Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

New molecularly-imprinted polymer for carnitine and its application as ionophore in potentiometric selective membranes

Carnitine (CRT) is a biological metabolite found in urine that contributes in assessingseveral disease conditions, including cancer. Novel quick screening procedures for CRT are therefore fundamental. This work proposes a novel potentiometric device where molecularly imprinted polymers (MIPs) were used as ionophores. The host-tailored sites were imprinted on a polymeric network assembled by radical polymerization of methacrylic acid (MAA) and trimethylpropane trimethacrylate (TRIM). Non-imprinted polymers (NIPs) were produced as control by removing the template from the reaction media. The selective membrane was prepared by dispersing MIP or NIP particles in plasticizer and poly(vinyl chloride), PVC, and casting this mixture over a solid contact support made of graphite. The composition of the selective membrane was investigated with regard to kind/amount of sensory material (MIP or NIP), and the need for a lipophilic additive. Overall, MIP sensors with additive exhibited the best performance, with near-Nernstian response down to ~ 1 × 10− 4 mol L− 1, at pH 5, and a detection limitof ~ 8 × 10− 5 mol L− 1. Suitable selectivity was found for all membranes, assessed by the matched potential method against some of the most common species in urine (urea, sodium, creatinine, sulfate, fructose and hemoglobin). CRT selective membranes including MIP materials were applied successfully to the potentiometric determination of CRT in urine samples.

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.

Host-Tailored Sensors for Leucomalachite Green Potentiometric Measurements

A new biomimetic sensor for leucomalachite green host-guest interactions and potentiometric transduction is presented. The artificial host was imprinted in methacrylic acid or acrylamido-2-methyl-1-propanesulfonic acid-based polymers. Molecularly imprinted particles were dispersed in 2-nitrophenyloctyl ether and trapped in poly(vinyl chloride). The potentiometric sensors exhibited a near-Nernstian response in steady state evaluations, with slopes and detection limits ranging from 45.8 to 81.2 mV and 0.28 to 1.01 , respectively. They were independent from the pH of test solutions within 3 to 5. Good selectivity was observed towards drugs that may contaminate water near fish cultures, such as oxycycline, doxycycline, enrofloxacin, trimethoprim, creatinine, chloramphenicol, and dopamine. The sensors were successfully applied to field monitoring of leucomalachite green in river samples. The method offered the advantages of simplicity, accuracy, applicability to colored and turbid samples, and automation feasibility.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0 mV decade−1 and 1.0 × 10−5 to 2.7 × 10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Biomimetic norfloxacin sensors made of molecularly-imprinted materials for potentiometric transduction

A biomimetic sensor for norfloxacin is presented that is based on host-guest interactions and potentiometric transduction. The artificial host was imprinted into polymers made from methacrylic acid and/or 2-vinyl pyridine. The resulting particles were entrapped in a plasticized poly(vinyl chloride) (PVC) matrix. The sensors exhibit near-Nernstian response in steady state evaluations, and detection limits range from 0.40 to 1.0 μg mL−1, respectively, and are independent of pH values at between 2 and 6, and 8 and 11, respectively. Good selectivity was observed over several potential interferents. In flowing media, the sensors exhibit fast response, a sensitivity of 68.2 mV per decade, a linear range from 79 μM to 2.5 mM, a detection limit of 20 μg mL−1, and a stable baseline. The sensors were successfully applied to field monitoring of norfloxacin in fish samples, biological samples, and pharmaceutical products.

Molecularly-Imprinted Materials for Potentiometric Transduction: Application to the Antibiotic Enrofloxacin

Enrofloxacin (ENR) is an antimicrobial used both in humans and in food producing species. Its control is required in farmed species and their surroundings in order to reduce the prevalence of antibiotic resistant bacteria. Thus, a new biomimetic sensor enrofloxacin is presented. An artificial host was imprinted in specific polymers. These were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibited a near-Nernstian response. Slopes expressing mV/Δlog([ENR]/M) varied within 48–63. The detection limits ranged from 0.28 to 1.01 µg mL−1. Sensors were independent from the pH of test solutions within 4–7. Good selectivity was observed toward potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin, and tetracycline. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ± 0.7%), fast response, good sensitivity (47 mV/Δlog([ENR]/M), wide linear range (1.0 × 10−5–1.0 × 10−3 M), low detection limit (0.9 µg mL−1), and a stable baseline for a 5 × 10−2 M acetate buffer (pH 4.7) carrier. The sensors were used to analyze fish samples. The method offered the advantages of simplicity, accuracy, and automation feasibility. The sensing membrane may contribute to the development of small devices allowing in vivo measurements of enrofloxacin or parent-drugs.

