Desenvolvimento de Anticorpos Pl��sticos para um Biomarcador do Cancro

O trabalho descrito compreende o desenvolvimento de um anticorpo pl��stico (MIP, do ingl��s Molecularly Imprinted Polymer) para o antig��nio carcinoembrion��rio (CEA, do ingl��s Carcinoembriogenic Antigen) e a sua aplica����o na constru����o de dispositivos port��teis, de tamanho reduzido e de baixo custo, tendo em vista a monitoriza����o deste biomarcador do cancro do colo-retal em contexto Point-of-Care (POC). O anticorpo pl��stico foi obtido por tecnologia de impress��o molecular orientada, baseada em eletropolimeriza����o sobre uma superf��cie condutora de vidro recoberto por FTO. De uma forma geral, o processo foi iniciado pela electropolimeriza����o de anilina sobre o vidro, seguindo-se a liga����o por adsor����o do biomarcador (CEA) ao filme de polianilina, com ou sem mon��meros carregados positivamente (Cloreto de vinilbenziltrimetilam��nio, VB). A ��ltima fase consistiu na electropolimeriza����o de o-fenilenodiamina (oPD) sobre a superf��cie, seguindo-se a remo����o da prote��na por clivagem de liga����es pept��dicas, com o aux��lio de tripsina. A efici��ncia da impress��o do biomarcador CEA no material polim��rico foi controlada pela prepara����o de um material an��logo, NIP (do ingl��s, Non-Imprinted Polymer), no qual nem a prote��na nem o mon��mero VB estavam presentes. Os materiais obtidos foram caracterizados quimicamente por t��cnicas de Infravermelho com Transformada de Fourier (FTIR, do ingl��s, Fourier Transform Infrared Spectroscopy) e microscopia confocal de Raman. Os materiais sensores preparados foram entretanto inclu��dos em membranas polim��ricas de Poli(cloreto de vinilo) (PVC) plastificado, para constru����o de sensores (biomim��ticos) seletivos a CEA, tendo-se avaliado a resposta anal��tica em diferentes meios. Obteve-se uma boa resposta potenciom��trica em solu����o tamp��o de ��cido 4-(2-hidroxietil)piperazina-1-etanosulf��nico (HEPES), a pH 4,4, com uma membrana seletiva baseada em MIP preparada com o mon��mero carregado VB. O limite de dete����o foi menor do que 42 pg/mL, observando-se um comportamento linear (versus o logaritmo da concentra����o) at�� 625 pg/mL, com um declive ani��nico igual a -61,9 mV/d��cada e r2>0,9974. O comportamento anal��tico dos sensores biomim��ticos foi ainda avaliado em urina, tendo em vista a sua aplica����o na an��lise de CEA em urina. Neste caso, o limite de dete����o foi menor do que 38 pg/mL, para uma resposta linear at�� 625 pg/mL, com um declive de -38,4 mV/d��cada e r2> 0,991. De uma forma geral, a aplica����o experimental dos sensores biomim��ticos evidenciou respostas exatas, sugerindo que os biossensores desenvolvidos prossigam estudos adicionais tendo em vista a sua aplica����o em amostras de indiv��duos doentes.

Desenvolvimento e aplica����o de um biossensor para monitoriza����o de creatinina, um biomarcador associado a doen��as emergentes

