Thesis (M.S.)--Rochester Institute of Technology, 2005. / Typescript. Includes bibliographical references (leaves 109-113).

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Submitted by CLAUDEMIR OLIVEIRA SOUZA null (clau_souza10@hotmail.com) on 2018-04-05T00:34:55Z No. of bitstreams: 1 Dissertação Claudemir final.pdf: 2598018 bytes, checksum: 6a02fac38da72d304450321a6bebe674 (MD5) / Approved for entry into archive by Ana Carolina Gonçalves Bet null (abet@iq.unesp.br) on 2018-04-05T20:46:17Z (GMT) No. of bitstreams: 1 souza_co_me_araiq_int.pdf: 2522583 bytes, checksum: 763389bef408c84daca17f39d0af0cd5 (MD5) / Made available in DSpace on 2018-04-05T20:46:17Z (GMT). No. of bitstreams: 1 souza_co_me_araiq_int.pdf: 2522583 bytes, checksum: 763389bef408c84daca17f39d0af0cd5 (MD5) Previous issue date: 2018-03-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As hemoproteínas desempenham um papel vital nos organismos. Dentre elas, as hemoglobinas extracelulares gigantes (Hbs) se destacam por sua alta cooperatividade, alta estabilidade oligomérica, grande afinidade para ligação com oxigênio e resistência à oxidação. Devido estas propriedades, as Hbs apresentam um grande potencial biotecnológico, como é o caso de estudos buscando protótipos de substituição sanguínea e elaboração de biossensores. Porém, o uso das hemoglobinas gigantes em biossensores para detecção de metais pesados ainda não é encontrado na literatura, este tipo de biossensor agiliza e diminui o custo das análises de empresas e agências de fiscalização ambiental, obtendo o resultado in loco. Diante do exposto, o objetivo deste trabalho foi verificar a sensibilidade da hemoglobina de Amynthas gracilis (HbAg) com os metais cobre e cádmio em diferentes valores de pH e imobilizá-la em substrato sólido com o auxílio dos seguintes polímeros: o catiônico polietilenoimina (PEI) e o aniônico hidrocloreto de polialilamina (PAH). Os estudos por absorção ótica, espalhamento de luz (LSI), dicroísmo circular (CD) e fluorescência da interação da HbAg em função da concentração do metal pesado cobre no pH 7,0 mostraram que a concentração de 30 μmol.L-1 é uma concentração crítica onde observa-se uma maior oxidação do grupo heme, agregação e dissociação da proteína, além de alteração em suas α- hélices. No pH 5,0 foi observada oxidação do grupo heme mas sem alterações nas estruturas secundárias da HbAg e formação de agregados. As análises dos espectros de absorção ótica, CD, fluorescência e LSI da HbAg na presença de diferentes concentrações do metal pesado cadmio não apresentaram mudanças significativas quando comparadas com o espectro da HbAg nativa. A imobilização da HbAg em PEI no pH 7,0 foi monitorada por absorção ótica e voltametria cíclica, entretanto a HbAg não foi possível imobilizar a HbAg no polímero PAH no mesmo valor de pH, sugerindo que a interação da HbAg com os biopolímeros se deve por interações eletrostáticas. A imobilização da HbAg em PEI manteve as características da hemoglobina em pH 7,0. A hemoglobina apresentou respostas ao cobre nas medidas de voltametria cíclica realizadas em concentrações de até 20 μmol.L-1 do metal, evidenciando o grande potencial de biossensoriamento do sensor fabricado. Assim, nestes estudos foi possível verificar que a HbAg na presença de cobre em pH 7,0 se oxida, agrega e dissocia, enquanto que em pH 5,0 ela apresenta apenas oxidação. Na presença de cádmio, a HbAg não apresentou nenhuma alteração através das técnicas avaliadas. Com isso, sugere-se que a HbAg pode ser utilizada como um biossensor de indicação de presença de concentrações micromolares do metal pesado cobre. / Hemoproteins are a group of proteins that play a vital role in organisms. Among them, giant extracellular hemoglobins (Hbs) stand