Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFU |
Texto Completo: | https://repositorio.ufu.br/handle/123456789/38849 https://doi.org/10.14393/ufu.di.2023.240 |
Resumo: | Respiratory syncytial virus (RSV) is an important respiratory pathogen that mainly affects children under the age of five. While RSV infections in healthy, immunocompetent adults usually result in mild uncomplicated cases, infants and the elderly often experience bronchiolitis, bronchitis, and pneumonia. Current detection methods for RSV include RTqPCR, immunochromatography, and indirect immunofluorescence assays, but they have disadvantages related to time, cost, and sensitivity. These limitations can be overcome by developing an electrochemical immunosensor, a low-cost, rapid, specific, and user-friendly device. Covalent binding is an effective methodology for antibody (Ab) immobilization, and the electrochemical modification of a graphite pencil lead working electrode (PGE) with conductive or non-conductive polymers that possess functional groups is of interest for this procedure. 3-aminophenylacetic acid (3APA) exhibits the desired characteristics and was chosen as the monomer to provide available carboxyl groups after polymerization. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were employed for electropolymerization and characterization of the formed material. The CV results demonstrated the formation of poly(3APA), with 30 cycles established as the optimal condition due to the best response observed in the system. Electrochemical and pH studies in CV allowed for the determination of the number of electrons and the proton-to-electron ratio, providing insight into the proposed mechanism of 3APA electropolymerization. The immunosensor was developed on this matrix by activating the carboxylic groups of poly(3APA) in a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / Nhydroxysuccinimide (NHS), followed by nucleophilic substitution reaction with the amino groups present in the antibody (Ab). After Ab immobilization, the PGE/poly(3APA) surface was blocked by glycine (Gly) to prevent non-specific interactions, followed by antigen (Ag) immobilization. Detection was performed using EIS, and the data were evaluated using equivalent circuits. The values of charge transfer resistance (Rtc) increased as the PGE was modified in the following order: PGE < PGE/poly(3APA) < PGE/poly(3APA)/Ab < PGE/poly(3APA)/Ab/Ag. The system was optimized to achieve the best detection conditions. The response variation (ΔRtc) between the complete system containing Ab/Ag and the system containing only Ab (n = 3) was utilized. For the preparation of the immunosensor, 100 ng of Ab was chosen, and the immobilization times for Ab and Gly were, respectively, 3 h and 50 min. The response time of the system, with Ag immobilization, was 30 min. Using the calibration curve of the optimized system, the calculated limit of detection (LOD) was 27.65 PFU/mL, and the limit of quantification (LOQ) was 92.15 PFU/mL, demonstrating that the proposed immunosensor is a viable alternative for the detection of RSV. |
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Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratórioDevelopment of an impedimetric immunosensor for the detection of respiratory syncytial virusVírus sincicial respiratórioRespiratory syncytial virusImunossensor eletroquímicoElectrochemical immunosensorÁcido 3- aminofenilacético3-aminophenylacetic acidEletropolimerizaçãoElectropolymerizationVoltametria cíclicaCyclic voltammetryEspectroscopia de impedância eletroquímicaElectrochemical impedance spectroscopyCNPQ::CIENCIAS BIOLOGICASCNPQ::CIENCIAS EXATAS E DA TERRA::QUIMICABiotecnologiaVírusAparelho respiratório - DoençasEspectroscopia de emissãoRespiratory syncytial virus (RSV) is an important respiratory pathogen that mainly affects children under the age of five. While RSV infections in healthy, immunocompetent adults usually result in mild uncomplicated cases, infants and the elderly often experience bronchiolitis, bronchitis, and pneumonia. Current detection methods for RSV include RTqPCR, immunochromatography, and indirect immunofluorescence assays, but they have disadvantages related to time, cost, and sensitivity. These limitations can be overcome by developing an electrochemical immunosensor, a low-cost, rapid, specific, and user-friendly device. Covalent binding is an effective methodology for antibody (Ab) immobilization, and the electrochemical modification of a graphite pencil lead working electrode (PGE) with conductive or non-conductive polymers that possess functional groups is of interest for this procedure. 