Este trabalho descreve o desenvolvimento de um material sensor para creatinina por impress��o molecular em estrutura polim��rica (MIP) e a sua aplica����o no desenvolvimento de um dispositivo de natureza potenciom��trica para a determina����o da mol��cula alvo em fluidos biol��gicos. A creatinina �� um dos biomarcadores mais utilizados no acompanhamento da doen��a renal, j�� que �� um bom indicador da taxa de filtra����o glomerular (TFG). Os materiais biomim��ticos desenhados para intera����o com a creatinina foram obtidos por polimeriza����o radicalar, recorrendo a mon��meros de ��cido metacr��clico ou de vinilpiridina e a um agente de reticula����o apropriado. De modo a aferir o efeito da impress��o da creatinina na resposta dos materiais MIP �� sua presen��a, foram tamb��m preparados e avaliados materiais de controlo, obtidos sem impress��o molecular (NIP). O controlo da constitui����o qu��mica destes materiais, incluindo a extra����o da mol��cula impressa, foi realizado por Espectroscopia de Raman e de Infravermelho com Transformada de Fourrier. A afinidade de liga����o entre estes materiais e a creatinina foi tamb��m avaliada com base em estudos cin��ticos. Todos os materiais descritos foram integrados em membranas selectivas de el��trodos seletivos de i��o, preparadas sem ou com aditivo i��nico lip��filo, de carga negativa ou positiva. A avalia����o das caracter��sticas gerais de funcionamento destes el��trodos, em meios de composi����o e pH diferentes, indicaram que as membranas com materiais impressos e aditivo ani��nico eram as ��nicas com utilidade anal��tica. Os melhores resultados foram obtidos em solu����o tamp��o Piperazine-N,N���-bis(2- ethanesulfonic acid), PIPES, de pH 2,8, condi����o que permitiu obter uma resposta quasi-Nernstiana, a partir de 1,6��10-5 mol L-1. Estes el��trodos demonstraram ainda uma boa selectividade ao apresentaram uma resposta preferencial para a creatinina quando na presen��a de ureia, carnitina, glucose, ��cido asc��rbico, albumina, cloreto de c��lcio, cloreto de pot��ssio, cloreto de s��dio e sulfato de magn��sio. Os el��trodos foram ainda aplicados com sucesso na an��lise de amostras sint��ticas de urina, quando os materiais sensores eram baseados em ��cido metacrilico, e soro, quando os materiais sensores utilizados eram baseados em vinilpiridina.

The materials, technique, conservation treatment and after-care of interior de um convento

Disserta����o apresentada na Faculdade de Ci��ncias e Tecnologia da Universidade Nova de Lisboa para a obten����o do grau de Mestre em Conserva����o e Restauro, especializa����o em pintura sobre tela

Haemoglobin smart plastic antibody material tailored with charged binding sites on silica nanoparticles: its application as an ionophore in potentiometric transduction

This work uses surface imprinting to design a novel smart plastic antibodymaterial (SPAM) for Haemoglobin (Hb). Charged binding sites are described here for the first time to tailor plastic antibody nanostructures for a large size protein such as Hb. Its application to design small, portable and low cost potentiometric devices is presented. The SPAM material was obtained by linking Hb to silica nanoparticles and allowing its ionic interaction with charged vinyl monomers. A neutral polymeric matrix was created around these and the imprinted protein removed. Additional materials were designed in parallel acting as a control: a neutral imprinted material (NSPAM), obtained by removing the charged monomers from the procedure, and the Non-Imprinted (NI) versions of SPAM and NSPAM by removing the template. SEM analysis confirmed the surface modification of the silica nanoparticles. All materials were mixed with PVC/plasticizer and applied as selective membranes in potentiometric transduction. Electromotive force (emf) variations were detected only for selective membranes having a lipophilic anionic additive in the membrane. The presence of Hb inside these membranes was evident and confirmed by FTIR, optical microscopy and Raman spectroscopy. The best performance was found for SPAM-based selective membranes with an anionic lipophilic additive, at pH 5. The limits of detection were 43.8 mg mL 1 and linear responses were obtained down to 83.8 mg mL 1, with an average cationic slope of +40 mV per decade. Good selectivity was also observed against other coexisting biomolecules. The analytical application was conducted successfully, showing accurate and precise results.

New modified electrochemical conductive paper support for BSA detection

Chemical sensors and biosensors are widely used to detect various kinds of protein target biomolecules. Molecularly Imprinted Polymers (MIPs) have raised great interest in this area, because these act as antibody-like recognition materials, with high affinity to the template molecule. Compared to natural antibodies, these are also of lower cost and higher stability. There are different types of supports used to carry MIP materials, mostly of these made of gold, favourably assembled on a Screen Printed Electrode (SPE) strategy. For this work a new kind of support for the sensing layer was developed: conductive paper. This support was made by modifying first cellulose paper with paraffin wax (to make it waterproof), and casting a carbon-ink on it afterwards, to turn it conductive. The SPAM approach previously reported in1 was employed herein to assemble to MIP sensing material on the conductive paper. The selected charged monomers were (vinylbenzyl) trimethlammonium chloride (positive charge) or vinylbenzoic acid (negative charge), used to generate binding positions with single-type charge (positive or negative). The non-specific binding area of the MIP layer was assembled by chronoamperometry-assisted polymerization (at 1 V, for 60, 120 or 180 seconds) of vinylbenzoate, cross-linked with ethylene glycol vinyl ether. The BSA biomolecules lying within the polymeric matrix were removed by Proteinase K action. All preparation stages of the MIP assembly were followed by FTIR, Raman spectroscopy and, electrochemical analysis. In general, the best results were obtained for longer polymerization times and positively charged binding sites (which was consistent with a negatively-charged protein under physiological pH, as BSA). Linear responses against BSA concentration ranged from 0.005 to 100 mg/mL, in PBS buffer standard solutions. The sensor was further calibrated in standard solutions that were prepared in synthetic or real urine, and the analytical response became more sensitive and stable. Compared to the literature, the detection capability of the developed device is better than most of the reported electrodes. Overall, the simplicity, low cost and good analytical performance of the BSA SPE device, prepared with positively charged binding positions, seems a suitable approach for practical application in clinical context. Further studies with real samples are required, as well as gathering with electronic-supporting devices to allow on-site readings.

