Molecular diagnostics is a fast growing field with new technologies being developed constantly. There is a demand for more sophisticated molecular tools able to detect a multitude of molecules on a single molecule level with high specificity, able to distinguish them from other similar molecules. This becomes very important for infectious diagnostics with the increasing antibiotic resistant viruses and bacteria, in gene based diagnostics and for early detection and more targeted treatments of cancer. For increased sensitivity, simplicity, speed and user friendliness, novel readouts are emerging, taking advantage of new technologies being discovered in the field of nanotechnology.  This thesis, based upon four papers, examines two novel electrical readouts for amplified single molecule detection. Target probing is based upon the highly specific amplification technique rolling circle amplification (RCA). RCA enables localized amplification resulting in a long single stranded DNA molecule containing tandem repeats of the probing sequence as product. Paper I demonstrates sensitive detection of bacterial genomic DNA using a magnetic nanoparticles-based substrate-free method where as few as 50 bacteria can be detected. Paper II illustrates a new sensor concept based on the formation of conducting molecular nanowires forming a low resistance circuit. The rolling circle products are stretched to bridge an electrode gap and upon metallization the resistance drops by several orders of magnitude, resulting in an extremely high signal to noise ratio. Paper III explores a novel metallization technique, demonstrating the efficient incorporation of boranephosphonate modified nucleotides during RCA.  In the presence of a silver ion solution, defined metal nanoparticles are formed along the DNA molecule with high spatial specificity. Paper IV demonstrates the ability to manipulate rolling circle products by dielectrophoresis. In the presence of a high AC electric field the rolling circle products stretch to bridge a 10 µm electrode gap.

<p>There is an urgent need to develop practical and effective systems to detect diseases, such as cancer, infectious diseases and cardiovascular diseases.</p><p>Nanotechnology is a new, maturing field that employs specialized techniques to detect and diagnose infectious diseases. To this end, there have been a wealth of techniques that have shown promising results, with fluorescence and surface-enhanced Raman scattering being two important optical modalities that are utilized extensively. The progress in this specialized niche is staggering and many research groups in academia, as well as governmental and corporate organizations, are avidly pursuing leads which have demonstrated optimistic results.</p><p>Although much fundamental science is still in the pipeline under the guise of both ex-vivo and in-vivo testing, it is ultimately necessary to develop diagnostic devices that are able to impact the greatest number of people possible, in a given population. Such systems make state-of-the-art technology platforms accessible to a large population pool. The development of such technologies provide opportunities for better screening of at-risk patients, more efficient monitoring of disease treatment and tighter surveillance of recurrence. These technologies are also intrinsically low cost, facilitating the large scale screening for disease prevention.</p><p>Fluorescence has long been established as the optical transduction method of choice, because of its wealth of available dyes, simple optical system, and long heritage from pathology. The intrinsic limitations of this technique, however, have given rise to a complementary, and more recent, modality: surface-enhanced Raman scattering (SERS). There has been an explosive interest in this technique for the wealth of information it provides without compromising its narrow spectral width.</p><p>A number of novel studies and advances are successively presented throughout this study, which culminate to an advanced SERS-based platform in the last chapter.</p><p>The finite element method algorithm is critically evaluated against analytical solutions as a potential tool for the numerical modeling of complex, three-dimensional nanostructured geometries. When compared to both the multipole expansion for plane wave excitation, and the Mie-theory with dipole excitation, this algorithm proves to provide more spatially and spectrally accurate results than its alternative, the finite-difference time domain algorithm.</p><p>Extensive studies, both experimental and numerical, on the gold nanostar and Nanowave substrate for determining their potential as SERS substrates, constituted the second part of this thesis. The tuning of the gold nanostar geometry and plasmon band to optimize its SERS properties were demonstrated, and significant 3-D modeling was performed on this exotic shape to correlate its geometry to the solution's exhibited plasmon band peak position and large FWHM. The Nanowave substrate was experimentally revived and its periodic array of E-field hotspots, which was until recently only inferred, was finally demonstrated via complex modeling.