Desenvolvimento de imunossensores eletroquímicos Baseados em nanopartículas de ouro recobertas por polímeros condutores Redox para o monitoramento de marcadores biológicos
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Data
2022-03-08
Autores
Oliveira, André Olean
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A presente tese reúne os estudos e esforços dedicados ao desenvolvimento de imunossensores eletroquímicos baseados em nanopartículas de ouro recobertas por polímeros condutores redox para o monitoramento de biomarcadores de interesses clínicos. Modificações genéticas e epigenéticas são fenômenos com ocorrência natural, sendo a principal responsável pela diversidade entre organismos. Contudo, fatores ambientais podem influenciar as taxas de mutações havendo como consequência doenças graves como o câncer. Assim, o desenvolvimento de tecnologias confiáveis e acessíveis que possibilitem o monitoramento de biomarcadores indicadores de anomalias genéticas pode auxiliar no diagnóstico precoce, aumentando significativamente as chances de recuperação. Por esse motivo, foram construídos dois imunossensores eletroquímicos baseados em espectroscopia de impedância eletroquímica (EIE) para detecção e quantificação dos biomarcadores de espécies de DNA metilado e facilitador de transporte de glicose 4 (GLUT-4). Os materiais nanocompósitos desenvolvidos foram obtidos por técnica de eletropolimerização em etapa única, tendo como base os monômeros de Bismark Brown Y (BBY) e Tionina (TH). Os filmes formados foram caracterizados por microscopia eletrônica de varredura de alta resolução (MEV-FEG), revelando nanoestruturas de ouro recobertas pelos polímeros condutores redox. Para imobilização dos anticorpos sobre a superfície dos nanocompósitos foi utilizada a estratégia de adsorção física para o poli(TH)-AuNPs e por ligação covalente para o poli(azo- BBY)-AuNPs. No melhor de nossos esforços, foi verificado que o mecanismo de detecção proposto na presente tese é inédito na literatura. Ele foi baseado na utilização do oxigênio dissolvido como mediador redox, não sendo preciso etapas extras para a marcação ou adição de mediadores redox na solução de análise. Por fim, verificou-se o desempenho analítico dos imunossensores, onde o imunossensor de poli(azo-BBY)-AuNPs apresentou limites de detecção de 0,64 pg mL-1 para espécies de DNA metilado enquanto o imunossensor de poli(TH)-AuNPs apresentou limite de detecção de 38,0 pg mL-1. Ambos os valores são satisfatórios para aplicação na análise de amostras de interesses clínico e acadêmico.
The present thesis the studies and efforts dedicated to the development of electrochemical immunosensors based on gold nanoparticles coated with redox conducting polymers for the detection of biomarkers of medical interest. Genetic and epigenetic modifications are naturally occurring phenomena, being the main responsible for the diversity among organisms. However, environmental factors can influence mutation rates resulting in serious diseases such as cancer. Thus, the development of reliable and accessible technologies that allow the monitoring of biomarkers showing genetic anomalies can help in early diagnosis, significantly increasing the chances of recovery. For this reason, two electrochemical immunosensors were constructed based on electrochemical impedance spectroscopy (EIS) for the detection and quantification of biomarkers of methylated DNA species and glucose transport facilitator 4 (GLUT-4). The nanocomposite materials developed were obtained by single-step electropolymerization technique, based on Bismarck Brown Y (BBY) and Thionine (TH) monomers. The formed films were characterized by field emission gun scanning electron microscopy (FEG-SEM), revealing gold particles covered by redox conducting polymers. For the immobilization of antibodies on the surface of the nanocomposites, the strategy of physical adsorption was used for poly(TH)-AuNPs and by covalent bonding for poly(azo-BBY)-AuNPs. In the best of our efforts, was verified that the detection mechanism proposed in the present thesis is unprecedented in the literature. It was based on the use of dissolved oxygen as a redox mediator, requiring no extra steps to label or add redox mediators to the analysis solution. Finally, the analytical performance of the immunosensors was verified, where the poly(azo-BBY)-AuNPs immunosensor presented detection limits of 0.64 pg mL-1 for methylated DNA species while the poly(TH)- AuNPs had a detection limit of 38.0 pg mL-1. Both values are satisfactory for application in the analysis of samples for clinical and academic interests.
The present thesis the studies and efforts dedicated to the development of electrochemical immunosensors based on gold nanoparticles coated with redox conducting polymers for the detection of biomarkers of medical interest. Genetic and epigenetic modifications are naturally occurring phenomena, being the main responsible for the diversity among organisms. However, environmental factors can influence mutation rates resulting in serious diseases such as cancer. Thus, the development of reliable and accessible technologies that allow the monitoring of biomarkers showing genetic anomalies can help in early diagnosis, significantly increasing the chances of recovery. For this reason, two electrochemical immunosensors were constructed based on electrochemical impedance spectroscopy (EIS) for the detection and quantification of biomarkers of methylated DNA species and glucose transport facilitator 4 (GLUT-4). The nanocomposite materials developed were obtained by single-step electropolymerization technique, based on Bismarck Brown Y (BBY) and Thionine (TH) monomers. The formed films were characterized by field emission gun scanning electron microscopy (FEG-SEM), revealing gold particles covered by redox conducting polymers. For the immobilization of antibodies on the surface of the nanocomposites, the strategy of physical adsorption was used for poly(TH)-AuNPs and by covalent bonding for poly(azo-BBY)-AuNPs. In the best of our efforts, was verified that the detection mechanism proposed in the present thesis is unprecedented in the literature. It was based on the use of dissolved oxygen as a redox mediator, requiring no extra steps to label or add redox mediators to the analysis solution. Finally, the analytical performance of the immunosensors was verified, where the poly(azo-BBY)-AuNPs immunosensor presented detection limits of 0.64 pg mL-1 for methylated DNA species while the poly(TH)- AuNPs had a detection limit of 38.0 pg mL-1. Both values are satisfactory for application in the analysis of samples for clinical and academic interests.
Descrição
Palavras-chave
Sensor de DNA Metilado, Sensor de GLUT-4, Imunossensor eletroquímico, Nanopartícula de ouro-polímero, Espectroscopia de Impedância Eletroquímica, DNA methylation sensor, Glut-4 sensor, Electrochemical immunosensor, Gold nanoparticle-polymers, Electrochemical impedance spectroscopy