Desenvolvimento de metodologias e dispositivos analíticos com deteção ótica para a indústria agroalimentar

Tese de doutoramento, Química (Química Física), Universidade de Lisboa, Faculdade de Ciências, 2016A indústria agroalimentar encontra-se em grande expansão e a sua monotorização é uma inevitabilidade mais que evidente. Com imposições ditadas por regulamentações ou pelo mercado, existe uma necessidade constante na implementação de sistemas de qualidade fiáveis, com tempos de resposta adequados e se possível de baixo custo. Esta necessidade, é o motor para o aparecimento de novas técnicas e equipamentos miniaturizados com vista à quantificação e deteção de compostos químicos e biológicos de interesse neste mercado. Neste contexto esta dissertação de doutoramento inserido no meio empresarial visou o estudo e desenvolvimento de sensores analíticos miniaturizados, para monotorização de diferentes parâmetros na indústria agroalimentar. O trabalho desenvolvido pode ser resumido em três grandes temas: contribuição para o desenvolvimento de uma plataforma para a construção de biossensores óticos; otimização de um equipamento e respetivo imunoensaio miniaturizado para a deteção de Ocratoxina A e desenvolvimento de sistemas analíticos para a quantificação de pH e azoto assimilável em vinho. Perspetivando a construção de uma plataforma de biossensores óticos, com todos os reagentes imobilizados, efetuou-se o estudo da modificação de superfícies (ITO e Au) recorrendo a polielectrólitos funcionalizados com ferroceno para a mediação da reação de eletroquimioluminescência do sistema luminol/H2O2. A mediação por diferentes derivados de ferroceno foi demonstrada em solução, e otimizada tendo em conta determinados fatores tais como pH, força iónica, concentração de reagentes (razão luminol/ferroceno) e método de perturbação eletroquímica no processo de eletroquimioluminescência. Sintetizou-se um polieletrólito catiónico funcionalizado com o grupo funcional ferroceno, tendo a sua adsorção electroestática sido avaliada em ITO e ouro previamente modificado com uma monocamada de 3-mercaptopropanosulfonato. A modificação dos elétrodos com recurso ao processo de montagem de camada por camada foi estudada, bem como a sua estabilidade a diferentes pH. Os elétrodos modificados aparentam uma elevada estabilidade eletroquímica quando avaliados em meios acídicos em oposição ao observado quando perturbados em soluções básicas, ideais para a intensificação do sinal ótico. No âmbito da participação no projeto europeu “Demotox” desenvolveu-se um protótipo pré-industrial para a deteção de Ocratoxina A (OTA) em cereais e vinho, recorrendo a um ensaio imunoenzimático em suporte sólido, baseado no princípio da interação específica entre anticorpo e antigénio (OTA). A miniaturização e alteração do formato do imunoensaio de competitivo indireto para competitivo direto bem como a otimização efetuada permitiu diminuir o tempo de ensaio, o número de passos no processo de deteção bem como revelar-se eficiente para a deteção de OTA em vinho no limite legal (2 ng/mL). A validação do equipamento, como sistema analítico semi-quantitativo de resposta binária (sim/não; contaminado/não-contaminado) foi realizado de acordo com as normas europeias em vigor, para cereais de alimentação animal, apresentando resultados bastante satisfatórios e passiveis de serem utilizados para o limite legal estabelecido 250 ppb de OTA. No terceiro tema abordado procedeu-se à construção de um elétrodo de pH de estado sólido e ao desenvolvimento de técnicas para a quantificação de azoto assimilável no vinho. A construção de um elétrodo de pH de estado sólido baseou-se na deposição eletroquímica de óxidos de irídio. A otimização do método de crescimento eletroquímico (potenciodinâmico e galvanoestático) foi efetuado contemplando o tempo de resposta e respetiva estabilidade dos depósitos obtidos, em soluções de diferentes pH. A resposta otimizada revelou um comportamento super-Nernstinian e boa linearidade com o pH em soluções tampão. O sinal obtido em amostras reais, vinho, apresenta uma elevada interferência de compostos redox presentes. Tal interferência foi minimizada pela deposição adicional de membranas de polifenol ou nafion, sem ser no entanto totalmente colmatada, estando em aberto a sua otimização. Para a determinação de iões amónio construiu-se um elétrodo seletivo de iões amónio (ISE) tendo sido avaliados vários parâmetros na sua construção, tais como: número de camadas da membrana seletiva, a presença e natureza de um condutor misto iónico/eletrónico. O elétrodo demonstrou excelente desempenho em solução tampão em oposição com o observado em amostras reais, devido à presença de uma elevada concentração de iões potássio no vinho. Com base nas propriedades de fluorescência de emissão de isoindolos provenientes da reação de o-ftaldeído, um nucleófilo e NH4 + ou α-aminoácidos, e utilizando como base a plataforma de medida Enosense® criada pela Lumisense,Lda desenvolveram-se