out for their high cooperativity, high oligomeric stability, high affinity for oxygen binding and resistance to oxidation. Due to these properties, the Hbs show a great biotechnological potential, as is the case of studies searching for prototypes of blood replacement and preparation of biosensors. However, the use of giant hemoglobins in biosensors for heavy metals detection is not yet reported on the literature, this kind of biosensors speeds up and lowers costs of analysis of companies and environmental inspection agencies, obtaining on the spot result. Thus, the aim of this work was verified the sensibility of HbAg with the metals copper and cadmium on different pH values and it's immobilization in solid surfaces with the polymers: the cationic polietilenimine (PEI) and anionic polyallylamine hydrochloride (PAH). Studies of optical absorption (UVVis), light scattering (LSI), circular dichroism (CD) and fluorescence of HbAg interaction with copper at pH 7.0 shown that 30 μmol.L-1 it’s a critical concentration where observed a great oxidation of group heme, aggregation and protein dissociation as well as alteration on your α-hélices. In pH 5.0 was observed oxidation of heme group but without alterations in HbAg secondary structure and no aggregates formation. UV-Vis spectra analysis, CD, fluorescence and LSI of HbAg at different concentrations of cadmium doesn’t shown significant changes on native HbAg spectra. The HbAg immobilization on PEI pH 7,0 has monitored by optical absorption and cyclic voltammetry, however it was not possible to immobilize the HbAg in the PAH polymer at the same pH value, suggesting that HbAg interaction with biopolymers is due to electrostatic ligations. The HbAg Immobilization in PEI maintained the hemoglobin characteristics at pH 7.0. The Hemoglobin shown responses to copper at concentrations of up to 20 μmol.L-1 of the metal in cyclic voltammetry measurements performed, demonstrated the great potential of the fabricated biosensor. Thus, in these studies it was possible to verify that HbAg on copper presence at pH 7.0 oxidizes, to aggregates and dissociates whereas at pH 5.0 it presents only oxidation. In the presence of cadmium, the HbAg showed no change through the techniques evaluated. Thereby, it is suggested that HbAg can be used as a biosensor indicating the presence of micromolar concentrations of copper heavy metal.

Orientador: Patricia Soares Santiago / Banca: Marli Leite de Moraes / Banca: Valquiria da Cruz Rodrigues Barioto / Resumo: As hemoproteínas desempenham um papel vital nos organismos. Dentre elas, as hemoglobinas extracelulares gigantes (Hbs) se destacam por sua alta cooperatividade, alta estabilidade oligomérica, grande afinidade para ligação com oxigênio e resistência à oxidação. Devido estas propriedades, as Hbs apresentam um grande potencial biotecnológico, como é o caso de estudos buscando protótipos de substituição sanguínea e elaboração de biossensores. Porém, o uso das hemoglobinas gigantes em biossensores para detecção de metais pesados ainda não é encontrado na literatura, este tipo de biossensor agiliza e diminui o custo das análises de empresas e agências de fiscalização ambiental, obtendo o resultado in loco. Diante do exposto, o objetivo deste trabalho foi verificar a sensibilidade da hemoglobina de Amynthas gracilis (HbAg) com os metais cobre e cádmio em diferentes valores de pH e imobilizá-la em substrato sólido com o auxílio dos seguintes polímeros: o catiônico polietilenoimina (PEI) e o aniônico hidrocloreto de polialilamina (PAH). Os estudos por absorção ótica, espalhamento de luz (LSI), dicroísmo circular (CD) e fluorescência da interação da HbAg em função da concentração do metal pesado cobre no pH 7,0 mostraram que a concentração