De nos jours, le domaine biomédical est en pleine croissance avec le développement de dispositifs thérapeutiques innovants, bas coût, pour le diagnostic, le traitement ou la prévention de maladies chroniques ou cardiovasculaires. Ces dernières années ont connu l’émergence des polymères semi-conducteurs, alternative intéressante aux matériaux inorganiques, présentant des propriétés uniques de conduction ionique et électronique. Tout d’abord, j’ai axé mes travaux de recherche sur le développement et l’optimisation d’une encre conductrice à base de PEDOT:PSS, parfait candidat comme matériau, pour la transduction des signaux biologiques en signaux électriques, compatible avec le process jet d’encre, pour la réalisation de dispositifs imprimés. Puis mes travaux se sont orientés vers la conception et l’étude d’électrodes imprimées sur supports papiers, tatous et textiles permettant des enregistrements long termes d’électrocardiogrammes (ECG) ou électromyogrammes (EMG), présentant des performances similaires aux électrodes commerciales, utilisant un système d’acquisition spécifique pour la mesure d’activités électriques de tissus musculaires. Puis dans un second temps, je me suis penchée sur l’impression sur support papier, de transistors organiques électrochimiques (OECTs) fonctionnalisés, afin de permettre la détection d’éléments biologiques ou chimiques comme l’alcool. Ces travaux proposent une nouvelle voie pour la conception de dispositifs innovants biomédicaux à bas couts, imprimés, permettant la personnalisation des produits pouvant être intégrés dans des dispositifs biomédicaux portables ou dans des vêtements « intelligents ». / With the evolution of microelectronics industry and their direct implementation in the biomedical arena, innovative tools and technologies have come to the fore enabling more reliable and cost-effective treatment. In this thesis I focus on the integration of the conducting polymer PEDOT:PSS with printing technologies toward the realization of performant biomedical devices. In the first part, I focus on the optimization of the conducting ink formulation. Following, I emphasize on the fabrication of inkjet printed PEDOT:PSS based biopotential electrodes on a wide variety of substrates (i.e., paper, textiles, tattoo paper) for use in electrophysiological applications such as electrocardiography (ECG) and electromyography (EMG). Printed electrodes on paper and printed wearable electrodes were fabricated and investigated for long-term ECG recordings. Then, conformable printed tattoo electrodes were fabricated to detect the biceps activity during muscle contraction and the conventional wiring was replaced by a simple contact between the tattoo and a similarly ink-jet printed textile electrode.In the last part, I present the potentiality of inkjet printing method for the realization of organic electrochemical transistor (OECTs) as high performing biomedical devices. A disposable breathalyzer comprised of a printed OECT and modified with alcohol dehydrogenase was used for the direct alcohol detection in breath, enabling future integration with wearable devices for real-time health monitoring. Their compatibility with printing technologies allows the realization of low-cost and large area electronic devices, toward next-generation fully integrated smart biomedical devices.

