Protein adsorption on solid surfaces is a common yet complicated phenomenon that is not fully understood. Self-assembled monolayers have been utilized in many studies, as well-defined model systems for studying protein-surface interactions in the atomic level. Various strategies, including the use of single component SAMs[1, 2], combinations of long and short alkanethiolates with methyl- and hydroxyl- terminal groups[3, 4], and using mixtures of alkanethiolates with similar chain length and varying terminal functional group [5] have been used to effectively control the surface wettability and determine the effect of surface composition and wettability on protein adsorption. In this dissertation we report key new findings on the effect of surface density of functional groups on protein adsorption phenomenon. In The first phase of this research, we developed a novel approach for preparation of low-density self-assembled monolayers(LD-SAMs) on gold surfaces, based on radical-initiated thiol-yne click chemistry. This approach provides exceptional adsorbate stability and conformational freedom of interfacial functional groups, and is readily adapted for low-density monolayers of varied functionality. The resulting monolayers have two distinct phases: a highly crystalline head phase adjacent to the gold substrate, and a reduced density tail phase, which is in contact with the environment. First, we investigated the feasibility of the proposed chemistry in solution-phase. In this approach, we synthesized “Y” shaped carboxylate-terminated thiol adsorbates via radical-initiated thiol-yne reaction. The LD-SAMs were then prepared through immersion of gold substrates into the solution of synthesized adsorbate molecules in hexane. The chemical structuring and electrochemical properties of resultant LD-SAMs were analyzed and compared with those of analogous traditional well-packed monolayers, using techniques such as Fourier transform infrared spectroscopy, ellipsometry, electrochemical impedance spectroscopy, reductive desorption, and contact angle goniometry. Characterization results indicated that resulting LD-SAMs have a lower average crystallinity, and higher electrochemical stability compared to well-packed monolayers. In addition, using a three-electrode system, we were able to show a reversible change in LD-SAM surface wettability, in response to an applied voltage. This remodeling capacity confirms the low density of the surface region of LD-SAM coatings. The second area of work was focused on using the developed chemistry in solid-phase. The solid-phase approach minimized the required synthesis steps in solution-phase method, and used the photo-initiated thiol-yne click-reaction for grafting of acid-terminated alkynes to thiol-terminated monolayers on a gold substrate to create similar LD-SAMs as what were prepared through solution-phase process. We characterized the resulting monolayers and compared them to analogous well-packed SAMs and the also low-density monolayers prepared through the solution phase approach. The results confirmed the proposed two-phase structure, with a well-packed phase head phase and a loosely-packed tail phase. In addition, the electrochemical studies, indicated that the resultant monolayers were less stable than the monolayers prepared via solution-phase, but they are yet significantly more stable than typical well-packed monolayers. The less stability of these monolayers were attributed to the partial desorption of adsorbates from the gold substrate due to UV irradiation during the grafting process. Building on the established chemistry, we studied the effect of lateral packing density of functional groups in a monolayer on the adsorption of Bovine serum albumin protein. we used surface plasmon resonance spectroscopy (SPR) and spectroscopic ellipsometry, to evaluate BSA adsorption on carboxylate‑, hydroxyl-, or alkyl- terminated LD-SAMs. It was found that for the LD-SAMs, the magnitude of protein adsorption is consistently higher than that of a pure component, well-packed SAM for all functionalities studied. In addition, it was seen that the magnitude of BSA adsorption the LD-SAMs, was consistently higher than that of a pure component, well-packed SAM for all functionalities studied. The difference of protein adsorption on LD-SAMs and SAMs can not be associated to difference in lateral packing density, unless we eliminate the impact of other contributing factors in protein adsorption such as surface energy. In order to better understand the impact of packing density on protein-surface interactions, we prepared the mixed SAMs of (carboxylate/alkyl) and (hydroxyl/alkyl) with matching surface energy as the carboxylate and hydroxyl terminated LD-SAMs. It was found that the energy-matched mixed SAMs of carboxylate and hydroxyl functionality adsorbed more protein than the LD-SAMs. However, an opposite trend was seen for the alkyl surfaces, where surface energies are comparable for LD-SAMs and pure component SAMs, indicating that BSA proteins have higher affinity for methyl- terminated LD-SAMs than well-packed SAMs.