</p><p>Novel gold- and silver- coated magnetic nanoparticles were synthesized after extensive tinkering of the experimental conditions. These plasmonics-active magnetic nanoparticles were small and displayed high stability, were easy to synthesize, exhibited a homogeneous distribution, and were easily functionalizable with Raman dye or thiolated molecules.</p><p>Finally, bowtie-shaped cobalt micromagnets were designed, modeled and fabricated to allow the controllable and reproducible concentrating of plasmonics-active magnetic nanoparticles. The external application of an oscillating magnetic field was accompanied by a cycling of the detected SERS signal as the nanoparticles were concentrated and re-dispersed in the laser focal spot. This constituted the first demonstration of magnetic-field modulated SERS; its simplicity of design, fabrication and operation opens doors for its integration into diagnostic devices, such as a digital microfluidic platform, which is another novel concept that is touched upon as the final section of this thesis.</p> / Dissertation

We solved the Landau-Lifshitz equations numerically to examine the time development of a system of magnetic particles. Constant or periodical external magnetic field has been applied. First, the system has been studied without dissipation. Local energy excitations (breathers) and chaotic transients have been found. The behaviour of the system and the final configurations can strongly depend on the initial conditions, and the strength of the external field at an earlier time. We observed some sudden switching between two remarkably different states. Series of bifurcations have been found. When a weak Gilbert-damping has been taken into account, interesting behaviour has been found even in the case of one particle as well: bifurcation series and period multiplication leading to chaos. For a system of antiferromagnetically coupled particles, highly nontrivial hysteresis loops have been produced. The dynamics of the magnetization reversal has been investigated and the characteristic time-scale of the reversal has been estimated. For more particles, the energy spectrum and the magnetization of the system exhibits fractal characteristics for increasing system size. Finally, energy have been pumped into the system in addition to the dissipation. For constant field, complicated phase diagrams have been produced. For microwave field, it has been found that the chaotic behaviour crucially depends on the parity of the number of the particles.

Magnetic particles have attracted increasing attention in fields ranging from separation processes to electromagnetic information storage an medical application. Various approaches for their synthesis have been developed and studied to satisfy the criteria of production. Improvement and optimization of size, stability, and functionality is of vital importance in biological applications. The main aspect of project, initially, was to study the application of aqueous functionalized magnetic nanoparticles coupled with high gradient magnetic separation technique for the removal of trace residue of organic contaminants from drinking water. However, the importance of synthesizing stable ferrofluid for this purpose became clear later and took precedence over the initial objective. Different approaches were adopted, such as the incorporation of poly(ethylene glycol) methacrylate, ethylenediamine, and chitosan, to enhance the stability of magnetic particles. However, these surface modifications had unfavorable effect on the stability of initial particles. In accord with the initial objective of the project, the possibility of utilization of β-cyclodextrin, as organic pollutant entrapment agent, was investigated in preliminary studies conducted on its interaction with a model compound, procaine hydrochloride. The outcomes of these experiments suggest its potential as a biocompatible removal agent for the elimination of organic pollutant in drinking water system, or other applications that require selective separation of organic compounds.

Orientador: José Ricardo de Arruda Miranda / Resumo: As nanopartículas magnéticas (NPMs) são uma classe de nanopartículas que se destacam em áreas da saúde, principalmente em aplicações teranósticas. O potencial das NPMs é prejudicado em função da absorção hepática, considerando que o fígado é uma rede complexa de células inter-relacionadas responsável pela captação do NPMs. Apesar de vários estudos concentrados na área, ainda é pouco compreendido como cada estrutura hepática opera no processo de retirada das NPMs da circulação sanguínea. As técnicas de imagem tem proporcionado avanços no entendimento de eventos fisiológicos, facilitando sua visualização. Atualmente, há uma gama de modalidades responsáveis pela detecção e imagiamento da biodistribuição das NPMs. Dentre essas técnicas de imagens, várias estão presentes apenas em hospitais ou em grandes centros de pesquisa. Esse trabalho tem como destaque, a aplicação de um novo método biomagnético, a Biosusceptometria AC (BAC), para a detecção in vivo de NPMs através de imagens. Fatores como ausência de radiação ionizante, versatilidade e alta resolução temporal são atributos do sistema frente às diversas técnicas de imagem. Neste estudo foi utilizado um novo arranjo do sistema, o sistema Multicanal BAC (ACB-MC), o qual permitiu a avaliação do processo de clearence das NPMs no sangue e seu posterior acúmulo no fígado em ratos Wistar. A partir das imagens dinâmicas obtidas e sua quantificação, foi proposto uma abordagem matemática a fim de auxiliar a farmacocinética de distribuiç... