Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.22/16004 |
Resumo: | A novel and disposable electrochemical biosensor for PCR-free and selective detection of Sola l 7, a non-specific lipid transfer protein (nsLTP) found in tomato seeds associated to severe symptoms of tomato-allergic patients, is reported in this work. The methodology involves the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sola l 7 allergen coding sequence with appropriate RNA probes designed and described for the first time in this work. Labeling was carried out with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with HRP onto the surface of magnetic beads. Amperometric transduction was performed upon magnetic capture of the resulting magnetic bioconjugates on screen-printed electrodes using the system H2O2/HQ. A comparison of the sandwich hybridization format with a direct approach as well as between different labeling strategies was performed. The LOD value achieved was 0.2 pM (5 amol in 25 μL). The biosensor was successfully applied to the selective analysis of the targeted Sola l 7 specific region directly in just 100 ng of non-fragmented denatured genomic DNA extracted from tomato seeds. |
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Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seedsSola l 7 allergen coding-sequencesSandwich hybridizationAbRNA/DNAScreen-printed electrodesAmperometryGenomic DNA extractA novel and disposable electrochemical biosensor for PCR-free and selective detection of Sola l 7, a non-specific lipid transfer protein (nsLTP) found in tomato seeds associated to severe symptoms of tomato-allergic patients, is reported in this work. The methodology involves the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sola l 7 allergen coding sequence with appropriate RNA probes designed and described for the first time in this work. Labeling was carried out with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with HRP onto the surface of magnetic beads. Amperometric transduction was performed upon magnetic capture of the resulting magnetic bioconjugates on screen-printed electrodes using the system H2O2/HQ. A comparison of the sandwich hybridization format with a direct approach as well as between different labeling strategies was performed. The LOD value achieved was 0.2 pM (5 amol in 25 μL). The biosensor was successfully applied to the selective analysis of the targeted Sola l 7 specific region directly in just 100 ng of non-fragmented denatured genomic DNA extracted from tomato seeds.The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2015-64402-C2-1-R, SAF2017-86483-R and the TRANSNANOAVANSENS Program from the Comunidad de Madrid (Grant P2018/NMT-4349) are gratefully acknowledged. M.F.B. is grateful to FCT grant SFRH/BPD/78845/2011,financed by POPH–QREN–Tipologia4.1–Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologiae Ensino Superior. The author M.A.P.B. is grateful to the authors J.M.R., M.F.B., S.C., J.M.P. for the scientific assistance and suggestions shared throughout the supervision of her Master’s project and Master’s thesis at University of Minho. The author M.A.P.B. also acknowledges the Department of Biology (DB) and the Centre of Molecular and Environmental Biology (CBMA) from University of Minho (UM), Braga,Portugal, by providing all the conditions leading to the Master's thesis in“Biophysics and Bionanosystems”. Financial support of M.A.P.B. forthis work was provided by a fellowship within the programme Erasmus+ scholarship/Portugal, which allowed performing the laboratorial work at the University Complutense of Madrid, Madrid, Spain. The author J.M.R. acknowledges CBMA-UM and DB-UM, Portugal, by the conditions provided. J.M.R. is also grateful to the Green Chemistry Laboratory (LAQV)–Research center Chemistry and Technology Network (REQUIMTE), and the Department of Chemistry and Biochemistry (DCB) from Faculty of Sciences, University of Porto(FCUP), Porto, Portugal, where currently he is researcher. J.M.R. acknowledges the financial support of the strategic programmes UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and PTDC/SAUNUT/30448/2017 (POCI-01-0145-FEDER-030448) and M.F.B. the financial support of the projects PTDC/QUI-QAN/30735/2017 and UID/QUI/50006/2019, funded by national funds through Fundação para a Ciência e Tecnologia [Foundation for Science and Technology](FCT-I.P.)