Reversible adsorption of nucleid acids on magnetic carriers was studied in this diploma thesis. Magnetic P(HEMA-co­-GMA) microspheres and magnetic glass particles were used. The aim of the study was to isolate DNA in suitable quality for polymerase chain reaction (PCR). Adsorption of DNA on magnetic carriers was achieved after DNA condensation by PEG and NaCl in separation mixture. PEGs of various molecular weight (600 and 6000 g/mol) and different concentrations of PEG in separation mixture (4, 8, 12, 16%) were used. Quantity of eluted DNA incerased with molecular weight and concentration of PEG in separation mixtures. Optimized experimental conditions were applied for the separation of DNA from chicken erythrocytes, purified DNA, DNA in crude lysates of bacterial cells of Lactobacillus paracasei ssp. paracasei CCDM 211/06 and from real samples (liquid dairy products, hard cheese). The presence of target DNA in eluates was tested using genus specific PCR (genus Lactobacillus) or species specific PCR (species Bifidobacterium longum) Aqueous two-phase system (liquid-liquid) was used for separation of DNA from real symplex, too. At first the condiotions aqueous two-phase systém creation were studied. It was created by 16% PEG of various molecular weight (600, 6000 g/mol) and by various concentration of ammonium sulphate. Reversible DNA adsorption on carboxyl group-containing magnetic nonporous P(HEMA-co-EDMA) microspheres for the isolation PCR-ready DNA from liquid dairy products containing PCR inhibitors was studied, too. The quality of isolated DNA was checked by PCR amplification.The presumption on the elimination of PCR inhibitors from DNA samples was confirmed.

La société Nanobactérie développe un traitement thermique innovant contre le cancer qui repose sur l‘utilisation de nanoparticules d‘oxyde de fer d‘origine bactérienne, appelées magnétosomes. Celles-ci sont injectées directement dans la tumeur puis activées par le champ magnétique alternatif. Cette activation crée une augmentation locale de la température provoquant la destruction de la tumeur, sans affecter les tissus sains environnants. Afin d‘éviter les problèmes de toxicité liés à la présence d‘endotoxines bactériennes à la surface des magnétosomes, un processus de purification est utilisé. Il permet l‘élimination de toute la membrane organique immunogène et de garder ainsi le minéral responsable de l‘activité thermique. Cependant, l‘élimination de cette membrane entraîne l‘agrégation des magnétosomes. La première étape de ce travail de thèse a donc consisté à stabiliser les magnétosomes purifiés, et l'‘identification du meilleur revêtement a été évaluée. Dans une deuxième partie, une nouvelle modalité de chauffage a été mise au point pour augmenter l‘efficacité de l‘hyperthermie magnétique dans la destruction de cellules cancéreuses. / The Nanobactérie company develops a novel strategy of cancer treatment using iron oxide nanoparticles of bacterial origin, called magnetosomes. These nanoparticles are injected directly into the tumor and then activated by an alternating magnetic field. Activated nanoparticles trigger a highly localized rise of temperature, inducing the destruction of the tumor without any adverse effects on adjacent healthy tissues. To avoid the problems of toxicity caused by the presence of bacterial endotoxin which present on the surface of magnetosomes extracted from bacteria, a process of purification is realized to eliminate all the immunogenic organic membrane and keep only the mineral responsible for the thermal activity. However, since elimination of this membrane causes the aggregation of the magnetosomes which become unstable in aqueous solution, the first part of this work consisted in stabilizing the purified magnetosomes by a modification of their surface. The identification of the best coating was then evaluated. Moreover, in the second part of this work, a new heating modality was assessed to increase the efficiency of the magnetic hyperthermia in the destruction of cancer cells.

