Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/12851 |
Resumo: | Citrus production is one of the world’s main agricultural activities, and Brazil is its largest producer. Considering the importance of this culture and its constant growth, some diseases may significantly affect world productivity. The Tristeza disease and the citrus canker, caused by the Citrus tristeza virus (CTV) and Xanthomonas citri subsp. citri (Xcc), respectively, are intensively monitored, since plant eradication is the main form of control. Thus, it is of the extreme interest to develop simple, fast, specific and low-cost analytical procedures for detecting these phytopathogens in the early stages. In this sense, this work proposed the development of electrochemical immunoassays for the determination of CTV capsid protein (CP-CTV) and trehalase (TREA) for the diagnosis of Tristeza disease and citrus canker, respectively. These sandwich-type immunoassays used screen-printed carbon electrodes and magnetic particles with biorecognition elements immobilized on their surface, which allowed the capture, separation, and pre-concentration of the analyte. The first approach was the development of a disposable microfluidic device to detect the CP-CTV protein, based on the redox reaction of hydroquinone/H2O2, that was enzymatically generated from the HRP label. The procedure presented a good linearity (r2 = 0.98), with a linear working range of 2.0 fg mL‒1 to 10.0 pg mL‒1, and a detection limit of 0.3 fg mL‒1. In the second approach, trehalase (TREA) was explored as a potential biomarker for the detection of citrus canker, employing an enzymatic and another non-enzymatic immunoassay. Thus, the enzymatic system used HRP as an electrochemical label, while the non-enzymatic immunoassay used gold nanoparticles for this same function. Comparatively, the two procedures presented good results, in which excellent linearities were obtained for the concentration ranges of 25.0 fg mL‒1 to 10.0 pg mL‒1 and 1.0 ‒ 62.5 pg mL‒1 for the enzymatic and non-enzymatic immunoassay, respectively. Besides, the detection limits for the two systems developed were similar, with values of 19.0 and 16.0 fg mL‒1. The third approach involved an immunosensor for multiplexed determination of CP-CTV and TREA proteins using a microfluidic device. In this multiplexed detection strategy, the use of the alkaline phosphatase enzyme as a label promoted good versatility and selectivity to the procedure when compared to the usual immunoassays. The linear intervals of the calibration curves for CP-CTV and TREA were 0.3 to 5.1 pg mL‒1 and 0.8 to 12.5 pg mL‒1, with detection limits of 0.1 and 0.6 pg mL‒1, respectively. Finally, the first and second approaches were applied to samples of healthy and infected plants, followed by comparison with the ELISA-based procedure, with no significant difference between the methods at the 95% confidence level, which demonstrated the applicability of the strategies. Besides, all devices developed showed good accuracy, high sensitivity, and low cost, hence being viable alternatives for the early detection of these diseases. |
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Freitas, Tayane AguiarFaria, Ronaldo Censihttp://lattes.cnpq.br/8659496864305621http://lattes.cnpq.br/25522379532589322020-05-28T23:54:28Z2020-05-28T23:54:28Z2020-03-06FREITAS, Tayane Aguiar. Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares. 2020. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2020. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12851.https://repositorio.ufscar.br/handle/ufscar/12851Citrus production is one of the world’s main agricultural activities, and Brazil is its largest producer. Considering the importance of this culture and its constant growth, some diseases may significantly affect world productivity. The Tristeza disease and the citrus canker, caused by the Citrus tristeza virus (CTV) and Xanthomonas citri subsp. citri (Xcc), respectively, are intensively monitored, since plant eradication is the main form of control. Thus, it is of the extreme interest to develop simple, fast, specific and low-cost analytical procedures for detecting these phytopathogens in the early stages. In this sense, this work proposed the development of electrochemical immunoassays for the determination of CTV capsid protein (CP-CTV) and trehalase (TREA) for the diagnosis of Tristeza disease and citrus canker, respectively. These sandwich-type immunoassays used screen-printed carbon electrodes and magnetic particles with biorecognition elements immobilized on their surface, which allowed the capture, separation, and pre-concentration of the analyte. The first approach was the development of a disposable microfluidic device to detect the CP-CTV protein, based on the redox reaction of hydroquinone/H2O2, that was enzymatically generated