Les biocapteurs optiques ont connu une évolution sans précédent au cours des dernières années, principalement en raison de la forte interaction entre la biotechnologie, l’optique et la chimie des matériaux. Dans cette thèse, deux différentes plates-formes de biocapteurs optiques ont été conçues pour la détection sensible et spécifique des biomolécules. Plus précisément, le premier système de détection optique est construit sur la base de la bioluminescence de cellules bactériennes d'Escherichia coli génétiquement modifiées. L’émission de lumière induite par cette interaction peut donc être utilisée pour la détection des substances toxiques. Le second système utilise des nanoparticules de métaux précieux (or et argent) aux propriétés plasmoniques accordables qui permettent de sonder les interactions des biomolécules spécifiques à l'interface nano-bio par la résonance plasmonique de surface (LSPR). Ces nanoparticules ont été obtenues par traitement thermique à haute température d’un film métallique déposé sur du verre à l’aide d’une grille de TEM ou déposé sur une couche de bactéries fixée sur le verre. Après une optimisation appropriée des nanostructures métalliques en termes de morphologie et de fonctionnalisation, une sensibilité élevée et une grande spécificité peuvent être simultanément obtenues avec ces immunocapteurs plasmonique. Ces deux plateformes ont été utilisées pour détecter des pesticides comme le carbofuran et l’atrazine / Optical biosensors have witnessed unprecedented developments over recent years, mainly due to the lively interplay between biotechnology, optical physics and materials chemistry. In this thesis, two different optical biosensing platforms have been designed for sensitive and specific detection of (bio)molecules. Specifically, the first optical detection system is constructed on the basis of bioluminescence derived from engineered Escherichia coli bacterial cells. Upon stressed by the toxic compounds, the bacterial cells produce light via a range of complex biochemical reactions in vivo and the resulted bioluminescent evolution thus can be used for toxicant detection. The bacterial bioluminescent assays are able to provide competitive sensitivity, while they are limited in the specificity. Therefore, the second optical detection platform is built on the localized surface plasmon resonance (LSPR) immunosensors. In this optical biosensor, the noble metal (gold and silver) nanoparticles with tunable plasmonic properties are used as transducer for probing the specific biomolecules interactions occurred in the nano-bio interface. These nanoparticles were obtained after a high temperature thermal treatment of an initially thin-metallic film deposited on a glass substrate through a TEM grid or on a bacteria layer fixed on the glass. After appropriate optimization on metal nanostructures morphology and surface biomodification, the applicable sensitivity and specificity can be both guaranteed in this LSPR immunosensor

Venir sonder de faibles quantités de molécules nécessite des capteurs ultra-sensibles. Il a été démontré que les capteurs plasmoniques pouvaient remplir ce rôle. Toutefois, même après trente ans de recherches, beaucoup de questions restent sans réponses. Dans cette étude nous tentons d'y répondre : que se passe-t-il lorsqu'une molécule s'adsorbe sur la surface d'une nanoparticule ? Lorsqu'une monocouche de molécule s'adsorbe ? Et que se passe-t-il pour les molécules suivantes ? Peut-on améliorer simplement la sensibilité et la lisibilité des nano-capteurs plasmoniques? Nous démontrons expérimentalement un comportement singulier lorsque la quantité de molécules dans le champ proche des nanoparticules est très faible, typiquement de quelques zeptogrammes. Afin de mesurer cette infime quantité de matière, des solutions d'amplification des signaux sont étudiées comme l'intégration de capteurs sur des micro-lentilles axicon, ou encore sur des nano-cavités de type Fabry-Perot. Nous avons développé les micro-lentilles axicon afin de palier la faible intensité du signal émanant de nanoparticules uniques. Elles ont pour but de redistribuer le champ électromagnétique, en faisceau de Bessel de faible ouverture numérique, donc facilement mesurable. Les nano-cavités optiques ont, quant à elles, étaient développées afin de diminuer l'amortissement des résonances plasmon et ainsi affiner les résonances et augmenter la lisibilité des capteurs.Toutes ces études ont un même but : détecter in-situ les marqueurs de maladies à des concentrations infinitésimales afin de traiter les patients avant les premiers symptômes / Ultra sensitive sensors are required to probe very low concentrations of molecules. It has been shown that plasmonic