Orientador: José Ricardo de Arruda Miranda / Resumo: Nanopartículas magnéticas (NPMs) têm sido alvo de vários estudos por conta de sua versatilidade, que se destaca quando comparadas a outros materiais. NPMs apresentam tamanho reduzido, alta susceptibilidade magnética e devido a possibilidade de alteração química de revestimento, apresentam um alto poder teranóstico. Entretanto, antes de aplicá-los clinicamente é de extrema importância conhecer seus destinos e possibilidades de interação no organismo. A quantificação da biodistribuição de NPMs por meio de suas características intrínsecas ainda se mostra como um desafio. A Biosusceptometria de corrente alternada (BAC) é uma técnica biomagnética que atua detectando a quantidade de materiais magnéticos em ambientes biológicos. Essa técnica, em relação às demais presentes no mercado, apresenta baixo custo e portabilidade. Baseado na problemática em torno da detecção e quantificação de NPMs, nesse estudo foi analisada a biodistribuição e eliminação de NPMs de ferrita de manganês revestidas com citrato e albumina via sistema BAC. Para isso, foram utilizados 100 ratos Wistar, separados em 2 grupos experimentais (NPMs revestidas com albumina e citrato) e 10 subgrupos, variando o tempo de eutanásia dos animais (1, 4, 12, 24 e 48 horas e 5, 10, 15, 30 e 60 dias após a administração). Para análise da biodistribuição, foram coletados 5 órgãos de interesse: fígado, pulmão, baço, rins e coração, além do sangue e, para análise da eliminação, foram coletadas as fezes dos animais. Foi possível ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Magnetic nanoparticles (MNPs) have been widely used in several studies due to their high versatility compared to other materials. MNPs presents small size, high magnetic susceptibility and, due to the possibility of biochemical alteration of its surface, enables a high level of theranostics applications. However, before applying them clinically, it is of paramount importance understand its destinations and possibilities of interaction in the organism. The biodistribution quantification of NPMs through their intrinsic characteristics still is a challenge. Alternating current biosusceptometry (ACB) is a biomagnetic technique that acts detecting a quantity of magnetic materials in biological samples. In comparison with the other techniques, ACB presents low cost and portability. Based on the lack of accessible techniques to study biodistribution and elimination process of MNPs, this work aimed to analyze the biodistribution and determine the elimination of manganese ferrite magnetic nanoparticles coated with citrate and albumin via BAC system. For this work, we used 100 Wistar rats, separated into 2 experimental groups (MNPs coated with albumin and citrate) and 10 subgroups, changing the euthanasia time point (1, 4, 12, 24 and 48 hours and 5, 10, 15, 30 and 60 days after administration). For biodistribution analysis, we collected the blood and 5 organs of interest: liver, lung, spleen, kidneys and heart, and, for elimination analysis, we collected the animal’s feces. It was poss... (Complete abstract click electronic access below) / Mestre

Magnetic nanoparticles have great potential in the biomedical and diagnostics field. Due to their small size, the particles have a high surface-to-volume ratio which enables for biofunctionalisation with different molecular probes. This makes itpossible to target them against a wide variety of biomarkers. In this project, the aim was to develop a magnetic nanoparticle-based DNA detection method with respectto sensitivity by employing circle-to-circle amplification, which is an extension of rolling circle amplification, in order to increase the assay sensitivity. The method provides high specificity due to the use of padlock probes for amplification. The project included testing and optimising the protocol used for DNA amplification and detection with a synthetic target, which involved testing different padlock probes, incubation times and incubation temperatures. Lastly, the method was tested on a biological target. It has recently been shown that specific aggregation occurs between magnetic nanoparticles and DNA, which enables for a visual readout strategy sincethe aggregates are visible to the naked eye. Initial testing of the method yielded asensitivity of about 100 attomoles. The achieved sensitivity after the optimisation work was 1 attomole of both synthetic and biological DNA targets. This is an improvement compared to the 400 attomoles that has previously been reported with one round of rolling circle amplification. The results can be used in further development of the naked-eye DNA detection method towards the realisation of a commercially attractive bioanalytical device.

