Au cours des dernières années, la possibilité de combiner la nanoélectronique et les biocapteurs a ouvert un champ très large et prometteur de la recherche, qui a le potentiel de révolutionner la biologie analytique et pour permettre le diagnostic envahissants et la médecine personnalisée. Plates-formes intégrées de biocapteurs nanoélectroniques peuvent fournir une compensation et l'étalonnage du matériel et des logiciels, une sensibilité améliorée en raison des très petites dimensions, parallélisme élevé, le coût remarquable et la réduction de la taille et les vastes marchés nécessaires à l'industrie des semi-conducteurs. Comme dans le cas de tous les capteurs intégrés nanoélectroniques, la conception fiable et abordable est possible que si des modèles précis sont disponibles pour élucider et quantitativement prédisent le processus de transduction du signal. Cependant, malgré les nombreux efforts, calibré modèles analytiques et numériques pour décrire avec précision la réponse du biocapteur sont encore souvent défaut. Animé par la volonté de combler cette lacune, dans ce travail, nous développons des modèles analytiques compacts et des outils complexes de simulation numérique pour l'étude de la chaîne de transduction dans des biocapteurs nanoélectroniques impédimétriques. En particulier, les ENBIOS simulateur 3D, entièrement développées et validées au cours de cette thèse, est un outil polyvalent qui peut être facilement étendu pour inclure de nouveaux effets physiques ou des descriptions plus sophistiqués d'électrolytes et analytes couplés à des dispositifs semi-conducteurs. Les modèles soulignent l'existence de deux fréquences de coupure pertinentes régissant le biocapteur réponse impédimétriques, ils révèlent les dépendances de la réponse du biocapteur à l'analyte et des conditions environnementales et ils révèlent la présence de signatures bien définis dans le signal d'impédance. Les outils analytiques et numériques sont soigneusement vérifiées et ensuite utilisés pour examiner plusieurs études de cas. La première que nous considérons est un réseau de impédimétriques nanoélectrode biocapteur. En collaboration avec l'Université de Twente, nous étudions sa réponse aux micro-particules conductrices et diélectriques dans des conditions expérimentales bien contrôlées. Nous montrons que les résultats de simulation sont en très bon accord avec les mesures et nous donnent un aperçu des conditions optimales de détection. En étudiant la réponse du biocapteur à de petites particules, comme des protéines, des virus ou de l'ADN, nous confirmons ensuite par simulation les avantages de la spectroscopie d'impédance à haute fréquence, en particulier la capacité des signaux de courant alternatif à une fréquence au-dessus de relaxation diélectrique de la fréquence de coupure de l'électrolyte pour surmonter la Debye criblage et de sonder le volume de l'électrolyte avec une sensibilité presque indépendante de la position et de la charge des particules et de concentration en sel. Dans un deuxième exemple notable nous considérons le cas d'une Silicon Nanowire (SiNW) biocapteur. Nous effectuons les mesures et simulations sur SiNWs dans le régime AC en collaboration avec le CEA / LETI et laboratoires de l'EPFL / CLSE. Nous démontrons le fonctionnement de SiNWs AC en particulier pour les applications de détection pH. Nous confirmons enfin avantage potentiel d'un biocapteur SiNW travailler à haute fréquence, afin d'augmenter la réponse à l'égard de l'opération DC. / In recent years the possibility to combine nanoelectronics and biosensing has opened a very broad and promising field of research, which holds the potential to revolutionize analytical biology and to enable pervasive diagnostics and personalized medicine. Integrated nanoelectronic biosensor platforms can provide compensation and calibration hardware and software, improved sensitivity due to the very small dimensions, high parallelism, remarkable cost and size reduction and the vast markets needed by the semiconductor industry. As in the case of all integrated nanoelectronic sensors, reliable and affordable design is possible only if accurate models are available to elucidate and quantitatively predict the signal transduction process. However, despite the numerous efforts, calibrated analytical and numerical models to accurately describe the biosensor response are often still lacking. Animated by the will to bridge this gap, in this work we develop compact analytical models and complex numerical simulation tools for the study of the transduction chain in impedimetric nanoelectronic biosensors. In particular, the 3D simulator ENBIOS, entirely developed and validated during this thesis, is a general-purpose tool that can be easily expanded to include new physical effects or more sophisticated descriptions of electrolytes and analytes coupled to semiconductor devices. The models point out the existence of two relevant cut-off