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Magnetic nanoparticles (MNPs) are widely used in healhty research areas, especially in terantic applications. The potential use of MNPS is diminished due to hepatic absorption, considering that the liver is a complex network of interrelated cells and the most responsible by MNPs uptake. Although there has been many studies in this area, it is still unknown how the liver removes MNPs from the bloodstream. Imaging techniques have provided more understanding of physiological events, enhancing their visualization. Currently, there is a range of modalities able to detect and image the biodistribution of MNPs. Among these techniques, several are restricted to large hospitals and research centers. In this work, our aim was to highlight the application of a new biomagnetic method, Biosusceptometry AC (BAC), for in vivo detection of MNPs through images. When compared to other systems, BAC has some advantages such as the absence of ionizing radiation, versatility and high temporal resolution. In this study, a new system arrangement, the Multicanal BAC system (ACB-MC) was used. This allowed the evaluation of the clearence process of MNPs in the blood and their subsequent accumulation in the liver. Those experiments were performed in Wistar rats. From the quantification of the dynamic images, a mathematical approach was proposed to investigate the pharmacokinetics of distribution and accumulation of MNPs. The ACB-MC system presented excellent temporal resolution and through sequential im... (Complete abstract click electronic access below) / Mestre

Magnetic hyperthermia (MH) is an anti-cancer treatment which exploits the heat produced by tumour-targeted magnetic nanoparticles (MNPs) subjected to an alternating magnetic field (AMF). A problem limiting the clinical use of MH, however, is the inability to adequately localise the MNPs at the tumour site. A cellular approach using mesenchymal stem cells (MSCs) as carriers has been proposed as these cells are believed to home to sites of tissue injury and tumour growth, however problems with MNPs uptake and toxicity retard progress and need to be overcome. The aim of this project was to find an alternative approach in MH treatment, creating engineered human MSCs able to biosynthesise MNPs. To achieve this goal, MSCs were transfected with either, or both, M. magneticum AMB-1 mms6 and mmsF genes. M. magneticum AMB-1 is a genus of magnetotactic bacteria, containing magnetosomes, which are lipidic organelles containing single crystals of magnetite. M. magneticum-AMB1 mms6 and mmsF genes are important for final crystal morphology and are known to play a role in crystal synthesis and growth respectively. The originality of this study was in using mms6 and mmsF genes, which were codon-optimized for mammalian expression, alone or in combination, for transfection of human MSCs, which have known tumour homing capacity. The transfected MNPs-bearing MSCs, able to migrate into the tumour tissue, were subjected to AMF in MH experiments in an attempt to induce cancer cell death. mms6 and mmsF gene expression, following transfection, was investigated in the human osteosarcoma cell line MG63 by reverse transcription polymerase chain reaction (RT-PCR). The cellular ultrastructure of transfected MG63 cells was investigated by transmission electron microscopy (TEM), revealing the presence of nanoparticles. The magnetism of transfected MG63 cells was proved by superconducting quantum interference device (SQUID) and supported by in vitro MH experiments. Then, human MSCs were transfected with mms6 and mmsF genes, alone or in combination. The effect of transfection experiments and MNPs synthesis on MSCs markers of stemness, cell proliferation and differentiation ability were investigated. The MTB genes expression in human MSCs was assessed by RT-PCR and cell magnetism was confirmed by SQUID, in vitro MH experiments and by magnetic force microscopy (MFM). Then, in vitro studies of MH were undertaken to establish whether mms6 transfected MSCs expressing MNPs supported a MH effect when exposed to an AMF. Cells were initially exposed to an AMF of 565.3 kHz frequency in monolayers and in 3D arrangements and cell death/viability was assessed. Subsequently, the effect of the same AMF on 3D models of mixed populations of mms6-expressing MSCs and cancer cells was assessed. The results indicate that viability of MNPs-expressing MSCs and adjacent cancer cells is reduced following AMF exposure. In vivo studies of MH were undertaken following intracardiac injection of mms6-expressing MSCs in tumour-bearing mice (epidermoid carcinoma). The expression of mms6-expressing MSCs inside mice organs was confirmed by RT-PCR, fluorescence microscopy and immunohistochemistry. The effect of the application of an AMF of 565.3 kHz on mice tumours was studied with different techniques (tumour size and volume measurement, multiphoton