Biomimetic Sensor Potentiometric System for Doxycycline Antibiotic Using a Molecularly Imprinted Polymer as an Artificial Recognition Element

Molecular imprinting is a useful technique for the preparation of functional materials with molecular recognition properties. A Biomimetic Sensor Potentiometric System was developed for assessment of doxycycline (DOX) antibiotic. The molecularly imprinted polymer (MIP) was synthesized by using doxycycline as a template molecule, methacrylic acid (MAA) and/or acrylamide (AA) as a functional monomer and ethylene glycol dimethacrylat (EGDMA) as a cross-linking agent. The sensing elements were fabricated by the inclusion of DOX imprinted polymers in polyvinyl chloride (PVC) matrix. The sensors showed a high selectivity and a sensitive response to the template in aqueous system. Electrochemical evaluation of these sensors under static (batch) mode of operation reveals near-Nernstian response. MIP/MAA membrane sensor was incorporated in flow-through cells and used as detectors for flow injection analysis (FIA) of DOX. The method has the requisite accuracy, sensitivity and precision to assay DOX in tablets and biological fluids.

Selective recognition in potentiometric transduction of amoxicillin by molecularly imprinted materials

The indiscriminate use of antibiotics in food-producing animals has received increasing attention as a contributory factor in the international emergence of antibiotic-resistant bacteria (Woodward in Pesticide, veterinary and other residues in food, CRC Press, Boca Raton, 2004). Numerous analytical methods for quantifying antibacterial residues in edible animal products have been developed over years (Woodward in Pesticide, veterinary and other residues in food, CRC Press, Boca Raton, 2004; Botsoglou and Fletouris in Handbook of food analysis, residues and other food component analysis, Marcel Dekker, Ghent, 2004). Being Amoxicillin (AMOX) one of those critical veterinary drugs, efforts have been made to develop simple and expeditious methods for its control in food samples. In literature, only one AMOX-selective electrode has been reported so far. In that work, phosphotungstate:amoxycillinium ion exchanger was used as electroactive material (Shoukry et al. in Electroanalysis 6:914–917, 1994). Designing new materials based on molecularly imprinted polymers (MIPs) which are complementary to the size and charge of AMOX could lead to very selective interactions, thus enhancing the selectivity of the sensing unit. AMOX-selective electrodes used imprinted polymers as electroactive materials having AMOX as target molecule to design a biomimetic imprinted cavity. Poly(vinyl chloride), sensors of methacrylic acid displayed Nernstian slopes (60.7 mV/decade) and low detection limits (2.9 × 10−5 mol/L). The potentiometric responses were not affected by pH within 4–5 and showed good selectivity. The electrodes were applied successfully to the analysis of real samples.

Otimização de um processo produtivo e formulação de um novo produto utilizando ferramentas estatísticas

Na tentativa de se otimizar o processo de fabrico associado a uma tinta base aquosa (TBA), para minimizar os desvios de viscosidade final verificados, e de desenvolver um novo adjuvante plastificante para betão, recorreu-se a métodos e ferramentas estatísticas para a concretização do projeto. Relativamente à TBA, procedeu-se numa primeira fase a um acompanhamento do processo de fabrico, a fim de se obter todos os dados mais relevantes que poderiam influenciar a viscosidade final da tinta. Através de uma análise de capacidade ao parâmetro viscosidade, verificou-se que esta não estava sempre dentro das especificações do cliente, sendo o cpk do processo inferior a 1. O acompanhamento do processo resultou na escolha de 4 fatores, que culminou na realização de um plano fatorial 24. Após a realização dos ensaios, efetuou-se uma análise de regressão a um modelo de primeira ordem, não tendo sido esta significativa, o que implicou a realização de mais 8 ensaios nos pontos axiais. Com arealização de uma regressão passo-a-passo, obteve-se uma aproximação viável a um modelo de segunda ordem, que culminou na obtenção dos melhores níveis para os 4 fatores que garantem que a resposta viscosidade se situa no ponto médio do intervalo de especificação (1400 mPa.s). Quanto ao adjuvante para betão, o objetivo é o uso de polímeros SIKA ao invés da matériaprima comum neste tipo de produtos, tendo em conta o custo final da formulação. Escolheram-se 3 fatores importantes na formulação do produto (mistura de polímeros, mistura de hidrocarbonetos e % de sólidos), que resultou numa matriz fatorial 23. Os ensaios foram realizados em triplicado, em pasta de cimento, um para cada tipo de cimento mais utilizado em Portugal. Ao efetuar-se a análise estatística de dados obtiveram-se modelos de primeira ordem para cada tipo de cimento. O processo de otimização consistiu em otimizar uma função custo associada à formulação, garantindo sempre uma resposta superior à observada pelo produto considerado padrão. Os resultados foram animadores uma vez que se obteve para os 3 tipos de cimentocustos abaixo do requerido e espalhamento acima do observado pelo padrão.

