In our modern society, we are surrounded by numerous sensors, constantly feeding us information about our physical environment. From small, wearable sensors that monitor our physiological status to large satellites orbiting around the earth, detecting global changes. Although, the performance of these sensors have been significantly improved during the last decades there is still a demand for faster and more reliable sensing systems with improved sensitivity and selectivity. The rapid progress in nanofabrication techniques has made a profound impact for the development of small, novel sensors that enables miniaturization and integration. A specific area where nanostructures are especially attractive is biochemical sensing, where the exceptional properties of nanomaterials can be utilized in order to detect and analyze biomolecular interactions.  The focus of this thesis is to investigate plasmonic nanoparticles composed of gold or silver and optimize their performance as signal transducers in optical biosensors. Metal nanoparticles exhibit unique optical properties due to excitation of localized surface plasmons, which makes them highly sensitive probes for detecting small, local changes in their surrounding environment, for instance the binding of a biomolecule to the nanoparticle surface. This is the basic principle behind nanoplasmonic sensing based on refractometric detection, a sensing scheme that offers real-time and label-free detection of molecular interactions.  This thesis shows that the sensitivity for detecting local refractive index changes is highly dependent on the geometry of the metal nanoparticles, their interaction with neighboring particles and their chemical composition and functionalization. An increased knowledge about how these parameters affects the sensitivity is essential when developing nanoplasmonic sensing devices with high performance based on metal nanoparticles.

O presente trabalho buscou investigar a formação da placa de aterosclerose através de caracterização da autofluorescência do tecido e do plasma na presença de marcadores fluorescentes. Para realizar o estudo, coelhos foram divididos em dois grupos: um grupo controle onde os animais foram submetidos a uma dieta normal e um grupo experimental onde os animais foram submetidos a uma dieta hipercolesterolêmica. Foram realizadas duas experimentações animais: na primeira os animais foram sendo eutanasiados ao longo do experimento e suas artérias foram coletadas. Na segunda os animais foram acompanhados por no máximo 80 dias. Durante o experimento apenas o sangue foi coletado e os animais foram eutanasiados no final do experimento. Dois marcadores fluorescentes foram utilizados no trabalho: o complexo európio-clorotetraciclina (EuCTc) e o corante tioflavina T (ThT). Analisouse inicialmente a fluorescência dos marcadores na presença do plasma dos coelhos tanto para o grupo controle, quanto para o grupo experimental em função dos tempos de dieta. Para o complexo EuCTc observou-se duas bandas de emissão, com excitação em 400 nm, uma característica da clorotetraciclina, em 515 nm e uma em 617 nm característica do íon európio. A análise da banda do íon európio indicou um incremento da banda de emissão do complexo na presença do plasma do grupo experimental em relação ao grupo controle. Para o corante ThT também foi observado um aumento na banda de emissão em 480 nm, com excitação em 413 nm, para o grupo experimental em comparação com o grupo controle. A potencialidade de utilização do complexo EuCTc e EuCTcMg (EuCTc na presença do íon magnésio) para marcação da placa de aterosclerose nas artérias, foi estudada através da análise de microscopia de fluorescência. Observou-se que a emissão do complexo melhora muito a visualização da placa quando comparada com a autofluorescência. Observou-se, através de microscopia de tempo de vida de fluorescência, que há uma transferência de energia entre os fluoróforos presentes na placa e os complexos EuCTc e EuCTcMg. Essa transferência de energia ocasionou em uma diminuição drástica no tempo de vida de fluorescência dos fluoróforos nessa região. Por fim, estudou-se a geração de segundo harmônico do colágeno na placa de aterosclerose, sendo obtidas diferenças na quantidade e organização do colágeno para os diferentes grupos experimentais. / This study aimed to investigate the formation of atherosclerotic plaque by the characterization of the autofluorescence of the tissue and plasma in the presence of fluorescent markers. For this study, rabbits were divided into two groups: a control group and an experimental group submitted to a hypercholesterolemic diet. The animal experimentation was performed twice, the first animals were being euthanized during the experiment and