frequencies governing the biosensor impedimetric response, they reveal the dependencies of biosensor response to the analyte and environmental conditions and they disclose the existence of well-defined signatures in the impedance signal. The analytical and numerical tools are carefully verified and then used to examine several case studies. The first one we consider is an impedimetric nanoelectrode array biosensor. In collaboration with Twente University, we study its response to conductive and dielectric micro-particles under well controlled experimental conditions. We show that the simulation results are in very good agreement with the measurements and we provide insight on optimum detection conditions. By studying the biosensor response to small particles, like proteins, viruses or DNA, we then confirm by simulation the advantages of high frequency impedance spectroscopy, in particular the ability of AC signals at frequency above electrolyte's dielectric relaxation cut-off frequency to overcome the Debye screening and to probe the electrolyte volume with sensitivity almost independent of the particle position and charge and of salt concentration. As a second notable example we consider the case of a Silicon Nanowire (SiNW) biosensor. We perform measurements and simulations on SiNWs in AC regime in collaboration with the CEA/LETI and EPFL/CLSE laboratories. We demonstrate the operation of SiNWs in AC in particular for pH sensing applications. We finally confirm potential advantage of a SiNW biosensor working at high frequency, in order to increase the response with respect to the DC operation. / Negli ultimi anni la possibilità di combinare nanoelettronica e biosensoristica ha apertoun campo di ricerca molto vasto e promettente, che ha il potenziale di rivoluzionare labiologia analitica e di consentire diagnostica pervasiva e medicina personalizzata. Lepiattaforme di biosensori nanoelettronici integrati sono potenzialmente in grado di fornirecompensazioni e calibrazioni hardware, firmware programmabili, una maggiore sensibilitàa causa delle ridotte dimensioni, elevato parallelismo, riduzione notevole dei costi e delledimensioni e i vasti mercati necessari per il settore dei semiconduttori. Come nel casodi tutti i sensori nanoelettronici integrati, un progetto affidabile e conveniente è possibilesolo se sono disponibili modelli accurati per comprendere e prevedere quantitativamente ilprocesso di trasduzione del segnale. Tuttavia, con l’eccezione di alcuni pionieristici sforzi,mancano ancora spesso modelli analitici e numerici calibrati per descrivere accuratamentela risposta della maggior parte dei concept di biosensori.Animati dalla volontà di colmare questa lacuna, in questo lavoro sviluppiamo modellianalitici compatti e complessi strumenti di simulazione numerica per lo studio della catenadi trasduzione in biosensori nanoelettronici impedimetrici. In particolare, il simulatore3D ENBIOS, interamente sviluppato e convalidato durante questa tesi, è uno strumentogenerale che può essere facilmente ampliato per includere nuovi effetti fisici o descrizionipiù sofisticate di elettroliti e analiti accoppiati ai dispositivi a semiconduttore. I modellirilevano l’esistenza di due frequenze di taglio rilevanti che regolano la risposta impedimet-rica del biosensore, rivelano le dipendenze della risposta del biosensore all’analita e allecondizioni ambientali e l’esistenza di firme ben definite nel segnale di impedenza.Gli strumenti analitici e numerici sono attentamente verificati e poi utilizzati per esam-inare diversi casi di studio. Il primo che consideriamo è un biosensore impedimetrico amatrice di nanoelettrodi. In collaborazione con l’Università di Twente, studiamo la suarisposta a micro-particelle conduttive e dielettriche in condizioni sperimentali ben con-trollate. I risultati della simulazione sono in ottimo accordo con le misure e ci fornisconoinformazioni sulle condizioni di rilevamento ottimali. Studiando la risposta del biosensorea piccole particelle, come proteine, virus o DNA, confermiamo quindi tramite simulazionii vantaggi della spettroscopia di impedenza ad alta frequenza, in particolare la capacitàdei segnali in AC a frequenza superiore alla frequenza di taglio di rilassamento dielettricodell’elettrolita di superare lo screening di Debye e di sondare il volume dell’elettrolita conuna sensibilità quasi indipendente da posizione e carica della particella e dalla concen-trazione salina.Come secondo esempio notevole consideriamo il caso di un biosensore a Nanofilo diSilicio (SiNW). Eseguiamo misure e simulazioni su SiNWs in regime AC in collaborazionecon i laboratori CEA / LETI ed EPFL / CLSE. Dimostriamo il funzionamento dei SiNWsin AC, in particolare per applicazioni di misura del pH. Infine, confermiamo i vantaggipotenziali di un biosensore a SiNW operante in alta frequenza, al fine di aumentarel’intensità della risposta rispetto al caso di funzionamento in DC.

