos filmes somente com polieletrólitos. Foi observada uma queda na resistividade dos filmes, com o aumento do número de bicamadas, sendo esta, mais acentuada com a presença dos CNTs. Os eletrodos contendo os filmes automontados apresentaram alta estabilidade eletroquímica, a presença dos CNTs aumentou a intensidade das correntes de pico e tornou os sistemas mais reversíveis. Os eletrodos que se mostraram mais sensíveis à detecção de peróxido de hidrogênio foram selecionados para imobilização da enzima Glicose Oxidase (GOx). Foram investigados os seguintes sistemas biossensores: (PANI/CNT)7, (PANI/PSS)1 e (QUIT/CNT)5 apresentando na mesma ordem: valores de sensibilidades de 190 nA/(mmol/L), 36 nA/(mmol/L) e 220 nA/(mmol/L); Limite de detecção de 2,2 µmol/L, 67,5 µmol/L e 8,5 µmol/L, e valores deconstante de Michealis-Menten(\'K IND.M\'POT.APP\' ) de 2,2 µmol/L, 67,5 µmol/L e 8,5 µmol/L. Os resultados indicam que a utilização dos CNTs é bastante promissora para fabricação de dispositivos biossensores para aplicação em Nanomedicina, e além disso, a escolha dos componentes para formação dos nanocompósitos exerce grande influência no desempenho do dispositivo. / Carbon Nanotubes (CNTs) are cylindrical carbon nanostructures exhibiting excellent electrical, thermal, optical and mechanical properties. The immobilization of CNTs in nanostructured thin films in conjunction with polymers using the Layer-by-Layer (LbL) technique provides a new alternative for development of nanocomposites to be used as sensors and biosensors. In this study we report the fabrication of LbL films containing CNTs functionalized with carboxylic acid immobilized in conjunction with polyaniline (PANI) or chitosan (QUIT). Films comprising PANI and sulfonated polystyrene (PSS) were also produced for comparison. Film growth was monitored by UV-Vis spectroscopy, cyclic voltammetry (CV) and quartz crystal microbalance (QCM) and the results showed a linear increase with the number of bilayers in PANI films, and an exponential growth for QUIT/CNT films. FTIR and Raman analyses revealed changes in the bands of nanocomposites compared to their precursor materials, indicating the interactions between the components in the multilayers. Morphological analysis of the films obtained by atomic force microscopy (AFM) showed that the roughness of the films increased with the number of bilayers. Raman mapping showed that the presence of CNTs generated a high heterogeneity in film surface in comparison to a films formed only by polyelectrolytes. Electrical resistivity of the films decrease upon increasing the number of bilayers, especially for CNTs-containing films. Furthermore, electrodes containing LbL films exhibited high electrochemical stability, in which the presence of CNTs increased the intensity of response signal. The electrodes that exhibited best performance toward hydrogen peroxide detection were employed for immobilization of glucose oxidase (GOx) and used as glucose biosensors. The systems (PANI/CNT)7, (PANI/PSS) 1 and (QUIT/CNT)5 exhibited a sensitivity of 190 nA/(mmol/L), 36 nA/(mmol/L) and 220 nA/(mmol/L), respectively. The detection limit was estimated at 2.2 mmol/L, 67.5 mmol/L and 8,5 mmol/L, whereas the Michaelis-Menten constant ( \'K IND.M\'POT.APP\') values was found to be 2.2 mmol/L, 67.5 mmol/L and 8.5 mmol/L, respectively, to the three systems employed. The results indicated that the use of CNTs in Layer-by-layer thin films is promising for use as biosensors. Furhtermore, we showed that the choice of the polyelectrolyte is a crucial parameter to tailor specific, high performance sensors.

Este trabalho teve como objetivo o desenvolvimento de biossensores eletroquímicos baseados na imobilização de duas enzimas diferentes em filmes de polipirrol (PPI) eletrodepositados, a fitase e a polifenol oxidase (PFO), esta última na forma de extrato bruto do fruto de abacate. Como a fitase hidrolisa cataliticamente ácido fítico (AF) em íons fosfatos, foram preparados biossensores por imobilização da enzima sobre filmes de PPI para a detecção indireta de ácido fítico via íons fosfatos. Foram utilizados dois métodos de imobilização; no primeiro, a enzima, fitase, foi imobilizada ao filme de PPI por imersão do filme em uma solução contendo a enzima por um período de 2 h, no segundo, a fitase foi encapsulada em lipossomos de dipalmitoil fosfatidil glicerol (DPPG) e depois foi imobilizada nos filmes de PPI depositados em eletrodos impressos. O segundo método se mostrou melhor para a detecção de ácido fítico, pois levou a um maior alcance linear e um baixo valor de limite de detecção. Neste caso, verificou-se que o DPPG preservou a integridade enzimática e levou a biossensores mais estáveis e sensíveis. Já para a PFO, que catalisa a oxidação de compostos fenólicos a quinonas, foram preparados biossensores para a detecção indireta de catecol. Para esta enzima, foram utilizados três métodos de imobilização: adsorção, ligação cruzada e confinamento, sendo o último que levou a melhores respostas. O método de confinamento consiste na adição da enzima, juntamente com o monômero, à solução de eletropolimerização, quando se procede com a metodologia normal de preparo dos filmes de PPI, que foram caracterizados por: microscopia de força atômica (AFM), microscopia eletrônica de varredura (MEV), espectroscopia de infravermelho por transformada de Fourier (FTIR) e espectroscopia de reflexão e absorção no infravermelho com modulação da polarização (PM-IRRAS). Estas técnicas de caracterização permitiram com que a presença da enzima fosse associada às modificações das características estruturais e morfológicas dos filmes de PPI. / This doctoral thesis reports on the development of electrochemical biosensors based on the immobilization of enzymes phytase and polyphenol oxidase (PPO) (the latter in the form of crude extract of avocado fruit) on electrodeposited polypyrrole films (PPY). As phytase catalytically hydrolyzes phytic acid (PA) in phosphate ions, biosensors were prepared by its immobilization on PPY films for the indirect detection of PA via phosphate ions. In the first method the enzyme was maintained on the PPY film for a period of 2 h, whereas in the second, it was encapsulated in Dipalmitoyl Phosphatidyl glycerol (DPPG) and immobilized on printed electrodes. The second system proved more viable for the detection of PA and showed broader linear range and low detection limit because DPPG preserved the integrity of the enzyme and produced more stable and sensitive biosensors. Regarding PPO, which catalyzes the oxidation of phenolic compounds to quinones, biosensors for the indirect detection of catechol via the formation of quinone in solution were prepared. Three methods of immobilization were used: adsorption, cross-linking and confinement. The latter yielded favorable results in comparison to other methods. The films were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) spectroscopy, fourier transform infrared (FTIR) spectroscopy and reflection absorption and infrared polarization modulation (PM-IRRAS) techniques and revealed the presence of the enzyme and a modification in the structural characteristics and morphology of the films.