Engenharia de superfícies para diagnóstico de dengue
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Data
2019-10-04
Autores
Cecchetto, Juliana
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Nesta tese foi desenvolvido um biossensor capacitivo redox para detecção e quantificação de diferentes concentrações do antígeno NS1 do vírus da dengue. Primeiramente, o sensor foi construído com eletrodos convencionais, em PBS e soro comercial com posterior validação do sistema, no qual, respondeu de maneira satisfatória. A próxima etapa foi a construção do biossensor realizado em eletrodos microfabricados, para possibilitar análises portáteis, porém este sistema não teve êxito, pela instabilidade da superfície dos mesmos. Em seguida, o sensoriamento foi realizado com amostras de soro de pacientes infectados com o vírus da dengue. A abordagem foi baseada na construção da superfície receptora (contendo anticorpo anti-NS1), utilizando SAMs mistas de tióis, (16-mercaptohexadecanóico, 16-MHDA) e (11-ferrocenil-undecanotiol, 11Fc) com o grupo redox, para medições capacitivas, que possibilitam a automontagem no eletrodo, sendo que cada etapa do processo de construção do biossensor foi monitorada por voltametria cíclica e espectroscopia de impedância/capacitância eletroquímica, com intuito de comparação com o sistema impedimétrico já analisado anteriormente. Pela técnica de espectroscopia de capacitância eletroquímica, a interface funcionalizada e sua interação com o analito, proteína NS1, geram uma variação do sinal de capacitância redox ( ), servindo dessa forma como sinal transdutor. Uma vez que em sua configuração os sensores capacitivos redox não usam espécie eletroativa em solução (pois ela está confinada na monocamada), estes são potencialmente adequados ao desenvolvimento de diagnósticos do tipo point-of-care (exame rápido com apenas um passo para detecção). Fatores como sensibilidade, reprodutibilidade, repetibilidade, limite de detecção e de quantificação foram avaliados e para amostras de soro comercial sem diluição, apresentaram LoD ~ 5 ng mL-1. Após, a metodologia foi modificada utilizando monocamada peptídica redox, mais adequada para análises em matriz biológica complexas, no qual foi testada em biblioteca de amostras de sangue de pacientes infectados ou não com o vírus da dengue, o que permitiu avaliar a aplicação clínica da metodologia proposta, evidenciando respostas relativas RR% para amostras negativas inferiores ao cut-off, enquanto que para amostras positivas variaram de ~ 6 a 9%. Esta tese teve seu propósito alcançado, com isso, a avaliação de um maior número de amostras de pacientes, por parcerias em laboratórios e hospitais será fundamental para conseguir-se vislumbrar uma nova ferramenta diagnóstica para biomarcadores de interesse clínico em um futuro próximo.
In this thesis a redox capacitive biosensor was developed to detect and quantify different concentrations of dengue virus NS1 antigen. Firstly, the sensor was constructed with conventional electrodes in PBS and commercial serum with subsequent system validation, in which it responded satisfactorily. The next step was the construction of the biosensor made in microfabricated electrodes, to enable portable analysis, but this system was unsuccessful due to their surface instability. Then, the sensing was performed with serum samples from patients infected with dengue virus. The approach was based on the construction of the receptor surface (containing anti-NS1 antibody) using mixed thiol SAMs, (16-mercaptohexadecanoic, 16-MHDA) and (11-ferrocenyl undecanothiol, 11Fc) with the redox group for measurements capacitive which enable self-assembly on the electrode, and each step of the biosensor construction process was monitored by cyclic voltammetry and electrochemical impedance/capacitance spectroscopy, in order to compare it with the impedimetric system already analyzed previously. Using the electrochemical capacitance spectroscopy technique, the functionalized interface and its interaction with the analyte, protein NS1, generate a variation of the redox capacitance signal ( ), thus serving as a transducer signal. Since redox capacitive sensors in their configuration do not use electroactive species in solution (as it is confined in the monolayer), they are potentially suitable for the development of point-of-care diagnostics (one-step rapid examination for detection). Factors such as sensitivity, reproducibility, repeatability, limit of detection and quantification were evaluated and for undiluted commercial serum samples presented LoD ~ 5 ng mL-1. Afterwards, the methodology was modified using redox peptide monolayer, more suitable for complex biological matrix analysis, which was tested in a library of blood samples from patients infected or not with dengue virus, which allowed to evaluate the clinical application of the methodology proposed, showing relative responses RR% for negative samples lower than the cut-off, while for positive samples ranged from ~ 6 to 9%. This thesis had its purpose achieved; therefore, the use of a greater number of samples by partnerships in laboratories and hospitals which will be fundamental for the development of a new diagnostic tool for biomarkers of clinical interest.
In this thesis a redox capacitive biosensor was developed to detect and quantify different concentrations of dengue virus NS1 antigen. Firstly, the sensor was constructed with conventional electrodes in PBS and commercial serum with subsequent system validation, in which it responded satisfactorily. The next step was the construction of the biosensor made in microfabricated electrodes, to enable portable analysis, but this system was unsuccessful due to their surface instability. Then, the sensing was performed with serum samples from patients infected with dengue virus. The approach was based on the construction of the receptor surface (containing anti-NS1 antibody) using mixed thiol SAMs, (16-mercaptohexadecanoic, 16-MHDA) and (11-ferrocenyl undecanothiol, 11Fc) with the redox group for measurements capacitive which enable self-assembly on the electrode, and each step of the biosensor construction process was monitored by cyclic voltammetry and electrochemical impedance/capacitance spectroscopy, in order to compare it with the impedimetric system already analyzed previously. Using the electrochemical capacitance spectroscopy technique, the functionalized interface and its interaction with the analyte, protein NS1, generate a variation of the redox capacitance signal ( ), thus serving as a transducer signal. Since redox capacitive sensors in their configuration do not use electroactive species in solution (as it is confined in the monolayer), they are potentially suitable for the development of point-of-care diagnostics (one-step rapid examination for detection). Factors such as sensitivity, reproducibility, repeatability, limit of detection and quantification were evaluated and for undiluted commercial serum samples presented LoD ~ 5 ng mL-1. Afterwards, the methodology was modified using redox peptide monolayer, more suitable for complex biological matrix analysis, which was tested in a library of blood samples from patients infected or not with dengue virus, which allowed to evaluate the clinical application of the methodology proposed, showing relative responses RR% for negative samples lower than the cut-off, while for positive samples ranged from ~ 6 to 9%. This thesis had its purpose achieved; therefore, the use of a greater number of samples by partnerships in laboratories and hospitals which will be fundamental for the development of a new diagnostic tool for biomarkers of clinical interest.
Descrição
Palavras-chave
Dengue, Diagnóstico virológico, Biossensores, Espectroscopia de impedância, Análise eletroquímica, Virological diagnosis, Biosensors, Impedance spectroscopy, Electrochemical analysis