nano-sensors could play this role. Nevertheless, even after thirteen years of research, a lot of questions remain unanswered.We will try to answer them in this study: what happens when a single molecule is adsorbed on a nanoparticle surface? In a monolayer? And what happens for the next layer of molecules? Can we easily enhance the sensitivity and the readability of sensors? We demonstrate experimentally a singular behavior when the quantity of molecules in the near-field region is very low, typically in the zeptogram level. To measure the low quantity of matter, different techniques to enhance the signal are studied: integration of sensor on axicon micro-lenses of Fabry-Perot like nano-cavities. We developed axicon micro-lenses to increase the intensity of unique nanoparticle signal. They redistribute the electromagnetic field into a Bessel beam with low numerical aperture, allowing an easy collection in far field. Nano-cavities have been designed to decrease the damping and refine the plasmonic resonance to increase the readability of the sensors. All these studies have the same target: to detect in-situ disease markers at very low concentrations in order to treat the patients before the first symptoms

The broader research area investigated during this programme was conceived from a goal to contribute towards solving the challenge of food security in the 21st century through the reduction of crop loss and minimisation of fungicide use. This is aimed to be achieved through the introduction of an empirical approach to agricultural disease monitoring. In line with this, the SYIELD project, initiated by a consortium involving University of Manchester and Syngenta, among others, proposed a novel biosensor design that can electrochemically detect viable airborne pathogens by exploiting the biology of plant-pathogen interaction. This approach offers improvement on the inefficient and largely experimental methods currently used. Within this context, this PhD focused on the adoption of multidisciplinary methods to address three key objectives that are central to the success of the SYIELD project: local spore ingress near canopies, the evaluation of a suitable model that can describe spore transport, and multivariate analysis of the potential monitoring network built from these biosensors. The local transport of spores was first investigated by carrying out a field trial experiment at Rothamsted Research UK in order to investigate spore ingress in OSR canopies, generate reliable data for testing the prototype biosensor, and evaluate a trajectory model. During the experiment, spores were air-sampled and quantified using established manual detection methods. Results showed that the manual methods, such as colourimetric detection are more sensitive than the proposed biosensor, suggesting the proxy measurement mechanism used by the biosensor may not be reliable in live deployments where spores are likely to be contaminated by impurities and other inhibitors of oxalic acid production. Spores quantified using the more reliable quantitative Polymerase Chain Reaction proved informative and provided novel of data of high experimental value. The dispersal of this data was found to fit a power decay law, a finding that is consistent with experiments in other crops. In the second area investigated, a 3D backward Lagrangian Stochastic model was parameterised and evaluated with the field trial data. The bLS model, parameterised with Monin-Obukhov Similarity Theory (MOST) variables showed good agreement with experimental data and compared favourably in terms of performance statistics with a recent application of an LS model in a maize canopy. Results obtained from the model were found to be more accurate above the canopy than below it. This was attributed to a higher error during initialisation of release velocities below the canopy. Overall, the bLS model performed well and demonstrated suitability for adoption in estimating above-canopy spore concentration profiles which can further be used for designing efficient deployment strategies. The final area of focus was the monitoring of a potential biosensor network. A novel framework based on Multivariate Statistical Process Control concepts was proposed and applied to data from a pollution-monitoring network. The main limitation of traditional MSPC in spatial data applications was identified as a lack of spatial awareness by the PCA model when considering correlation breakdowns caused by an incoming erroneous observation. This resulted in misclassification of healthy measurements as erroneous. The proposed Kriging-augmented MSPC approach was able to incorporate this capability and significantly reduce the number of false alarms.