Magister Scientiae - MSc (Biotechnology) / It has been estimated that the world population will reach about 10 billion by the year 2050 and in order to accommodate the increased demand of food, the world agricultural production needs to rise by 70 % in the year 2030. However, the realisation of the goal in food production is hindered by limited arable land caused by urbanisation, salinisation, desertification and environmental degradation. Furthermore, abiotic and biotic stresses affect plant growth and development, which lead to major crop losses. The long term goal of this study is to improve food security by producing genetically engineered agricultural crops that will be tolerant to diverse stresses. This research aims at developing stress tolerant crops through the determination of important signalling molecules and second messengers, such as nitric oxide (NO), carbon monoxide (CO) and calcium ion (Ca2+), which can bind to plant proteins such as AtNOGC1 in order to induce stress tolerance in plants.

Malgré l'enthousiasme croissant pour les nanotechnologies, les nanoparticules (NPs) peuvent interagir avec les systèmes biologiques et affecter leur comportement, et pourraient ainsi présenter un danger pour les écosystèmes et l’Homme. Il est donc essentiel de connaître leurs mécanismes d'interactions afin non seulement de prévenir leurs risques potentiels, mais également de bénéficier de leurs propriétés uniques, par exemple dans la conception des biocapteurs. Dans ce contexte, nous étudions la cytotoxicité des NPs de silice, de tailles et charges diverses, sur les propriétés des bactéries Escherichia coli et Bacillus subtilis, au moyen de la microscopie à force atomique et des tests de viabilité. Les NPs chargées négativement (NPs-) de diamètre inférieur à un diamètre critique φc, 50 - 80 nm, (i) mènent à l'isolation des bactéries E. coli, (ii) induisent une "sphérification" de la cellule initialement en bâtonnet, (iii) provoquent des lésions dans la membrane externe et une réorganisation de sa structure. Pour la bactérie B. subtilis, seule la dégradation de la structure du peptidoglycane a été observée. Cependant, pour les deux souches, une activité antibactérienne a été démontrée pour les NPs- en dessous de φc, qui peuvent conduire à la lyse cellulaire tandis que, au-dessus de φc, les NPs- n’ont aucun effet sur la population, la morphologie ou la structure bactérienne. En ce qui concerne les NPs chargées positivement, elles conduisent, quel que soit leur diamètre, à une forte agrégation des cellules, en raison des interactions électrostatiques, et tendent à favoriser la formation d'invaginations membranaires, ne menant pas nécessairement à la lyse cellulaire. Cette étude fondamentale a mené au développement d’un biocapteur électrochimique pour la détection de bactéries, application notable pour des problèmes biomédicaux, environnementaux et de défense. Les NPs, intégrées à ces outils, offrent un mode de détection rapide, très sensible et peu coûteux. Expérimentalement, une multicouche de polyélectrolytes a été utilisée pour immobiliser des NPs inoffensives (φ = 100 nm), auxquelles sont ensuite fixés des anticorps spécifiques, afin d'améliorer la détection finale de la bactérie E. coli. L’ensemble des étapes a été optimisé par le procédé du spin coating et étudié à l'aide de mesures de microbalance à quartz et de voltametrie cyclique. L’intégration de NPs au biocapteur a permis une détection linéaire et non saturée des bactéries E. coli dans une large gamme de concentration (jusqu’à 10^9 CFU/mL) pour une limite de détection de 10^6 CFU/mL. / Despite the growing enthusiasm for nanotechnologies, nanoparticles (NPs) might put environmental safety and human health at risk, as they can interact with biological systems and affect their behavior. It is therefore essential to know their mechanisms of interactions in order not only to prevent their potential risks but also to benefit from their unique properties, such as in biosensors design. In this context, we study the cytotoxicity of silica NPs, with diverse sizes and charges, on the properties of Escherichia coli and Bacillus subtilis bacteria, by means of atomic force microscopy and viability