microscopy, haematoxylin and eosin staining, and activated Caspase 3 expression), to understand if MNPs created inside mms6- expressing MSCs, following AMF exposure, could lead to cancer cell death. Results indicate that mice tolerate the treatment well, however no appreciable tumour reduction or necrosis was evident. Overall the results suggest that mms6 transfection alone confers the highest magnetisation to MSCs compared to mmsF alone or mms6+mmsF co-transfected, and that mms6 expression in human MSCs does not have an adverse effect on important cell functions. mms6-expressing MSCs, when exposed to an AMF, show reduced viability and enhanced cell cytotoxicity in vitro. When co-cultured with cancer cells in 3D models in vitro, mms6-expressing MSCs are able to reduce viability of adjacent cancer cells confirming the potential applicability of mms6- expressing MSCs for MH treatment. In vivo proof of concept experiments show that mms6-expressing MSCs can locate to the tumour tissue, and mms6-expressing intracardiac injected MSCs mice exposed to AMF tolerate the treatment well. However, the number of mms6-expressing MSCs able to localize to the tumour tissue in this experiment was too low to give an appreciable tumour reduction, so more experiments are needed to enhance the experimental protocol. A number of improvements are required to progress this novel technique towards clinical application. Gene transfection and MNPs production need to be optimised, the best frequency for MH needs to be established and MSCs delivery to the tumour has to be significantly increased to allow concentration of MNPs. The study has helped to increase our knowledge on the creation of magnetic human MSCs to potentially use these cells in MH cancer treatment.

Submitted by Milena Rubi (milenarubi@ufscar.br) on 2016-11-03T17:25:54Z No. of bitstreams: 1 SAMPAIO_Guilherme_2016.pdf: 12947275 bytes, checksum: e37468f50e4625a4fdc9875a5cf3f85f (MD5) / Approved for entry into archive by Milena Rubi (milenarubi@ufscar.br) on 2016-11-03T17:26:13Z (GMT) No. of bitstreams: 1 SAMPAIO_Guilherme_2016.pdf: 12947275 bytes, checksum: e37468f50e4625a4fdc9875a5cf3f85f (MD5) / Approved for entry into archive by Milena Rubi (milenarubi@ufscar.br) on 2016-11-03T17:26:21Z (GMT) No. of bitstreams: 1 SAMPAIO_Guilherme_2016.pdf: 12947275 bytes, checksum: e37468f50e4625a4fdc9875a5cf3f85f (MD5) / Made available in DSpace on 2016-11-03T17:26:28Z (GMT). No. of bitstreams: 1 SAMPAIO_Guilherme_2016.pdf: 12947275 bytes, checksum: e37468f50e4625a4fdc9875a5cf3f85f (MD5) Previous issue date: 2016-05-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The comprehension of the phenomena related to magnetism allowed advances in various fields of knowledge and several modern applications that are now part of our daily lives. Specifically, the magnetic nanoparticles (NPs) has aroused a great interest among researchers in recent decades. This curiosity due to its unique properties is due to its small size and surface interactions. One of the main physical phenomena that appear when we reduce the size of a magnetic particle is called superparamagnetism. In this work, we have looked for a description and understanding of the mathematical formalism attached to magnetic phenomena at nanoscale through the application of the Langevin’s function to magnetic NPs in superparamagnetic regime and non-interacting model. Accordingly, we use theoretical simulations of magnetization curves as a function of applied field to study some parameters and verify its compatibility with magnetic characterization data obtained for samples of NPs magnetite and maghemite. In this way, we can get relevant information to the analysis of the magnetic behavior of nanoparticulate systems. Finally, we have lunched the potential of these magnetic NPs in biomedical applications both in diagnostic techniques and in disease treatments. / A compreensão dos fenômenos ligados ao magnetismo permitiu avanços nas mais variadas áreas do conhecimento e diversas aplicações modernas que hoje fazem parte do nosso cotidiano. Especificamente, as nanopartículas (NPs) magnéticas têm despertado um grande interesse por parte dos pesquisadores nas últimas décadas. Essa curiosidade devido às suas propriedades diferenciadas é decorrente de suas dimensões reduzidas e de interações de superfície. Um dos principais fenômenos físicos que surgem ao se reduzir o tamanho de partículas magnéticas é o chamado superparamagnetismo. Neste trabalho, procuramos por uma descrição e entendimento do formalismo matemático ligado ao fenômeno magnético em nanoescala por meio da aplicação do modelo proposto por Langevin a NPs magnéticas em regime superparamagnético não-interagente. Nesse sentido, utilizamos de simulações teóricas de curvas de magnetização em função do campo aplicado a fim de estudar alguns parâmetros e verificar sua compatibilidade com dados de caracterização magnética obtidos para amostras de NPs de magnetita e maghemita. Desta forma, podemos obter informações relevantes para a análise do comportamento magnético desses sistemas nanoparticulados. Por fim, evidenciamos as potencialidades dessas NPs magnéticas nas aplicações biomédicas tanto em técnicas de diagnóstico quanto em tratamentos de doenças.