dois métodos de medida, um para a determinação de iões amónio e outro para a determinação de aminoácidos em amostras de vinho.The agrifood industry is in expansion and its monitoring is an inevitability more than evident. With impositions dictated simultaneously by legal regulations and market, there is a constant need for the implementation of reliable control quality systems, with adequate response times and whenever possible with low cost. This need, is the drive for the development of new miniaturized analytical techniques and equipments aimed for the quantification and detection of chemical and biological compounds of interest in this market. In this context this doctoral dissertation aimed at the study and development of miniaturized analytical sensors for monitoring different parameters in the agribusiness industry. The work can be subdivided into three main themes: contribution to the development of a platform for the construction of optical biosensors, optimization of an equipment and corresponding miniaturized immunoassay for the detection of Ochratoxin A and development of analytical systems for the measurement of pH and yeast assimilable nitrogen in wine. Aiming at the construction of a platform for optical biosensors, with all the reagents immobilized on an electrode, a study was conducted on surface modification (IndiumTin Oxide (ITO) and Au) by ferrocene functionalized polyelectrolytes for the mediation of the electrochemiluminescence reaction between luminol and H2O2. The mediation of distinct ferrocenes derivatives was demonstrated in solution, and the experimental conditions were optimized considering the pH, ionic strength, concentration of reagents (molar fraction luminol/ferrocene) and the applied electrochemical method in the ECL process. The electrostatic adsorption of the synthetized ferrocene functionalized cationic polyelectrolyte was evaluated in ITO and gold surfaces, where the latter was previously modified with a self-assembled monolayer of (3-mercaptopropyl)sulfonate monolayer. The modification of the electrode surfaces with thin films, formed via the adsorption of a layer-by-layer assembly of charged polyelectrolytes, revealed a high electrochemical stability in acidic media as opposed to the observed when studied in basic solutions, which are ideal for the intensification of the optical signal. In the context of Lumisense participation in a European project "Demotox", a preindustrial prototype was developed for the detection of Ochratoxin A (OTA) in cereals and wine, by means of an enzyme-linked immunosorbent assay in solid support, based on the principle of specific interaction between antibody and antigen (OTA). The miniaturization and the format change of the immunoassay from an indirect competitive to a direct competitive immunoassay, as well as its performance optimization, allowed to decrease the total assay time, the number of involved steps, and also enabled OTA detection in wine at the legal limit (2 ng/mL). The equipment validation, as an analytical semi-quantitative system based on a binary response (yes/no; contaminated/non-contaminated) was performed in accordance to European standards for cereals used for animal feed, presenting very satisfactory results and allow it to be used for the legal limit established for OTA of 250 ppb. The third theme concerned the construction of a solid state pH probe, as well as the development of optical methods for the quantification of yeast assimilable nitrogen in wine. The pH probe was constructed based on the electrochemical deposition of iridium oxides. The optimization of the electrochemical growth methods (potentiodynamic and galvanostatic) was performed by considering the stability and response time of the obtained deposits, at solutions with different pH. The optimized answer showed super-Nernstinan response with good linearity in buffer solutions. The signal obtained in real wine samples reveals a high interference of the redox compounds present in wine matrix. Such interference was slightly minimized by adding a membrane of polyphenol or nafion, without being totally surpassed, opening space for further optimization. An ion solid state selective electrode probe was developed for the determination of ammonium ions in wine. A number of parameters have been optimized during the electrode construction, such as number of layers of the selective membrane, the presence and nature of a ionic/electronic conductive layer. The probe demonstrated excellent performance in buffer solution in opposition with the observed in real samples, due to the presence of a high concentration of potassium ions in the wine. Based on the fluorescence properties of the isoindole products, formed during the reaction of o-phthalaldehyde, a nucleophile and NH4 + or α-amino acids, and using Enosense® platform, developed by Lumisense Lda for optical detection, two analytical methodologies were successfully developed, one for the determination of ammonia ions