Sol-Gel Chemistry in Biosensing Devices of Electrical Transduction: Application to CEA Cancer Biomarker

Sol-gel chemistry allows the immobilization of organic molecules of biological origin on suibtable solid supports, permitting their integration into biosensing devices widening the possibility of local applications. The present work is an application of this principle, where the link between electrical receptor platform and the antibody acting as biorecognition element is made by sol-gel chemistry. The immunosensor design was targeted for carcinoembryonic antigen (CEA), an important biomarker for screening the colorectal cancer, by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SVW). The device displayed linear behavior to CEA in EIS and in SWV assays ranging from 0.50 to 1.5ng/mL, and 0.25 to 1.5ng/mL, respectively. The corresponding detection limits were 0.42 and 0.043 ng/mL. Raman spectroscopy was used to characterize the surface modifications on the conductive platform (FTO glass). Overall, simple sol-gel chemistry was effective at the biosensing design and the presented approach can be a potential method for screening CEA in point-of-care, due to the simplicity of fabrication, short response time and low cost. - See more at: http://www.eurekaselect.com/127192/article#sthash.m1AWhINx.dpuf

Protein-responsive polymers for point-of-care detection of cardiac biomarker

This work describes a novel use for the polymeric film, poly(o-aminophenol) (PAP) that was made responsive to a specific protein. This was achieved through templated electropolymerization of aminophenol (AP) in the presence of protein. The procedure involved adsorbing protein on the electrode surface and thereafter electroploymerizing the aminophenol. Proteins embedded at the outer surface of the polymeric film were digested by proteinase K and then washed away thereby creating vacant sites. The capacity of the template film to specifically rebind protein was tested with myoglobin (Myo), a cardiac biomarker for ischemia. The films acted as biomimetic artificial antibodies and were produced on a gold (Au) screen printed electrode (SPE), as a step towards disposable sensors to enable point-of-care applications. Raman spectroscopy was used to follow the surface modification of the Au-SPE. The ability of the material to rebind Myo was measured by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The devices displayed linear responses to Myo in EIS and SWV assays down to 4.0 and 3.5 ��g/mL, respectively, with detection limits of 1.5 and 0.8 ��g/mL. Good selectivity was observed in the presence of troponin T (TnT) and creatine kinase (CKMB) in SWV assays, and accurate results were obtained in applications to spiked serum. The sensor described in this work is a potential tool for screening Myo in point-of-care due to the simplicity of fabrication, disposability, short time response, low cost, good sensitivity and selectivity.

A new biomimetic sensor for detecting carnitine, a potential biomarker in ovarian cancer

1st ASPIC International Congress

The potential of molecular imprinting as a biosensing devices for monitoring the CEA cancer biomarker

6th Graduate Student Symposium on Molecular Imprinting

Sol-gel biomimetic material designed to target CEA cancer biomarker

1st ASPIC International Congress

Effect of selenization conditions on the growth and properties of Cu2ZnSn(S,Se)4 thin films

The opto-electronic properties of copper zinc tin sulfide can be tuned to achieve better cell efficiencies by controlled incorporation of selenium. In this paper we report the growth of Cu2ZnSn(S,Se)4 (CZTSSe) using a hybrid process involving the sequential evaporation of Zn and sputtering of the sulfide precursors of Cu and Sn, followed by a selenization step. Two approaches for selenization were followed, one using a tubular furnace and the other using a rapid thermal processor. The effects of annealing conditions on the morphological and structural properties of the films were investigated. Scanning electron microscopy and energy dispersive spectroscopy were employed to investigate the morphology and composition of the films. Structural analyses were done using X-ray diffraction (XRD) and Raman spectroscopy. Structural analyses revealed the formation of CZTSSe. This study shows that regardless of the selenization method a temperature above 450 ��C is required for conversion of precursors to a compact CZTSSe layer. XRD and Raman analysis suggests that the films selenized in the tubular furnace are selenium rich whereas the samples selenized in the rapid thermal processor have higher sulfur content.