This PhD project aimed to investigate the possibility of using Odorant Binding Proteins (OBPs) as sensing layers of chemical sensors, for the detection of organic compounds in both vapour and liquid phases. OBPs are small soluble proteins present in high concentrations in the olfactory system of vertebrates and insects. OBPs are attractive in the biosensor field since they can bind odorants and pheromones in a reversible way. They are resistant to high temperatures and protease activity and they can be easily expressed in large amounts. OBPs belonging to different species of mammals and insects were utilised for developing biosensors relied on different transduction mechanisms. Recombinant OBPs were grafted on the gold electrode of transducers by using Self-assembled monolayers (SAMs) of alkanethiols. The efficiency of the immobilisation method was proved by using electrochemical techniques. Quartz crystal microbalances (QCMs), screen-printed electrodes (SPEs) and interdigitated electrodes (IDEs) were employed for developing three types of OBP-based biosensors. I. QCMs functionalised with OBPs were tested against pheromones (i.e. bombykol and bombykal) and volatile compounds found in foodstuffs (i.e. pyrazine derivatives and geosmin) in vapour phase. The QCM based biosensors showed a good degree of selectivity and a detection limit of the order of parts per billion, in air. II. In liquid phase, impedimetric biosensors based on SPEs also showed a good selectivity and sensitivity being able to detect analyte concentrations of the order of 10-9 M. III. OBPs immobilised on the gold electrodes of IDEs were instead tested against S-(+) carvone vapour, proving that the binding activity of the proteins was preserved in vapour phase and can be quantified as variation of capacitance. The developed OBP biosensors showed good selectivity, sensitivity and stability over time in both liquid and vapour phase. The responses of the sensors were reversible, allowing to the device to be used several times. Moreover, the biosensors were label-free, hence the interaction between OBPs and ligand was directly detected without using auxiliary probes/species. With these findings, we envisage the use of our biosensors in several applications, including monitoring of the quality of food along the transportation and storage, controlling of pests and useful insects in agriculture, or as analytical devices for studying the dynamics in binding processes.

In this research the integration of nanostructures and micro-scale devices was investigated using silica nanowires to develop a simple yet robust nanomanufacturing technique for improving the detection parameters of chemical and biological sensors. This has been achieved with the use of a dielectric barrier layer, to restrict nanowire growth to site-specific locations which has removed the need for post growth processing, by making it possible to place nanostructures on pre-pattern substrates. Nanowires were synthesized using the Vapor-Liquid-Solid growth method. Process parameters (temperature and time) and manufacturing aspects (structural integrity and biocompatibility) were investigated. Silica nanowires were observed experimentally to determine how their physical and chemical properties could be tuned for integration into existing sensing structures. Growth kinetic experiments performed using gold and palladium catalysts at 1050 ˚C for 60 minutes in an open-tube furnace yielded dense and consistent silica nanowire growth. This consistent growth led to the development of growth model fitting, through use of the Maximum Likelihood Estimation (MLE) and Bayesian hierarchical modeling. Transmission electron microscopy studies revealed the nanowires to be amorphous and X-ray diffraction confirmed the composition to be SiO2 . Silica nanowires were monitored in epithelial breast cancer media using Impedance spectroscopy, to test biocompatibility, due to potential in vivo use as a diagnostic aid. It was found that palladium catalyzed silica nanowires were toxic to breast cancer cells, however, nanowires were inert at 1µg/mL concentrations. Additionally a method for direct nanowire integration was developed that allowed for silica nanowires to be grown directly into interdigitated sensing structures. This technique eliminates the need for physical nanowire transfer thus preserving nanowire structure and performance integrity and further reduces fabrication cost. Successful nanowire integration was physically verified using Scanning electron microscopy and confirmed electrically using Electrochemical Impedance Spectroscopy of immobilized Prostate Specific Antigens (PSA). The experiments performed above serve as a guideline to addressing the metallurgic challenges in nanoscale integration of materials with varying composition and to understanding the effects of nanomaterials on biological structures that come in contact with the human body.

