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This thesis describes work carried out on the research, development and implementation of new processes for the fabrication of surface plasmon waveguide biosensors. Fabrication of surface plasmon resonance (SPR) based waveguides embedded in a thick CYTOP cladding with the incorporation of fluidic channels was achieved with improved quality and operability compared to previous attempts. The fabrication flow was modified in key areas including lithography for feature definition, gold evaporation and the upper cladding deposition procedure. The combined result yielded devices with sharper resolution of waveguides, gold surfaces with minimal aberrations, reduced surface roughness and minimization of waveguide deformation due to reduction of solvent diffusion into the lower cladding. The fabricated waveguides consisted of a thin, 35 nm, patterned gold film, embedded in a thick, 18 µm, CYTOP fluoroploymer cladding. The gold devices were exposed by O2 plasma etching through the upper cladding to form fluidic channels for the facilitation of flow of an index matched sensing medium. Optical and physical characterization of devices revealed structures of significantly improved quality over previous attempts, rendering the platform competitive for biosensing applications.

Biomedical sciences are in need of more versatile and more sensitive approaches for research and also for diagnostic purposes. In particular, intracellular detection and imaging of disease relevant proteins is a challenge. Although the state of the art method of intracellular imaging is fluorescence, it suffers from several drawbacks. Raman is an alternative imaging modality and this work investigates the use of different Raman techniques for detection and imaging of cellular constituents. In one aspect of the work, surface-enhanced Raman spectroscopy using gold nanoshells excitable at a wavelength of 780 nm was investigated. Initially the investigation of the uptake of the 150 nm diameter nanoparticles showed that NS are taken up voluntarily by a non-standard en- docytosis mechanism into mammalian fibroblast cells. Furthermore it was shown that internalized particles have no detrimental in uence on cell growth or cell viability. That these nanoparticles are non toxic was further confirmed by testing for markers of apoptosis and necrosis. Preliminary surface-enhanced Raman spectroscopy (SERS) studies produced spectra from intracellular compartments with an enhancement factor of 1010. To yield high specificity of the intracellular Raman protein sensor, two different approaches were studied. The first is based on the application of DNA aptamers which form a stacked G-quadruplex on target protein binding. A SERS sensor based on the well characterized Thrombin binding aptamer (TBA) yielded high reproducibility, high target specificity, and a limit of detection down to 0.1 fM. Further studies on a similar stacked G-quadruplex forming aptamer confirmed that observed detection signal is produced by the aptamer assuming its secondary structure but also showed that the stabilization and formation of the G-quadruplex secondary structure is strongly buffer dependent. A second sensing approach was based on a peptide (a3(IV)NC1) influential in Goodpasture's syndrome, an autoimmune disease. With the help of this peptide we found that an intracellular redoxpotential of -200 mV is necessary to make it accessible for the protease Cathepsin D. We found that SERS sensing has the ability to study the binding of Cathepsin D, its activity and with the help of a synthesized amino-acid SERS library the direct detection of the remaining peptide products. Finally this work concludes with imaging the changes of lipid droplet structure and distribution in fibroblast cells during the infection process of the murine cytomegalovirus (MCMV) in fixed and in living cells by coherent anti-Stokes Raman based on a Synchro-lock phase coupled setup. This showed that CARS imaging is able to non-invasively investigate the changes of lipid structures during different stages of the infection process and therefore promises to be a valuable tool in biological research.