L’objectif de cette thèse est la fabrication d’un microdispositif magnétique pour la détection et la manipulation d’éléments biologiques à base de nanoparticules magnétiques en conditions microfluidiques. Il a pour but d’intégrer des fonctions de base de contrôle et détection magnétique, pour atteindre des mesures spécifiques, stables, rapides et reproductibles. En effet, la technique d’immunodosage couplée à des nanoparticules magnétiques, bien connue dans la littérature, nécessite un contrôle du déplacement de ces dernières pour les fonctionnaliser efficacement et créer un complexe biologique encapsulant une molécule cible (biomarqueur). Dans notre cas une molécule modèle pour le domaine de la biodéfense a été utilisée : l’ovalbumine. Pour contrôler le champ magnétique nécessaire pour la capture des complexes magnétiques, nous avons opté pour l’utilisation de microbobines intégrées aux dispositifs fluidiques et comparé cette technique originale avec d’autres plus conventionnelles. Pour détecter un complexe biologique, la fluorescence est largement utilisée en biologie, mais cette technique ne permet pas une intégration complète pour un dispositif autonome. Dans cette optique, nous proposons la détection des complexes à base de nanoparticules magnétiques en relevant la variation de l’inductance d’un microcircuit magnétique refermant une chambre microfluidique contenant ces complexes immunologiques. Le dimensionnement des microbobines de contrôle par simulation a permis de déterminer les paramètres permettant d’obtenir le champ magnétique le plus adapté au contrôle des complexes biologiques. Dans le cas des microbobines utilisées pour la détection, des branches magnétiques micrométriques ont été insérées autour des microbobines pour créer un circuit de détection magnétique encore plus sensible. La réalisation de ces dispositifs a impliqué l’intégration de matériaux et de structures de nature fortement hétérogène, et leur assemblage a nécessité de résoudre de nombreux verrous technologiques. L’enjeu a été de déterminer l’ensemble des étapes successives et nécessaires pour un procédé de microfabrication fiable et reproductible. Pour montrer l’intérêt des dispositifs de capture des nanoparticules magnétiques, des tests immunologiques ont été réalisés tout d’abord en microtubes pour les comparer à ceux réalisés dans un circuit fluidique à l’aide d’aimant externe puis de microbobines intégrées. Dans ce dernier cas, une optimisation considérable a été validée en termes de réduction de temps d’incubation, de reproductibilité des mesures et de limites de détection équivalentes à l’état de l’art pour l’ovalbumine. Pour le dispositif de détection magnétique, des premières expériences de caractérisation électrique ainsi que des études en concentration de nanoparticules magnétiques ont été réalisées et comparées aux résultats obtenus par simulation. Pour la preuve de concept, un démonstrateur de détection de complexes magnétiques a été également finalisé validant la possibilité d’intégration du microcircuit magnétique dans un dispositif fluidique. Il a validé également l’obtention d’une gamme de sensibilité remarquable corrélée à la présence des complexes magnétiques. Ses caractéristiques ont été confrontées à celles obtenues par les simulations et discutées en tenant compte de toutes les étapes critiques du procédé de microfabrication. / RésuméThe objective of this thesis is the fabrication of a magnetic microdevice for the detection and manipulation of biological elements based on magnetic nanoparticles under microfluidic conditions. It aims to integrate basic functions of control and magnetic detection, to achieve specific, stable, fast and reproducible measurements. Indeed, the immunoassay technique coupled to magnetic nanoparticles, well known in the literature, requires a control of the displacement of magnetic nanoparticles to effectively functionalize them and create a biological complex to encapsulate a target molecule (biomarker). In our case, a model molecule for the field of biodefense was used: ovalbumin. To control the magnetic field which is necessary for the capture of magnetic complexes, we opted for the use of microcoils integrated in the fluidic devices and compared this original technique with other more conventional ones. To detect a biological complex, fluorescence is widely used in biology, but this technique does not allow a complete integration for an autonomous device. In this context, we propose the detection of complexes based on magnetic nanoparticles by observing the variation of the inductance of a magnetic microcircuit closing a microfluidic chamber containing these immunological complexes. The design of the control microcoils by simulation made it possible to determine the parameters allowing to obtain the most adapted magnetic field to the control of the biological complexes. In the case of microcoils used for detection, micrometric magnetic branches were inserted around the microcoils to create an even more sensitive magnetic sensing circuit. The realization of these devices involved the integration of materials and structures of highly heterogeneous nature, and their assembly has required to solve many technological locks. The challenge was to determine all the successive and necessary steps for a reliable and reproducible microfabrication process. To show the interest of magnetic nanoparticle capture devices, immunoassays were first performed in microtubes to compare with those made in a fluid circuit using external magnet and integrated microcoils. In the latter case, considerable optimization has been validated in terms of reduction of incubation time, reproducibility of measurements and detection limits equivalent to the state of the art for ovalbumin. For the magnetic detection device, first experiments of electrical characterization as well as concentration studies of magnetic nanoparticles were carried out and compared to the results obtained by simulation. For the proof of concept, a demonstrator of detection of magnetic complexes was also finalized validating the possibility of integration of the magnetic microcircuit in a fluidic device. It has also validated obtaining a remarkable range of sensitivity correlated with the presence of magnetic complexes. Its characteristics were compared to those obtained by the simulations and discussed taking into account all the critical steps of the microfabrication process.