tests. Negatively charged NPs (NPs-) with a diameter φ lower than a critical diameter φc, 50 - 80 nm, (i) lead to the isolation of E. coli bacteria, (ii) induce a "spherification" of the cell initially rod shaped, and (iii) cause the formation of pore-like lesions in the outer membrane and a reorganization of its structure. For B. subtilis bacteria, only the degradation of the peptidoglycane’s structure was observed. Though, for both strains, an antibacterial activity was shown for NPs- below φc, which potentially lead to the cell lysis whereas, above φc, NPs- have no effect on population, morphology or bacterial structure. As positively charged NPs are concerned, whatever their diameter, they lead to a strong aggregation of the cells, due to electrostatic interactions, and tend to favor the formation of membrane invaginations, not necessarily involving cell lysis. This fundamental study has been used to develop an electrochemical biosensor for bacteria, which are of great importance for biomedical, environmental and defense issues. NPs involved in such tools offer a fast, high-sensitive and low-cost way of detection. A polyelectrolyte multilayer was used to immobilize harmless NPs (φ = 100 nm), which are, then, functionalized with specific antibodies, in order to enhance the final detection of E. coli bacteria. All steps were optimized by a spin coating process and studied through quartz microbalance and cyclic voltametry measurements. Integrating NPs in this biosensor resulted in a linear and unsaturated detection of E. coli bacteria in a wide range of concentration (until 10^9 CFU/mL) and a limit of detection of 10^6 CFU/mL.

The cholinesterase (ChE) play an important role in the organophosphates and carbamates detection substances that are of high interest for environmental control, because these compounds are present in most pesticides applied to crops. The development of ChE biosensors to detect these contaminants with greater speed, sensitivity and selectivity, has the ability to quantify from a suitable transducer, the reduction of enzyme activity caused by contaminants. This study aimed to purify and characterize AChE of Prochilodus brevis brain for use in electrochemical tests. Gradients of pH, ionic strength and temperature, showed high activities in pH 8,5, 0,08M at ionic strength, 28Cº of temperature optimum and 37ºC of thermal stability for cell-free extract. Was applied salting out method in fractions 0-70% followed by 0-40% of (NH4)2SO4 as a refinement to liquid chromatography. The purification protocol 1 showed specific activity of 1.41 U/mg in 0-70% and 0-40% was 1.94 U/mg. The purification protocol 2, the 0-70% fraction had specific activity of 0.31 U/mg and 0-40% with 1.77 U/mg. After Sephacryl S-200 chromatography, showed specific activity for protocols 1 and 2 of 0,128U/mg and 0.2869 U/mg, respectively. The next stage of the Protocol 2 with DEAE-Sepharose was eluted peak with specific activity of 0.01326 U/mg. In the purification protocol 3, 0-70% have specific activity 0.310 U / mg, followed by 1,774 U/mg in 0-40%. After Sephacryl S-100, was obtain two peaks with a specific activity of 0.194 U/mg (ChE1) and 0.0873 U/mg (ChE2). SDS-PAGE of ChE 1 showed somewhat visible band of approximately 67kDa. Inhibition assays with ChE 1 had higher specificity for BW 284c51. Electrochemical tests with cell-free extract, dialyzed, ChE1 and ChE2, showed thiocholine (TCh) oxidation with better limits of detection and quantification limits for ChE1 and ChE2. The molecular modeling experiments showed favorable results in complex formation ChE-NTCPM. The study showed the isolation of ChE with catalytic activity for both enzymes, AChE and BuChE, with favorable adsorption properties in NTCPM in the development of enzymatic biosensor for environmental monitoring. