Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata
Autor(a) principal: | |
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Data de Publicação: | 2013 |
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/1822/24332 |
Resumo: | The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmol L−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The short-term assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μg L−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples. |
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Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitataBioassayFluorescein diacetate (FDA)Growth phaseMicroplate assayMetabolic activitySelenastrum capricornutumScience & TechnologyThe present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmol L−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The short-term assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μg L−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.The authors thank the Fundacao para a Ciencia e a Tecnologia (FCT) through the Portuguese Government for their financial support of this work through the grant PEST-OE/EQB/LA0023/2011 to IBB. Manuela D. Machado gratefully acknowledges the postdoctoral grant from FCT (SFRH/BPD/72816/2010).SpringerKluwer Academic PublishersUniversidade do MinhoMachado, Manuela D.Soares, Eduardo V.20132013-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/24332eng0049-69791573-293210.1007/s11270-012-1358-3info: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:RCAAP2023-07-21T12:24:11Zoai:repositorium.sdum.uminho.pt:1822/24332Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:18:07.411465Repositório 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 |
Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata |
title |
Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata |
spellingShingle |
Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata Machado, Manuela D. Bioassay Fluorescein diacetate (FDA) Growth phase Microplate assay Metabolic activity Selenastrum capricornutum Science & Technology |
title_short |
Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata |
title_full |
Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata |
title_fullStr |
Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata |
title_full_unstemmed |
Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata |
title_sort |
Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata |
author |
Machado, Manuela D. |
author_facet |
Machado, Manuela D. Soares, Eduardo V. |
author_role |
author |
author2 |
Soares, Eduardo V. |
author2_role |
author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Machado, Manuela D. Soares, Eduardo V. |
dc.subject.por.fl_str_mv |
Bioassay Fluorescein diacetate (FDA) Growth phase Microplate assay Metabolic activity Selenastrum capricornutum Science & Technology |
topic |
Bioassay Fluorescein diacetate (FDA) Growth phase Microplate assay Metabolic activity Selenastrum capricornutum Science & Technology |
description |
The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmol L−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The short-term assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μg L−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013 2013-01-01T00:00:00Z |
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/1822/24332 |
url |
http://hdl.handle.net/1822/24332 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
0049-6979 1573-2932 10.1007/s11270-012-1358-3 |
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 |
Springer Kluwer Academic Publishers |
publisher.none.fl_str_mv |
Springer Kluwer Academic Publishers |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
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1799132635749941248 |