their arteries were collected. In the second experiment, the animals were followed for a maximum of 80 days and only during the experiment the blood was collected. The animals were euthanized at the end of experimentation. Two fluorescent markers were used in this study: europiumchlortetracycline complex (EuCTc) and the dye Thioflavin T (ThT). Firstly, it was analyzed the markers fluorescence in the presence of rabbits plasma for both, the control and the experimental groups with different diet times. For EuCTc complex, it was observed two bands of emission with excitation at 400 nm, first, a characteristic of chlortetracycline at 515 nm and at 617 nm characteristic of the europium ion. Analyzing only the band of europium ion, it was observed a greater increase of the complex in the presence of plasma in the experimental group. For ThT dye the emission band at 480 nm with excitation at 413 nm for the experimental group, in comparison with the control group. I was analysed the possibility to use EuCTc and EuCTcMg complex (EuCTc the Mg ion present) for marking the atherosclerotic plaque in arteries by fluorescence microscopy analysis. The results showed that the emission of the complex increase dramatically compared to the autofluorescence. Also, there was evidence of energy transfer between the fluorophores present of the plaque and EuCTc and EuCTcMg complex by fluorescence lifetime microscopy. This energy transfer generated a drastic decrease in the fluorescence lifetime of fluorophores. Finally, the generation of second harmonic of collagen in the atherosclerotic plaque was studied and it was obtained differences in the amount of collagen and organization in the different experimental groups.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-02T20:43:30Z No. of bitstreams: 1 LaianeSouto.pdf: 1543926 bytes, checksum: b4812ad4c2c00b039863648640d563e8 (MD5) / Made available in DSpace on 2017-06-02T20:43:30Z (GMT). No. of bitstreams: 1 LaianeSouto.pdf: 1543926 bytes, checksum: b4812ad4c2c00b039863648640d563e8 (MD5) Previous issue date: 2016-12-13 / Microcystin (MC-LR) are a class hepatotoxins produced by cyanobacteria in surface water. scientific records show that the MC-LR, inhibits the action of intracellular proteins, alkaline phosphatases, but has also been proven to increase the enzyme acetylcholinesterase activity (AChE) by the MC-LR action. Therefore, this study aimed to develop biosensors amperometric based AChE enzyme for indirect detection of MC-LR. For construction of the working electrode, a powder graphite paste containing hidroxicetilcelulose (HEC), bovine serum albumin (BSA) and glutaraldehyde (Glu) was prepared. The slurry was incorporated into the AChE enzyme extracted bovine erythrocyte (EB) and electric eel (EE) as well as enzymes derived from Drosophila melanogaster was tested genetically modificadas.Também enzyme butyrylcholinesterase (BChE) obtained from human serum. A portion of the sensitive paste was deposited in the screen-printed sensor working electrode and performed characterization tests involving differential pulse voltammetry and cyclic voltammetry. cronoamperométricas readings were performed and the percentage built on activation curves (% RA) as a function of the concentration of MC-LR. Some operating conditions, such as working potential, electrochemical mediator, medium pH and substrate concentration, were optimized. enzyme activation assays showed that the EE-AChE enzyme showed best results percent relative activation (% RA) (> 10%), these values being directly proportional to the concentration of MC-LR. The biosensor developed proved accurate (CV ~ 8.32%), sensitive (Ld 0.27 μg .L-1 and LQ 0.91 μg .L-1) and accurate (recovery rates varying from 73 to 105%). The initial study proved to be the right biosensor to verify the presence of MC-LR in aquatic environments, this being then used in monitoring pollutant seven points of Bacanga River, an important aquatic ecosystem of San Luis, MA. The results indicated that there was no significant contamination. / Microcistinas (MC-LR) são uma classe de hepatotoxinas produzidas por cianobactérias em águas de superfície. Registros científicos comprovam que a MC-LR inibe a ação de proteínas intracelulares, as fosfatases alcalinas, mas também foi comprovado que sua ação aumenta a atividade da enzima acetilcolinesterase (AChE). Portanto, esse trabalho objetivou desenvolver biossensores amperométricos à base da enzima AChE para detecção indireta da MC-LR. Para construção do eletrodo de trabalho, foi preparada uma pasta de grafite em pó contendo hidroxicetilcelulose (HEC), soroalbumina bovina (BSA) e glutaraldeído (Glu). A pasta foi incorporada às enzimas AChE extraídas do eritrócito bovino (EB) e da enguia