Re-Use Assessment of Thermoset Composite Wastes as Aggregate and Filler Replacement for Concrete-Polymer Composite Materials: A case study regarding GFRP pultrusion wastes

Glass fibre-reinforced plastics (GFRP), nowadays commonly used in the construction, transportation and automobile sectors, have been considered inherently difficult to recycle due to both the cross-linked nature of thermoset resins, which cannot be remoulded, and the complex composition of the composite itself, which includes glass fibres, polymer matrix and different types of inorganic fillers. Hence, to date, most of the thermoset based GFRP waste is being incinerated or landfilled leading to negative environmental impacts and additional costs to producers and suppliers. With an increasing awareness of environmental matters and the subsequent desire to save resources, recycling would convert an expensive waste disposal into a profitable reusable material. In this study, the effect of the incorporation of mechanically recycled GFRP pultrusion wastes on flexural and compressive behaviour of polyester polymer mortars (PM) was assessed. For this purpose, different contents of GFRP recyclates (0%, 4%, 8% and 12%, w/w), with distinct size grades (coarse fibrous mixture and fine powdered mixture), were incorporated into polyester PM as sand aggregates and filler replacements. The effect of the incorporation of a silane coupling agent was also assessed. Experimental results revealed that GFRP waste filled polymer mortars show improved mechanical behaviour over unmodified polyester based mortars, thus indicating the feasibility of GFRP waste reuse as raw material in concrete-polymer composites.

Otimização da Hidrofugação do Couro Wetwhite

O processo de hidrofugação é definido como um tratamento efetuado com gorduras e agentes químicos, nomeadamente polímeros e acrílicos que dão características de lubrificação e propriedades hidrofóbicas ao material, cujas etapas são adicionadas na fase de recurtume. Um dos objetivos principais foi a obtenção de um couro com alto grau hidrofóbico, isto é um couro com baixo poder de absorção da água, resistência a sua penetração e conservação da permeabilidade ao vapor de água, sem a utilização do sal de crómio na fase de fixação, e mais pelas problemáticas ambientais que existem na atualidade com o uso deste metal no mundo dos couros. É de salientar que, o único parâmetro que foi possível analisar, de acordo com a disponibilidade do laboratório foi a percentagem de absorção da água. Portanto, para este trabalho escrito foram desenhadas uma série de ensaios, onde se testaram inúmeros produtos utilizados na indústria de produção dos couros, alterando-se fatores de temperatura, dosagem, posição dos produtos aplicados, tempos, entre outros parâmetros, de modo a obter um couro com melhores resultados na percentagem de absorção. A hidrofugação é um processo bastante delicado e mais quando se tenta produzir um couro isento de metais e com alto poder hidrofóbico. Os resultados obtidos experimentalmente apontam para uma percentagem de absorção de cerca de 30% para espaços de tempo de 10 minutos, utilizando sempre sais de alumínio na etapa de fixação, pelo que ainda se requer mais investigação para obter um couro wet-white sem adição de sais metálicas e com alto poder de absorção.

Produção de biodiesel a partir de pastas de gordura e resíduo de glicerol

O principal objectivo deste trabalho consistiu na produção de biodiesel a partir de resíduos fornecidos pela Empresa SOCIPOLE, S.A. Esta, forneceu, pastas de gordura (depósito formado nos óleos usados), só aproveitadas pela empresa para a produção de sabões, e resíduo de glicerol proveniente da produção de biodiesel. Com as pastas de gordura, foi feito um estudo onde se variaram alguns parâmetros, tais como: o tipo de reactor, catalisador, temperatura, agitação, tempo de reacção, razão molar gordura:metanol e condições de secagem. O estudo mais pormenorizado foi efectuado com o NaOH, como catalisador. Foram feitos vários ensaios para os tempos de reacção de 30minutos, 1hora e 2horas, a 65ºC e razão molar gordura:metanol 1:6, tendo-se obtido respectivamente 86,7; 88,1 e 87,5% para o teor de ésteres. No caso da resina catiónica forte como catalisador, aparentemente não houve reacção ao fim de 12h, enquanto no ensaio que utilizou o ácido sulfúrico, o teor de ésteres foi de 82,6%, ao fim de 12h com razão molar gordura:metanol igual a 1:11. Quanto à produção de biodiesel a partir do resíduo de ácidos gordos resultante da purificação do resíduo de glicerol, foi feito um estudo preliminar usando diferentes catalisadores, uma resina catiónica forte, ácido sulfúrico e zircónia sulfatada. Este resíduo continha cerca de 60% de ácidos gordos e aproximadamente 39% de ésteres metílicos. Dos três catalisadores usados, o que permitiu obter maior teor de ésteres foi a zircónia sulfatada, tendo-se obtido 82,9% trabalhando a 60ºC durante 18horas com agitação de 230rpm e razão molar ácidos gordos:metanol de 1:12 . O ensaio em que se obteve menor percentagem de ésteres (56,4%) foi com resina catiónica. Com as duas matérias-primas obteve-se sempre um teor de ésteres, no produto final, abaixo dos 96,5% estabelecidos pela Norma Europeia EN 14103. Estes resultados reflectem provavelmente a composição complexa das matérias-primas. As pastas de gordura podem conter compostos não esterificáveis, solúveis no biodiesel, ou mais dificilmente esterificáveis nas condições operacionais usadas. Já o resíduo de ácidos gordos apresenta uma cor escura o que indica a existência de compostos corados, talvez polímeros. Portanto, para se tirar conclusões fundamentadas é necessário fazer análises mais completas das pastas de gordura e do resíduo de ácidos gordos, assim como ao biodiesel produzido de forma a identificar as possíveis contaminações existentes nos produtos com esta proveniência.