No description available.

In this dissertation a study of enhanced hyperthermia for cancer treatment through the use of magnetic nanoparticles is presented. Hyperthermia has been in use for many years, as a potential alternative method in cancer treatment, and high frequency microwave radiation has been used successfully to raise the tumor temperature to around 42°C in superficial tumors without causing damage to surrounding healthy tissues. Magnetic fluid hyperthermia involves the use of magnetic nanoparticles injected into the tumor before exposure to microwave radiation. The magnetic energy in the nanoparticles is converted into heat allowing for a more rapid rise of temperature in the tumor to the desired level. In addition, the nanoparticles allow the electromagnetic absorption to be focused in the tumor and can be used to treat deep tumors in organs, such as the liver. Iron oxide magnetic nanoparticles were considered for this study as they are non-toxic and bio-compatible. For the case of breast cancer, the values for the temperature and specific absorption rate (SAR) in the tumor and in the healthy tissue were obtained through simulations and validated by measurement done on phantom models. Various characteristics of the nanoparticles such as radius, magnetic susceptibility and concentration were considered. In order to take the effect of the blood flow, which causes cooling and helps maintain the body temperature, various blood perfusion rates for a tumor in the liver were studied. A human male model in SEMCAD X, in which blood flow can be adjusted, was used for simulations. The tumor was injected with the nanoparticles and the change in temperature upon exposure to electromagnetic radiation was observed. The simulated results were compared with measured results on a liver phantom model in which saline solution was used to model blood flow. There was good agreement between the measured and simulated results.

No / Cd3P2 quantum dots (QDs) have been synthesized in both aqueous and high boiling point surfactant solutions via a gas-bubbling method. The synthesized QDs exhibit photoluminescent wavelengths spanning across the visible red to the near-infrared (NIR) spectral region. Two types of shell materials, SiO2 nanobeads and PS micro-spheres, have been employed to encapsulate the Cd3P2 QDs which provide protecting layers against physiological solutions. The coating layers are proven to enhance the optical and chemical stability of Cd3P2 QDs, and make the fluorescent particles capable of sustaining long-term photo-oxidation. To demonstrate the applicability of the bio-labelling, the fluorescent composite particles (PS@QDs, SiO2@QDs) were injected into a culture medium of colorectal carcinoma (LoVo) cells. The results demonstrated that the PS@QDs exhibited a brighter fluorescence, but the SiO2@QDs provided a better photostability which consequently led to long-term cancer cell detection as well as a much lower release of toxic Cd2+ into the PBS solutions.

No description available.