from the HRP label. The procedure presented a good linearity (r2 = 0.98), with a linear working range of 2.0 fg mL‒1 to 10.0 pg mL‒1, and a detection limit of 0.3 fg mL‒1. In the second approach, trehalase (TREA) was explored as a potential biomarker for the detection of citrus canker, employing an enzymatic and another non-enzymatic immunoassay. Thus, the enzymatic system used HRP as an electrochemical label, while the non-enzymatic immunoassay used gold nanoparticles for this same function. Comparatively, the two procedures presented good results, in which excellent linearities were obtained for the concentration ranges of 25.0 fg mL‒1 to 10.0 pg mL‒1 and 1.0 ‒ 62.5 pg mL‒1 for the enzymatic and non-enzymatic immunoassay, respectively. Besides, the detection limits for the two systems developed were similar, with values of 19.0 and 16.0 fg mL‒1. The third approach involved an immunosensor for multiplexed determination of CP-CTV and TREA proteins using a microfluidic device. In this multiplexed detection strategy, the use of the alkaline phosphatase enzyme as a label promoted good versatility and selectivity to the procedure when compared to the usual immunoassays. The linear intervals of the calibration curves for CP-CTV and TREA were 0.3 to 5.1 pg mL‒1 and 0.8 to 12.5 pg mL‒1, with detection limits of 0.1 and 0.6 pg mL‒1, respectively. Finally, the first and second approaches were applied to samples of healthy and infected plants, followed by comparison with the ELISA-based procedure, with no significant difference between the methods at the 95% confidence level, which demonstrated the applicability of the strategies. Besides, all devices developed showed good accuracy, high sensitivity, and low cost, hence being viable alternatives for the early detection of these diseases.A citricultura é uma das principais atividades agrícolas, sendo o Brasil o maior produtor de citros do mundo. Considerando a relevância desta cultura e seu constante crescimento, algumas doenças podem afetar significativamente a produtividade mundial. As doenças Tristeza dos citros e o cancro cítrico, causadas pelo Citrus tristeza virus (CTV) e Xanthomonas citri subsp. citri (Xcc), respectivamente, são intensivamente monitoradas, uma vez que a erradicação das plantas é a principal forma de controle. Deste modo, é de extremo interesse o desenvolvimento de procedimentos analíticos simples, rápidos, específicos e de baixo custo para detecção desses fitopatógenos em estágios iniciais. Nesse sentido, esse trabalho propôs o desenvolvimento de imunoensaios eletroquímicos para determinação da proteína do capsídeo do CTV (CP-CTV) e trealase (TREA) para o diagnóstico da Tristeza dos citros e cancro cítrico, respectivamente. Esses imunoensaios do tipo sanduíche utilizaram eletrodos serigrafados e partículas magnéticas com elementos de biorreconhecimento imobilizados em sua superfície, que auxiliaram na captura, separação e pré-concentração do analito. A primeira abordagem foi o desenvolvimento de um dispositivo descartável microfluídico para detectar a proteína CP-CTV, baseado na reação redox da hidroquinona/H2O2, devido à presença da enzima peroxidase (HRP). O procedimento apresentou boa linearidade (r2 = 0.98), com faixa linear de trabalho de 2,0 fg mL‒1 a 10,0 pg mL‒1 e limite de detecção de 0,3 fg mL‒1. Na segunda abordagem, foi explorada a TREA como um potencial biomarcador proteico para detecção do cancro cítrico, por meio de um imunoensaio enzimático e outro não-enzimático. Assim, o sistema enzimático utilizou a HRP como marcador eletroquímico, enquanto o imunoensaio não-enzimático usou nanopartículas de ouro para essa mesma função. Comparativamente, os dois procedimentos apresentaram bons resultados, nos quais foram obtidas ótimas linearidades para as faixas de concentração de 25,0 fg mL‒1 a 10,0 pg mL‒1 e 1,0 - 62,5 pg mL‒1 para o imunoensaio enzimático e não-enzimático, respectivamente. Além disso, os limites de detecção para os dois sistemas desenvolvidos foram similares, com valores de 19,0 e 16,0 fg mL‒1. A terceira abordagem envolveu um imunossensor para determinação multiplexada das proteínas CP-CTV e TREA usando um dispositivo microfluídico. Nesta estratégia de detecção multiplexada, o uso da enzima fosfatase alcalina como marcador eletroquímico promoveu boa versatilidade ao procedimento em relação aos imunoensaios usuais. Os intervalos lineares das curvas de calibração para CP-CTV e TREA foram de 0,3 a 5,1 pg mL‒1 e 0,8 a 12,5 pg mL‒1, com limites de detecção de 0,1 pg mL‒1 e 0,6 pg mL‒1, respectivamente. Finalmente, a primeira e a segunda abordagem foram aplicadas em amostras de plantas sadias e infectadas, seguida pela comparação com o procedimento ELISA, não havendo diferença significativa entre os métodos ao nível de 95% de confiança, o que demonstrou a aplicabilidade das estratégias. Além disso, todos os dispositivos desenvolvidos apresentaram boa precisão, alta sensibilidade e baixo custo, sendo alternativas viáveis para detecção precoce dessas doenças.