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The £\-fetoprotein (AFP) is an important tumor marker for detecting hepatocellular carcinoma (HCC), the second major cancer killers in Taiwan. This thesis utilized a high-sensitivity low-cost quartz crystal microbalance (QCM) to replace the conventional biochemical analysis to detect the extreme low concentration of AFP in human serum. In general, QCM-based biosensors or immunosensors require smaller volume of sample and fewer responding time than conventional analysis system. Once the AFP antibody has been successfully adsorbed on the surface of QCM, any AFP antigen bonded with it will change the output frequency of the QCM sensor. This research investigates the influence of the mass-sensitivity of QCM-AFP sensor on three surface-modification methods: (i) Adsorption method, (ii) Cystamine-Glutaldehyde (CA-GA) method and (iii) Cystamine (CA) method. The effects of the concentration of AFP antibody on the sensitivity also will be studied. The adsorption method shows an unstable adhesion than the other methods. The CA-GA method and CA method presents respectively the maximum binding ratio of AFP antibody/antigen pair at 0.1 mg/mL and 1.0 mg/mL AFP-antibody concentration. Under such condition, the high sensitivity and linearity of the CA-GA and CA methods equal to 0.142 (Hz mL mg-1)/99.8% and 0.35 (Hz mL mg-1)/98.2% can be demonstrated. Finally, this thesis uses a commerical AFM system to analyze the effective bonding area of the CA-GA and CA methods. The CA method shows a higher effective surface area than that of CA-GA method so it can present a higher mass-sensitivity of AFP-antigen.

O câncer colorretal é uma das malignidades com maior incidência no Brasil e, na maioria dos casos, tem poucos sintomas visíveis até a doença estar em um estágio avançado. Pensando em simplificar o procedimento do exame e fazer um diagnóstico mais rápido para pacientes com câncer colorretal, este estudo teve como objetivo desenvolver um processo de diagnóstico simplificado baseado na caracterização de um biossensor eletroquímico em que fitas simples de miRNAs de interesse foram imobilizadas na superfície de eletrodos de ouro com objetivo de fazer a detecção de sua fita complementar utilizando-se dos métodos de espectroscopia de impedância eletroquímica e microbalança de cristal de quartzo. A sequência que foi imobilizada foi uma sequência de DNA que corresponde ao miRNA-224, cuja elevada expressão foi recentemente correlacionada com a agressividade e a incidência de metástase em pacientes com câncer colorretal. Os dados obtidos através da técnica de EIS foram comparados a dois modelos físicos de circuito equivalente, o circuito Randles clássico e um circuito Randles modificado, onde o capacitor foi substituído por um elemento de fase constante, tentando modelar de maneira mais eficiente a dupla camada que se forma na região de contado do eletrodo com a solução eletrolítica. O modelo modificado apresentou uma janela de funcionamento de 40,11% enquanto o circuito original apresentou uma janela de funcionamento de 23,98%; o que juntamente com o embasamento teórico do modelo de dupla camada fez com que o circuito modificado tenha sido escolhido para modelar os dados dos experimentos feitos na sequência. Tendo em vista que a imobilização das fitas simples de DNA na superfície do eletrodo de ouro é o passo chave para a obtenção de uma resposta satisfatória no biossensor, foram feitos testes alterando a proporção de DNA e espaçadores na fase de funcionalização a fim de determinar qual a proporção que otimiza a resposta do sensor. Comparando os resultados obtidos para as proporções de ssDNA:tiol total na etapa de funcionalização do eletrodo de 1:3, 1:4 e 1:5, a proporção 1:4 que corresponde a 20% de amostra no buffer de imobilização foi a que levou a uma maior variação percentual dos valores de Rct devido às hibridizações. Considerando esses como sendo os parâmetros otimizados para o sistema, foi construída então uma curva de caracterização do sensor, e o limite de detecção encontrado foi de 10pM; um valor considerado alto comparado com outros experimentos semelhantes, entretanto deve-se lembrar que o sistema aqui estudado não fez o uso de elementos amplificadores, como nanopartículas ou intercaladores de DNA. Para fins de comparação entre técnicas, foi conduzida uma análise semelhante utilizando uma microbalança de cristal de quartzo. Inicialmente foram utilizados os parâmetros encontrados como otimizados para a EIS, e posteriormente foram usados parâmetros mais próximos aos otimizados para essa técnica encontrados na literatura. Em ambos os casos foi possível observar variações referentes à hibridização das fitas complementares, entretanto como esperado, a resposta se mostrou mais expressiva para o segundo caso. Essas diferenças de resposta puderam ser relacionadas aos diferentes princípios de funcionamento dos sensores nas duas técnicas, mostrando que para a QCM o fator que influencia na resposta está relacionado à quantidade total de amostra imobilizada na superfície do eletrodo, enquanto que para a técnica de EIS o mais relevante é o quão compacta a camada se mostra. / Colorectal cancer is one of the most prevalent malignancies in Brazil and, in most cases, has few visible symptoms until the disease is at an advanced stage. In order to simplify the examination procedure and to make a faster diagnosis for patients with colorectal cancer, this study aimed to develop a simplified diagnostic process based on the characterization of an electrochemical