Thesis submitted in fulfilment of the requirements for the degree of Doctor Technologiae: Chemical Engineering, Faculty of Engineering at the Cape Peninsula University of Technology - Cape Town, South Africa / Polycyclic aromatic hydrocarbons (PAHs) are one of the most common and recalcitrant environmental contaminants – known for their potential toxicity, mutagenicity, and carcinogenicity to humans. Biosurfactant application can enhance the biodegradation of PAHs. The main object of this work was to explore the novelty of biosurfactant produced by the isolated strains of Bacillus sp and Pseudomonas aeruginosa grown exclusively on Beta vulgaris, and the modification of the zeolites nanoparticles by the biosurfactant, for enhanced biodegradation of PAHs in soil. Novel biosurfactant-producing strains were isolated from hydrocarbon-contaminated environments, while several agrowaste were screened as primary carbon sources for the expression of biosurfactants, which were quantified using various standardized methods......

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2018-09-10T18:22:00Z No. of bitstreams: 2 Dissertação- Michelly Christine dos Santos - 2012.pdf: 1834625 bytes, checksum: af88a6d02e2b5362de31315d2c4ff78c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-09-11T11:11:24Z (GMT) No. of bitstreams: 2 Dissertação- Michelly Christine dos Santos - 2012.pdf: 1834625 bytes, checksum: af88a6d02e2b5362de31315d2c4ff78c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-09-11T11:11:24Z (GMT). No. of bitstreams: 2 Dissertação- Michelly Christine dos Santos - 2012.pdf: 1834625 bytes, checksum: af88a6d02e2b5362de31315d2c4ff78c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-10-11 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Magnetic nanoparticles with two different chemical compositions, magnetite and zinc doped manganese ferrite were synthesized, functionalized with lauric acid bilayers and dispersed into water yielding stable aqueous colloidal suspensions at physiological pH. Bioactive molecules of different sizes, Amphotericin B and curcumin, were associated to the lauric acid bilayers of the suspended nanoparticles. X-ray diffraction analyses reveled a diffraction pattern characteristic of the cubic spinel crystalline phase for both samples. The nanoparticles average size estimated by Debye-Scherrer’s formula was 9 nm for zinc doped manganese ferrite and 8 nm for magnetite. The hysteresis curves showed that the nanoparticles are super paramagnetic at room temperature with saturation magnetization of 44.2 emug-1 for zinc doped manganese ferrite and 51.5 emug-1 for magnetite. The IR analysis showed the presence of lauric acid associated to the nanoparticles, and the TG curves showed a higher amount of lauric acid bonded to zinc doped manganese ferrite. The hydrodynamic diameters of the nanoparticles indicate that they are dispersed into the suspension as aggregates, and that the association of bioactive molecules did not affect the hydrodynamic sizes of the aggregates. The IR spectra confirmed the presence of bioactive molecules in the formulations and the UV-VIS spectra showed that these molecules are solubilized into the lauric acid bilayers in their monomeric forms. The functionalized nanoparticles containing the bioactive molecules present less negative zeta potential than the lauric acid functionalized nanoparticles, indicating that the bioactive molecules interact with the medium of the suspensions. The evaluation of aggregation of the nanoparticles in cell culture medium showed that the nanoparticles aggregation is significantly reduced by the addition of proteins (FBS and BSA) in cell culture medium. The study of interaction of the nanoparticles with bovine serum albumin before and after the association with the bioactive molecules showed that these systems present great affinity for BSA, with binding constants (Kb) in the range of 105 -106M-1. These results suggest that the nanoparticles functionalized