Orientadora: Mônica Alonso Cotta / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-31T00:06:01Z (GMT). No. of bitstreams: 1 Silva_AldelianeMariada_M.pdf: 11574559 bytes, checksum: 5c39733d3a4441b98e7edbef8adbd795 (MD5) Previous issue date: 2016 / Resumo: Este trabalho apresenta resultados de nossa investigação sobre as propriedades elétricas do biossensor do tipo transistor de efeito de campo (FET, do inglês Field Effect Transistor) baseado em fosfeto de índio (InP). A estrutura deste biossensor consiste em um filme fino de InP do tipo-n crescido por Epitaxia de Feixe Químico (CBE, do inglês Chemical Beam Epitaxy) sobre um substrato de InP semi-isolante. No nosso biosensor, o contato da porta foi substituído por uma camada de biomoléculas carregadas de interesse para a detecção, funcionalizadas na camada de óxido do InP. O campo elétrico associado a estas biomoléculas pode modular o canal de condução. O sistema de interação específica utilizado foi a hibridização de fitas de ssDNA (single stranded DNA) complementares, onde os oligonucleotídeos receptores (probe) ssDNA foram imobilizados covalentemente na superfície da amostra. Este procedimento foi realizado através da oxidação com plasma de O2, seguida da funcionalização utilizando etanolamina e polietileno glicol (PEG), que serve como linker para a imobilização de receptores na superfície. As medidas elétricas de detecção foram feitas com as moléculas de target diluídas em buffer TRIS. A hibridização do DNA provoca um aumento na densidade de cargas na superfície, que consequentemente aumenta a largura da região de depleção no semicondutor, variando a resistência medida. A resposta do biossensor corresponde à variação da resistência em função da concentração de target. O biossensor apresentou sensibilidade para medidas de concentrações entre 10 pM e 30 pM, onde ocorre a saturação, e o tempo de resposta, no qual encontramos a estabilização do sinal medido, foi de aproximadamente 20 min. Variando a concentração de portadores e a espessura da camada semicondutora, verificamos alterações no limite de saturação (até ?M) e na sensibilidade do dispositivo. O controle destas propriedades, porém, mostrou-se limitado devido à variações na dopagem residual do semicondutor, e por isso discutimos aqui alternativas à geometria do dispositivo. Analisamos também a camada funcionalizada através de medidas de topografia e potencial de superfície usando métodos de microscopia de varredura por sonda (SPM, do inglês Scanning Probe Microscopy). Pudemos identificar a variação no potencial de superfície associada à imobilização do PEG e do DNA probe, mas não obtivemos resolução para o DNA target. Esta técnica permitiu porém verificar a estratificação de quatro níveis de potencial de superfície, no caso onde a funcionalização resultou em camadas mais espessas do que os valores típicos (~2 nm de espessura), em pequenas áreas do semicondutor / Abstract: This dissertation presents our results for the electrical properties investigation of Indium Phosphide (InP) based Field Effect Transistor (FET) biosensor. The structure of this biosensor consists of a thin n-type InP film grown by Chemical Beam Epitaxy (CBE) on a semi-insulating InP substrate. In our biosensor, the gate contact has been replaced by charged biomolecules of interest for detection, functionalized to the InP oxide layer. The electric field associated with these biomolecules provides the conduction channel modulation. The specific interaction system used here was the hybridization of single stranded-DNA (ssDNA) complementary oligonucleotides, for which the ssDNA receivers (probes) were covalently immobilized on the sample surface. The functionalization was carried out by oxidation with O2 plasma, followed by grafting biomolecules using ethanolamine and polyethylene glycol (PEG), which act as a linker for immobilizing the receptors on the surface. Electrical detection measurements were made with the target molecules diluted in TRIS buffer. DNA hybridization causes an increase in the surface charge density; consequently the semiconductor depletion width increases, affecting the measured resistance. The biosensor response function corresponds to the resistance variation as a function of target concentration. Our biosensor showed measured sensitivity to concentrations between 10 pM and 30 pM, for which signal saturation occurs. The response time, for which the measured signal stabilization was observed, was approximately 20 min. By varying the carrier concentration and the thickness of the semiconductor layer, we observed changes in the saturation limit (up ?M) and device sensitivity. The control of these properties, however, is limited due to variations in the residual doping of the semiconductor. Therefore we discuss here alternative device geometries. We also analyzed the functionalized layer by topography and surface potential measurements obtained using scanning probe microscopy (SPM) methods. We were able to identify the change in surface potential associated with the immobilization of PEG and probe DNA, but not for the target DNA. These techniques have however shown four surface potential levels in the case when the functionalization resulted in non-uniform layers, thicker than the typical values (~ 2 nm), in small areas of the semiconductor / Mestrado / Física / Mestra em Física / 165741/2014-7 / CNPQ