biosensor in which single strands of miRNAs of interest were immobilized on the surface of gold electrodes with the objective of detecting their complementary strand using the methods of electrochemical impedance spectroscopy and quartz crystal microbalance. The sequence that was immobilized was a DNA sequence corresponding to miRNA-224, whose high expression was recently correlated with the aggressiveness and incidence of metastasis in patients with colorectal cancer. The data obtained through the EIS technique were compared to two equivalent circuit models, the classic Randles circuit and a modified Randles circuit, where the capacitor was replaced by a constant phase element, trying to more efficiently model the double layer that is formed in the interface between the electrode and the electrolytic solution. The modified model presented an operating range of 40.11% while the original circuit presented an operating range of 23.98%; which along with the theoretical understanding of the double-layer model made the modified circuit to be chosen as the model to fit the data of the experiments realized in the sequence. Considering that the immobilization of the simple DNA strands on the surface of the gold electrode is the key step to obtain a satisfactory response in the biosensor, tests were performed by altering the ratio of DNA and spacers in the functionalization phase in order to determine which the ratio that optimizes the sensor response. Comparing the results obtained for the proportions of ssDNA:total thiol in the functionalization stage of the electrode of 1:3, 1:4 and 1:5, the ratio 1:4 corresponding to 20% of sample in the immobilization buffer was the one that led to a greater percentage variation of Rct values due to hybridizations. Considering these as the optimized parameters for the system, a sensor characterization curve was built, and the limit of detection found was 10pM; a value considered high compared to other similar experiments, however it must be remembered that the system studied here did not make use of amplifying elements, such as nanoparticles or DNA intercalators. For purposes of comparing techniques, a similar analysis was conducted using a quartz crystal microbalance. Initially, the parameters found as optimized for the SIS were used, and later parameters that were closer to those optimized for this technique found in the literature were. In both cases it was possible to observe variations related to the hybridization of the complementary tapes, however, as expected, the response was more expressive for the second case. These differences in optimization parameters for the two techniques could be related to the different principles of sensor operation, showing that for the QCM the factor that influences the response is related to the total amount of sample immobilized on the surface of the electrode, while for the EIS technique the most relevant parameter is how compact the layer is.

The Quartz crystal microbalance (QCM) is a very sensitive mass-detecting device which is based on changes in to the vibrational frequency of quartz crystals after adsorption of substances to a modified crystal surface. In this study a QCM-based biosensor was developed for the rapid diagnosis of influenza viruses and its suitability and limitations were compared with currently available diagnostic methods on 67 clinical samples (nasal washes) received during the 2005 Australian winter. The type-specific and conserved viral M1 proteins of both A/PR/8/34 and B/Lee/40 viruses were used to prepare polyclonal antisera for the development of an ELISA. The limits of detection of ELISAs for the detection of purified A/PR/8/34 and B/Lee/40 ƒnviruses were 20ƒÝg/mL ƒnand 14 ƒÝg/mL using polyclonal antibodies, and 30 ƒÝg/mL and 20 ƒÝg/mL for monoclonal antibodies, respectively. The limit for detection of each virus was 104 pfu/mL, irrespective of whether antisera or monoclonal antibodies were used for capture. Non-purified cell culture-grown preparations of either virus could be detected at 103 pfu/mL The QCM utilised the same reagents used in ELISAs. However, a number of parameters were then further optimised to improve the sensitivity of the tests. These included blocking of non-specific binding, examination of the effects of flow-cell compression, the role of pH, flow rate, antibody concentration and the addition of protein A to the crystal surfaces of the biosensor. The lowest virus concentration that could be detected with the QCM was 104 pfu/mL for egg-grown preparations of both A/PR/8/34 and B/Lee/40, which could be detected within 30 min. However, conjugation of 13 nm gold nanoparticles to a second detector antibody resulted in a 10-fold increase in sensitivity and a detection limit of 103 pfu/mL that could be determined within 1 h. The direct detection of the influenza viruses in nasal samples was not possible by QCM because of the significant frequency fluctuation that was probably caused by the viscosity of the samples. Therefore, an additional culture step of 12 h was required, which increased the processing time to 2 days. The QCM/nanoparticle method was shown to be as sensitive as the standard cell culture method, and the QCM method as sensitive as the shell vial method. The QCM and QCM/nanoparticle methods were shown to be 81 and 87% as sensitive and both were 100% as specific as the real-time polymerase chain reaction. However, for use in rapid diagnosis, improvements are required to remove frequency fluctuation resulting from the direct use of nasal samples.

