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Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line

Cell-based biosensors (CBBs) are becoming important tools for biosecurity a lications and rapid diagnostics in food microbiology for their unique capability of detecting hazardous materials. Pollutants, such as heavy metals and chemicals, are now considered as a global threat and are associated with...

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Detalles Bibliográficos
Autores principales: Rahimirad, Nazanin, Kavoosi, Saeedeh, Shirzad, Hadi, Sadeghizadeh, Majid
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Shaheed Beheshti University of Medical Sciences 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706743/
https://www.ncbi.nlm.nih.gov/pubmed/31531052
http://dx.doi.org/10.22037/ijpr.2019.1100687
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author Rahimirad, Nazanin
Kavoosi, Saeedeh
Shirzad, Hadi
Sadeghizadeh, Majid
author_facet Rahimirad, Nazanin
Kavoosi, Saeedeh
Shirzad, Hadi
Sadeghizadeh, Majid
author_sort Rahimirad, Nazanin
collection PubMed
description Cell-based biosensors (CBBs) are becoming important tools for biosecurity a lications and rapid diagnostics in food microbiology for their unique capability of detecting hazardous materials. Pollutants, such as heavy metals and chemicals, are now considered as a global threat and are associated with detrimental health outcomes. Fast and accurate detection of pollutants is essential to reduce these threats. In this study, the enhancer sequence of human cytomegalovirus (hCMV) IE genes cloned upstream of luc gene in PGL4.26 plasmid, in order to increase basal luciferase activity. This recombinant vector was transfected into Huh7 cell line and after 21 days of treatment with Hygromycin B selectable marker, stable cell line was generated. After several passages, cells containing this vector showed high luciferase activity in normal conditions without any induction following to overexpression of luc gene. Huh7-CMV-luc cell line was able to detect the slightest changes in ATP level, due to the effect of different toxins on the cell which disrupt cellular respiration and ATP production processes. In order to investigate the sensitivity of the cell line, the cells were incubated with 0.1-10 μM of chemical toxins affecting ATP production. These toxins affect luciferase activity in a dose dependent manner, with maximal sensitivity approximately about 0.2 μM to toxin concentrations. Additionally, this biosensor provided a rapid detection as early as 4 h in response to the toxicants. Whole cell biosensors like huh7-CMV-luc cell line can be considered as a powerful tool for the sensitive and efficient monitoring of general toxins, drugs, and environmental pollutants.
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spelling pubmed-67067432019-09-17 Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line Rahimirad, Nazanin Kavoosi, Saeedeh Shirzad, Hadi Sadeghizadeh, Majid Iran J Pharm Res Original Article Cell-based biosensors (CBBs) are becoming important tools for biosecurity a lications and rapid diagnostics in food microbiology for their unique capability of detecting hazardous materials. Pollutants, such as heavy metals and chemicals, are now considered as a global threat and are associated with detrimental health outcomes. Fast and accurate detection of pollutants is essential to reduce these threats. In this study, the enhancer sequence of human cytomegalovirus (hCMV) IE genes cloned upstream of luc gene in PGL4.26 plasmid, in order to increase basal luciferase activity. This recombinant vector was transfected into Huh7 cell line and after 21 days of treatment with Hygromycin B selectable marker, stable cell line was generated. After several passages, cells containing this vector showed high luciferase activity in normal conditions without any induction following to overexpression of luc gene. Huh7-CMV-luc cell line was able to detect the slightest changes in ATP level, due to the effect of different toxins on the cell which disrupt cellular respiration and ATP production processes. In order to investigate the sensitivity of the cell line, the cells were incubated with 0.1-10 μM of chemical toxins affecting ATP production. These toxins affect luciferase activity in a dose dependent manner, with maximal sensitivity approximately about 0.2 μM to toxin concentrations. Additionally, this biosensor provided a rapid detection as early as 4 h in response to the toxicants. Whole cell biosensors like huh7-CMV-luc cell line can be considered as a powerful tool for the sensitive and efficient monitoring of general toxins, drugs, and environmental pollutants. Shaheed Beheshti University of Medical Sciences 2019 /pmc/articles/PMC6706743/ /pubmed/31531052 http://dx.doi.org/10.22037/ijpr.2019.1100687 Text en This is an Open Access article distributed under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Rahimirad, Nazanin
Kavoosi, Saeedeh
Shirzad, Hadi
Sadeghizadeh, Majid
Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line
title Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line
title_full Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line
title_fullStr Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line
title_full_unstemmed Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line
title_short Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line
title_sort design and application of a bioluminescent biosensor for detection of toxicity using huh7-cmv-luc cell line
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706743/
https://www.ncbi.nlm.nih.gov/pubmed/31531052
http://dx.doi.org/10.22037/ijpr.2019.1100687
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