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / As colinesterases (ChE) desempenham um papel importante na detecção de organofosforados e carbamatos que são substâncias de alto interesse para o controle ambiental, pois estes compostos estão presentes na maioria dos pesticidas aplicados em lavouras. O desenvolvimento de biossensores a base de ChE para a detecção destes contaminantes com maior rapidez, sensibilidade e seletividade, tem a capacidade de quantificar, a partir de um transdutor adequado, a diminuição da atividade enzimática causada pelos contaminantes. Este trabalho teve como objetivo de purificar e caracterizar AChE de cérebro de Prochilodus brevis para o emprego em testes eletroquímicos. Os gradientes de pH, força iônica e temperatura, mostraram maiores atividades em pH 8,5, força iônica de 0,08M, temperatura ótima 28°C e estabilidade térmica de 37°C para extrato livre de células. Foi aplicado o método de precipitação salina em frações de 0-70% seguido de 0-40% de (NH4)2SO4 como refinamento para cromatografia líquida. O protocolo de purificação 1 apresentou atividade específica de 1,41 U/mg em 0-70%, enquanto 0-40% apresentou 1,94 U/mg. O protocolo de purificação 2, a fração 0-70% teve atividade específica de 0,31 U/mg e 0-40% de 1,77 U/mg. Após a cromatografia de sephacryl S-200 apresentou atividade específica para os protocolos 1 e 2 de 0,128U/mg e 0,2869 U/mg, respectivamente. Na continuidade do protocolo 2 com DEAE-sepharose o pico eluído apresentou atividade específica de 0,01326 U/mg. No protocolo de purificação 3, 0-70% tem atividade específica 0,310 U/mg, seguido de 1,774 U/mg em 0-40%. Após Sephacryl S-100 foi obtido dois picos com atividade específica de 0,194 U/mg (ChE1) e 0,0873 U/mg (ChE2). Eletroforese SDS-PAGE de ChE1 apresentou banda pouco visível de aproximadamente 67KDa. Os ensaios de inibição com ChE1 apresentaram maior especificidade para BW 284c51. Os testes eletroquímicos com extrato livre de célula, dialisado, ChE1 e ChE2, mostraram oxidação de tiocolina (TCh) com melhores limites de detecção e quantificação para ChE1 e ChE2. Os experimentos de modelagem molecular apresentaram resultados favoráveis na formação do complexo ChE-NTCPM. O estudo mostrou o isolamento de ChE com atividade catalítica para as duas enzimas, AChE e BuChE, com propriedades de adsorção favoráveis em NTCPM no desenvolvimento de biossensor enzimático para monitoramento ambiental.

The gold nanoparticles (NPAus) and silver (NPAgs) exhibit a variety of effects and applications in the biomedical field. In this study, silver and gold nanoparticles were synthesized and characterized in order to evaluate its effect on wound healing and sensing eotaxin, an analyte of great relevance to inflammatory events. . In a first case, NPAgs were synthesized by chemical reduction method and dispersed in copaiba oil. It was observed that all NPAgs had spherical shapes and dispersed in copaiba oil. This composition NPAgs more copaiba oil was more effective to heal skin wounds than Dermazine, a reference medicinal product. With gold nanoparticles (NPAu) were synthesized by chemical methods by citrate reduction. Then, these nanostructures are functionalized for making a biosensor, with the purpose of identifying the analyte of choice (eotaxin) by identifying the displacement of the surface plasmon resonance band. The results with this prototype of the great potential of biosensor proved to detection of eotaxin in small quantities 15μL. The particles used in this study were characterized by UV-vis spectroscopy and transmission electron microscopy, while those associated with quartz are characterized with UV-vis spectroscopy and atomic force microscopy. Together, this study revealed the potential installed in UFAL for synthesis, characterization and application in the biomedical field of metal nanoparticles. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As nanopartícula de ouro (NPAu) e de prata (NPAg) exibem uma variedade de efeitos e aplicações na área biomédica. No presente estudo, nanopartículas de prata e ouro foram sintetizadas e caracterizadas com objetivo avaliar seu efeito sobre a cicatrização de feridas e sensoriamento de eotaxina, um analito de grande relevância para eventos inflamatórios. Em um primeiro caso, NPAg foram sintetizadas por método químico de redução, e dispersas em óleo de copaiba. Observou-se que todas NPAg tinham formas esféricas e se dispersaram no óleo de copaiba. Essa composição NPAg mais óleo de copaiba mostrou-se mais eficaz para cicatrizar feridas cutâneas do que a Dermazine, um medicamento de referência. Com as