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Made available in DSpace on 2014-06-12T15:52:16Z (GMT). No. of bitstreams: 2 arquivo4515_1.pdf: 709397 bytes, checksum: 32ca7a05bac132d3232f3cc45576b1ac (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As determinações da troponina cardíaca T (TnT) podem contribuir para o diagnóstico e tratamento de infarto agudo do miocárdio e para a estratificação dos riscos dos pacientes com síndromes coronárias agudas no que respeita ao risco relativo de mortalidade. Neste trabalho, um sistema de microbalança de quartzo por injeção de fluxo baseado na alteração de freqüência elétrica em resposta à ligação antígeno-anticorpo foi empregado para determinação da TnT cardíaca. As variações de freqüências foram registradas por um contador de freqüência acoplado a um microcomputador. O anticorpo monoclonal específico foi imobilizado sobre a superfície de um eletrodo de cristal de quartzo por ligação irreversível via monocamadas auto-organizadas. O adsorbato composto por filme de alcanotiól foi formado incubando uma solução de 2-aminoetanotiol (cisteamina) por 2 h, seguido por glutaraldeído a 2,5% (v/v). Em seguida, anticorpos monoclonais anti- troponina T (anti-TnT) foram covalentemente imobilizados sobre o eletrodo de ouro do cristal de quartzo e foi usada uma solução de glicina (10mM) como agente bloqueante. Com o imunossensor desenvolvido foi possivel medir concentrações de troponina T com limite de detecção de 0,025 ng/mL. A superfície do sensor pode ser regenerada por injeção de uma solução do dodecil-sulfato de sódio 1% (p/v). A determinação da TnT foi realizada em amostras de soros humanos permitindo seu uso nas aplicações clínicas para diagnóstico do IAM

The electrochemical biosensor has been extensively used due to its capacity for rapid and accurate detection of a wide variety of target molecules or biomarkers. DNA hybridization sensors are based on the increase of negative charge on the electrode surface after the DNA target hybridize to the immobilized probes. The development of this platform requires first an understanding of the immobilization process and optimization of surface probe density. In this thesis the electron transfer is investigated on a label-free DNA hybridization detection by its intrinsic charge. The investigation using different immobilization buffers shows a strong dependence on their composition and concentration, and also the influence of the probe and spacer co-immobilized to obtain an organized and compact self-assembled monolayer. The probe density is determined using the chronocoulometry method with hexaammineruthenium (III) chloride, where the value is calculated from the number of cationic redox molecules electrostatically associated with the anionic DNA backbone and presented a linear relationship between thiol molar fraction and probe density from 2 to 5 x 1012 molecules/cm2. The effect of hybridization was determined using electrochemical impedance spectroscopy using negatively charged ferri/ferrocyanide redox couple in solution. After probe surface density optimization, the maximum shift of charge transfer resistence (20%) upon 1 M complementary sequence was obtained with around 25% probe fraction immobilized vii viii on surface. This electrochemical platform developed was able to detected 100 pM of target sequence and distinguish mismatched sequences. The limit of detection is higher when compared to the literature, however, this system can be further improved by amplifing the signal. The same platform is reproduced in the quartz crystal microbalance system and with field-effect transistor, comparing the different detections. The same platform is tested using two different HER2 aptamer sequences. Biological aspects are explored for a better understanding of the system / O biossensor eletroquímico foi amplamente utilizado devido à sua capacidade de Detecção rápida e precisa de uma grande variedade de moléculas alvo ou biomarcadores. Os sensores de hibridização do DNA são baseados no aumento da carga negativa na Superfície do eletrodo depois que o alvo do DNA se hibridar com as sondas imobilizadas. o O desenvolvimento desta plataforma exige primeiro uma compreensão da imobilização Processo e otimização da densidade da sonda de superfície. Nesta tese, o elétron A transferência é investigada em uma detecção de hibridação de DNA sem rótulas por sua intrínseca carregar. A investigação usando diferentes