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES: Código de Financiamento 001porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Química - PPGQUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessTristeza dos citrosCancro cítricoDispositivo microfluídicoImunoensaios eletroquímicosPartículas magnéticasEletrodos serigrafadosTristeza diseaseCitrus cankerDisposable microfluidicElectrochemical immunoassayMagnetic beadsScreen-printed electrodeCIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA ANALITICA::ELETROANALITICADesenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivaresDevelopment of electrochemical immunoassays for the determination of biomarkers applied in the diagnosis of diseases in cultivars.info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALtese-Tayane Freitas.pdftese-Tayane Freitas.pdfTese de doutorado Tayane Freitas- versão finalapplication/pdf9122094https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/12851/4/tese-Tayane%20Freitas.pdf7b67575e627bcc1820289da559e9d10cMD54Carta-comprovante_homologacao-Tayane Freitas.pdfCarta-comprovante_homologacao-Tayane Freitas.pdfCarta comprovante da versão final de tese- Tayane Freitasapplication/pdf140261https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/12851/5/Carta-comprovante_homologacao-Tayane%20Freitas.pdf9569decd2d88953beff975bd3a09a3c5MD55CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/12851/6/license_rdfe39d27027a6cc9cb039ad269a5db8e34MD56TEXTtese-Tayane Freitas.pdf.txttese-Tayane Freitas.pdf.txtExtracted texttext/plain219919https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/12851/7/tese-Tayane%20Freitas.pdf.txtf74cc5334038958509dd16aec1b2b74bMD57Carta-comprovante_homologacao-Tayane Freitas.pdf.txtCarta-comprovante_homologacao-Tayane Freitas.pdf.txtExtracted texttext/plain1352https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/12851/9/Carta-comprovante_homologacao-Tayane%20Freitas.pdf.txtc2f8705c32a9fcdcd6224fb683cda903MD59THUMBNAILtese-Tayane Freitas.pdf.jpgtese-Tayane Freitas.pdf.jpgIM Thumbnailimage/jpeg9030https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/12851/8/tese-Tayane%20Freitas.pdf.jpg7d5524e1118256bfa5a0569c66b6a957MD58Carta-comprovante_homologacao-Tayane Freitas.pdf.jpgCarta-comprovante_homologacao-Tayane Freitas.pdf.jpgIM Thumbnailimage/jpeg11847https://{{ getenv "DSPACE_HOST" "repositorio.ufscar.br" }}/bitstream/ufscar/12851/10/Carta-comprovante_homologacao-Tayane%20Freitas.pdf.jpg9825bf84f79fd95af13aef20bb1e5969MD510ufscar/128512020-07-08 22:08:12.268oai:repositorio.ufscar.br:ufscar/12851Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222020-07-08T22:08:12Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares |
dc.title.alternative.eng.fl_str_mv |
Development of electrochemical immunoassays for the determination of biomarkers applied in the diagnosis of diseases in cultivars. |
title |
Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares |
spellingShingle |
Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares Freitas, Tayane Aguiar Tristeza dos citros Cancro cítrico Dispositivo microfluídico Imunoensaios eletroquímicos Partículas magnéticas Eletrodos serigrafados Tristeza disease Citrus canker Disposable microfluidic Electrochemical immunoassay Magnetic beads Screen-printed electrode CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA ANALITICA::ELETROANALITICA |
title_short |
Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares |
title_full |
Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares |
title_fullStr |
Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares |
title_full_unstemmed |
Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares |
title_sort |
Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares |
author |
Freitas, Tayane Aguiar |
author_facet |
Freitas, Tayane Aguiar |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/2552237953258932 |
dc.contributor.author.fl_str_mv |
Freitas, Tayane Aguiar |
dc.contributor.advisor1.fl_str_mv |
Faria, Ronaldo Censi |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/8659496864305621 |
contributor_str_mv |
Faria, Ronaldo Censi |
dc.subject.por.fl_str_mv |
Tristeza dos citros Cancro cítrico Dispositivo microfluídico Imunoensaios eletroquímicos Partículas magnéticas Eletrodos serigrafados |
topic |
Tristeza dos citros Cancro cítrico Dispositivo microfluídico Imunoensaios eletroquímicos Partículas magnéticas Eletrodos serigrafados Tristeza disease Citrus canker Disposable microfluidic Electrochemical immunoassay Magnetic beads Screen-printed electrode CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA ANALITICA::ELETROANALITICA |
dc.subject.eng.fl_str_mv |