nanopartículas de ouro (NPAu) foram sintetizadas por método químico de redução por citrato. Em seguida, estas nanoestruturas foram funcionalizadas para confecção de um biossensor, com finalidade de identificar o analito de escolha (eotaxina) por meio da identificação do deslocamento da banda de ressonância de plasmon de superfície. Os resultados com este prótotipo do biosensor se mostraram de grande potencial para deteccção da eotaxin em pequenas quantidades 15µL. As partículas utilizadas neste estudo foram caracterizadas por espectroscopia de UV-vis e microscopia eletrônica de transmissão, enquanto que aquelas associadas ao quartzo são caracterizadas com espectroscopia UV-vis e microscopia de força atômica. Em conjunto, este estudo revelou o potencial instalado na UFAL para síntese, caracterização e aplicação na área biomédica de nanopartículas metálicas.

miRNAs are small regulatory RNAs, which function as post-transcriptional mRNA regulators. They direct ribonucleoprotein complexes to cognate mRNA to repress them by translational inhibition and degradation. miRNAs regulate thousands of mRNAs in mammals and have been recognized as regulatory factors in most cellular and developmental processes. Dysregulation of the miRNA pathway can lead to severe defects and diseases. Interestingly, a unique situation exists in mouse oocytes, where all the miRNA pathway components are present, yet the pathway is dispensable and nonfunctional, the molecular foundation of this phenomenon and its significance still remain unclear. In spite of the pronounced effects of the miRNA pathway in gene regulation in somatic cells, study strategies of the pathway bare limitations. Current methods for studying the activity of the miRNA pathway employ corelative studies (such as NGS) or reporter assays, which have relatively low throughput and are prone to artifacts. Here, I present design and development of a new strategy for directly monitor global miRNA pathway activity and integrity in near physiological conditions in living cells, which could also be employed in vivo for studies of mouse oocytes. The strategy is based on fluorescently tagged endogenous proteins of the...

Nowadays, to diagnose a lung cancer, a bronchoscopy is performed and a biological sample is extracted and analyzed by the anatomical pathology department of the hospital. Currently, there are no commercially available techniques allowing a real-time, in vivo, label-free diagnosis of lung cancer. The PREDICTION project aims to develop a biosensing tool gathering all the attributes mentioned above by combining optics, biochemistry and mechanics. The role of my research is to focus on the mechanics and to develop an instrumented catheter, acting as a shield of the biosensor. The choice of the material and the design were made based on the optical properties (visible under fluoroscopy) and the mechanical characteristics (trade-off between rigidity and compliance). In order to provide a stable measurement, the distal extremity of the instrumented catheter was shaped in the form of a conical needle. A window was patterned on the side of the instrumented catheter to expose the biosensor to the targeted tissue. The instrumented catheter was designed to be able to embed one biosensor and one control fibre. Its measurement integrity has been validated through in vitro and ex vivo experiments. In order to improve navigation outside the scope of the working channel of the bronchoscope, i.e. add one degree of freedom to the catheter, Shape Memory Polymers were investigated. Two prototypes were designed. The first prototype combines a soft pneumatic actuator with a shape memory polymer strip acting as a stiffness tuner. The Shape Memory Polymer structure proved to be efficient to fix the shape of the soft pneumatic actuator and also to increase the force it can provide. The second prototype combines a catheter with a Shape Memory Polymer strip. The experimental results proved the ability of the Shape Memory Polymer strip to develop a force high enough to bend a catheter with an adequate bending angle for in vivo lung navigation. To conclude, the work produced during this PhD resulted in the development of an instrumented catheter allowing real time, ex vivo, label-free diagnosis of lung cancer. Further work should be done on the instrumented catheter dimensions and sterilization to apply these results to in vivo diagnosis. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished

