Síntese e aplicação de polímeros com impressão molecular (MIP) e uso de aptâmeros na construção de dispositivos de análise rápida do tipo fluxo lateral para determinação de tetraciclina
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2021-06-22
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Universidade Estadual Paulista (Unesp)
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Os contaminantes emergentes são uma das classes de compostos liberados no meio ambiente que ocasionam poluição no ambiente terrestre, aquático e atmosférico; além de também estarem presentes nos alimentos e águas para consumo humano. Os fármacos, principalmente os antibióticos, estão entre os mais encontrados no meio ambiente. A tetraciclina pertence a uma classe de antibióticos abundantemente aplicados em uso humano e veterinário devido seu amplo espectro de ação e, por conseguinte, resíduos desse medicamento são facilmente detectados no ambiente. Como a exposição contínua a medicamentos pode ser prejudicial à saúde humana, métodos rápidos e seletivos para detectá-los são essenciais. Testes rápidos do tipo fluxo lateral foram desenvolvidos para determinação de tetraciclina: em um dos testes foi utilizado polímeros molecularmente impressos (MIP) e no outro aptâmeros, ambos os componentes como fase de reconhecimento seletivo. Inicialmente foram sintetizados três tipos diferentes de MIP: tradicional, core-shell e oco (de alta porosidade). Os materiais foram caracterizados por técnicas como microscopia eletrônica de varredura e espectroscopia no infravermelho. Após otimizadas as melhores condições de análise, o MIP oco apresentou resultados superiores para identificação da tetraciclina: capacidade máxima de ligação de 5,6 mg g-1 e constante de afinidade de 0,2 L mg-1. O MIP de alta porosidade também apresentou resultados satisfatórios nos ensaios de seletividade, com retenção sempre superior para tetraciclina frente a outros fármacos. Foram avaliados diferentes tipos de amostras fortificadas com tetraciclina, e nelas o MIP apresentou recuperação na faixa de 74 ‒ 96%; com faixa linear de trabalho de 4,5 ‒ 155 μmol L-1 e limites de detecção e quantificação de 4 e 13 μmol L-1, respectivamente. Devido aos bons resultados, o MIP de alta porosidade foi aplicado como fase de reconhecimento em um dos sistemas de fluxo lateral. Nesse sistema a resposta visual indicativa da presença do analito, após ligado ao MIP, baseou-se em uma reação de complexação entre a tetraciclina e o cátion Fe3+, formando um composto de coordenação de coloração amarelo escuro. Esse sistema também foi quantificado por meio do tratamento de imagens obtidas das tiras de fluxo lateral usando um smartphone. Depois de otimizados todos os parâmetros, a identificação qualitativa foi obtida em 10 minutos de corrida do teste. A eficiência dos testes foi avaliada na aplicação de amostras fortificadas com o analito e por meio dos ensaios quantitativos obteve-se valores de recuperação na faixa de 73 ‒ 104%; com faixa linear de trabalho de 45 ‒ 180 μmol L-1 e limites de detecção e quantificação na ordem de 17 e 45 μmol L-1, respectivamente. Outro teste rápido para identificação de tetraciclina foi construído utilizando aptâmeros como biomoléculas de reconhecimento seletivo. A resposta colorimétrica desse sistema baseou-se na aplicação de nanopartículas de ouro modificadas com os aptâmeros. A modificação das nanopartículas com o aptâmero de ligação à tetraciclina foi realizada com sucesso por meio da interação entre ouro e aptâmero marcado com grupo tiol. Os ensaios preliminares para construção da linha de teste (nanopartículas de SiO2 modificadas com tetraciclina) e linha de controle (fita de aptâmero complementar ao de ligação) mostraram que o sistema é viável, necessitando de mais ensaios de otimização. Portanto, os MIPs e os sistemas de fluxo lateral desenvolvidos se mostraram bem-sucedidos na identificação e quantificação da tetraciclina.
