Desenvolvimento de um biossensor para Ácido Glutâmico

A presente dissertação tem com objetivo o desenvolvimento de um biossensor com base nos polímeros de impressão molecular para a deteção de uma molécula alvo, o ácido glutâmico que é convertido em glutamina pela glutamina sintetase, recorrendo à potenciometria. Nas células neoplásicas a glutamina não é sintetizada podendo-se considerar que o ácido glutâmico é um potencial agente anti-cancro. A técnica de impressão molécular utilizada foi a polimerização em bulk, combinando a acrilamida e a bis acrilamida com o ácido glutâmico. Para se verificar se a resposta potenciométrica obtida era de facto da molécula alvo foram preparados em paralelo com os sensores, materiais de controlo, ou seja, moléculas sem impressão molécular (NIP). Para se controlar a constituíção química dos vários sensores nomeadamente, do NIP e do polímero de impressão molecular (MIP) antes e após a remoção bem como a molécula foram realizados estudos de Espetroscopia de Infravermelhos de Transformada de Fourier (FTIR), Scanning electron microscope (SEM) e Espetroscopia de Raios X por dispersão em energia (EDS). Os materiais desenvolvidos foram aplicados em várias membranas que diferiam umas das outras, sendo seletivas ao ião. A avaliação das características gerais das membranas baseou-se na análise das curvas de calibração, conseguidas em meios com pHs diferentes, comparando os vários elétrodos. O pH 5 foi o que apresentou melhor resultado, associado a uma membrana que continha um aditivo, o p-tetra-octilphenol, e com o sensor com percentagem de 3%. Posto isto, testou-se em material biológico, urina, com as melhores características quer em termos de sensibilidade (18,32mV/década) quer em termos de linearidade (1,6x10-6 a 1,48x10-3 mol/L). Verificou-se ainda que aplicando iões interferentes na solução, estes não interferem nesta, podendo ser aplicados na amostra sem que haja alteração na resposta potenciométrica. O elétrodo é capaz de distinguir o ácido glutâmico dos restantes iões presentes na solução.

Materiais e dispositivos sensores para a Norfloxacina

O trabalho consistiu no desenvolvimento e caracterização de sensores potenciométricos com base em polímeros de impressão molecular para a determinação de um antibiótico, a norfloxacina, em aquacultura. A simplicidade, o baixo custo e a interação rápida e reversível dos sensores potenciométricos com os analitos fizeram com que este fosse o tipo de sensor escolhido. O material sensor foi obtido por tecnologia de impressão molecular, baseada em polimerização em bulk, em que a NOR foi a molécula molde e foram utilizados como monómeros para autoconstrução dos sensores o pirrol, isoladamente, ou em conjunto com partículas de sílica gel funcionalizadas com 3-aminopropil. Também foi obtido material sensor, para controlo, em que a molécula molde NOR não estava presente (NIP). As características dos materiais sensores foram sujeitas a análise de microscopia eletrónica SEM e análise por espectrómetro de infravermelhos com transformada de Fourier. Os materiais sensores foram incluídos em membranas poliméricas, que seriam incorporadas em elétrodos. A avaliação do desempenho dos elétrodos foi feita através de curvas de calibração em diferentes meios (PBS, MES e HEPES). Também foi efetuada com sucesso a análise da sensibilidade dos elétrodos em água dopada. As diversas avaliações e análises efetuadas levaram a concluir que o MIP de pirrol com aditivo aniónico, foi o material sensor testado que permitiu obter melhores propriedades de resposta.

Insights into cell wall synthesis and cell division in Staphylococcus aureus

Dissertation presented to obtain the Ph.D degree in Biology

Separation of bioactive peptides by electrodialysis with ultrafiltration membranes: membrane characteristics, ex-situ and in-situ digestion and their impact on peptide migration

Dissertation presented to Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa for obtaining the master degree in Membrane Engineering

Overexpressing BDNF in Neural Stem Cells using non-viral gene delivery strategies

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Smart windows with permanent memory effect

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Role of surfactants in filtration and fouling of colloidal silica

The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities

Aqueous biphasic system based on cholinium ionic liquids: extraction of biologically active phenolic acids

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the plant kingdom. Due to their biological and pharmacological properties, they have been playing an important role in phytotherapy and consequently techniques for their separation and purification are in need. This thesis aims at exploring new sustainable separation processes based on ionic liquids (ILs) in the extraction of biologically active phenolic acids. For that purpose, three phenolic acids with similar chemical structures were selected: cinnamic acid, p-coumaric acid and caffeic acid. In the last years, it has been shown that ionic liquids-based aqueous biphasic systems (ABSs) are valid alternatives for the extraction, recovery and purification of biomolecules when compared to conventional ABS or extractions carried out with organic solvents. In particular, cholinium-based ILs represent a clear step towards a greener chemistry, while providing means for the implementation of efficient techniques for the separation and purification of biomolecules. In this work, ABSs were implemented using cholinium carboxylate ILs using either high charge density inorganic salt (K3PO4) or polyethylene glycol (PEG) to promote the phase separation of aqueous solutions containing three different phenolic acids. These systems allow for the evaluation of effect of chemical structure of the anion on the extraction efficiency. Only one imidazolium-based IL was used in order to establish the effect of the cation chemical structure. The selective extraction of one single acid was also researched. Overall, it was observed that phenolic acids display very complex behaviours in aqueous solutions, from dimerization to polymerization and also hetero-association are quite frequent phenomena, depending on the pH conditions. These phenomena greatly hinder the correct quantification of these acids in solution.