elétrica (EE), bem como enzimas geneticamente modificadas extraídas da Drosophila melanogaster. Também foi testada a enzima butirilcolinesterase (BChE) obtida de soro humano. Uma porção da pasta sensivel foi depositada no eletrodo de trabalho de sensores serigrafados, e realizados testes de caracterização envolvendo voltametria de pulso diferencial e voltametria cíclica. Leituras cronoamperométricas foram realizadas, em seguida, construídas curvas da ativação relativa percentual (AR%) em função da concentração da MC-LR. As seguintes condições operacionais: potencial de trabalho, mediador eletroquímico, pH do meio e concentração do substrato, foram otimizadas. Os ensaios de ativação enzimática revelaram que a enzima AChE (EE) apresentou melhores resultados de ativação relativa percentual (AR %) (>10%), sendo esses valores diretamente proporcionais à concentração da MC-LR. O biossensor desenvolvido mostrou-se preciso Coeficiente de Variação (CV ~ 8,32%), sensível Limite de detecção e Quantificação (LD 0,27 μgL-1 e LQ 0,91 μgL-1) e exato (índices de recuperação variando de 73 a 105%). O estudo revelou ser o biossensor adequado à verificação da presença de MC-LR em ambientes aquáticos, tendo sido este então utilizado no monitoramento do poluente em sete pontos do Rio Bacanga, um importante ecossistema aquático de São Luis, MA. Os resultados indicaram não haver uma importante contaminação desse poluente nas áreas investigadas.

Neste trabalho são elaborados biossensores para a detecção amperométrica de glicose. Para isso, imobilizou-se a enzima glicose oxidase em matrizes de polímeros condutores. Foram construídos sensores utilizando-se poli(pirrol) e poli(N-metilpirrol). Com o objetivo de substituir o oxigênio molecular na etapa de transdução do sinal, o ferroceno foi incorporado dentro do polímero condutor. Para isso, os polímeros foram elaborados utilizando misturas água-etanol como meio de polimerização. A inclusão do ferroceno no sensor resulta em maior sensibilidad à glicose (4,33 µA Mm-1 cm-2 para o biossensor preparado a partir da mistura água-etanol contendo o ferroceno e de 0,23 µA mM-1 cm-2 para o sensor sem ferroceno ). Por outro lado, permite o funcionamento do sensor a potenciais menores que no caso do sensor sem ferroceno (0,4 V para o sensor com ferroceno vs. 0,65 V para o caso sem ferroceno). O deslocamento do potencial de detecção para valores menos positivos não foi suficiente para evitar as interferências causadas pelos íons ascorbato e ureato. Para isto, mostrou-se 100% efetivo o recobrimento dos sensores com uma película de Nafion®. A sobreoxidação do poli(pirrol) também mostrou potencialidade para a eliminação de interferentes, embora o processo resulte na perda de sensibilidade, provavelmente causada pela desnaturação da enzima. Quando usado o poli(N-metilpirrol) como suporte para a enzima, obtiveram se melhores respostas, causadas pela possibilidade de se preparar filmes mais espessos, consequentemente de imobilizar maior quantidade de enzima, sem observar perda de resposta causada por problemas difusionais. / The present work describes the elaboration of a biosensor for glucose detection. The enzyme, glucose oxidase, was immobilized in different conducting polymers. Two different polymers were used: polypyrrole and poly(N-methilpyrrole ). With the aim of replacing the molecular oxygen in the transduction step, ferrocene has been immobilized within the conducting polymer. Once the ferrocenium was insoluble in water, in order to develop a different route, the electropolymerization was carried out in a mixture of water and ethanol (1:1). This procedure leads to a polymer with a poor electroactivity, detected by Raman experiments. The ferrocene addition in the sensor increases the sensitivity to the glucose determination (4,33 µA mM-1 cm-2 for the biosensor with ferroecene and 0,23 µA mM-1 cm-2 for the sensor without ferrocene). Alternatively, the sensor containing ferrocene allows to operate at less positive potentials than that one without ferrocene (+ 0,40 V and + 0,65 V, respectively). This potential shift was not enough to inhibit the interference caused by ascorbate and ureate ions. One method to avoid the interference problem was to recover the sensor with a very thin layer of Nafion. Also poly(pyrrole) overoxidation is a very efficient method to eliminate this interference, but this process leads to a sensitivity decrease dueto enzyme denaturation. A better response was observed for sensor assembled using the poly(Nmethyl-pyrrole) as the support for enzyme immobilization. This behavior was provoked by the thicker of polymer film formed leading to higher amount of immobilized enzyme. Even though, no diminution in the response was caused by diffusion problems.