Este trabalho teve como objetivo estudar e caracterizar filmes de nanocompósitos de polipirrol dopado com índigo carmim e dodecilsulfato combinados com nanopartículas de ouro para aplicação como biossensores eletroquímicos de pesticida. Primeiramente sintetizou-se três filmes a 10 e 25 ºC por voltametria cíclica: polipirrol dopado com índigo carmim (PPi-IC), polipirrol dopado com índigo carmim contendo nanopartículas de ouro (PPi-IC-NPAu) e polipirrol dopado com índigo carmim e dodecilsulfato de sódio, contendo nanopartículas de ouro (PPi-IC-DS-NPAu). Nesses filmes foram realizadas caracterizações estruturais, morfológicas, ópticas e eletroquímicas, utilizando espectroscopia no infravermelho, espectroscopia Raman, microscopia eletrônica de varredura, espectroscopia UV-VIS-NIR e voltametria cíclica. Os filmes sintetizados a 10 ºC foram modificados com a enzima acetilcolinesterase (AChE), sendo posteriormente caracterizados por microscopia eletrônica de varredura, espectroscopia Raman e voltametria cíclica. Os resultados obtidos mostraram que a interação da enzima com os filmes de polipirrol é dependente de sua composição. A presença de DS não favoreceu a sua imobilização, sendo assim, os filmes de PPi-IC e PPi-IC-NPAu foram os materiais mais adequados para a imobilização física da AChE, sendo capazes de detectar o pesticida metil paration (MP) através da porcentagem de pesticida inibido pela ação da AChE em relação ao cloreto de acetiltiocolina (ATCl). Essa detecção foi realizada por voltametria cíclica, onde a resposta do biossensor é medida a partir da diminuição da corrente produzida. Isso ocorre porque o MP se encontra em contato com a AChE e inibe a ação da mesma, impedindo a hidrólise da ATCl, o que faz diminuir a corrente, pois não há produção de tiocolina, que é produto da hidrólise e uma espécie eletroativa. Os filmes apresentaram sensibilidades (% de inibição por mmol L-1 de pesticida) de 1,9×106 (R2 0,976) e 2,7×106 (R2 0,964) e limites de detecção de 4,15 e 14,8 ng L-1, para PPi-IC-AChE e PPi-IC-NPAu-AChE respectivamente. / The goal of this work was to study and characterize nanocomposite films of gold nanoparticles with indigo carmine and dodecylsulfate doped polypyrrole for application as electrochemical biosensor for pesticide. Initially, three films were synthesized by cyclic voltammetry at 10 and 25 ºC: polypyrrole doped with indigo carmine (PPy-IC), polypyrrole doped with indigo carmine containing gold nanoparticles (PPy-IC-AuNP) and polypyrrole doped with indigo carmine and sodium dodecylsulfate containing gold nanoparticles (PPy-IC-DS-AuNP). These films were studied by structural, morphological, optical and electrochemical characterization, using infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, UV-VIS-NIR spectroscopy and cyclic voltammetry. The films synthesized at 10 ºC were modified with acetylcholinesterase (AChE) enzyme; followed by characterization with scanning electron microscopy, Raman spectroscopy and cyclic voltammetry The results showed a preferential immobilization of the enzymes on films without DS, therefore, PPy-IC and PPy-IC-AuNP were the most suitable materials for the physical immobilization of AChE and for detection of methyl parathion (MP) pesticide through the percentage of pesticide inhibited by the action of AChE on acetylthiocholine chloride (ATCl). This detection was carried out by cyclic voltammetry, where the biosensor response is measured as a decrease of the produced current. The decrease in current occurs because MP reaches the AChE, inhibiting the action of the enzyme, avoiding the hydrolysis of ATCl, leading to the decrease of current as no thiocholine (an electroactive specie, product of the hydrolysis) is produced. The films presented sensitivities (inhibition percentage per mmol L-1) of 1.9×106 (R2 0.976) and 2.7×106 (R2 0.964) and detection limits of 4.15 and 14.8 ng L-1 for PPy-IC-AChE and PPy-IC-AuNP-AChE respectively.