de 30 μmol.L-1 é uma concentração crítica onde observa-se uma maior oxidação do grupo heme, agregação e dissociação da proteína, além de alteração em suas α- hélices. No pH 5,0 foi observada oxidação do grupo heme mas sem alterações nas estruturas se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hemoproteins are a group of proteins that play a vital role in organisms. Among them, giant extracellular hemoglobins (Hbs) stand out for their high cooperativity, high oligomeric stability, high affinity for oxygen binding and resistance to oxidation. Due to these properties, the Hbs show a great biotechnological potential, as is the case of studies searching for prototypes of blood replacement and preparation of biosensors. However, the use of giant hemoglobins in biosensors for heavy metals detection is not yet reported on the literature, this kind of biosensors speeds up and lowers costs of analysis of companies and environmental inspection agencies, obtaining on the spot result. Thus, the aim of this work was verified the sensibility of HbAg with the metals copper and cadmium on different pH values and it's immobilization in solid surfaces with the polymers: the cationic polietilenimine (PEI) and anionic polyallylamine hydrochloride (PAH). Studies of optical absorption (UVVis), light scattering (LSI), circular dichroism (CD) and fluorescence of HbAg interaction with copper at pH 7.0 shown that 30 μmol.L-1 it's a critical concentration where observed a great oxidation of group heme, aggregation and protein dissociation as well as alteration on your α-hélices. In pH 5.0 was observed oxidation of heme group but without alterations in HbAg secondary structure and no aggregates formation. UV-Vis spectra analysis, CD, fluorescence and LSI of HbAg at different concentrations of... (Complete abstract click electronic access below) / Mestre

Luminescent transition metal complexes have attracted tremendous interest in the analytical field. Most luminescent metal complexes possess long emission lifetimes in the visible region, and their phosphorescence can be readily distinguished from short-lived background auto-fluorescence. Moreover, their large Stokes shift can prevent self-quenching, while their modular synthesis allows their properties to be readily tuned without labor-intensive synthetic protocols. These properties render transition metal complexes as promising candidates for the development of biosensors. In this study, I aimed to explore different kinds of Ir(III) complexes that can be used as biosensors to monitor DNA secondary structures or protein conformations. Chapter 1 gives a brief introduction regarding the luminescence mechanism of transition metal complexes, and the properties, structures and preparations of Ir(III) complexes are also introduced. Chapters 24 report the syntheses and screening of Ir(III) complexes, and the use of these Ir(III) complexes to monitor the secondary structure of DNA, such as G-quadruplex, G-triplex and i-motif DNA. Chapter 2 reports the G-quadruplex-selective properties of two luminescent Ir(III) complexes 2.1 and 2.9 for the detection of ochratoxin A (OTA) and nicking endonuclease in aqueous solution, respectively; Chapter 3 explores a novel Ir(III) complex 3.8 which is highly specific for G-triplex DNA, and was thus employed for Mung Bean nuclease activity detection; Chapter 4 introduces the Ir(III) complex 4.1 that exhibited a high signal enhancement to i-motif DNA, and was therefore used for terminal deoxynucleotidyl transferase (TdT) activity detection. Chapter 5 describes the two novel cyclometalated Ir(III) complexes 5.1 and 5.21 that were used to monitor two kinds of proteins, human serum albumin (HSA) and beta-amyloid(140) (Aβ140), respectively.