<p>This thesis deals with the synthesis and characterization ofnovel polyhydroxyl surfactants. The first part describes thesynthesis of a number of stereoisomers of a polyhydroxylsurfactant, and the second part concerns surface chemicalcharacterization.</p><p>A stereodivergent route for preparation of the hydrophilichead group was developed, featuring consecutive stereoselectivedihydroxylations of a diene. This afforded in total fourdifferent polyhydroxyl head groups. These surfactant headgroups were natural and unnatural sugar analogues, and wereused for the coupling with two different hydrophobic tailgroups.</p><p>Three of these surfactants were used to investigate thechiral discrimination in Langmuir monolayers at an air-waterinterface. The isotherms showed a remarkable difference incompressibility between surfactants of diastereomericrelationship and also a pronounced chiral discriminationbetween racemic and enantiomerically pure surfactants favoringheterochiral discrimination.</p>

Continuous scaling down of the dimensions of electronic devices has made present day computers more powerful. In the front end of line, the minimum lateral dimensions in a transistor have shrunk from 45 nm in 2007 to 22 nm currently, and the gate oxide film thickness is two to three monolayers. This reduction in dimensions makes surface preparation an increasingly important part of the device fabrication process. The atoms or molecules that terminate surfaces function as passivation layers, diffusion barriers, and nucleation layers. In the back end of line, metal layers are deposited to connect transistors. We demonstrate a reproducible process that deposits a monolayer of aminopropyltrimethoxysilane molecules less than one nanometer thick on a silicon dioxide surface. The monolayer contains a high density of amine groups that can be used to deposit Pd and Ni and subsequently Co and Cu to serve as the nucleation layer in an electroless metal deposition process. Because of the shrinking device dimensions, there is a need to find new transistor channel materials that have high electron mobilities along with narrow band gaps to reduce power consumption. Compound III--V channel materials are candidates to enable increased performance and reduced power consumption at the current scaled geometries. But many challenges remain for such high mobility materials to be realized in high volume manufacturing. For instance, low defect density (1E7 /cm²) III--V and Ge on Si is the most fundamental issue to overcome before high mobility materials become practical. Unlike Si, dry etching of III-V semiconductor surfaces is believed to be difficult and uncontrollable. Therefore, new wet etching chemistries are needed. Si has been known to passivate by etching in hydrofluoric acid, but similar treatments on III--Vs are known to temporarily hydrogen passivate the surfaces. However, any subsequent exposure to the ambient reoxidizes the surface, resulting in a chemically unstable and high defect density interface. This work compares old and new wet etching chemistries and investigates new methods of passivating the III--V semiconductors.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes the application of single molecule spectroscopy and tracking to investigations of the nanoscale properties of thin-film chemical gradients and the transport dynamics of molecules dispersed within and upon these gradients. Chemical gradients are surface bound materials that incorporate gradually changing chemical and/or physical properties. A continuous and gradual change in the properties of gradients are expected and often required for their intended applications, which range from directed growth of cell colonies to combinatorial materials science. In reality, such conditions are almost never met due to spontaneous demixing and dewetting processes that can lead to properties variations on microscopic length scales. A better understanding on the properties of chemical gradients on microscopic length scales will aid in the production of better engineered materials. Single molecule spectroscopy (SMS) allows for gradient properties to be probed on nanometer-to-micrometer length scales. In this dissertation, quantitative measurements of gradient polarity (i.e., dielectric properties) are made along a sol-gel derived thin film that incorporates a macroscopic polarity gradient. These measurements report on the microscopic heterogeneity of the gradient film, and point to the occurrence of phase separation of the polar and nonpolar components along the gradient. Single molecule tracking (SMT) provides an important means to examine the dynamics of molecular mass transport in thin films and on surfaces. In this dissertation, SMT is employed to study mass transport in thin water films condensed over monolayer wettability gradients under ambient environments. The results show that the rate and the mechanism of molecular transport depend on the surface wettability, and on the ambient relative humidity. Finally, wettability gradients have been broadly used to drive the transport of liquid droplets. In this dissertation, these applications are extended to achieve spontaneous stretching of DNA by the propulsion of liquid droplets along the gradient. Single molecule fluorescence imaging of DNA stretched along these gradients demonstrates that hydrophobic surfaces play an important role in DNA stretching. The study also shows the surface tension force acting at the gradient-droplet contact line (interface) to be responsible for DNA elongation and alignment. Overall, single molecule methods have been shown to be highly useful for better understanding the properties of chemical gradients as described in this dissertation.