Emerging pollutants are a class of compounds released into the environment that cause pollution in terrestrial, aquatic and atmospheric environment; also, they can be found in food and water for human consumption. Drugs, especially antibiotics, are among the most found in the environment. Tetracycline belongs to a class of antibiotics widely applied in human and veterinary use due to its broad spectrum of action and, therefore, residues of this antibiotic are easily found in soil, water and food. Since continued exposure to drugs can be harmful to human health, rapid and selective methods to detect them are essential. Lateral flow tests were developed to determine tetracycline: in one of the tests molecularly imprinted polymers (MIP) were used and in the other system were applied aptamers, both components as a selective recognition phase. Initially, three different types of MIP were synthesized: traditional, core-shell and hollow (with high porosity). All materials were characterized by techniques such as scanning electron microscopy and infrared spectroscopy. After optimizing the analysis conditions, hollow MIP presented superior results for the identification of tetracycline: maximum binding capacity of 5.6 mg g-1 and 0.2 L mg-1 affinity constant. Hollow MIP also showed good results in selectivity tests, with higher retention for tetracycline compared to other drugs. Different types of samples fortified with tetracycline were evaluated and MIP showed recovery in the range of 74 - 96%, with linear range of 4.5 ‒ 155 μmol L-1 and the limits of detection and quantification of 4 e 13 μmol L-1, respectively. Due to the good results, the hollow MIP was applied as a recognition phase in one of the lateral flow systems. In this system, visual response indicating the presence of the analyte, after being recognized by the MIP, was based on a complexation reaction between tetracycline and Fe3+ cation, forming a coordinating compound with a dark yellow colour. This system was also quantified through the treatment of lateral flow strips images obtained using a smartphone. After optimizing all the necessary parameters, qualitative identification was obtained in 10 minutes of running. The efficiency of the tests was evaluated in the application of samples fortified with the analyte and through the quantitative results was obtained recovery values in the range of 73 - 104%; with linear range of 45 ‒ 180 μmol L-1 and the limits of detection and quantification of 17 e 45 μmol L-1, respectively. Another rapid test for tetracycline identification was structured using aptamers as selective recognition biomolecules. The colorimetric response of this system was based on the application of gold nanoparticles modified with aptamers. Nanoparticles modifications with the tetracycline-binding aptamer was successfully accomplished through the interaction between gold and aptamer thiol labelled aptamer. Preliminary tests for the construction of the test line (SiO2 nanoparticles modified with tetracycline) and control line (complementary aptamer strand) showed that the elution and colorimetric response were viable, requiring further optimization tests. Therefore, MIPs and lateral flow tests were successful in the identification and quantification of tetracycline.
Emerging pollutants are a class of compounds released into the environment that cause pollution in terrestrial, aquatic and atmospheric environment; also, they can be found in food and water for human consumption. Drugs, especially antibiotics, are among the most found in the environment. Tetracycline belongs to a class of antibiotics widely applied in human and veterinary use due to its broad spectrum of action and, therefore, residues of this antibiotic are easily found in soil, water and food. Since continued exposure to drugs can be harmful to human health, rapid and selective methods to detect them are essential. Lateral flow tests were developed to determine tetracycline: in one of the tests molecularly imprinted polymers (MIP) were used and in the other system were applied aptamers, both components as a selective recognition phase. Initially, three different types of MIP were synthesized: traditional, core-shell and hollow (with high porosity). All materials were characterized by techniques such as scanning electron microscopy and infrared spectroscopy. After optimizing the analysis conditions, hollow MIP presented superior results for the identification of tetracycline: maximum binding capacity of 5.6 mg g-1 and 0.2 L mg-1 affinity constant. Hollow MIP also showed good results in selectivity tests, with higher retention for tetracycline compared to other drugs. Different types of samples fortified with tetracycline were evaluated and MIP showed recovery in the range of 74 - 96%, with linear range of 4.5 ‒ 155 μmol L-1 and the limits of detection and quantification of 4 e 13 μmol L-1, respectively. Due to the good results, the hollow MIP was applied as a recognition phase in one of the lateral flow systems. In this system, visual response indicating the presence of the analyte, after being recognized by the MIP, was based on a complexation reaction between tetracycline and Fe3+ cation, forming a coordinating compound with a dark yellow colour. This system was also quantified through the treatment of lateral flow strips images obtained using a smartphone. After optimizing all the necessary parameters, qualitative identification was obtained in 10 minutes of running. The efficiency of the tests was evaluated in the application of samples fortified with the analyte and through the quantitative results was obtained recovery values in the range of 73 - 104%; with linear range of 45 ‒ 180 μmol L-1 and the limits of detection and quantification of 17 e 45 μmol L-1, respectively. Another rapid test for tetracycline identification was structured using aptamers as selective recognition biomolecules. The colorimetric response of this system was based on the application of gold nanoparticles modified with aptamers. Nanoparticles modifications with the tetracycline-binding aptamer was successfully accomplished through the interaction between gold and aptamer thiol labelled aptamer. Preliminary tests for the construction of the test line (SiO2 nanoparticles modified with tetracycline) and control line (complementary aptamer strand) showed that the elution and colorimetric response were viable, requiring further optimization tests. Therefore, MIPs and lateral flow tests were successful in the identification and quantification of tetracycline.
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Palavras-chave
Tetraciclina, Materiais biomiméticos, DNA, Materiais porosos, Antibacterianos