Ho, Yee Man Martina = 硒化鎘三維納米結構之製作及其感應脫氧核糖核酸之應用潛能 / 何綺雯. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references. / Abstract also in Chinese. / Ho, Yee Man Martina = Xi hua ge san wei na mi jie gou zhi zhi zuo ji qi gan ying tuo yang he tang he suan zhi ying yong qian neng / He Qiwen. / Chapter Chapter 1 --- Introduction / Chapter 1 --- Photovoltaic properties of CdSe --- p.1 / Chapter 1.1 --- Quantum size effect --- p.1 / Chapter 1.2 --- Synthesis of CdSe nanostructures --- p.3 / Chapter 1.3 --- Electrochemical sensing of CdSe nanostructures --- p.4 / Chapter 1.3.1 --- Surface passivation and functionalization of CdSe nanostructures --- p.5 / Chapter 1.4 --- Electronic properties of nanocrystalline semiconductor electrode --- p.6 / Chapter 1.4.1 --- Band alignment --- p.6 / Chapter 1.4.2 --- Interfacial charge transfer process --- p.9 / Chapter 1.4.3 --- Surface traps and adsorbed molecules --- p.10 / Chapter 1.4.4 --- DNA molecules as a capping group --- p.11 / Chapter 1.5 --- Literatures review in DNA sensing --- p.12 / Chapter 1.6 --- Present study --- p.14 / Chapter 1.6.1 --- Objective --- p.14 / Chapter 1.6.2 --- General methodology --- p.15 / Chapter Chapter 2 --- Experimental / Chapter 2.1 --- Introduction into the instrumentation of this project --- p.21 / Chapter 2.2 --- CHI Electrochemical workstation --- p.22 / Chapter 2.2.1 --- Linear sweep voltammetry --- p.24 / Chapter 2.2.2 --- Cyclic voltammetry --- p.24 / Chapter 2.2.3 --- Multiple potential step --- p.25 / Chapter 2.3 --- CEM Microwave-assisted chemical synthesizer --- p.27 / Chapter 3.1 --- Morphological examination by scanning electron microscopy --- p.28 / Chapter 3.2 --- Elemental analysis by energy dispersive x-ray spectroscopy --- p.30 / Chapter 3.3 --- Crystal structure analysis by x-ray diffraction --- p.31 / Chapter 3.4 --- Surface compositional analysis by x-ray photoelectron spectroscopy --- p.32 / Chapter 3.5 --- Transmission electron microscopy --- p.34 / Chapter Chapter 3 --- Synthesis of 3D nanostructured CdSe multipod electrodes / Chapter 3.1 --- Introduction into the synthesis of CdSe MP electrode --- p.35 / Chapter 3.2 --- Recipe for the synthesis of CdSe NPs --- p.36 / Chapter 3.3 --- The synthesis of CdSe MPs --- p.37 / Chapter 3.3.1 --- Tuning the experimental parameters: Reaction temperature --- p.37 / Chapter 3.3.2 --- Tuning the experimental parameters: Reaction hold time --- p.46 / Chapter 3.3.3 --- Tuning in experimental parameters: Precursor molar ratio --- p.50 / Chapter 3.4 --- The fabrication of MP CdSe on a conductive substrate --- p.54 / Chapter 3.4.1 --- The electrodeposition of CdSe thin films on ITO/glass substrates --- p.55 / Chapter 3.4.2 --- The growth of CdSe MPs on CdSe/ ITO/glass --- p.57 / Chapter 3.5 --- The characterization of MP CdSe electrode --- p.57 / Chapter Chapter 4 --- Electrical and opto-electric characteristics of CdSe MP electrodes and their applications as platforms for the DNA recognition / Chapter 4.1 --- Introduction to the property characterization of CdSe MP electrodes --- p.62 / Chapter 4.2 --- DNA surface attachment --- p.64 / Chapter 4.2.1 --- Mechanism of DNA surface anchoring --- p.65 / Chapter 4.3 --- I-V characterization in PBS --- p.69 / Chapter 4.3.1 --- Experimental procedures of the I-V tests in PBS --- p.70 / Chapter 4.3.2 --- Results and discussions of I-V tests in PBS --- p.72 / Chapter 4.3.2.1 --- Exercising as-prepared CdSe MP electrode --- p.74 / Chapter 4.3.2.2 --- I-V characteristics of CdSe MP electrodes before and after ssDNA attachment --- p.75 / Chapter 4.3.2.3 --- I-V characteristics of CdSe MP electrodes before and after the dsDNA attachment --- p.76 / Chapter 4.3.2.4 --- "Photo-response of bare CdSe MP, ssDNA/CdSe MP and dsDNA/CdSe electrodes" --- p.77 / Chapter 4.4 --- "Photovoltaic I-V measurement in I3""/I"" redox electrolyte" --- p.79 / Chapter 4.4.1 --- Experimental procedures --- p.79 / Chapter 4.4.2 --- Results and discussions --- p.80 / Chapter 4.5 --- Possible application implied by the results --- p.88 / Chapter 4.5.1 --- DNA base pair mismatch identification --- p.91 / Chapter 4.5.2 --- Field-assisted DNA hybridization acceleration process --- p.92 / Chapter Chapter 5 --- Conclusions / Chapter 5.1 --- Conclusions --- p.95