abstract: Biogenic silica nanostructures, derived from diatoms, possess highly ordered porous hierarchical nanostructures and afford flexibility in design in large part due to the availability of a great variety of shapes, sizes, and symmetries. These advantages have been exploited for study of transport phenomena of ions and molecules towards the goal of developing ultrasensitive and selective filters and biosensors. Diatom frustules give researchers many inspiration and ideas for the design and production of novel nanostructured materials. In this doctoral research will focus on the following three aspects of biogenic silica: 1) Using diatom frustule as protein sensor. 2) Using diatom nanostructures as template to fabricate nano metal materials. 3) Using diatom nanostructures to fabricate hybrid platform. Nanoscale confinement biogenetic silica template-based electrical biosensor assay offers the user the ability to detect and quantify the biomolecules. Diatoms have been demonstrated as part of a sensor. The sensor works on the principle of electrochemical impedance spectroscopy. When specific protein biomarkers from a test sample bind to corresponding antibodies conjugated to the surface of the gold surface at the base of each nanowell, a perturbation of electrical double layer occurs resulting in a change in the impedance. Diatoms are also a new source of inspiration for the design and fabrication of nanostructured materials. Template-directed deposition within cylindrical nanopores of a porous membrane represents an attractive and reproducible approach for preparing metal nanopatterns or nanorods of a variety of aspect ratios. The nanopatterns fabricated from diatom have the potential of the metal-enhanced fluorescence to detect dye-conjugated molecules. Another approach presents a platform integrating biogenic silica nanostructures with micromachined silicon substrates in a micro/nano hybrid device. In this study, one can take advantages of the unique properties of a marine diatom that exhibits nanopores on the order of 40 nm in diameter and a hierarchical structure. This device can be used to several applications, such as nano particles separation and detection. This platform is also a good substrate to study cell growth that one can observe the reaction of cell growing on the nanostructure of frustule. / Dissertation/Thesis / Doctoral Dissertation Materials Science and Engineering 2014

abstract: The American Diabetes Association reports that diabetes costs $322 billion annually and affects 29.1 million Americans. The high out-of-pocket cost of managing diabetes can lead to noncompliance causing serious and expensive complications. There is a large market potential for a more cost-effective alternative to the current market standard of screen-printed self-monitoring blood glucose (SMBG) strips. Additive manufacturing, specifically 3D printing, is a developing field that is growing in popularity and functionality. 3D printers are now being used in a variety of applications from consumer goods to medical devices. Healthcare delivery will change as the availability of 3D printers expands into patient homes, which will create alternative and more cost-effective methods of monitoring and managing diseases, such as diabetes. 3D printing technology could transform this expensive industry. A 3D printed sensor was designed to have similar dimensions and features to the SMBG strips to comply with current manufacturing standards. To make the sensor electrically active, various conductive filaments were tested and the conductive graphene filament was determined to be the best material for the sensor. Experiments were conducted to determine the optimal print settings for printing this filament onto a mylar substrate, the industry standard. The reagents used include a mixture of a ferricyanide redox mediator and flavin adenine dinucleotide dependent glucose dehydrogenase. With these materials, each sensor only costs $0.40 to print and use. Before testing the 3D printed sensor, a suitable design, voltage range, and redox probe concentration were determined. Experiments demonstrated that this novel 3D printed sensor can accurately correlate current output to glucose concentration. It was verified that the sensor can accurately detect glucose levels from 25 mg/dL to 400 mg/dL, with an R2 correlation value as high as 0.97, which was critical as it covered hypoglycemic to hyperglycemic levels. This demonstrated that a 3D-printed sensor was created that had characteristics that are suitable for clinical use. This will allow diabetics to print their own test strips at home at a much lower cost compared to SMBG strips, which will reduce noncompliance due to the high cost of testing. In the future, this technology could be applied to additional biomarkers to measure and monitor other diseases. / Dissertation/Thesis / Masters Thesis Bioengineering 2017