with lauric acid bilayers, pure or associated to amphotericin B and curcumin, can be bioconjugated to proteins producing formulations with higher colloidal stability in biological media. / Nanopartículas magnéticas com duas diferentes composições químicas, magnetita e ferrita de manganês dopada com zinco, foram sintetizadas, funcionalizadas com bicamadas de ácido láurico e dispersas em meio aquoso produzindo suspensões coloidais estáveis em pH fisiológico. Posteriormente, foram associadas moléculas bioativas de tamanhos diferentes, Anfotericina B e curcumina, nas bicamadas de ácido láurico funcionalizadas nos dois tipos de nanopartículas em suspensão. A caracterização estrutural e composicional das nanopartículas foi realizada por difração de raios X e análise química dos teores de íons metálicos. Os difratogramas mostraram um padrão de difração referente à formação de material constituído por uma única fase cristalina cúbica do tipo espinélio. Os tamanhos médios estimados pela fórmula de Debye-Scherrer foram 9 e 8 nm para as nanopartículas de ferrita de manganês dopadas com zinco e de magnetita, respectivamente. Os teores dos íons metálicos indicaram um pequeno desvio na estequiometria final dos sólidos em relação à estequiometria inicial. As técnicas utilizadas para caracterizar as nanopartículas funcionalizadas antes e após a associação da Anfotericina B e da curcumina foram IV, UV-VIS, TG, VSM e ainda foram realizadas medidas de potencial zeta e de diâmetro hidrodinâmico. As curvas de histerese indicaram a formação de nanopartículas superparamagnéticas à temperatura ambiente com magnetização de saturação de 44,2 emug-1 para a ferrita de manganês dopada com zinco e 51,6 emug-1 para a magnetita. As análises de IV mostraram que as nanopartículas foram funcionalizadas com ácido láurico e as curvas de TG indicaram que as nanopartículas de ferrita de manganês dopadas com zinco contêm maior quantidade de ácido láurico do que as nanopartículas de magnetita. Os valores de diâmetros hidrodinâmicos das nanopartículas indicaram que as mesmas estão dispersas na suspensão na forma de agregados e que a associação das moléculas bioativas não influenciou nos tamanhos hidrodinâmicos dos agregados. Os espectros de IV comprovaram a presença das moléculas bioativas nas formulações e a análise dos espectros de UV-VIS mostrou que as moléculas bioativas estão solubilizadas nas bicamadas de ácido láurico em suas formas monoméricas. As nanopartículas funcionalizadas associadas às moléculas bioativas apresentaram potencial zeta menos negativo que as nanopartículas funcionalizadas, indicando que as moléculas bioativas interagem com o meio das suspensões. O estudo de agregação de nanopartículas em meio de cultura celular mostrou que a adição de proteínas contribui significativamente para a diminuição da agregação das nanopartículas neste meio. O estudo de interação entre a albumina de soro bovino (BSA) e as nanopartículas funcionalizadas antes e após a associação das moléculas bioativas mostrou que os sistemas têm grande afinidade com a BSA, com constantes de ligação (Kb) da ordem de 105–106 M-1. Esses resultados sugerem que as nanopartículas funcionalizadas com bicamadas de ácido láurico, puras ou associadas à Anfotericina B e à curcumina podem ser bioconjugadas a essa proteína produzindo formulações com maior estabilidade coloidal em meios biológicos.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-06-27T18:26:30Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO Daiane Laise da Silvadf.pdf: 1941460 bytes, checksum: ee592cf258d3acc5df19b753d3b621b6 (MD5) / Made available in DSpace on 2016-06-27T18:26:30Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO Daiane Laise da Silvadf.pdf: 1941460 bytes, checksum: ee592cf258d3acc5df19b753d3b621b6 (MD5) Previous issue date: 2015-03-16 / CNPQ / Os métodos empregados para o isolamento de lectinas são diversos e podem combinar diferentes processos que em sucessivas etapas chegam a purificar estas moléculas. Suportes