A caracterização das propriedades ópticas de moléculas de DNA, tais como a absorção e a fluorescência por excitação, são importantes para a determinação de parâmetros usados no desenvolvimento de biossensores fotônicos. O estudo da absorção óptica do DNA, obtido por diferentes métodos tem se mostrado muito eficiente na determinação do grau de pureza do material genético obtido por amplificação, ou extração e purificação do DNA total. Por outro lado, a fluorescência por excitação a partir de um marcador cromóforo é uma técnica importante em processos de quantificação de massa, em técnicas tais como a eletroforese em gel de agarose. Devido à baixa fluorescência de moléculas de DNA na região visível do espectro, entre 500-600nm, utiliza-se destes marcadores que se ligam à molécula e que são opticamente ativos nesta região para detecção de sua emissão de fluorescência. Neste trabalho foi realizado um estudo da fluorescência do DNA, obtido a partir de uma amplificação por transcriptase reversa do RNA (RT-PCR), na região de 400nm a 600nm, sem adição de marcador e utilizando excitação por um e dois fótons (405nm e 800nm) através da técnica de microscopia confocal. As amostras contendo solução de dsDNA (237ng/μL) foram depositadas sobre um filme de prata de 200nm de espessura que também é crescido previamente sobre um substrato de vidro. Sobre o filme metálico é fabricado nanoestruturas metálicas por de litografia por feixe de íons com um microscópio de duplo feixe FEI Quanta 3D 200i. As nanoestruturas são formadas por arranjos concêntricos de anéis com diâmetros de até 20 μm, largura 50nm e separados por 400nm. Quando a excitação do material genético ocorre sobre a nanoestrtutura o laser gera na nanoestrutura plasmon-polaritons de superfície (SPP) que interagem com as moléculas de dsDNA na solução. Observa-se que nas regiões onde as nanoestruturas são fabricadas que a intensidade de fluorescência da macromolécula é muito maior do que a obtida fora da estrutura e sobre o filme metálico. Os efeitos da interação entre SPPs e as moléculas aumentam a atividade óptica (taxa de emissão) e podem servir como base para a fabricação de sensores fotônicos ultrasensíveis. Concluindo, os efeitos dos campos plasmônicos sobre o fluoróforo são significativos e foram observados pela diminuição do tempo de vida das moléculas e o aumento da sua fluorescência. / The characterization of the optical properties of DNA molecules, such as absorption and excitation fluorescence, is important to the determination of the parameters used for the preparation of photonic biosensors. The study of optical absorption of DNA, obtained through different methods, currently has a high sensitivity to determine the degree of purity of the genetic material obtained by amplification, extraction and purification of the total DNA. On the other hand, excitation fluorescence using a marker is an important technique in mass quantification processes together with techniques such as agarose gel electrophoresis. Because of low fluorescence of DNA molecules, in the visible region of the spectrum, between 500-600nm, the use of labels that bind to the molecule are critically for the detection of their fluorescence emission. In this work we studied the DNA fluorescence, obtained from a RNA reverse transcriptase (RT-PCR) amplification, in the region from 400nm to 600nm, without the addition of a marker as a fluorophore and using confocal microscopy with one and two photons (405nm and 800nm). The solutions of dsDNA (237ng/μL) were dropped on a silver film with 200nm tackiness deposited on a glass substrate. In the silver film nanostructures were fabricated ion beam lithography with FEI Quanta 3D 200i dual beam microscope. The nanostructures are formed by concentric arrangements of rings with diameters up to 20 μm, width 50nm and separated by 400nm. When the excitation of the genetic material occurs on a nanostructure an excited surface plasmon-polaritons (SPP) is responsible for the DNA excitation. It is observed in these regions an increase of the fluorescence intensity many times higher than one obtained out of the nanostructure on the silver film. The effects of the interaction between SPPs and molecules increase the optical activity of the molecule (emission rate) and can serve as the basis of photonic sensors. Concluding, the effects of the plasmon fields on the fluorophore are significant and were observed by decreasing the life time of the molecules and the increasing of their fluorescence.