Selective synthesis under microwave irradiation of new monomers for potential applications in PDLC films

Dissertation to obtain the degree of master in Bioorganic

Valorização da Drêche Cervejeira: Fermentação de Pentoses

O presente trabalho tem como objetivo a otimização da etapa de fermentação dos açúcares obtidos a partir da drêche cervejeira para produção do bioetanol através da utilização das leveduras Pichia stipitis NCYC 1541 e Kluyveromyces marxianus NCYC 2791 como agentes fermentativos. O meio de cultura usado para manter as culturas destas leveduras foi Yeast Extract Peptone Dextrose (YEPD). O principal propósito deste trabalho foi o de encontrar alternativas aos combustíveis fósseis, pautando-se por soluções inofensivas para o meio ambiente e sustentáveis. Assim, o trabalho está dividido em quatro etapas: 1) caraterização química e biológica da drêche; 2) pré-tratamento ácido e hidrólise enzimática para primeiramente quebrar as moléculas de lenhina que envolvem os polímeros de celulose e hemicelulose e em seguida romper as ligações poliméricas destas macromoléculas por ação enzimática e transforma-las em açúcares simples, respetivamente, obtendo-se então a glucose, a maltose, a xilose e a arabinose; e, por último, 3) otimização da etapa de fermentação da glucose, maltose e das pentoses que constitui a condição essencial para se chegar à síntese do bioetanol de um modo eficiente e sustentável e 4) a recuperação do bioetanol produzido por destilação fracionada. A quantificação dos açúcares libertados no processo foi feita recorrendo a análises por cromatografia líquida de alta eficiência (HPLC). Neste estudo foram identificados e quantificados cinco açúcares: Arabinose, Glucose, Maltose, Ribose e Xilose. Na etapa de pré-tratamento e hidrólise enzimática foram usados os ácidos clorídrico (HCl) e nítrico (HNO3) com a concentração de 1% (m/m), e as enzimas Glucanex 100g e Ultraflo L. Foram testadas seis condições de pré-tratamento e hidrólise enzimática, alterando os parâmetros tempo de contacto e razão enzimas/massa de drêche, respetivamente, e mantendo a temperatura (50 ºC), velocidade de agitação (75 rpm) e concentração dos ácidos (1% (m/m)). No processamento de 25 g de drêche seca com 0,5 g de Glucanex, 0,5 mL de Ultraflo e um tempo de reação de 60 minutos para as enzimas foi obtida uma eficiência de 15%, em hidrolisado com 6% da celulose. Realizou-se a fermentação do hidrolisado resultante do pré-tratamento ácido e hidrólise enzimática de drêche cervejeira e de meios sintéticos preparados com os açúcares puros, usando as duas estirpes selecionadas para este estudo: Pichia stipitis NCYC 1541 e Kluyveromyces marxianus NYCY 2791. As eficiências de fermentação dos açúcares nos meios sintéticos foram superiores a 80% para ambas as leveduras. No entanto, as eficiências de fermentação do hidrolisado da drêche foram de 45,10% pela Pichia stipitis e de 36,58 para Kluyveromyces marxianus, para um tempo de fermentação de 72 horas e à temperatura de 30 °C. O rendimento teórico em álcool no hidrolisado da drêche é de 0,27 g/g, três vezes maior do que o real (0,0856 g/g), para Pichia stipitis e de 0,19 g/g seis vezes maior do que o real (0,0308 g/g), para a Kluyveromyces marxianus.