Cette thèse s’inscrit dans le domaine de la spintronique moléculaire. Elle s’intéresse plus précisément aux nouvelles opportunités de façonnage de la polarisation de spin qui découlent de l'hybridation métal ferromagnétique/molécule à l'interface : le nouveau concept de « spinterface ».Dans une première partie nous présentons l’étude de nanojonctions tunnel magnétiques à base de monocouches auto-assemblées (SAMs). Ce système est un des plus prometteur dans l’optique de moduler les propriétés des dispositifs de spintronique par ingénierie chimique, tel un LEGO moléculaire. Nous y présentons la fonctionnalisation de la manganite demi-métallique (La,Sr)MnO3 (LSMO) avec des SAMs d’acides alkylphosphoniques et la fabrication de nanojonctions LSMO/SAMs/Co avec une surface de quelque 10 nm2. Une magnétorésistance de 30% à 50% est observée dans la majorité des dispositifs avec une magnétorésistance tunnel (TMR) jusqu'à 250 % à basse température. Un point remarquable est aussi le comportement très robuste du signal avec la tension: environ 20% de TMR est encore observée au-dessus d’une tension de 1 V. L'influence de la longueur de la chaîne moléculaire a été aussi étudiée et représente un premier pas vers la modulation des dispositifs au niveau moléculaire. Dans une deuxième partie nous présentons l’étude des dispositifs organiques à base de métaux ferromagnétiques à haute TC (température de Curie) et semi-conducteurs organiques. Nous avons réalisé des vannes de spin de Co/Alq3/Co avec des sections de 50 ou 100 µm et fabriquées in-situ par « shadow mask ». Des mesures à température ambiante ont permis d’observer -4% de magnétorésistance (MR) dans une vanne de spin Co/Alq3/Co et +8% MR dans une vanne de spin de Co/MgO/Alq3/Co. Le rôle des deux interfaces sur les propriétés de polarisation de spin des dispositifs est aussi étudié et détaillé. Une forte hybridation métal/molécule dépendant du spin à l'interface inferieure de Co/Alq3, présentant un effet de spinterface (inversion de la polarisation en spin), est observée. Ces études montrent que les effets de spinterface, comme l’inversion de la polarisation de spin, peuvent persister dans un dispositif jusqu’à température ambiante. / This thesis targets the field of molecular spintronics and more particularly the new spin polarization tailoring opportunities, unachievable with inorganic materials, which arise from the ferromagnetic metal/molecule hybridization at the interface.: the new concept of Spinterface.In a first part we investigate Self-Assembled Monolayers (SAMs) based magnetic tunnel nanojunctions. This system appears to be a highly promising candidate to engineer the properties of spintronics devices at the molecular level since SAMs are the equivalent of a molecular LEGO building unit. We present the functionalization of the half-metallic manganite (La,Sr)MnO3 (LSMO) with alkyl phosphonic acids SAMs and the fabrication of LSMO/SAMs/Co magnetic tunnel nanojunctions with an area of few 10 nm2. MR of 30% to 50% is observed in most of the devices, while we report even up to 250% tunnel magnetoresistance (TMR) at low temperature. The most striking point is the robustness of the signal with bias voltage with still 20% TMR observed in the volt range. The influence of the molecular chain length is also investigated and represents a first step towards achieving molecular tailoring.In a second part we develop organic spintronics devices relying on high Curie temperature metallic ferromagnetic electrodes and standard organic semiconductor such as Co/Alq3/Co organic spin valves (OSVs). Junctions have a large area (section of 50 or 100 µm) and are fabricated in-situ by shadow mask. Magnetoresistance (MR) effects at room temperature are investigated with -4% MR observed in Co/Alq3/Co OSVs and +8% MR in Co/MgO/Alq3/Co OSVs. The role of the two interfaces on the spin polarization properties of the devices is also investigated. A stronger spin-dependent hybridization is found to occur at the bottom Co/Alq3 interface inverting the spin polarization on the first molecular layer. The observation of spin polarization inversion at room temperature demonstrates that spinterface effects can strive up to room temperature.