and another for the determination of amino acids in wine samples. The agrifood industry is in expansion and its monitoring is an inevitability more than evident. With impositions dictated simultaneously by legal regulations and market, there is a constant need for the implementation of reliable control quality systems, with adequate response times and whenever possible with low cost. This need, is the drive for the development of new miniaturized analytical techniques and equipments aimed for the quantification and detection of chemical and biological compounds of interest in this market. In this context this doctoral dissertation aimed at the study and development of miniaturized analytical sensors for monitoring different parameters in the agribusiness industry. The work can be subdivided into three main themes: contribution to the development of a platform for the construction of optical biosensors, optimization of an equipment and corresponding miniaturized immunoassay for the detection of Ochratoxin A and development of analytical systems for the measurement of pH and yeast assimilable nitrogen in wine. Aiming at the construction of a platform for optical biosensors, with all the reagents immobilized on an electrode, a study was conducted on surface modification (IndiumTin Oxide (ITO) and Au) by ferrocene functionalized polyelectrolytes for the mediation of the electrochemiluminescence reaction between luminol and H2O2. The mediation of distinct ferrocenes derivatives was demonstrated in solution, and the experimental conditions were optimized considering the pH, ionic strength, concentration of reagents (molar fraction luminol/ferrocene) and the applied electrochemical method in the ECL process. The electrostatic adsorption of the synthetized ferrocene functionalized cationic polyelectrolyte was evaluated in ITO and gold surfaces, where the latter was previously modified with a self-assembled monolayer of (3-mercaptopropyl)sulfonate monolayer. The modification of the electrode surfaces with thin films, formed via the adsorption of a layer-by-layer assembly of charged polyelectrolytes, revealed a high electrochemical stability in acidic media as opposed to the observed when studied in basic solutions, which are ideal for the intensification of the optical signal. In the context of Lumisense participation in a European project "Demotox", a preindustrial prototype was developed for the detection of Ochratoxin A (OTA) in cereals and wine, by means of an enzyme-linked immunosorbent assay in solid support, based on the principle of specific interaction between antibody and antigen (OTA). The miniaturization and the format change of the immunoassay from an indirect competitive to a direct competitive immunoassay, as well as its performance optimization, allowed to decrease the total assay time, the number of involved steps, and also enabled OTA detection in wine at the legal limit (2 ng/mL). The equipment validation, as an analytical semi-quantitative system based on a binary response (yes/no; contaminated/non-contaminated) was performed in accordance to European standards for cereals used for animal feed, presenting very satisfactory results and allow it to be used for the legal limit established for OTA of 250 ppb. The third theme concerned the construction of a solid state pH probe, as well as the development of optical methods for the quantification of yeast assimilable nitrogen in wine. The pH probe was constructed based on the electrochemical deposition of iridium oxides. The optimization of the electrochemical growth methods (potentiodynamic and galvanostatic) was performed by considering the stability and response time of the obtained deposits, at solutions with different pH. The optimized answer showed super-Nernstinan response with good linearity in buffer solutions. The signal obtained in real wine samples reveals a high interference of the redox compounds present in wine matrix. Such interference was slightly minimized by adding a membrane of polyphenol or nafion, without being totally surpassed, opening space for further optimization. An ion solid state selective electrode probe was developed for the determination of ammonium ions in wine. A number of parameters have been optimized during the electrode construction, such as number of layers of the selective membrane, the presence and nature of a ionic/electronic conductive layer. The probe demonstrated excellent performance in buffer solution in opposition with the observed in real samples, due to the presence of a high concentration of potassium ions in the wine. Based on the fluorescence properties of the isoindole products, formed during the reaction of o-phthalaldehyde, a nucleophile and NH4 + or α-amino acids, and using Enosense® platform, developed by Lumisense Lda for optical detection, two analytical methodologies were successfully developed, one for the determination of ammonia ions and another for the determination of amino acids in wine samples