magnéticos como nanopartículas de óxidos de ferro podem ser empregados a fim de conseguir o isolamento de proteínas e enzimas através do uso da separação magnética. O objetivo deste trabalho foi avaliar se nanopartículas magnéticas revestidas com levana (MNPs-levana) podem ser usadas como matriz de purificação de lectinas a partir de extratos brutos (EB) de sementes de Cratylia mollis e Canavalia ensiformis. As MNPs-levana obtidas pelo método de síntese por co-precipitação foram incubadas com extrato bruto (EB) de sementes de Cratylia mollis e Canavalia ensiformis. A determinação de proteínas, atividade hemaglutinante (HA), inibição da atividade hemaglutinante (HAI) e SDS-PAGE confirmaram a purificação das lectinas Cramoll 1,4 e Concanavalina A. Para caracterização dos materiais, MNPs-levana foram comparadas com nanopartículas magnéticas (MNPs) e MNPs-levana após incubação com extratos brutos (MNPs-levana com lectinas ligadas), pelas análises MEV, EDX e MET. MEV mostrou diferenças entre as superfícies das nanopartículas, com aspectos lisos e rugosos em MNPs, superfícies rugosas em MNPs-levana e superfícies planas e rugosas em MNPs-levana após incubação com extrato bruto. Análise por MET mostraram diferenças entre as dimensões maiores (M) e menores (m) das nanopartículas sintetizadas (MNPs com MD = 15 nm  3nm e mD = 12 nm  3nm; e MNPs-levancom MD = 23 nm 5 nm e 18 nm mD = 4 nm) e revelaram um perfil homogêneo de agregação e forma esférica das nanopartículas. MNPs-levana também apresentarampossuir estrutura “núcleo-concha”, devido à presença da levana. Análise de EDX realizada mostrou picos mais elevados de C e O, conforme segue: MNPs-levana com lectinas ligadas>MNPs-levan>MNPs. Também se observou a presença de Fe em todas as amostras.As MNPs-levana mostraram ser matrizes de purificação eficientes, com vantagens como a possibilidade de reutilização (quatro vezes); baixo custo e simplicidade, quando comparadas com os métodos geralmente usados para purificar essas lectinas. / The methods used for lectins isolation are diverse and can combine several processes in successive steps to purify these molecules.Magnetic supports such as iron oxides nanoparticles can be employed to achieve the isolation of proteins and enzymes through the use of magnetic separation. The aim of this work was to evaluate if magnetic nanoparticles coated by levan (MNPs-levan) can be used as purification matrix of lectins from Cratylia mollis and Canavalia ensiformis seeds crude extracts (CE). The MNPs-levan obtained by co-precipitation synthesis method were incubated with crude extracts (CE) of Cratylia mollis and Canavalia ensiformis seed. Protein determination, hemaglutinanting activity (HA), hemaglutinanting activity inhibition (HAI)and SDS-PAGE confirmed the purification of lectins Cramoll 1,4 and Concanavalin A. For material characterization, MNPs-levanwere compared with iron oxide magnetic nanoparticles (MNPs), and MNPs-levan after incubation with crude extracts (MNPs with bonded lectins), by SEM, EDX and TEM analyses. SEM analysis showed differences between nanoparticles surfaces, with smooth and rough aspects in MNPs, rough surfaces in MNPs-levan, and flat and rough surfaces in MNPs-levan after CE incubation. TEM analysis showed differences between the large and small dimensionsof nanoparticles synthesized (MNPs with LD=15 nm +- 3nm and SD= 12 nm +- 3nm; and MNPs-levan with LD= 23 nm +-5 nm and SD= 18 nm +- 4 nm) and an homogeneous profile of aggregation and spherical shape.MNPs-levan also presented a core-shell structure due to the levan presence.EDX analysis performed showed higher peaks of C and O as follows: MNPs-levan with bonded lectins > MNPs-levan> MNPs. Also were observed the presence of Fe in all samples due to the presence of magnetite. The MNPs-levan showed to be an efficient purification matrix, with advantages as possibility of fourfold reuse, low cost and simplicity when compared with methods generally used to purificate these same lectins