Production and characterization of electrospun composite fibers: confinement of thermosensitive microgels

Materials engineering focuses on the assembly of materials´ properties to design new products with the best performance. By using sub-micrometer size materials in the production of composites, it is possible to obtain objects with properties that none of their compounds show individually. Once three-dimensional materials can be easily customized to obtain desired properties, much interest has been paid to nanostructured poly-mers in order to build biocompatible devices. Over the past years, the thermosensitive microgels have become more common in the framework of bio-materials with potential applicability in therapy and/or diagnostics. In addition, high aspect ratio biopolymers fibers have been produced using the cost-effective method called electrospinning. Taking advantage of both microgels and electrospun fibers, surfaces with enhanced functionalities can be obtained and, therefore employed in a wide range of applications. This dissertation reports on the confinement of stimuli-responsive microgels through the colloidal electro-spinning process. The process mainly depends on the composition, properties and patterning of the precur-sor materials within the polymer jet. Microgels as well as the electrospun non-woven mats were investigated to correlate the starting materials with the final morphology of the composite fibers. PNIPAAm and PNIPAAm/Chitosan thermosensitive microgels with different compositions were obtained via surfactant free emulsion polymerization (SFEP) and characterized in terms of chemical structure, morphology, thermal sta-bility, swelling properties and thermosensitivity. Finally, the colloidal electrospinning method was carried out from spinning solutions composed of the stable microgel dispersions (up to a concentration of about 35 wt. % microgels) and a polymer solution of PEO/water/ethanol mixture acting as fiber template solution. The confinement of microgels was confirmed by Scanning Electron Microscopy (SEM). The electrospinning process was statistically analysed providing the optimum set of parameters aimed to minimize the fiber diameter, which give rise to electrospun nanofibers of PNIPAAm microgels/PEO with a mean fiber diameter of 63 ± 25 nm.

Coupling organic substrate removal to methane production in a fully biological microbial electrolysis cell

Microbial electrolysis cells (MECs) are an innovative and emerging technique based on the use of solid-state electrodes to stimulate microbial metabolism for wastewater treatment and simultaneous production of value-added compounds (such as methane). This research studied the performance of a two-chamber MEC in terms of organic matter oxidation (at the anode) and methane production (at the cathode). MEC‟s anode had been previously inoculated with an activated sludge, whereas the cathode chamber inoculum was an anaerobic sludge (containing methanogenic microorganisms). During the experimentation, the bioanode was continuously fed with synthetic solutions in anaerobic basal medium, at an organic load rate (OLR) of around 1 g L-1 d-1, referred to the chemical oxygen demand (COD). At the beginning (Run I), the feeding solution contained acetate and subsequently (Run II) it was replaced with a more complex solution containing soluble organic compounds other than acetate. For both conditions, the anode potential was controlled at -0.1 V vs. standard hydrogen electrode, by means of a potentiostat. During Run I, over 80% of the influent acetate was anaerobically oxidized at the anode, and the resulting electric current was recovered as methane at the cathode (with a cathode capture efficiency, CCE, accounting around 115 %). The average energy efficiency of the system (i.e., the energy captured into methane relative to the electrical energy input) under these conditions was over 170%. However, reactor‟s performance decreased over time during this run. Throughout Run II, a substrate oxidation over 60% (on COD basis) was observed. The electric current produced (57% of coulombic efficiency) was also recovered as methane, with a CCE of 90%. For this run the MEC‟s average energy efficiency accounted for almost 170 %. During all the experimentation, a very low biomass growth was observed at the anode whereas ammonium was transferred through the cationic membrane and concentrated at the cathode. Tracer experiments and scanning electron microscopy analyses were also carried out to gain a deeper insight into the reactor performance and also to investigate the possible reasons for partial loss of performance. In conclusion, this research suggests the great potential of MEC to successfully treat low-strength wastewaters, with high energy efficiency and very low sludge production.

A continuous solvent- and oil-free method to prepare injectable PEGylated fibrinogen cell-laden microparticles

Injectable biomaterials with in situ cross-linking reactions have been suggested to minimize the invasiveness associated with most implantation procedures. However, problems related with the rapid liquid-to-gel transition reaction can arise because it is difficult to predict the reliability of the reaction and its end products, as well as to mitigate cytotoxicity to the surrounding tissues. An alternative minimally invasive approach to deliver solid implants in vivo is based on injectable microparticles, which can be processed in vitro with high fidelity and reliability, while showing low cytotoxicity. Their delivery to the defect can be performed by injection through a small diameter syringe needle. We present a new methodology for the continuous, solvent- and oil-free production of photopolymerizable microparticles containing encapsulated human dermal fibroblasts. A precursor solution of cells in photo-reactive PEG-fibrinogen (PF) polymer was transported through a transparent injector exposed to light-irradiation before being atomized in a jet-in-air nozzle. Shear rheometry data provided the cross-linking kinetics of each PF/cell solution, which was then used to determine the amount of irradiation required to partially polymerize the mixture prior to atomization. The partially polymerized drops fell into a gelation bath for further polymerization. The system was capable of producing cell-laden microparticles with high cellular viability, with an average diameter of between 88.1 µm to 347.1 µm and a dispersity of between 1.1 and 2.4, depending on the parameters chosen.