The superior properties of carbon nanotubes (CNTs) are best manifest in bulk materials when the CNTs are organized in tandem and embedded in a continuous matrix. Decorating the CNTs with magnetic nanoparticles (MNPs) facilitates their expedient organization with a magnetic field. One of the most convenient methods for their decoration is to first treat the CNTs with oxidative acids, and then coprecipitated MNPs in situ. This method results magnetized CNTs that are covalently functionalized with the MNPs. The associated destruction in the CNTs required running a comparative study of this protocol to identify the influence of the acid treatment on the decoration of multiwalled CNTs (MWNTs). Further, we explore means to tune the physical properties of these magnetized CNTs (mMWNTs) by varying the (1) MNP material composition, and (2) MNP:MWNT (w/w) magnetization weight ratio (γ). The resulted composite materials (mMWNTs) are utilized to synthesize a novel and hitherto unreported class of colloidal suspensions (MCCs) for which the dispersed phase, which consists of MWNTs decorated with MNPs, is both magnetoresponsive and electrically conductive. Synthesis of the dispersed phase merges processes for producing ferrofluids and mMWNTs. Later, these MCCs are adapted and engineered to produce a biological ink containing MWNTs that are twice functionalized, first with MNPs and thereafter with the anti-c-Myc monoclonal antibodies (Abs). The ink is pipetted and dynamically self-organized by an external magnetic field into a dense electrically conducting sensor strip that measures the decrease in current when a sample containing c-Myc antigens (Ags) is deposited on it. On the other side, a nondestructive methods to magnetize MWNTs and provide a means to remotely manipulate them is through the electroless deposition of magnetic nickel nanoparticles on their surfaces. The noncovalent bonds between Ni nanoparticles and MWNTs produce a Ni-MWNT hybrid material (NiCH) that is electrically conductive and has an enhanced magnetic susceptibility and elastic modulus. Raising γ (Ni:MWNT weight ratios) increases the coating layer thickness, which influences the NiCH magnetic properties and tunes its elastic modulus. The NiCH was used to fabricate Ni-MWNT macrostructures and tune their morphologies by changing the direction of an applied magnetic field. Leveraging the hydrophilic Ni-MWNT outer surface, a water-based conductive ink was created and used to print a conductive path that had an electrical resistivity of 5.9 Ωm, illustrating the potential of this material for printing electronic circuits. Further, the NiCHs are introduced into an epoxy matrix at low 0.25-1% volume fractions and aligned along the direction of an applied magnetic field, which produces anisotropic bulk properties. However, nanoparticles aligned in perpendicular directions in sequential layers result in an effectively isotropic composite material. Furthermore, the subsequent annealing of the NiCH in the presence of air oxidizes nickel to nickel oxide whereas carbon is released as gaseous carbon dioxide, which leads to a novel approach for the fabrication of nickel oxide nanotubes (NiONTs) based on MWNTs as a sacrificial template. New chelating polyelectrolytes are used as dispersing agents to achieve high colloidal stability both for NiCH and NiONTs. A gravimetric specific capacitance of 245.3 F g-1 and areal capacitance of 3.28 F cm-2 at a scan rate of 2 mV s-1 is achieved with an electrode fabricated using nickel oxide nanotubes as the active element with a mass loading of 24.1 mg/cm2. / Thesis / Doctor of Philosophy (PhD) / The superior properties of carbon nanotubes (CNTs) are best manifested in bulk materials when the CNTs are organized axially and in tandem, and embedded in a continuous matrix. Decorating the CNTs with magnetic nanoparticles (MNPs) facilitates their organization through “action from a distance” with a magnetic field. The attachment of MNPs to the surfaces of CNTs can be realized through covalent or non-covalent (i.e. physical) bonding. This work develops both methodologies to investigate how the physical properties of magnetized CNT (mCNT) can be tuned and produce new CNT-based nanostructures for particular applications. First, mCNTs are utilized to synthesize a hitherto unreported class of colloidal suspensions based on which a magnetic bio-ink is fabricated to print a fast-response biological sensor. Next, nickel-coated CNTs prepared using electroless deposition are used in the form of a filler at low volume fractions in an epoxy matrix, where they are aligned along multiple-direction using a magnetic field, producing either anisotropic or isotropic bulk properties on demand. Finally, subsequent annealing of nickel-coated CNTs in air oxidizes nickel to nickel oxide while carbon is released in the form of gaseous carbon dioxide. This leads to another novel approach for the fabrication of nickel oxide nanotubes, which are demonstrated to be an alternate viable material to fabricate electrodes for use in supercapacitors.