Polychlorinated biphenyls (PCBs) are persistent environmental chemicals. Mono-hydroxylated polychlorinated biphenyls (OH-PCBs) are PCB metabolites found commonly in human blood, environmental water and sediment samples. Detection of small amounts of PCBs and their OH-PCB metabolites in biological matrices from epidemiological and laboratory studies remains a challenge. The application of aptamers is studied as a means to identify and quantify PCBs and OH-PCBs. Aptamers are single stranded short oligonucleotides that arrange into unique shape of three-dimensional structures when binding to their target. Like antibodies they have high affinity and specificity for their specific target. The hypothesis is that aptamers can identify PCBs and PCB metabolites in environmental and biological samples. To test this hypothesis, three different OH-PCBs, 4’-OH-PCB3, 4-OH-PCB72 and 2-OH-PCB106 along with 4-OH-biphenyl as a control, were covalently attached to beads with carboxylic acid groups on their surface. Several methods were explored to characterize covalent binding of OH-PCBs to the beads: FTIR-spectroscopy, Dynamic Light Scattering (DLS) and Zeta-Potential (ZP) measurements. The beads were then used in in vitro assays to test binding of two different aptamers specific to OH-PCBs. In this study, these aptamers were tested for the ability to distinguish structurally different OH-PCB congeners and other environmental pollutants. In future studies, aptamers can be selected for a PCB metabolite of interest, 4’-OH-PCB3, via a modified form of Systemic Evolution of Ligands by Exponential Enrichment (SELEX). Single stranded DNA (ssDNA) aptamers generated will be applied as a biosensor for the detection and quantification of traces of 4’-OH-PCB3.

This dissertation presents the synthesis and application of nanodiamond based materials for electrochemical biosensors. In this research work, nanodiamond particles have been used to prepare doped and undoped nanocrystalline diamond films, and conducting polymer composites for enhanced biosensing. The performance of the synthetized materials towards sensing applications was evaluated against glucose amperometric biosensing. Besides, cholesterol biosensing was attempted to prove the capabilities of the platform as a generic biosensing substrate. Biosensors have been proved to provide reliable detection and quantification of biological compounds. The detection of biological markers plays a key factor in the diagnosis of many diseases and, even more importantly, represents a major aspect in the survival rate for many patients. Among all of the biosensors types, electrochemical biosensors have demonstrated the best reliability to cost ratio. Amperometric biosensors, for example, have been used for decades as point of care sensing method to monitor different conditions such as glucose. Despite the amount the research presented, the sensitivity, selectivity, stability, low cost and robustness are always driving forces to develop new platforms for biosensor devices. In the first phase of this dissertation, we synthesized undoped and nitrogen doped nanocrystalline diamond films. The synthetic material was thoroughly studied using different material characterization techniques and taken through a chemical functionalization process. The functionalization process produced a hydrogen rich surface suitable for enzymatic attachment. Glucose oxidase was covalently attached to the functionalized surface to form the biosensing structure. The response of the biosensor was finally recorded following voltammetry and amperometric techniques under steady state and dynamic conditions. The experimental results demonstrated that conductivity induced by the doping process enhanced the sensitivity of the sensing structure with respect to the undoped substrate. Also, the functionalization procedure showed strong bonding to avoid enzyme leaching during the measurements. Later, in the second phase of this dissertation, the nanodiamond particles were used as filler for conducting polymer composites. The objective for developing these composite materials was to overcome the high resistivity observed for nanocrystalline films. The experimental results demonstrated that the inclusion of nanodiamond particles increased the sensitivity of the overall structure towards the quantification of glucose with respect to the nanocrystalline films and the bare polymer. Besides, the experiment showed a noticeable enhancement in the signal-to-noise