There are significant worries about the impact of heavy metal pollutants in soils during urbanisation and industrialisation in the developing world. Routine chemical analysis of soils is used to characterise the concentration of metals translocated by point source and diffuse actions. This fails to put in context the bioavailability or potency of these analytes. Biosensors offer a novel and direct method for hazard assessment of soils impacted with anthropogenic contamination. However, few significant examples of thorough applications of the biosensor in environment have been confirmed. This thesis makes use of comprehensively characterised biosensors for the applications to different environmental matrices. The objective was to apply and evaluate the performance of biosensors in determining the bioavailable fraction of heavy metals in the environment in conjunction with chemical analysis data. Both constitutively marked and metal induced biosensors were optimised and applied, but only the constitutively marked biosensor was responsive to a range of soils and elements of concern. As the matrix that the biosensors were exposed to became increasingly complex, the biosensors' performance became more difficult to interpret. For the metal induced biosensor, the response to standard aqueous samples was predictable as the relationship between chemical analysis data and biosensor predicted data was strong. By contrast, the performance of the metal induced biosensor was poor in soil samples which were historically contaminated (Chapter 9). This could become impacted by the soil characterisation, such as the presence of eo-contaminants, soil associated carbon or variations in soil pH. The constitutively-marked biosensors offered greater predictability of response and this is the reason why these are widely adopted in soil applications. Bacterial biosensors provide a useful tool for assessing the bioavailable fraction of analytes in soils and for complementing chemical analysis. If matrix matched control samples can be collected, then this technology can be applied to a wide range of contrasting soils with a suite of contaminants to aid in the development of generic soil hazard evaluation.

Orientador: Maria Del Pilar Taboada Sotomayor / Banca: Olga Maria Mascarenhas de Faria Oliveira / Banca: Antonio Aparecido Pupim Ferreira / Banca: Cesar Ricardo Teixeira Tarley / Banca: Rosa Amalia Fireman Dutra / Resumo: O presente trabalho apresenta o desenvolvimento de biossensor baseado em extrato de açaí e sensor biomimético baseado em polímeros de impressão molecular, para a determinação eletroquímica da hexazinona-HXZ (3-ciclohexila-6-(dimetilamino)-1-metil-1,3,5-triazina-2,4(1H,3H)-diona) empregando eletrodos de pasta de carbono e de ouro. O sensor biomimético à base de pasta de carbono foi construído usando polímeros biomiméticos, MIPs (Moleculary Imprinted Polymer). As sínteses dos MIPs e dos respectivos polímeros de controle NIPs, foram realizadas pelos métodos de bulk e de precipitação. O comportamento eletroquímico do sensor biomimético foi estudado por voltametria cíclica e a quantificação da hexazinona foi realizada utilizando voltametria adsortiva de pulso diferencial com redissolução catódica (DPAdCSV). Sob estas condições, obteve-se uma faixa de resposta para HXZ, entre 2,0 x 10-11 e 1,1 x 10-10 mol L-1, e um limite de detecção de 5,8 x 10-12 mol L-1, o sensor biomimetico foi aplicado para a determinação de hexazinona em agua utilizada em uma plantação de cana de açucar, obtendo-se uma recuperação de 98%. No biossensor, a base do extrato de açaí usou-se um eletrodo de ouro cuja superfície foi modificada com monocamadas auto-arranjadas (SAM) de cistamina sobre a qual foi imobilizada a enzima peroxidase extraída da polpa de açaí (Euterpe oleracea). A voltametria de onda quadrada foi a técnica utilizada na otimização e quantificação do herbicida, obtendo-se uma resposta para HXZ de 2,0 x 10-5 até 1,1, x 10-4 mol L-1 e limite de detecção de 6,6 x 10-6 mol L-1. Os resultados obtidos mostram que os sensores propostos são promissores para a quantificação da hexazinona. / Abstract: The present work describe the development of electrochemical biosensors and biomimetec sensor for the determination of hexazinone-HXZ (3-ciclohexyl-6- (dimethylamino)-1-methyl-1,3,5-triazine-2,4(1H,3H)-dione) employing carbon paste and gold electrodes. The biomimetic sensor based on carbon paste was prepared using molecularly imprinted polymers (MIPs). The syntheses of MIPs and the respective control polymers, NIPs, were performed by bulk and precipitation methods. The