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Abstract(s)
As alergias alimentares afetam uma percentagem considerável da população mundial (cerca de 10%) e tendem a aumentar, constituindo um fator preocupante para a segurança alimentar e para a saúde pública. Não existem tratamentos para a alergia alimentar, sendo as únicas opções a prevenção ou a não ingestão do produto. Deste modo, os rótulos devem indicar obrigatoriamente informação sobre os alergénios. Por este motivo, a avaliação rigorosa da presença de alergénios em produtos alimentares e a deteção de quantidades vestigiais é de extrema relevância. O desenvolvimento de metodologias analíticas capazes de detetar e quantificar alergénios em amostras alimentares, de forma rápida e in situ, com elevada sensibilidade, precisão e exatidão, pode auxiliar a indústria alimentar na rotulagem e garantir a conformidade com a legislação. Neste sentido, a presente dissertação reporta o desenvolvimento de um imunossensor eletroquímico para a análise do alergénio do amendoim, Ara h 1. Foram utilizados elétrodos serigrafados de carbono (SPCE) como plataformas transdutoras, nanoestruturados com nanodiamantes (NDs), e posteriormente biomodificados com um anticorpo de captura anti-Ara h 1. Na metodologia, efetuada em formato sandwich, após a ligação do alergénio ao anticorpo de captura, ocorre a interação entre um anticorpo de deteção biotinilado e o alergénio. Esta interação é reconhecida utilizando uma marca enzimática (fosfatase alcalina) conjugada com estreptavidina (S-AP). Após a adição do substrato enzimático juntamente com nitrato de prata, o sinal analítico é obtido através da oxidação de um dos produtos de reação (prata metálica) por voltametria de varrimento linear (LSV). Após a otimização das condições experimentais, o desempenho analítico do imunossensor foi avaliado, sendo estabelecida uma gama de linearidade entre 25 e 500 ng/mL de Ara h 1, com um limite de deteção de 0,78 ng/mL. Os resultados obtidos demonstram uma boa precisão (coeficiente de variação 𝑉𝑥0 (%) ≈ 10), num tempo total de ensaio de 2 horas e 20 minutos. A análise do analito em amostras alimentares foi realizada com sucesso e o método foi validado utilizando a técnica Enzyme-Linked Immunosorbent Assay (ELISA). As análises efetuadas permitem inferir que o imunossensor desenvolvido possibilita a deteção de quantidades vestigiais de amendoim em amostras alimentares, tendo elevado potencial de aplicação no controlo de qualidade em empresas e/ou fornecedores da área alimentar e por entidades reguladoras.
Food allergies affect a significant percentage of the world population (about 10%) with a tendency to increase, representing a worrying condition for food safety and public health. There are no treatments for food allergy and the only options are prevention or avoidance of food intake. Therefore, product labels must declare and clearly indicate information about allergens. For this reason, the rigorous evaluation of the presence of allergens in food products and the detection of trace amounts is of utmost importance. The development of analytical methodologies that can detect and quantify allergens in food samples in situ, with high sensitivity, precision, and accuracy, can support the food industry to efficiently label and ensure compliance with legislation. Thus, the present dissertation reports the development of an electrochemical immunosensor for the analysis of a major peanut allergen, Ara h 1. Screenprinted carbon electrodes (SPCE) were used as transducers, nanostructured with nanodiamonds (NDs), and biomodified with the capture antibody anti-Ara h 1. In a sandwich format methodology, the interaction between a biotinylated detection antibody and the allergen occurs after the specific recognition of the allergen by the capture antibody. The antibody-antigen interaction is recognized using an enzyme (alkaline phosphatase) conjugated with streptavidin (S-AP). The analytical signal is obtained by adding the enzymatic substrate, previously mixed with silver nitrate, followed by the oxidation of one of the reaction products (metallic silver) using linear sweep voltammetry (LSV). With the optimized experimental conditions, the analytical performance of the immunosensor was evaluated and a linear range was established between 25 and 500 ng/mL of Ara h 1, with a detection limit of 0.78 ng/mL. The obtained results confirm a good precision (coefficient of variation 𝑉𝑥0 (%) ≈ 10), in a total assay time of 2 hours and 20 minutes. The analysis of Ara h 1 in food samples was successfully performed and the method was validated using an Enzyme-Linked Immunosorbent Assay (ELISA). The obtained data demonstrate that the developed immunosensor enables the detection of trace amounts of peanuts in food samples, with a high potential for application in quality control in food companies and/or suppliers and by regulatory entities.
Food allergies affect a significant percentage of the world population (about 10%) with a tendency to increase, representing a worrying condition for food safety and public health. There are no treatments for food allergy and the only options are prevention or avoidance of food intake. Therefore, product labels must declare and clearly indicate information about allergens. For this reason, the rigorous evaluation of the presence of allergens in food products and the detection of trace amounts is of utmost importance. The development of analytical methodologies that can detect and quantify allergens in food samples in situ, with high sensitivity, precision, and accuracy, can support the food industry to efficiently label and ensure compliance with legislation. Thus, the present dissertation reports the development of an electrochemical immunosensor for the analysis of a major peanut allergen, Ara h 1. Screenprinted carbon electrodes (SPCE) were used as transducers, nanostructured with nanodiamonds (NDs), and biomodified with the capture antibody anti-Ara h 1. In a sandwich format methodology, the interaction between a biotinylated detection antibody and the allergen occurs after the specific recognition of the allergen by the capture antibody. The antibody-antigen interaction is recognized using an enzyme (alkaline phosphatase) conjugated with streptavidin (S-AP). The analytical signal is obtained by adding the enzymatic substrate, previously mixed with silver nitrate, followed by the oxidation of one of the reaction products (metallic silver) using linear sweep voltammetry (LSV). With the optimized experimental conditions, the analytical performance of the immunosensor was evaluated and a linear range was established between 25 and 500 ng/mL of Ara h 1, with a detection limit of 0.78 ng/mL. The obtained results confirm a good precision (coefficient of variation 𝑉𝑥0 (%) ≈ 10), in a total assay time of 2 hours and 20 minutes. The analysis of Ara h 1 in food samples was successfully performed and the method was validated using an Enzyme-Linked Immunosorbent Assay (ELISA). The obtained data demonstrate that the developed immunosensor enables the detection of trace amounts of peanuts in food samples, with a high potential for application in quality control in food companies and/or suppliers and by regulatory entities.
Description
Keywords
Imunossensor eletroquímico Elétrodo serigrafado de carbono (SPCE) Nanodiamantes Alergia alimentar Amendoim Alergénio Voltametria de varrimento linear Produtos alimentares Electrochemical immunosensor Screen-printed carbon electrode (SPCE) Nanodiamonds Food allergy Peanut Ara h 1 Allergen Linear sweep voltammetry Food products