The present research focused on the development of a new methodology to assess the strength of the interaction between vaccine antigens and elicited polyclonal antibodies through SPR biosensors. Quantifying the binding strength of polyclonal antibodies is of first importance to evaluate the quality of the vaccine as well as to increase the scientific knowledge of immune protection mechanisms. To now the development of such tool has been complicated by the non-specific binding caused by high protein abundance in the blood and serum samples but also by the way of interpreting the data resulting from multi-interaction events measured at the same time. At first, we unsuccessfully tried to segregate the individual affinity contribution of each antibody population by measuring the signal as the sum of singular interactions. Differentiation of the singular contribution would have needed the fulfillment of the “additivity” hypothesis, meaning that each antibody bind identically alone or in mixture with other antibody. This hypothesis was not met and mathematical assessment by the sum of singular contribution led to fitting results that did not reflect the biological reality. It was therefore decided to switch the analysis method and to measure the end association binding level reached by the different samples injected at the same specific antibody content. The dissociation behavior was interpreted by the percentage of binding after long and fixed dissociation time. In a first application, we compared the antibodies elicited by two different commercially available vaccines and we showed that the binding interaction was not concentration dependent as, highly different levels were reached when injecting identical antibody concentration. No statistical significant difference was observed between both vaccines. Research firstly focused on the decrease of the non-specific binding and we found that ionic strength was a key parameter, increasing the buffer salt concentration reduced the non-specific binding without diminishing the binding strength. The sample composition was also a key parameter and purifying the IgG allowed to decrease dramatically the undesired binding events. A second application aimed at showing the equivalence between two different antigen constructions for two antibodies population. Even if identical antigen level immobilization is a challenge, the methodology is completely suitable to perform a 2-dimensional comparison (ligand and analyte). A last application was dedicated to the comparison between D and Q-pan Flu vaccines, and results showed that there was no statistical evidence of significant differences between both vaccines. End association level correlated well with haemagglutination inhibition assay at least when serum samples were not diluted at the same antibody content. This last application also showed that throughput may be extended to more than 50 samples per 80 hours / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

abstract: Demand for biosensor research applications is growing steadily. According to a new report by Frost & Sullivan, the biosensor market is expected to reach $14.42 billion by 2016. Clinical diagnostic applications continue to be the largest market for biosensors, and this demand is likely to continue through 2016 and beyond. Biosensor technology for use in clinical diagnostics, however, requires translational research that moves bench science and theoretical knowledge toward marketable products. Despite the high volume of academic research to date, only a handful of biomedical devices have become viable commercial applications. Academic research must increase its focus on practical uses for biosensors. This dissertation is an example of this increased focus, and discusses work to advance microfluidic-based protein biosensor technologies for practical use in clinical diagnostics. Four areas of work are discussed: The first involved work to develop reusable/reconfigurable biosensors that are useful in applications like biochemical science and analytical chemistry that require detailed sensor calibration. This work resulted in a prototype sensor and an in-situ electrochemical surface regeneration technique that can be used to produce microfluidic-based reusable biosensors. The second area of work looked at non-specific adsorption (NSA) of biomolecules, which is a persistent challenge in conventional microfluidic biosensors. The results of this work produced design methods that reduce the NSA. The third area of work involved a novel microfluidic sensing platform that was designed to detect target biomarkers using competitive protein adsorption. This technique uses physical adsorption of proteins to a surface rather than complex and time-consuming immobilization procedures. This method enabled us to selectively detect a thyroid cancer biomarker, thyroglobulin, in a controlled-proteins cocktail and a cardiovascular biomarker, fibrinogen, in undiluted human serum. The fourth area of work involved expanding the technique to produce a unique protein identification method; Pattern-recognition. A sample mixture of proteins generates a distinctive composite pattern upon interaction with a sensing platform consisting of multiple surfaces whereby each surface consists of a distinct type of protein pre-adsorbed on the surface. The utility of the "pattern-recognition" sensing mechanism was then verified via recognition of a particular biomarker, C-reactive protein, in the cocktail sample mixture. / Dissertation/Thesis / Ph.D. Electrical Engineering 2011