ratio and the mechanical stability of the sensing platform due to the nanodiamond addition. The best structures from the previous experiments were further grafted with iron oxide nanoparticles to attempt signal amplification. Initial experiments with nanodiamond based composited showed similar current for low glucose concentrations for two different active electrochemical sensing areas. This observation indicates that more area is still available to transport signal and to enhance even further the sensing action. Oxidation of iron oxide nanoparticles after initial enzymatic decomposition of glucose has been proved to provide higher current for the same glucose concentration; thus, creating amplification effect for the signal. Finally, the toxicity of the nanomaterial synthesized during this dissertation was evaluated in mammalian cells. The advances in biosensing techniques indicate the potential application of amperometric platform for continuous implantable devices; hence, the toxicity of the materials becomes a key aspect of the platform design.

Along with an increasing understanding of the harmful effects on the environment of a wide range of pollutants has come the need for more sensitive, faster and less expensive detection methods of identification and quantitation. Many environmental pollutants occur in low levels and often in complex matrices thus analysis can be difficult, time consuming and costly. Because of the availability and easy cultivation of the microorganisms with potentially high specificity, there is considerable interest in the use of living microorganisms as the analytical component (the biocomponent) of sensors for pollutants. While a number of biosensors using bacteria have been developed, yeast has been comparatively rarely used as the biocomponent. Yeast are attractive because they are easy to culture and they are eukaryotes which means their biochemistry is in many respects closer to that of higher organisms. This thesis describes the development of whole cell bioassays that use yeast cells as a sensing element and redox mediators to probe the intracellular redox reactions to monitor the catabolic activity of the yeast resulting from the external substrate, steady-state voltammetry is utilised as the electrochemical detection technique. The isogenic differential enzyme analysis (IDEA) concept of Lincoln Ventures Limited, lead NERF funded research consortium uses bacteria that have been cultured using specific organic pollutants as the carbon source which are the biocomponent in sensors. The use of wild type yeast Arxula adeninivorans that has the ability to use a very wide variety of substrates as sources of carbon and nitrogen was used as an alternative to bacteria to validate the “IDEA” concept. Naphthalene and di-butyl phthalate were chosen as model target contaminant molecules. The performance, detection limits and the usefulness of yeast based biosensor applications for environmental analysis are discussed. This thesis also describes the development and optimisation of a simple, cost effective in vivo estrogens bioassay for the detection of estrogens using either genetically modified or a wild type yeast Saccharomyces cerevisiae. In this study, catabolic repression by glucose was exploited to achieve specificity to estrogens in complex environmental samples that eliminates the requirement for conventional sample preparation. This is the first time that the use of wild type yeast to quantify estrogens has been reported. The attractive features of the bioassay are its use of a non-GMO organism, its speed, its high specificity and sensitivity with a detection limit of 10-15 M. The similarity of binding affinities for major estrogens to those of human estrogens receptors makes this in vivo estrogen bioassay very useful for analytical/screening procedures. The electrochemical detection method also makes it easy to interface with a variety of electronic devices.

This thesis presents a novel algorithmic A/D converter to be used in a biosensor. The converter is capable of a conversion rate of 1.5 bits/phase and hence the required conversion time is reduced. The proposed architecture is analyzed for non-ideal effects and compared with existing algorithmic A/D architectures. The converter needs only one op-amp, 4 comparators and 3 capacitors. Power reduction techniques are discussed with respect to the biosensor and the ADC. The ADC is designed for fabrication in a CMOS 0.18μm process. / Graduation date: 2004