Les procédés lithographiques sont de nos jours très utilisés au sein de l’industrie de la microélectronique pour réaliser des matériaux fonctionnels de taille nanométrique. L’obtention de composants de taille de plus en petite (<100 nm) nécessite la mise en œuvre de nouveaux procédés de fabrication. Les travaux de recherches réalisés dans cette thèse portent sur l’étude d’un nouveau concept de lithographie par microcopie à force atomique (AFM). L'objectif principal est d'utiliser la pointe d’un AFM comme outil pour promouvoir des réactions catalysées sur une zone bien définie d’une surface greffée. De cette manière, diverses molécules ont pu être greffées sélectivement et spatialement pour conduire à des objets finis en 3 dimensions. Afin de mieux comprendre le mécanisme réactionnel, différents paramètres physico-chimiques ont été étudiés dans la première partie : vitesse de balayage de la pointe, force appliquée, distance interligne lors de la gravure du motif, largeur de lignes limites, durée de vie de la pointe catalytique et influence de la flexibilité du catalyseur présent sur la pointe. La deuxième partie consiste à la réalisation de nanostructures avec des molécules aux propriétés physico-chimiques particulières (optique, électrique, catalytique) ainsi qu’à la construction de nanostructures tridimensionnelles. Quelques résultats marquants sont l’obtention d’une largeur de ligne limite de 25 nm, d’une surface de greffage minimum de 480 µm² et d’une structure de forme pyramidale composée de trois niveaux moléculaires distincts. / Lithography processes are widely used in the microelectronics industry for the realization of functional materials of nanometric size. To obtain components increasingly small (<100 nm) the development of new manufacturing processes is requires. The research presented in this thesis concerns the study of a new concept of lithography by Atomic Force Microscopy (AFM). The main objective is to use the tip of an AFM as a tool to promote catalysed reactions on a well defined zone of a grafted surface. In this way, diverse molecules could be grafted selectively and spatially to form three dimensioned objects. To better understand the reaction mechanism, different physico-chemical parameters were studied in the first part: the scanning speed of the tip, the strength applied, the interline spacing during the engraving pattern, the width lines limits, the life time of the catalytic tip and the influence of the catalyst flexibility coated to the tip. The second part consists in the realization of nanostructures with molecules that have particular physico-chemical properties (optical, electric, catalytic) and the construction of three-dimensional nanostructures. Some pertinent results are the achievement of line width of 25 nm, a minimum grafted surface of 480 µm² and the formation of a structure of pyramidal shape constituted of three different molecular levels.

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.

Proteínas Inativadoras de Ribossomos (RIPs) são rRNA N-glicosilases capazes de inibir a síntese protéica pela remoção de uma adenina específica do RNA ribossomal. São geralmente classificadas em tipo 1 e tipo 2, sendo as últimas divididas em altamente tóxicas e não tóxicas. A maior parte das RIPs tipo 2 identificadas pertence a espécies de dicotiledôneas, como é o caso da pulchellina. As cadeias tóxicas das RIPs possuem uma região C-terminal hidrofóbica conservada, a qual se atribui a capacidade de interação com a membrana do retículo endoplasmático (RE), durante o transporte retrógrado da toxina para o citosol. Neste trabalho duas abordagens diferentes foram aplicadas para o estudo das RIPs tipo 2: identificação e caracterização de novos integrantes desta família de proteínas, e investigação da interação da cadeia-A da pulchellina (PAC) com sistemas miméticos da membrana celular. Na primeira abordagem, uma busca in silico em bancos de dados genéticos públicos permitiu identificar quatro novas RIPs do tipo 2 de monocotiledôneas. A análise da estrutura primária das proteínas identificadas mostrou a ocorrência de mutações em alguns dos principais aminoácidos que formam o sítio ativo nas RIPs, indicando uma possível perda de função. O representante de Saccharum officinarum (cana-de-açúcar) foi então analisado em maior detalhe, sendo sua cadeia-A clonada (soRIPA), expressa em sistema heterólogo e caracterizada em termos de atividade e estrutura secundária. Os ensaios in vitro mostraram que a soRIPA não foi capaz