Nesta dissertação, a polifenol oxidase (PFO) como extrato bruto de abacate (Persea americana) foi imobilizada em filmes de polipirrol (PPI) sintetizados eletroquimicamente utilizando o glutaraldeído (GA) como um agente de ligação entrecruzada. Os filmes PPI e PPI/PFO-GA foram caracterizados por eletroquímica, principalmente voltametria cíclica, sendo avaliadas a eletroatividade e a reversibilidade eletroquímica. A detecção de compostos fenólicos em soluções padrão foi feita por cronoamperometria, tendo um controle sobre a concentração dos compostos. O processo de transferência de massa foi monitorado com uma microbalança de cristal de quartzo eletroquímica. Os resultados indicaram uma boa reprodutibilidade das medidas na detecção dos compostos fenólicos. A estabilidade do biossensor em uma solução tampão manteve-se durante 27 dias, um resultado aceitável já que é encontrado na literatura um tempo de vida estável para sistemas semelhantes em tomo de 30 dias / In this dissertation, polyphenol oxidase (PPO) as crude extract of avocado (Persea americana) was immobilized on electrochemical1y synthesized polypyrrole (PPY) films using glutaraldehyde (GA) as a crosslinking agent. PPY and PPY/PPO-GA films were electrochemical1y characterized, mainly by cyclic voltametry, where electroactivity and electrochemical reversibility were evaluated. The detection of phenolic compounds in standard samples was made by chronoamperometry with a control over the compound concentration. The process of mass transfer was monitored with an electrochemical quartz crystal microbalance (EQCM). Our results indicated a good repeatability of the measurements for the detection of phenolic compounds. The stability of biosensor in a buffer solution has remained for 27 days, a result acceptable since it is found in the literature a time of life stable for similar systems around 30 days

Neste trabalho, foram fabricados biossensores amperométricos a partir do uso da polifenol oxidase (PFO), obtida do abacate como fonte enzimática, imobilizada em filmes de polipirrol (PPI) e que foram eletrossintetizados em meio de três diferentes eletrólitos de suporte (NaCl e NaClO4 e NaDDS). O método de imobilização enzimática foi o da adsorção, sendo a solução de enzima adicionada à solução com o pirrol e o eletrólito durante o processo de eletropolimerização. Os filmes de PPI/PFO foram caracterizados por técnicas eletroquímicas, principalmente por voltametria cíclica. A detecção de compostos fenólicos (catecol e pirogalol) foi realizada pela técnica de cronoamperometria após se variar a concentração do analito. A morfologia dos filmes foi estudada por microscopia de força atômica (AFM), sendo observado que a presença da enzima no filme polimérico assim como o uso de diferentes eletrólitos de suporte levou a diferenças na superfície dos filmes. Além disto, verificou-se que o biossensor construído a partir do uso do NaCl, apresentava uma resposta mais eficiente, ou seja, ele foi capaz de detectar catecol e pirogalol em um menor limite de detecção. / In this study amperometric biosensors were manufactured from the use of polyphenol oxidase (PPO) obtained from avocado as a source of enzyme immobilized in polypyrrole (PPY) films that were electrosynthesized with three different support electrolytes (NaCl, NaClO4 and NaDDS). The method of enzyme immobilization was the adsorption. The PPO was added in the solution containing pyrrole and electrolyte during electropolymerization. The PPY/PPO films were characterized by electrochemical techniques mainly by cyclic voltammetry. Detection of phenolic compounds (catechol and pyrogallol) was performed by the technique of chronoamperometry after varying the concentration of the analyte. The morphology of the films was studied by the atomic force microscopy (AFM) and observed that the presence of the enzyme in the polymer film and the use of different electrolytes support led to differences in the surface of films. However it was found that the biosensor constructed from the use of NaCl showed more efficient response and it was able to detect catechol and pyrogallol in a lower limit of detection.