Organic-inorganic nanostructured multilayers for calcium phosphate biomineralization

The weak fixation of biomaterials within the bone structure is one of the major reasons of implants failures. Calcium phosphate (CaP) coatings are used in bone tissue engineering to improve implant osseointegration by enhancing cellular adhesion, proliferation and differentiation, leading to a tight and stable junction between implant and host bone. It has also been observed that materials compatible with bone tissue either have a CaP coating or develop such a calcified surface upon implantation. Thus, the development of bioactive coatings becomes essential for further improvement of integration with the surrounding tissue. However, most of current applied CaP coatings methods (e.g. physical vapor deposition), cannot be applied to complex shapes and porous implants, provide poor structural control over the coating and prevent incorporation of bioactive organic compounds (e.g. antibiotics, growth factors) because of the used harsh processing conditions. Layer-by-layer (LbL) is a versatile technology that permits the building-up of multilayered polyelectrolyte films in mild conditions based on the alternate adsorption of cationic and anionic elements that can integrate bioactive compounds. As it is recognized in natureâ s biomineralization process the presence of an organic template to induce mineral deposition, this work investigate a ion based biomimetic method where all the process is based on LbL methodology made of weak natural-origin polyelectrolytes. A nanostructured multilayer component, with 5 or 10 bilayers, was produced initially using chitosan and chondroitin sulphate polyelectrolyte biopolymers, which possess similarities with the extracellular matrix and good biocompatibility. The multilayers are then rinsed with a sequential passing of solutions containing Ca2+ and PO43- ions. The formation of CaP over the polyelectrolyte multilayers was confirmed by QCM-D, SEM and EDX. The outcomes show that 10 polyelectrolyte bilayer condition behaved as a  better site for initiating the formation of CaP as the precipitation occur at earlier stages than in 5 polyelectrolyte bilayers one. This denotes that higher number of bilayers could hold the CaP crystals more efficiently. This work achieved uniform coatings that can be applied to any surface with access to the liquid media in a low-temperature method, which potentiates the manufacture of effective bioactive biomaterials with great potential in orthopedic applications.

Phenanthroimidazoles as fluorimetric and colourimetric chemosensors in aqueous solution

2,4,5-Triaryl-imidazoles are versatile compounds with application in medicine, due to their biological activity, and materials sciences, for their interesting optical properties. These properties can be tuned by careful selection of substituents at positions 2, 4 and 5: replacement of the aryl group by an heterocyclic group results in larger π-conjugated systems with improved optical properties for application in nonlinear optics, OLEDs, DNA intercalators, and chemosensors. Moreover, it is expected that introducing more conjugation and rigidity into the resulting system will further improve its properties. The development of chromo/fluorescent probes that are capable of detecting ions with high sensitivity and selectivity in aqueous media is currently a topic of strong interest and the design of heteroditopic receptors that contain two or more different binding sites for the simultaneous complexation of cationic and anionic guests is a emerging field of supramolecular chemistry. In this communication, we report the synthesis of new phenanthroimidazoles substituted at position 2 with arylthienyl or arylfuryl moieties possessing substituents of different electronic character, in order to tune the chromo/fluoro response in the presence of relevant anions and metal cations. Their photophysical properties and chemosensory ability were studied in acetonitrile and mixtures of acetonitrile and water, and selective detection of cyanide was achieved in aqueous mixtures for some of the derivatives.

Assessment of liposome disruption to quantify drug delivery in vitro

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34,580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.

One-step in situ synthesis of polyamide microcapsules with inorganic payload and their transformation into responsive thermoplastic composite materials

Well-dispersed loads of finely powdered metals, metal oxides, several carbon allotropes or nanoclays are incorporated into highly porous polyamide 6 microcapsules in controllable amounts via an original one-step in situ fabrication technique. It is based on activated anionic polymerization (AAP) of ε-caprolactam in a hydrocarbon solvent performed in the presence of the respective micro- or nanosized loads. The forming microcapsules with typical diameters of 25-50 µm entrap up to 40 wt% of load. Their melt processing produces hybrid thermoplastic composites. Mechanical, electric conductivity and magnetic response measurements show that transforming of in situ loaded microcapsules into composites by melt processing (MP) is a facile and rapid method to fabricate materials with high mechanical resistance and electro-magnetic characteristics sufficient for many industrial applications. This novel concept requires low polymerization temperatures, no functionalization or compatibilization of the loads and it is easy to scale up at industrial production levels.