3-aminophenylacetic acid (3APA) exhibits the desired characteristics and was chosen as the monomer to provide available carboxyl groups after polymerization. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were employed for electropolymerization and characterization of the formed material. The CV results demonstrated the formation of poly(3APA), with 30 cycles established as the optimal condition due to the best response observed in the system. Electrochemical and pH studies in CV allowed for the determination of the number of electrons and the proton-to-electron ratio, providing insight into the proposed mechanism of 3APA electropolymerization. The immunosensor was developed on this matrix by activating the carboxylic groups of poly(3APA) in a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / Nhydroxysuccinimide (NHS), followed by nucleophilic substitution reaction with the amino groups present in the antibody (Ab). After Ab immobilization, the PGE/poly(3APA) surface was blocked by glycine (Gly) to prevent non-specific interactions, followed by antigen (Ag) immobilization. Detection was performed using EIS, and the data were evaluated using equivalent circuits. The values of charge transfer resistance (Rtc) increased as the PGE was modified in the following order: PGE < PGE/poly(3APA) < PGE/poly(3APA)/Ab < PGE/poly(3APA)/Ab/Ag. The system was optimized to achieve the best detection conditions. The response variation (ΔRtc) between the complete system containing Ab/Ag and the system containing only Ab (n = 3) was utilized. For the preparation of the immunosensor, 100 ng of Ab was chosen, and the immobilization times for Ab and Gly were, respectively, 3 h and 50 min. The response time of the system, with Ag immobilization, was 30 min. Using the calibration curve of the optimized system, the calculated limit of detection (LOD) was 27.65 PFU/mL, and the limit of quantification (LOQ) was 92.15 PFU/mL, demonstrating that the proposed immunosensor is a viable alternative for the detection of RSV.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorCNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas GeraisDissertação (Mestrado)O vírus sincicial respiratório (RSV) é um importante patógeno respiratório que acomete principalmente crianças menores de cinco anos. Apesar de na maioria dos casos de infecção em pessoas adultas e imunocompetentes causar quadros leves sem complicações, em crianças e idosos é frequente o aparecimento de bonquiolite, bronquite e pneumonia. As formas de detecção do RSV incluem RT-qPCR, ensaio imunocromatográfico e ensaio de imunofluorescência indireto que apresentam desvantagens relacionadas ao tempo, preço e sensibilidade que podem ser contornadas pelo desenvolvimento de um imunossensor eletroquímico, um dispositivo de baixo custo, rápido, específico e de fácil manuseio. A ligação covalente é uma metodologia efetiva para a imobilização do anticorpo (Ab), de modo que a modificação eletroquímica do eletrodo de trabalho de carbono grafite de lapiseira (PGE), com polímeros condutores ou não, que apresentem grupos funcionais, são interessantes para esse procedimento. O ácido 3-aminofenilacético (3APA) apresenta as características desejadas e foi o monômero escolhido, com intuito de apresentar grupos carboxilas disponíveis após a formação polimérica. As técnicas de voltametria cíclica (CV) e de espectroscopia de impedância eletroquímica (EIS) foram usadas para a eletropolimerização e caracterização do material formado. Os CVs demonstraram que houve formação de poli(3APA) e 30 ciclos foram estabelecidos por apresentar a melhor resposta para o sistema. Estudos eletroquímicos e de pH em CV possibilitaram determinar o número de elétrons e a razão próton/elétron para apresentar a proposta do mecanismo de eletropolimerização do 3APA. O imunossensor foi desenvolvido sobre esta matriz com a ativação dos grupos carboxílicos do poli(3APA) em solução de 1-etil-3-(3-dimetilaminopropil)carbodiimida (EDC) / N-hidroxisuccinimida (NHS) seguido da reação de substituição nucleofílica com os grupos amino presentes no Ab. Após a imobilização de Ab, o PGE/poli(3APA) teve sua superfície bloqueada por glicina (Gly) para impedir interações inespecíficas, seguido da imobilização do antígeno (Ag). As detecções ocorreram por EIS e os dados foram avaliados pelos circuitos equivalentes. Houve o aumento nos valores de resistência à transferência de carga (Rtc) conforme o PGE foi modificado, na seguinte ordem: PGE < PGE/poli(3APA) < PGE/poli(3APA)/Ab < PGE/poli(3APA)/Ab/Ag. O sistema foi otimizado buscando as melhores condições para detecção. Foi usada a variação de resposta (ΔRtc) entre o sistema completo contendo Ab/Ag e o sistema