/Ministério da Ciência, Tecnologia e Ensino Superior [Ministry of Science, Technology and Higher Education] (MCTES), and Fundo Europeu de Desenvolvimento Regional [European RegionalDevelopment Fund] (FEDER), under the scope of the COMPETE2020–Programa Operacional Competitividade e Internacionalização[COMPETE2020–Competitiveness and InternationalizationOperational Program, POCI]. S.B. acknowledges the financial support from MINECO through the“Juan de la Cierva” program.ElsevierRepositório Científico do Instituto Politécnico do PortoPereira-Barros, Magda A.Barroso, M. FátimaMartín-Pedraza, LauraVargas, EvaBenedé, SaraVillalba, MayteRocha, João M.Campuzano, SusanaPingarrón, José M.2020-06-25T10:53:05Z2019-05-072019-05-07T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.22/16004eng0956-566310.1016/j.bios.2019.05.011info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-03-13T13:01:40Zoai:recipp.ipp.pt:10400.22/16004Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T17:35:44.198019Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds |
title |
Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds |
spellingShingle |
Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds Pereira-Barros, Magda A. Sola l 7 allergen coding-sequences Sandwich hybridization AbRNA/DNA Screen-printed electrodes Amperometry Genomic DNA extract |
title_short |
Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds |
title_full |
Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds |
title_fullStr |
Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds |
title_full_unstemmed |
Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds |
title_sort |
Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds |
author |
Pereira-Barros, Magda A. |
author_facet |
Pereira-Barros, Magda A. Barroso, M. Fátima Martín-Pedraza, Laura Vargas, Eva Benedé, Sara Villalba, Mayte Rocha, João M. Campuzano, Susana Pingarrón, José M. |
author_role |
author |
author2 |
Barroso, M. Fátima Martín-Pedraza, Laura Vargas, Eva Benedé, Sara Villalba, Mayte Rocha, João M. Campuzano, Susana Pingarrón, José M. |
author2_role |
author author author author author author author author |
dc.contributor.none.fl_str_mv |
Repositório Científico do Instituto Politécnico do Porto |
dc.contributor.author.fl_str_mv |
Pereira-Barros, Magda A. Barroso, M. Fátima Martín-Pedraza, Laura Vargas, Eva Benedé, Sara Villalba, Mayte Rocha, João M. Campuzano, Susana Pingarrón, José M. |
dc.subject.por.fl_str_mv |
Sola l 7 allergen coding-sequences Sandwich hybridization AbRNA/DNA Screen-printed electrodes Amperometry Genomic DNA extract |
topic |
Sola l 7 allergen coding-sequences Sandwich hybridization AbRNA/DNA Screen-printed electrodes Amperometry Genomic DNA extract |
description |
A novel and disposable electrochemical biosensor for PCR-free and selective detection of Sola l 7, a non-specific lipid transfer protein (nsLTP) found in tomato seeds associated to severe symptoms of tomato-allergic patients, is reported in this work. The methodology involves the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sola l 7 allergen coding sequence with appropriate RNA probes designed and described for the first time in this work. Labeling was carried out with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with HRP onto the surface of magnetic beads. Amperometric transduction was performed upon magnetic capture of the resulting magnetic bioconjugates on screen-printed electrodes using the system H2O2/HQ. A comparison of the sandwich hybridization format with a direct approach as well as between different labeling strategies was performed. The LOD value achieved was 0.2 pM (5 amol in 25 μL). The biosensor was successfully applied to the selective analysis of the targeted Sola l 7 specific region directly in just 100 ng of non-fragmented denatured genomic DNA extracted from tomato seeds. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-05-07 2019-05-07T00:00:00Z 2020-06-25T10:53:05Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.22/16004 |
url |
http://hdl.handle.net/10400.22/16004 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
0956-5663 10.1016/j.bios.2019.05.011 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Elsevier |
publisher.none.fl_str_mv |
Elsevier |
dc.source.none.fl_str_mv |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799131448113889280 |