electrochemical behavior of biomimetic sensor was studied by cyclic voltammetry and the quantification of hexazinone was performed using differential pulse adsorptive cathodic stripping voltammetry (DPAdCSV). Under these conditions response range was obtained for HXZ between 2.0 x 10-11 e 1.1 x 10-10 mol L-1 and detection limit of 5.8 x 10-12 mol L-1. the biomimetic sensor was applied to real samples of a sugar plantation, the recovery was 98% The In the second sensor gold electrode was used whose surface was modified with self-assembled monolayers (SAM) of cystamine on which was immobilized peroxidase enzyme extracted from the pulp of Açai (Euterpe oleracea). The square wave voltammetry technique was used in the optimization and quantification of the herbicide having response for HXZ 2.0 x 10-5 up to 1.1 x 10-4 mol L-1 and detection limit of 6.6 μmol L-1. The results obtained show that the proposed sensors are promising for the quantification of this pesticide. / Doutor

"Electrochemistry, based on the study of an electrochemical reaction at the interface between an electrode and an electrolyte, is having a profound effect on the development of different fields of science and engineering including battery, fuel cell, electrochemical sensor, electrochromic display, electrodeposition, electroplating, electrophoresis, corrosion, and so on. The performance of the electrochemical reaction depends strongly on the nature of the employed electrode such as structure, chemical composition, and surface morphology. Nanomaterials, notable for their extremely small feature size (normally in the range of 1-100 nm), exhibit new properties which are different from those of bulk materials due to their small size effect. In past decade, nanomaterials have been widely used to develop new strategies for designing electrode and its surface morphology for electrocatalysis and electrochemical sensing applications. My work is aimed at exploring the application of low dimensional nanomaterials (nanotubes and nanoparticles) in electrocatalysis and electrochemical biosensors. Electrocatalysis plays an important role in energy and industrial applications. As one of the most attractive support materials for electrocatalyst, carbon nanotubes have been extensively reported to enhance the performance of various electrochemical catalytic reactions. In recent years, carbon nanotubes with a bamboo-like structure due to nitrogen doping have become a hot topic of increased interest in the field of electrocatalysis because of the unique bamboo shaped structure associated properties. In this work, bamboo shaped carbon nanotubes, synthesized by chemical vapor deposition method, were investigated for ethanol/methanol electro-oxidation, respectively. Small sized platinum nanoparticles (Pt NPs) were dispersed onto BCNT surface through an impregnation method. The role of nitrogen doping in the formation of bamboo shaped structure and its effect in the electrochemical performance of CNTs were discussed. The electrochemical studies showed that the as-prepared Pt/BCNTs electrocatalysts indeed exhibited a remarkable enhancement in catalytic activity for methanol/ethanol oxidation compared to that of the Pt/commercial CNT electrocatalysts. In order to further investigate the potential of using BCNTs as bioelectrocatalyst support materials, a hybrid organic-inorganic nanocomposite film of BCNTs/ploymer was constructed to immobilize an enzyme horseradish peroxidase (HRP) to examine the direct electrochemical behavior of the enzyme towards electrocatalysis process of H2O2. The results indicated that the immobilized HRP onto the film retains its good bioelectrocatalytic activity to H2O2. The defective sites on the BCNTs surface induced by nitrogen doping could help to promote the direct electron transfer between enzyme and the electrode. The BCNT/polymer film structure provides a vast array of new opportunities to use BCNTs as building units for bioelectrochemical and biomedical applications. Compared to carbon nanotubes, TiO2 nanotubes have much better biocompatibility and show greater potential as implant materials. The advantages of TiO2 nanotube array include high biocompatibility, good corrosion resistance in biological environments and highly ordered one dimensional nanotubular geometry. Herein, a well performing non-enzymatic electrochemical glucose biosensor by using CuO nanoparticle decorated TiO2 nanotube array electrode was developed. Well-aligned TiO2 nanotube arrays were successfully synthesized by electrochemical anodization. Highly uniform CuO nanoparticles were electrodeposited