contendo somente Ab (n = 3). Para o preparo do imunossensor foram escolhidos 100 ng de Ab, e o tempo de imobilização de Ab e Gly foram de, respectivamente, 3 h e 50 min. O tempo de resposta do sistema, com a imobilização de Ag foi de 30 min. Com a curva de calibração do sistema otimizado, o LOD calculado foi de 27,65 PFU/mL e o LOQ de 92,15 PFU/mL, demonstrando que o imunossensor proposto é uma alternativa viável para a detecção de RSV.Universidade Federal de UberlândiaBrasilPrograma de Pós-graduação em BiotecnologiaFranco, Diego Leonihttp://lattes.cnpq.br/6817236390376119Freitas, Guilherme Ramos Oliveira ehttp://lattes.cnpq.br/4014066906990228Ferreira, Lucas Francohttp://lattes.cnpq.br/6643874023907971Silva, Tallita Stéfanne e2023-08-07T11:42:58Z2023-08-07T11:42:58Z2023-06-23info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfSILVA, Tallita Stéfanne e. Desenvolvimento de um imunossensor impedimétrico para a detecção do Vírus Sincicial Respiratório. 2023. 84 f. Dissertação (Mestrado em Biotecnologia) - Universidade Federal de Uberlândia, Patos de Minas, 2023. DOI http://doi.org/10.14393/ufu.di.2023.240https://repositorio.ufu.br/handle/123456789/38849https://doi.org/10.14393/ufu.di.2023.240porhttp://creativecommons.org/licenses/by-nc-nd/3.0/us/info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFUinstname:Universidade Federal de Uberlândia (UFU)instacron:UFU2023-08-08T06:17:32Zoai:repositorio.ufu.br:123456789/38849Repositório InstitucionalONGhttp://repositorio.ufu.br/oai/requestdiinf@dirbi.ufu.bropendoar:2023-08-08T06:17:32Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)false |
dc.title.none.fl_str_mv |
Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório Development of an impedimetric immunosensor for the detection of respiratory syncytial virus |
title |
Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório |
spellingShingle |
Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório Silva, Tallita Stéfanne e Vírus sincicial respiratório Respiratory syncytial virus Imunossensor eletroquímico Electrochemical immunosensor Ácido 3- aminofenilacético 3-aminophenylacetic acid Eletropolimerização Electropolymerization Voltametria cíclica Cyclic voltammetry Espectroscopia de impedância eletroquímica Electrochemical impedance spectroscopy CNPQ::CIENCIAS BIOLOGICAS CNPQ::CIENCIAS EXATAS E DA TERRA::QUIMICA Biotecnologia Vírus Aparelho respiratório - Doenças Espectroscopia de emissão |
title_short |
Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório |
title_full |
Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório |
title_fullStr |
Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório |
title_full_unstemmed |
Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório |
title_sort |
Desenvolvimento de um imunossensor impedimétrico para a detecção do vírus sincicial respiratório |
author |
Silva, Tallita Stéfanne e |
author_facet |
Silva, Tallita Stéfanne e |
author_role |
author |
dc.contributor.none.fl_str_mv |
Franco, Diego Leoni http://lattes.cnpq.br/6817236390376119 Freitas, Guilherme Ramos Oliveira e http://lattes.cnpq.br/4014066906990228 Ferreira, Lucas Franco http://lattes.cnpq.br/6643874023907971 |
dc.contributor.author.fl_str_mv |
Silva, Tallita Stéfanne e |
dc.subject.por.fl_str_mv |
Vírus sincicial respiratório Respiratory syncytial virus Imunossensor eletroquímico Electrochemical immunosensor Ácido 3- aminofenilacético 3-aminophenylacetic acid Eletropolimerização Electropolymerization Voltametria cíclica Cyclic voltammetry Espectroscopia de impedância eletroquímica Electrochemical impedance spectroscopy CNPQ::CIENCIAS BIOLOGICAS CNPQ::CIENCIAS EXATAS E DA TERRA::QUIMICA Biotecnologia Vírus Aparelho respiratório - Doenças Espectroscopia de emissão |
topic |
Vírus sincicial respiratório Respiratory syncytial virus Imunossensor eletroquímico Electrochemical immunosensor Ácido 3- aminofenilacético 3-aminophenylacetic acid Eletropolimerização Electropolymerization Voltametria cíclica Cyclic voltammetry Espectroscopia de impedância eletroquímica Electrochemical impedance spectroscopy CNPQ::CIENCIAS BIOLOGICAS CNPQ::CIENCIAS EXATAS E DA TERRA::QUIMICA Biotecnologia Vírus Aparelho respiratório - Doenças Espectroscopia de emissão |
description |