de depurinar ribossomos eucariotos. Porém, os ensaios de inibição da síntese proteica mostraram uma possível atividade inibitória da soRIP, que precisa ainda ser confirmada. A presença dos transcritos no banco do SUCEST sugere que estes genes não sejam pseudogenes, embora não tenha sido possível purificar a proteína a partir de extratos de folhas. Isto indica que se a soRIP está sendo traduzida, deve sofrer um rápido turnover, tornando difícil a sua detecção e purificação ou, ainda, que a ausência de sítios de ligação à galactose funcionais na cadeia-B impediu sua purificação por cromatografia de afinidade à galactose. A outra abordagem no estudo das RIPs tipo 2 foi centrada na cadeia-A recombinante da pulchellina (rPAC), estudando sua interação com monocamadas de Langmuir. Foram construídos 3 mutantes da rPAC, cada um com diferentes deleções na região C-terminal visando determinar a região responsável pela interação com a membrana do RE. A cinética de adsorção e pressão superficial exercida pela rPAC sobre a monocamada, assim como o estudo com os mutantes demontraram que a proteína interage fortemente com a monocamada fosfolipídica e que esta interação in vitro é dependente da presença da região C-terminal. De forma geral, os resultados obtidos neste trabalho contribuíram com novas informações sobre esta família de proteínas, identificando e analisando novos integrantes e, ainda, adicionando detalhes do mecanismo funcional do tráfego das toxinas RIPs. / Ribosome Inactivating Proteins (RIPs) are rRNA N-glycosilases which are able to inhibit the protein synthesis by removing a specific adenine from the ribosomal RNA. They are usually classified as type 1 and type 2, being the latter divided into highly toxic and nontoxic. The majority of type 2 RIPs currently identified are found in species of dicotyledons, as the pulchellin. The toxic chain of RIPs has a conserved hydrophobic C-terminal region, which is believed to be responsible for the interaction with the lipid membrane of the endoplasmic reticulum ER during the retrograde transport of the toxin to the cytosol. In this work, two different approaches were applied in the study of type 2 RIPs: identification and characterization of new members of this protein family, and investigation of the interaction of the pulchellin\'s A-chain (PAC) with systems that mimic the cellular membrane. In the first approach, an in silico search in public genetic databases was performed and allowed us to identify four new type 2 RIPs in monocots. The primary structure analysis of the identified proteins showed the presence of mutations in key amino acids that form the active site of RIPs, indicating a possible interference on its catalytic activity. The representative of Saccharum officinarum (sugar cane) was then analyzed in greater detail. Its A-chain clone (soRIPA) was expressed in a heterologous system and characterized in terms of activity and secondary structure. In vitro experiments showed that soRIPA was not able to perform the depurination of eukaryotic ribosomes. However, the inhibition of protein synthesis assays presented a possible low inhibitory activity of the soRIP, which still needs to be further investigated. The presence of transcripts on the bank of SUCEST indicates that these genes are not pseudogenes, although it was not possible to purify the protein from leaf extracts. If the soRIP is being translated, this may indicate that it undergoes a quick turnover, preventing its detection and purification. It is also possible that the absence of functional galactose binding sites in the B-chain has prevented its purification by galactose affinity chromatography. Our second approach to the study of type 2 RIPs was focused on the recombinant pulchellin A-chain (rPAC), by investigating its interaction with Langmuir monolayers. We have constructed three mutants of rPAC, each one with different deletions at the C-terminal to determine the region responsible for interaction with the membrane of the ER. The adsorption kinetic and surface pressure applied by rPAC on the monolayer, as well as the study of the mutants, have demonstrated that the protein has a strong interaction with the phospholipid monolayer and that this interaction in vitro is dependent on the presence of the C-terminal. The results of this work have provided new information about the type 2 RIP protein family, identifying and analyzing new members, and also bringing new details about the functional mechanism of the RIP\'s toxin traffic.

