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O consumo crescente de compostos farmacêuticos tem levado à libertação de inúmeros compostos não metabolizados para o meio ambiente, sobretudo através de fluidos biológicos, como a urina, ou outros compostos como fezes. Os efluentes resultantes devem ser tratados em Estação de Tratamento de Águas Residuais, porém estas instalações não têm capacidade ou infraestruturas adequadas para a remoção destes compostos. O trimetoprim (TMP) é um agente antimicrobiano e antiparasítico vastamente prescrito/administrado, existindo um elevado consumo por parte da população. Devido à sua alta prevalência em corpos aquáticos, este fármaco é considerado um poluente emergente. Deste modo, tornase imprescindível o desenvolvimento de métodos analíticos inovadores para a análise e deteção de TMP, os quais devem ter características vantajosas comparativamente aos métodos tradicionalmente usados. Os biossensores são uma alternativa atrativa devido à sua rápida resposta, fácil miniaturização e portabilidade. Neste trabalho foi desenvolvido um imunossensor eletroquímico para análise de TMP em amostras alimentares provenientes do meio aquático. Procedeu-se à construção de um sistema eletroquímico que contempla uma célula eletroquímica de tamanho reduzido, um elétrodo de trabalho de papel de fibra de carbono (CP) e um elétrodo de carbono vítreo (GCE) utilizado como elétrodo de referência acoplado ao elétrodo auxiliar. Este sistema permitiu o uso de um volume de eletrólito reduzido relativamente menor comparativamente aos sistemas convencionais. Para a construção do imunossensor, imobilizou-se 10 µL de anticorpo anti-TMP policlonal (concentração de 100 µg/mL), durante 20 minutos. Seguiu-se a adição de TMP à célula eletroquímica (método de adição padrão), sendo efetuada a medição em 5 mL de solução tampão PBS, utilizando como técnica a voltametria de onda quadrada (SWV). As características analíticas do imunoensaio foram avaliadas numa gama de concentrações entre 0,5 µM a 3,5 µM, verificando-se a linearidade entre 1,2 µM e 2,8 µM. Os limites de deteção e quantificação (LOD e LOQ, respetivamente) foram calculados, obtendo-se um LOD de 0,25 µM e um LOQ de 0,84 µM. Após a validação do imunossensor, foram analisadas amostras alimentares de bacalhau. Foi efetuado o estudo de recuperação após fortificação das amostras previamente tratadas de acordo com um procedimento estabelecido para o TMP e obtiveram-se recuperações entre 88 e 113%. O biossensor desenvolvido demonstrou resultados promissores para a análise de TMP em peixes. Apesar de serem necessárias melhorias adicionais, este método demonstra ter potencial para o desenvolvimento de um imunossensor capaz de efetuar a deteção rápida, eficaz e utilizando um sistema de tamanho reduzido comparativamente aos tradicionalmente empregues.
The rising of pharmaceutical compounds consumption has led to the release of many nonmetabolized compounds to the environment, mainly through biological fluids, like urine or even other compounds like feces. The resulting effluents must be treated in Wastewater Treatment Plants, although, these facilities do not have the capacity or suitable infrastructures capable of removing these compounds. TMP is an antimicrobial and antiparasitic agent that is commonly administrated. Given this, its prevalence is already high on water bodies due to the high consumption by the population, being then considered an emergent pollutant. It is then imperative the development of innovative analytical methods for the detection and analysis of this drug, which must have advantageous characteristics compared to those traditionally used. The biosensors are an attractive alternative due to their fast response, easy miniaturization, and portability. This work consisted of the development of an electrochemical immunosensor for the TMP analysis in food samples, using the Square Wave Voltammetry technique. A reduced size electrochemical cell was used with a system consisting of a carbon paper electrode and a glassy carbon electrode as the reference electrode coupled as auxiliary electrode. This system allowed the use of a relatively reduced electrolyte volume compared to those traditionally used. For the immunosensor construction, 10 µL of anti-TMP polyclonal antibody were immobilized (100 µg/mL concentration), for 20 minutes. Then, the TMP administration took place by the standard addition method, assessing a concentration range between 0,5 µM and 3,5 µM, which resulted in a linearity between 1,2 µM and 2,8 µM. It was then calculated the limit of detection (LOD) and limit of quantification (LOQ), being these 0,25 µM and 0,84 µM, respectively. After the immunosensor validation, cod samples were analyzed. A recovery assessment after fortification of the samples was performed according to the established procedure for the TMP, and recoveries between 88 and 113% were obtained. The biosensor showed promising results for the TMP analysis in fish. Although some additional improvements are necessary, this method demonstrates potential for the development of an immunosensor capable of performing rapid and effective detections, using a reduced size system compared to the conventionally used.
The rising of pharmaceutical compounds consumption has led to the release of many nonmetabolized compounds to the environment, mainly through biological fluids, like urine or even other compounds like feces. The resulting effluents must be treated in Wastewater Treatment Plants, although, these facilities do not have the capacity or suitable infrastructures capable of removing these compounds. TMP is an antimicrobial and antiparasitic agent that is commonly administrated. Given this, its prevalence is already high on water bodies due to the high consumption by the population, being then considered an emergent pollutant. It is then imperative the development of innovative analytical methods for the detection and analysis of this drug, which must have advantageous characteristics compared to those traditionally used. The biosensors are an attractive alternative due to their fast response, easy miniaturization, and portability. This work consisted of the development of an electrochemical immunosensor for the TMP analysis in food samples, using the Square Wave Voltammetry technique. A reduced size electrochemical cell was used with a system consisting of a carbon paper electrode and a glassy carbon electrode as the reference electrode coupled as auxiliary electrode. This system allowed the use of a relatively reduced electrolyte volume compared to those traditionally used. For the immunosensor construction, 10 µL of anti-TMP polyclonal antibody were immobilized (100 µg/mL concentration), for 20 minutes. Then, the TMP administration took place by the standard addition method, assessing a concentration range between 0,5 µM and 3,5 µM, which resulted in a linearity between 1,2 µM and 2,8 µM. It was then calculated the limit of detection (LOD) and limit of quantification (LOQ), being these 0,25 µM and 0,84 µM, respectively. After the immunosensor validation, cod samples were analyzed. A recovery assessment after fortification of the samples was performed according to the established procedure for the TMP, and recoveries between 88 and 113% were obtained. The biosensor showed promising results for the TMP analysis in fish. Although some additional improvements are necessary, this method demonstrates potential for the development of an immunosensor capable of performing rapid and effective detections, using a reduced size system compared to the conventionally used.
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Keywords
TMP Poluentes emergentes Imunossensor Eletroquímico CP SWV Amostras alimentares Emergent pollutants Immunosensor Electrochemical Food samples