Respiratory syncytial virus (RSV) is an important respiratory pathogen that mainly affects children under the age of five. While RSV infections in healthy, immunocompetent adults usually result in mild uncomplicated cases, infants and the elderly often experience bronchiolitis, bronchitis, and pneumonia. Current detection methods for RSV include RTqPCR, immunochromatography, and indirect immunofluorescence assays, but they have disadvantages related to time, cost, and sensitivity. These limitations can be overcome by developing an electrochemical immunosensor, a low-cost, rapid, specific, and user-friendly device. Covalent binding is an effective methodology for antibody (Ab) immobilization, and the electrochemical modification of a graphite pencil lead working electrode (PGE) with conductive or non-conductive polymers that possess functional groups is of interest for this procedure. 3-aminophenylacetic acid (3APA) exhibits the desired characteristics and was chosen as the monomer to provide available carboxyl groups after polymerization. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were employed for electropolymerization and characterization of the formed material. The CV results demonstrated the formation of poly(3APA), with 30 cycles established as the optimal condition due to the best response observed in the system. Electrochemical and pH studies in CV allowed for the determination of the number of electrons and the proton-to-electron ratio, providing insight into the proposed mechanism of 3APA electropolymerization. The immunosensor was developed on this matrix by activating the carboxylic groups of poly(3APA) in a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / Nhydroxysuccinimide (NHS), followed by nucleophilic substitution reaction with the amino groups present in the antibody (Ab). After Ab immobilization, the PGE/poly(3APA) surface was blocked by glycine (Gly) to prevent non-specific interactions, followed by antigen (Ag) immobilization. Detection was performed using EIS, and the data were evaluated using equivalent circuits. The values of charge transfer resistance (Rtc) increased as the PGE was modified in the following order: PGE < PGE/poly(3APA) < PGE/poly(3APA)/Ab < PGE/poly(3APA)/Ab/Ag. The system was optimized to achieve the best detection conditions. The response variation (ΔRtc) between the complete system containing Ab/Ag and the system containing only Ab (n = 3) was utilized. For the preparation of the immunosensor, 100 ng of Ab was chosen, and the immobilization times for Ab and Gly were, respectively, 3 h and 50 min. The response time of the system, with Ag immobilization, was 30 min. Using the calibration curve of the optimized system, the calculated limit of detection (LOD) was 27.65 PFU/mL, and the limit of quantification (LOQ) was 92.15 PFU/mL, demonstrating that the proposed immunosensor is a viable alternative for the detection of RSV. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-08-07T11:42:58Z 2023-08-07T11:42:58Z 2023-06-23 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
SILVA, Tallita Stéfanne e. Desenvolvimento de um imunossensor impedimétrico para a detecção do Vírus Sincicial Respiratório. 2023. 84 f. Dissertação (Mestrado em Biotecnologia) - Universidade Federal de Uberlândia, Patos de Minas, 2023. DOI http://doi.org/10.14393/ufu.di.2023.240 https://repositorio.ufu.br/handle/123456789/38849 https://doi.org/10.14393/ufu.di.2023.240 |
identifier_str_mv |
SILVA, Tallita Stéfanne e. Desenvolvimento de um imunossensor impedimétrico para a detecção do Vírus Sincicial Respiratório. 2023. 84 f. Dissertação (Mestrado em Biotecnologia) - Universidade Federal de Uberlândia, Patos de Minas, 2023. DOI http://doi.org/10.14393/ufu.di.2023.240 |
url |
https://repositorio.ufu.br/handle/123456789/38849 https://doi.org/10.14393/ufu.di.2023.240 |
dc.language.iso.fl_str_mv |
por |
language |
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http://creativecommons.org/licenses/by-nc-nd/3.0/us/ info:eu-repo/semantics/openAccess |
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http://creativecommons.org/licenses/by-nc-nd/3.0/us/ |
eu_rights_str_mv |
openAccess |
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application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Uberlândia Brasil Programa de Pós-graduação em Biotecnologia |
publisher.none.fl_str_mv |
Universidade Federal de Uberlândia Brasil Programa de Pós-graduação em Biotecnologia |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFU instname:Universidade Federal de Uberlândia (UFU) instacron:UFU |
instname_str |
Universidade Federal de Uberlândia (UFU) |
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UFU |
institution |
UFU |
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Repositório Institucional da UFU |
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Repositório Institucional da UFU |
repository.name.fl_str_mv |
Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU) |
repository.mail.fl_str_mv |
diinf@dirbi.ufu.br |
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1805569618062868480 |