Monocamadas auto-organizadas formadas pela quimissorção de alcanotióis sobre ouro apresentam estruturas bem definidas, organizadas e reprodutíveis. As propriedades de SAMs, aliadas à facilidade de síntese, têm atraído o interesse da comunidade cientifica o que ocasionou um grande avanço na área de nanotecnologia, especialmente em engenharia de superfícies. Neste trabalho, avaliou-se a modificação de um substrato de ouro com uma mistura dos ácidos 3-mercaptopropiônico e 11-mercaptoundecanóico. Com isso procurou-se controlar a configuração da superfície da SAM a fim de se obter um arranjo com ilhas de 3amp circundadas por 11amu, similar a um conjunto de ultramicroeletrodos. A SAM mista foi produzida por incubação de um substrato de ouro em solução etanólica contendo ambos os tiois. O estudo do processo redox do par [Fe(CN)6]4-/[Fe(CN)6]3- demonstrou que monocamadas de 3amp se comportam como a superfície de ouro não modificado, apresentando os mesmos valores de corrente de pico (Ip), potencial de pico (Ep) e resistência de transferência de carga (Rtc), pois permitem que ocorra a transferência eletrônica por efeito de tunelamento quântico dos elétrons através da monocamada. Entretanto monocamadas de 11amu demonstram comportamento isolante, apresentando uma Rtc 250 vezes maior e Ip significativamente menores que a observada para a SAM-3amp. As cargas de dessorção redutiva e variação de massa obtidas com a MECQ para a SAM mista evidenciaram alto recobrimento da superfície e um mecanismo de adsorção que varia com o tempo de incubação do substrato em solução contendo os tiois. Assim, a configuração da superfície com domínios de 3amp bem estabelecidos é alcançada com 20 horas de incubação do substrato. O perfil observado com a voltametria cíclica, aliado aos dados obtidos com a EIE comprova a existência de segregação de fases na SAM mista contendo regiões recobertas pelo 3amp (raio médio de 4,3 &micro;m) circundadas por 11amu (com separação média de 75,42 &micro;m). O estudo do comportamento eletroquímico do fisetin confirma o surgimento de propriedades diferenciadas com a SAM mista, onde observou-se apenas um processo redox sem que o analito permanecesse adsorvido na superfície da SAM. Todavia para a SAM-3amp e ouro não modificado foi observado dois processos redoxes seguido de adsorção irreversível do produto da reação na superfície eletródica. Utilizando a SAM mista como sensor eletroquímico para determinação de fisetin em água alcançou-se um limite de detecção de 1,67 x10-8 mol L-1. / Self-assembled monolayers obtained by chemiosorption of alkanethiols on gold present well-defined, organized and reproducible structures. The unique properties exhibited by such surfaces, allied to the great facility to obtain, have attracted the interest of the scientific community and caused a significant advance in the nanotechnology research, especially in the surface engineering one. In this work, the modification of gold substrate with a mixture of 3-mercaptopropionic acid and 11-mercaptoundecanoic acid was evaluated. The aim was control the surface of the SAMs in order to obtain an island arrangement of 3mpa isolated by 11mua, similar to an array of ultramicroelectrodes. The mixed-SAM was built by the incubation of Au substrate in an ethanolic solution containing both thiols. The redox couple [Fe(CN)6]4-/[Fe(CN)6]3- study demonstrated that pure 3mpa monolayers behaves like the bare gold surface, presenting barely the same parameters of peak current (Ip), peak potential (Ep) and charge transfer resistance (Rct), since it allows the electronic transference to occurs by quantum tunneling effect through the monolayer. On the other hand the 11mua monolayers showed an insulating behavior and a Rct value nearly 250 times greater and, consequently, Ip values significantly less than that for 3mpa. The reductive desorption charges and mass changes obtained with an EQCM for the mixed-SAM indicated the high coverage of gold surface and a adsorption mechanism that depends on the incubation time of the substrate in the thiols solution. The surface configuration with 3mpa domains is reached after 20 hours of immersion. The cyclic voltammetric profile, together with the data from electrochemical impedance spectroscopy , proved the existence of phases segregation in the mixed-SAM with surface regions covered solely by 3mpa (with mean radius of 4.3 &micro;m) surrounded by 11mua with a mean separation of 75.42 &micro;m. The electrochemical behavior of the flavonoid fisetin confirms the rising of differentiated properties with the mixed-SAM, where only one electrochemical process was able to be observed, without irreversible adsorption of the analyte. For 3mpa and bare gold surfaces, two (or more) electrochemical processes were observed together with the blocking of the electrode surface by irreversible adsorption of the reaction product. The utilization of the mixed-SAM with electrochemical sensor for fisetin determination in pure water yielded a detection limit of 1.67 x10-8 mol L-1.