Les systèmes MEMS et NEMS permettent, par résonance mécanique, des mesures de masse avec une sensibilité et une résolution propices à la caractérisation d'objets de taille micro- et nanométrique. Ces dispositifs, adaptés à une intégration dans des systèmes d'analyse miniatures plus complexes, sont d'intérêt pour la recherche biomédicale et la détection de particules. Toutefois la caractérisation en milieu liquide reste à ce jour délicate, principalement à cause de phénomènes dissipatifs associés à la mise en mouvement du fluide environnant le dispositif vibrant.Afin de lever ce verrou, l’équipe au sein de laquelle s’est déroulée cette thèse a développé des MEMS fluidiques sous forme de plaques minces mises en vibration dans leur plan de manière à limiter l'excitation du fluide environnant. Chaque plaque comporte un canal microfluidique permettant la circulation d'un liquide dont la masse moyenne est précisément déterminée par la fréquence de résonance du système. A terme, l'ambition de ces systèmes est de parvenir à révéler, par un décalage en fréquence, le passage au sein de la plaque vibrante d’une particule unique transportée par le liquide.Deux objectifs ont été atteints dans le cadre de cette thèse. D'une part, le comportement de ces structures en présence de divers liquides a été finement caractérisé ce qui a permis d’évaluer leurs performances réelles en fonction des conditions d'excitation. La résolution mesurée pour ces capteurs est de l’ordre de quelques g.L-1, pour une sensibilité d’environ 100 Hz.(g.L-1)-1.D'autre part, une nouvelle génération de capteurs aux caractéristiques innovantes a été conçue en vue d’abaisser le seuil de détection en diminuant la masse des résonateurs et en améliorant le bruit en fréquence.Ce manuscrit sera articulé autour de quatre chapitres. Le premier propose un état de l’art des techniques existantes pour la caractérisation de particules en fluide, et détaille ensuite les solutions MEMS et NEMS développées à cette fin dans la littérature. Le second chapitre livre les résultats issus de la caractérisation d’une première génération de MEMS fluidiques. Le troisième décrit les observations et mesures réalises, et propose des perspectives d’amélioration de ces composants ainsi que de leur protocole de caractérisation. Enfin, on présente dans le dernier chapitre une nouvelle génération de NEMS conçue et fabriquée au cours de cette thèse ; pour finir sont discutés les choix réalisés et les perspectives d’évolution attendues pour ces composants. / MEMS and NEMS allow sensitive and precise mass detection consistent with micro- and bio- objects analysis. These systems are promising for biomedical research and particle metrology, and can be easily integrated in miniaturized multifunctional systems. Thererfore, characterization in liquid media remains tricky due to viscous dissipation consequent to the movement induced in the fluidic environment.In order to overcome this technological lock, our laboratory previously designed and fabricated specific MEMS devices for fluidic analysis; these thin plate resonators with and embedded microchannel are actuated in liquid media, with four capacitive electrodes providing both actuation and detection. The circulating fluid mass can be precisely measured by monitoring the device’s resonant frequency. The long-term objective is to be able to detect and weigh one single particle transported by the fluid.Two main objectives were fulfilled during these three years. First, the MEMS behaviour in presence of various liquids was evaluated, providing a fine-grained analysis of their performances as mass sensors. The measured resolution of our sensors is about a few g.L-1 with a sensitivity of 100 Hz.(g.m-3)-1.Meanwhile, a new generation of NEMS sensors with innovative features was designed; the objective is to decrease the effective mass and reduce the frequency noise, both for a better mass resolution.This thesis includes four chapters. The first one consists in a review of the existing techniques for particles characterization in fluid as well as MEMS and NEMS solutions for particles metrology described in the litterature. The second part of the manuscript presents the results of the experimental characterizations carried out on the first generation of sensors. The third chapter gathers the conclusions of these measurements and gives an outlook on possible improvements on both the design and the characterization of the sensors. At last, the fourth part describes the new generation of devices and discusses their characteristics in terms of expected resolution and applications.