Este trabalho tem como principal objetivo comparar as respostas de biossensores amperomé-tricos preparados a partir do uso de uma enzima, a tirosinase (polifenoloxidase, PFO), quando nas formas purificada e adquirida comercialmente e de extrato bruto enzimático do fruto do abacate (Persea Americana). As soluções de PFO apresentaram valores de atividades enzimá-ticas de 479 unidades de enzimas ativas por mililitro (UA mL-1) (purificada) e 259 UA mL-1 (extrato bruto). A enzima nas duas formas (pura e como extrato bruto) foi imobilizada quimi-camente em substratos de Au modificados com monocamadas automontadas (self-assembly monolayers, SAMs) de ácido mercaptopropiônico (MPA) pela técnica de impressão por mi-crocontato (CP). Os biossensores foram preparados sobre substratos de Au modificados por CP com um molde elastomérico de polidimetilsiloxano (PDMS), cujas trilhas paralelas de 100 m foram produzidas pela cura polimérica sobre um molde mestre de GaAs. A réplica de PDMS se mostrou fiel em relação ao molde mestre nos picos e vales, mas apresentou defeitos na borda dos moldes. Com o objetivo de otimizar o funcionamento dos biossensores, eles fo-ram caracterizados pelas técnicas de voltametria cíclica e cronoamperometria com um sistema de análise de injeção em fluxo (FIA) em meio de catecol a diferentes concentrações. Os po-tenciais de oxidação do catecol nos eletrodos de Au modificados foram observados em 418 e 365 mV, em pHs ótimos de funcionamento de 7,0 e 7,2, e com limites de detecção de 6,65 nmol L-1 e 4,65 nmol L-1 para os biossensores com a enzima purificada e com o extrato bruto, respectivamente. A saturação dos sensores teve início a uma concentração de catecol de 0,02 mol L-1. Com estes resultados, mostramos que é possível o preparo de biosensores com um baixo custo, eficientes e miniaturizadas a partir do uso da PFO na forma extrato bruto do fruto do abacate para a detecção de compostos fenólicos, tal como catecol, abrindo assim a possibi-lidade de uso destes biossensores na análise e no monitoramento de pesticidas presentes no solo e na água. / The main objective of this master thesis is to compare the performance of amperometric biosensors prepared using the purified tirosinase (PPO) enzyme, (commercially acquired) and the PPO present in the enzymatic crude extract from the avocado fruit (Persea Americana). The PPO solutions had 479 units of active enzyme per milliliter (UA mL-1) (purified) and 259 UA mL-1 (crude extract). Both forms of the enzyme (purified and crude extract) were chemically immobilized on gold substrates patterned with 3-mercaptopropionic acid (MPA) self assembled monolayers (SAMs), using the microcontact printing (CP) technique. The biosensors were prepared on gold subtracts patterned using CP with a polydimethylsiloxane (PDMS) elastomeric mold, that was shaped in the form as tracks using a gallium arsenate master mold. The PDMS mold was quite similar to the master mold in its peaks and valleys, but defects were found on the edges. In order to optimize the parameters of the biosensors , they were characterized using cyclic voltammetry and chronoamperometry techniques in a FIA system by cathecol injections at different concentrations. The oxidation potentials for the cathecol analysis using the patterned biosensors were observed at 418 and 365 mV, the optimum pH were 7,0 and 7,0, with detection limits of 6,65 nmol L-1 and 4,65 nmol L-1 for the purified enzyme and crude extract biosensors, respectively. The biosensors saturation point started at 0,02 mol L-1 of cathecol concentration. With this results, we demonstrate that it is possible to use miniaturized, efficient, low cost biosensors based on tyrosine from the avocado´s fruit crude extract to detect phenolic compounds, as the cathecol. This expands the possibility of using this biosensors in the analysis and monitoring pesticides in water and soil.