Este trabalho descreve a preparação, a caracterização e o uso de um biossensor bienzimático confeccionado com as enzimas glicose oxidase e frutose dehidrogenase imobilizadas em camadas auto-organizadas ou self-assembled monolayers (SAMs) de cistamina para a quantificação de açúcares em alimentos. Após o preparo do eletrodo de ouro com a SAM de cistamina, biossensores foram construídos e para obtenção de melhores respostas, condições foram otimizadas, tais como: concentração do mediador tetratiafulvaleno (TTF), porcentagem de glutaraldeído, temperatura e tempo de vida do biossensor. Com as condições estabelecidas, fez-se então, a determinação analítica da D-glicose e da D-frutose em eletrólito puro pelo método da adição de padrão e os resultados foram obtidos por voltametria cíclica e cronoamperometria. A corrente de pico de oxidação do mediador de elétrons (TTF) aumentou proporcionalmente com o aumento da concentração e não ocorreram deslocamentos nos potenciais de pico. Os limites de detecção (LD) foram encontrados por meio do desvio padrão da média aritmética de dez amperogramas do branco no potencial equivalente aos dos picos de oxidação do mediador de elétrons TTF, juntamente com o valor do coeficiente angular da curva analítica. Após a obtenção da curva analítica o biossensor foi aplicado diretamente em amostras de refrigerante dietético e não dietético, bem como em amostras de adoçantes comerciais, onde foram realizados testes comparativos da resposta dos biossensores. Para o eletrólito puro e amostras de refrigerante dietético e de adoçantes, ou seja, onde não há presença de D-glicose e D-frutose, notou-se a ausência de corrente de pico, enquanto que para as amostras de refrigerante não dietético, houve um valor significativo de resposta de corrente, indicando a presença dos açúcares em estudo. Com o propósito de verificar a influência de interferentes e o efeito de matriz, foi construída uma curva analítica para a D-glicose e para a D-frutose, em amostras de refrigerante dietético e adoçantes, onde foram obtidas as menores quantidade destes açúcares. Para o refrigerante não dietético, foi determinado o valor inicial dos açúcares presentes nas amostras. Pode-se afirmar que a utilização dos biossensores baseados nas enzimas GOx e FDH mostraram-se eficientes para a determinação dos açúcares D-glicose e D-frutose nas amostras analisadas (com diferença significativa nos valores de corrente), apresentando uma resposta rápida, além da eliminação do efeito da matriz. A utilização do mediador de elétrons (TTF) possibilitou a reação em potencial próximo de zero, diminuindo o efeito de interferentes e evitando a desnaturação das enzimas. / This work describes the preparation, characterization and application of a bienzymatic biosensor based in the glucose oxidaze and fructose dehydrogenase immobilized on self-assembled monolayer of cystamine for sugar quantification in foodstuff. After the modification of the gold electrode with cystamine, the biosensors were developed and optimized for best responses. Optimization parameters were: mediator tetrathiafulvalene (TTF) concentration, glutaraldehyde percentage, temperature and life time of the biosensor. With the best conditions established, the analytical determinations of d-glucose and d-fructose in pure phosphate buffer were conducted by the standard additions method and the results obtained by cyclic voltammetry and chronoamperometry. The oxidation peak current related to the TTF voltammetric behavior raised proportionally to the increasing concentration of d-glucose or d-fructose, in a given and constant peak potential. The methodology detection limits were found using the standard deviation of ten chronoamperograms of the blank solution, in the potential value corresponding to that of TTF oxidation, and the slope of the analytical curve. After the analytical curve acquirement the biosensor was directly applied in samples of diet or non-diet softdrinks, as well as in commercial sweeteners samples, with comparative tests of the biosensor responses. For pure electrolyte and for diet foodstuff samples, i.e., were there is not expectation for d-glucose or d-fructose existence, it was detected the lack of the voltammetric peak associated with the mediator oxidation. In non-diet samples, a pronounced voltammetric peak was obtained, testifying the presence of sugar in the electrolytes under study. The matrix effect was verified by means of an analytical curve obtained for both analytes (d-glucose and d-fructose), in diet and sweeteners samples, properly spiked with known amounts of each analyte. It can be concluded that the utilization of the biosensor based in GOx and FDH showed to be efficient for d-glucose and d-fructose determinations in the analysed samples, with a fast response time and elimination of the matrix effect. The mediator promoted the electrochemical reaction to occur in potentials very close to zero, minimizing the interferences or the enzyme denaturation.