onto TiO2 nanotube arrays through a two-step method and used to electrocatalyze the glucose oxidation. The proposed electrode produced a high sensitivity of 239.9 ìA mM-1 cm-2 and a low detection limit of 0.78 ìM with good stability, reproducibility, selectivity and fast response time, suggesting its potential to be developed as a low-cost nano-biosensor for glucose measurements in human fluids. The final work of this thesis presents a simple sandwich-type electrochemical impedance immunosensor with antitoxin heavy-chain-only VH (VHH) antibodies labeled gold nanoparticles as the amplifying probe for detecting Clostridium difficile toxins. Gold nanoparticles (Au NPs) with diameter of ~13-15 nm were synthesized and characterized by transmission electron microscopy and UV-vis spectra. The electron transfer resistance of the working electrode surface was used as parameter in the measurement of the biosensor. With the increase of the concentration of toxins from 1pg/mL to 100 pg/mL, a linear relationship was observed between the relative electron transfer resistance and toxin concentration. In addition, the detection signal was enhanced due to the amplification effect. This proposed method achieved a limit of detection for TcdA and TcdB as 0.61 pg/mL and 0.60 pg/mL, respectively. The pilot study with spiked clinical stool samples showed promising results, indicating the designed biosensor has a great potential in clinical applications."

Orientador: Valber de Albuquerque Pedrosa / Resumo: Doenças tropicais são aquelas causadas por agentes infecciosos ou parasitas e consideradas endêmicas em populações na sua maioria de baixa renda, residente em países tropicais e subtropicais. Dentre essas doenças temos a dengue, que acomete milhares de pessoas levando a mortes todos os anos. A dengue é transmitida através da picada do mosquito contaminado e a movimentação populacional a torna de fácil disseminação em todo o território nacional. Atualmente, os exames para diagnóstico desta doença são realizados em laboratórios, principalmente testes sorológicos. Porém, esta metodologia apresenta como limitação o custo elevado além dos resultados levarem dias para que fiquem prontos. Por isso, se faz necessário o desenvolvimento de uma nova metodologia confiável e de baixo custo para um rápido diagnóstico. Atualmente, nanobiossensores vem se destacando como uma ferramenta analítica que pode identificar a presença de patógenos utilizando como base nanoestruturas. Logo, o objetivo do presente projeto foi desenvolver e padronizar uma nova metodologia para o diagnóstico rápido e eficaz do vírus da dengue, utilizando diferentes nanoestruturas modificadas com a deposição de anticorpos específicos e aptâmeros. As metodologias foram comparadas mediante as técnicas de ressonância plasmônica de superfície localizada, ressonância plasmônica de superfície, microscopia eletrônica de transmissão, espectroscopia de impedância eletroquímica, espalhamento dinâmico de luz e análise do software I... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tropical diseases are those caused by infectious agents or parasites and are considered endemic in mostly low-income populations residing in tropical and subtropical countries. Among these diseases we have the dengue that causes thousands of deaths each year. Dengue is transmitted through the bite of the contaminated mosquito and the population movement makes it easily spread throughout the national territory. Currently, the diagnostic test of this disease is performed in laboratories, being serological tests. However this methodology presents as a limitation the higher cost and the delay for the result come out. Therefore, it is necessary to develop a new, reliable and low-cost methodology for rapid diagnosis. Nowadays, nanobiosensors have been highlighted with analytical tool that can identify the presence of pathogens using as base nanostructures. The objective of the present project was to develop and standardize a new methodology for a fast and effective diagnosis of dengue virus using using different nanostructures modified with the deposition of specific antibodies and aptamers. The methodology was compared by localized surface plasmon resonance, surface plasmon resonance, transmission electron microscopy, electrochemical impedance spectroscopy, dynamic light scattering techniques and ImageJ software analysis. The results obtained with the techniques presented some advantages over the traditional methods of detection, such as fast analysis and low cost, showing to be h... (Complete abstract click electronic access below) / Doutor