buffers de imobilização mostra uma forte Dependência de sua composição e concentração, e também a influência da Sonda e espaçador co-imobilizados para obter uma auto-montagem organizada e compacta Monocamada. A densidade da sonda é determinada usando o método de cronoculometria Com cloreto de hexaamminerutenio (III), onde o valor é calculado a partir do Número de moléculas redox catiónicas associadas eletrostática com o aniónico DNA backbone e apresentou uma relação linear entre a fração molar tiol E densidade de sonda de 2 a 5 x 1012 moléculas / cm2. O efeito da hibridação Foi determinado usando espectroscopia de impedância eletroquímica usando negativamente Casal redox ferri / ferrocianeto carregado em solução. Após a densidade da superfície da sonda Otimização, a mudança máxima da resistência à transferência de carga (20%) após 1 M A sequência complementar foi obtida com cerca de 25% de fração de sonda imobilizada Vii Viii Na superfície. Esta plataforma eletroquímica desenvolvida foi capaz de detectar 100 pM Da sequência alvo e distinguir sequências incompatíveis. O limite de detecção É maior quando comparado à literatura, no entanto, esse sistema pode ser mais Melhorou ampliando o sinal. A mesma plataforma é reproduzida no Sistema de microbalança de cristal de quartzo e com transistor de efeito de campo, comparando o Detecções diferentes. A mesma plataforma é testada usando dois diferentes HERMETO aptamer Sequências. Os aspectos biológicos são explorados para uma melhor compreensão do sistema

RNA aptamers that bind to a wide range of targets with high affinity and specificity have been identified via the in vitro systematic evolution of ligands by exponential enrichment (SELEX). However, the process is quite unpredictable due in part to binding that occurs not only on the targets themselves but also on any of the other functional groups, moieties, or surfaces. Recent modelling work has shown that this level of “background binding” is a key parameter in the performance of aptamer selection processes. One strategy to minimize the amount of background binding is to pre-block those possible binding sites with a non-amplifiable nucleic acid molecule, such as yeast tRNA. It is also known that binding buffer conditions have strong effect on the binding affinity of nucleic acids. However, there are no detailed studies and little quantitative information available to guide the design of aptamer selection processes. In this study, the binding ability of yeast tRNA, which has comparable size with most RNA aptamer libraries, on both silicon dioxide and poly (ethylene terephthalate glycol) (PET-G) surfaces was studied using Quartz Crystal Microbalance with Dissipation (QCM-D). Silicon dioxide surface is a commonly used substrate for QCM-D tests on the adsorption behaviour of different nucleic acid. PET-G is a commonly used polymer substrate for the fabrication of microfluidic devices, which are advanced techniques for aptamer selection. The presence of specific divalent cations, for example Mg2+ over Ca2+, in binding buffers greatly enhanced the binding of yeast tRNA on silicon dioxide surfaces and PET-G surfaces. Proper NaCl concentration (100 mM) and MgCl2 concentration (5 mM) is necessary to enhance yeast tRNA binding on both surfaces. Yeast tRNA binding ability on silicon dioxide surfaces show more dependence on binding buffer pH than on PET-G surfaces. / Thesis / Master of Applied Science (MASc)

Lubricants are used in numerous applications to control friction and protect moving parts from fatigue. These fluids consist of a variety of surface active chemistries competing for the surface to provide performance. In order to develop fluids that meet the ever-increasing requirements (from legislation and manufacturers), techniques that can provide insight into surface adsorption, in real time, and relate it back to performance are critical. The objective of this work is to determine if Quartz Crystal Microbalance with Dissipation (QCM-D) is an effective technique to investigate surfactant adsorption in regimes that are common to the transportation lubricant industry. QCM-D is employed to quantify the mass, characterize the morphology, and quantify the kinetics of adsorption of common friction modifiers. The adsorption information is then compared to macroscopic properties (friction and corrosion prevention) to determine if this technique can aid in formulating future lubricants.