Tristeza disease Citrus canker Disposable microfluidic Electrochemical immunoassay Magnetic beads Screen-printed electrode |
dc.subject.cnpq.fl_str_mv |
CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA ANALITICA::ELETROANALITICA |
description |
Citrus production is one of the world’s main agricultural activities, and Brazil is its largest producer. Considering the importance of this culture and its constant growth, some diseases may significantly affect world productivity. The Tristeza disease and the citrus canker, caused by the Citrus tristeza virus (CTV) and Xanthomonas citri subsp. citri (Xcc), respectively, are intensively monitored, since plant eradication is the main form of control. Thus, it is of the extreme interest to develop simple, fast, specific and low-cost analytical procedures for detecting these phytopathogens in the early stages. In this sense, this work proposed the development of electrochemical immunoassays for the determination of CTV capsid protein (CP-CTV) and trehalase (TREA) for the diagnosis of Tristeza disease and citrus canker, respectively. These sandwich-type immunoassays used screen-printed carbon electrodes and magnetic particles with biorecognition elements immobilized on their surface, which allowed the capture, separation, and pre-concentration of the analyte. The first approach was the development of a disposable microfluidic device to detect the CP-CTV protein, based on the redox reaction of hydroquinone/H2O2, that was enzymatically generated from the HRP label. The procedure presented a good linearity (r2 = 0.98), with a linear working range of 2.0 fg mL‒1 to 10.0 pg mL‒1, and a detection limit of 0.3 fg mL‒1. In the second approach, trehalase (TREA) was explored as a potential biomarker for the detection of citrus canker, employing an enzymatic and another non-enzymatic immunoassay. Thus, the enzymatic system used HRP as an electrochemical label, while the non-enzymatic immunoassay used gold nanoparticles for this same function. Comparatively, the two procedures presented good results, in which excellent linearities were obtained for the concentration ranges of 25.0 fg mL‒1 to 10.0 pg mL‒1 and 1.0 ‒ 62.5 pg mL‒1 for the enzymatic and non-enzymatic immunoassay, respectively. Besides, the detection limits for the two systems developed were similar, with values of 19.0 and 16.0 fg mL‒1. The third approach involved an immunosensor for multiplexed determination of CP-CTV and TREA proteins using a microfluidic device. In this multiplexed detection strategy, the use of the alkaline phosphatase enzyme as a label promoted good versatility and selectivity to the procedure when compared to the usual immunoassays. The linear intervals of the calibration curves for CP-CTV and TREA were 0.3 to 5.1 pg mL‒1 and 0.8 to 12.5 pg mL‒1, with detection limits of 0.1 and 0.6 pg mL‒1, respectively. Finally, the first and second approaches were applied to samples of healthy and infected plants, followed by comparison with the ELISA-based procedure, with no significant difference between the methods at the 95% confidence level, which demonstrated the applicability of the strategies. Besides, all devices developed showed good accuracy, high sensitivity, and low cost, hence being viable alternatives for the early detection of these diseases. |
publishDate |
2020 |
dc.date.accessioned.fl_str_mv |
2020-05-28T23:54:28Z |
dc.date.available.fl_str_mv |
2020-05-28T23:54:28Z |
dc.date.issued.fl_str_mv |
2020-03-06 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
FREITAS, Tayane Aguiar. Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares. 2020. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2020. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12851. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/12851 |
identifier_str_mv |
FREITAS, Tayane Aguiar. Desenvolvimento de imunoensaios eletroquímicos para determinação de biomarcadores aplicados no diagnóstico de doenças em cultivares. 2020. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2020. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12851. |
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https://repositorio.ufscar.br/handle/ufscar/12851 |
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por |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ info:eu-repo/semantics/openAccess |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ |
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openAccess |
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Universidade Federal de São Carlos Câmpus São Carlos |
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Programa de Pós-Graduação em Química - PPGQ |
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UFSCar |
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Universidade Federal de São Carlos Câmpus São Carlos |
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