Printed electronics for ubiquitous computing applications

Disserta����o para obten����o do Grau de Doutor em Qu��mica

Treatment of a nineteenth century male portrait in oil including the characterisation of materials, technique and a study of the lead soap aggregation in the paint composite

Disserta����o para obten����o do Grau de Mestre em Conserva����o e Restauro

The brushstroke and materials of Amadeo de Souza-Cardoso combined in an authentication tool

Nowadays, authentication studies for paintings require a multidisciplinary approach, based on the contribution of visual features analysis but also on characterizations of materials and techniques. Moreover, it is important that the assessment of the authorship of a painting is supported by technical studies of a selected number of original artworks that cover the entire career of an artist. This dissertation is concerned about the work of modernist painter Amadeo de Souza-Cardoso. It is divided in three parts. In the first part, we propose a tool based on image processing that combines information obtained by brushstroke and materials analysis. The resulting tool provides qualitative and quantitative evaluation of the authorship of the paintings; the quantitative element is particularly relevant, as it could be crucial in solving authorship controversies, such as judicial disputes. The brushstroke analysis was performed by combining two algorithms for feature detection, namely Gabor filter and Scale Invariant Feature Transform. Thanks to this combination (and to the use of the Bag-of-Features model), the proposed method shows an accuracy higher than 90% in distinguishing between images of Amadeo���s paintings and images of artworks by other contemporary artists. For the molecular analysis, we implemented a semi-automatic system that uses hyperspectral imaging and elemental analysis. The system provides as output an image that depicts the mapping of the pigments present, together with the areas made using materials not coherent with Amadeo���s palette, if any. This visual output is a simple and effective way of assessing the results of the system. The tool proposed based on the combination of brushstroke and molecular information was tested in twelve paintings obtaining promising results. The second part of the thesis presents a systematic study of four selected paintings made by Amadeo in 1917. Although untitled, three of these paintings are commonly known as BRUT, Entrada and Coty; they are considered as his most successful and genuine works. The materials and techniques of these artworks have never been studied before. The paintings were studied with a multi-analytical approach using micro-Energy Dispersive X-ray Fluorescence spectroscopy, micro-Infrared and Raman Spectroscopy, micro-Spectrofluorimetry and Scanning Electron Microscopy. The characterization of Amadeo���s materials and techniques used on his last paintings, as well as the investigation of some of the conservation problems that affect these paintings, is essential to enrich the knowledge on this artist. Moreover, the study of the materials in the four paintings reveals commonalities between the paintings BRUT and Entrada. This observation is supported also by the analysis of the elements present in a photograph of a collage (conserved at the Art Library of the Calouste Gulbenkian Foundation), the only remaining evidence of a supposed maquete of these paintings. The final part of the thesis describes the application of the image processing tools developed in the first part of the thesis on a set of case studies; this experience demonstrates the potential of the tool to support painting analysis and authentication studies. The brushstroke analysis was used as additional analysis on the evaluation process of four paintings attributed to Amadeo, and the system based on hyperspectral analysis was applied on the painting dated 1917. The case studies therefore serve as a bridge between the first two parts of the dissertation.

Structural, optical and magnetic properties of pulsed laser deposited Co-doped ZnO films

Zn1���xCoxO films with different Co concentrations (with x=0.00, 0.10, 0.15, and 0.30) were grown by pulsed laser deposition (PLD) technique. The structural and optical properties of the films were investigated by grazing incidence X-ray diffraction (GIXRD), Raman spectroscopy and photoluminescence (PL). The magnetic properties were measured by conventional magnetometry using a SQUID and simulated by ab-initio calculations using Korring���Khon���Rostoker (KKR) method combined with coherent potential approximation (CPA). The effect of Co-doping on the GIXRD and Raman peaks positions, shape and intensity is discussed. PL studies demonstrate that Co-doping induces a decrease of the bandgap energy and quenching of the UV emission. They also suggest the presence of Zn interstitials when x���0.15. The 10% Co-doped ZnO film shows ferromagnetism at 390 K with a spontaneous magnetic moment ���4��10���5 emu and coercive field ���0.17 kOe. The origin of ferromagnetism is explained based on the calculations using KKR method.