Nanoelectrodes are defined as electrodes which have a critical dimension on the order of nanometres. Due to their smaller dimensions they have a reduced iR drop and enhanced mass transport, which results in the rapid establishment of an enhanced steady-state diffusion profile and a greater Faradaic current density, along with a smaller relative double layer capacitance, which together give a significantly increased signal to noise ratio compared to macroelectrodes. This potentially makes nanoelectrodes better sensors and analytical tools than macroelectrodes in terms of their having lower limits of detection and faster detection times. However, due to difficulties with fabrication most nanoelectrode designs are highly irreproducible which has inhibited their characterisation and commercial development. The Mount group has previously reported the design, fabrication and characterisation of a novel nanoelectrode design in conjunction with Engineers from the Scottish Microelectronic Centre (SMC). Microsquare Nanoband Edge Electrode arrays (MNEEs) consist of an array of cavities with nanoscale Pt bands (formed by sandwiching the metal between insulating layers) exposed around their perimeter. MNEEs are fabricated using a photolithographic process so can be reproducibly made in large quantities to high fidelity. The purpose of this work is to develop our understanding of the fundamental electrochemical behaviour of MNEEs for biosensing. First, a quantitative analysis of the cyclic voltammograms (CVs) and Electrochemical Impedance Spectroscopy (EIS) of macroelectrodes, microelectrodes and MNEE are compared and discussed. Second, their fundamental response is compared in terms of their biosensing properties by using a pre-established impedimetric biosensing protocol developed on macroelectrodes. This protocol uses a PNA probe to detect the mecA cassette of methicillin resistant staphylococcus aureus (MRSA). The procedure has been optimised and compared for macroelectrodes, microelectrodes and MNEE so as to compare their performances as biosensors. It was observed that MNEE’s: (a) form thiol films faster than electrodes with larger dimensions, determined by kinetic studies of 6-mercaptohexan-1-ol film formation (b) form films with different packing structures dependant on the electrode bulk to edge ratio (c) can detect the same concentration of target in less time than larger electrodes because of their increased sensitivity. The film packing has also been quantitatively investigated using EIS and it can be seen that films formed n MNEE were better able to incorporate target DNA into their more splayed out structure. Unique to this project has been the establishment of a protocol to form heterogeneous carbazole-alanine hydrogel matrices on nanoelectrodes, whose polymerisation is initiated by a pH swing at the electrode surface induced by the oxidation of hydroquinone. The gels growth pattern follows the diffusion field at the electrode and can be monitored using EIS. This also gives a measure of the permeability of the gel by fitting to the correct equivalent circuit. The gel structure has been imaged using light microscopy, confocal microscopy and scanning electrochemical microscopy (SEM). The results give a visual demonstration that MNEE has enhanced diffusion at the corners of the cavities, which is in agreement with previously published simulations, and give evidence as to the onset of hemispherical diffusion and the conditions at which the diffusion field between neighbouring electrodes begin to overlap, a phenomenon which can be observed visually and correlated to changes in the EIS data. Hydrogels have been grown chronopotentiometrically at different currents and the permittivity (through the diffusion coefficients) has been measured of redox couples through gels grown at different speeds. It was found that the hierarchical structure of the hydrogels can be tuned; potentially opening the door to a new breed of tuneable, biocompatible anti-biofouling matrices on bio-functionalised electrodes. The system was characterised using the same MRSA detection protocol as optimised for the MNEE and the target DNA was found to be able to permeate through the hydrogels and bind to the probe, which resulted in a significant change in impedance.

abstract: Advances in miniaturized sensors and wireless technologies have enabled mobile health systems for efficient healthcare. A mobile health system assists the physician to monitor the patient's progress remotely and provide quick feedbacks and suggestions in case of emergencies, which reduces the cost of healthcare without the expense of hospitalization. This work involves development of an innovative mobile health system with adaptive biofeedback mechanism and demonstrates the importance of biofeedback in accurate measurements of physiological parameters to facilitate the diagnosis in mobile health systems. Resting Metabolic Rate (RMR) assessment, a key aspect in the treatment of diet related health problems is considered as a model to demonstrate the importance of adaptive biofeedback in mobile health. A breathing biofeedback mechanism has been implemented with digital signal processing techniques for real-time visual and musical guidance to accurately measure the RMR. The effects of adaptive biofeedback with musical and visual guidance were assessed on 22 healthy subjects (12 men, 10 women). Eight RMR measurements were taken for each subject on different days under same conditions. It was observed the subjects unconsciously followed breathing biofeedback, yielding consistent and accurate measurements for the diagnosis. The coefficient of variation of the measured metabolic parameters decreased significantly (p < 0.05) for 20 subjects out of 22 subjects. / Dissertation/Thesis / M.S. Electrical Engineering 2012