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Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay

In this work, we describe the use of a new truncated aptamer for the determination of ofloxacin (OFL), being a principal quinolone commonly used in both human and animal healthcare. Since the affinity of a 72-mer ssDNA sequence has been previously described without further investigations, this paper...

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Autores principales: Ben Aissa, Sondes, Mastouri, Mohamed, Catanante, Gaëlle, Raouafi, Noureddine, Marty, Jean Louis
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761777/
https://www.ncbi.nlm.nih.gov/pubmed/33287135
http://dx.doi.org/10.3390/antibiotics9120860
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author Ben Aissa, Sondes
Mastouri, Mohamed
Catanante, Gaëlle
Raouafi, Noureddine
Marty, Jean Louis
author_facet Ben Aissa, Sondes
Mastouri, Mohamed
Catanante, Gaëlle
Raouafi, Noureddine
Marty, Jean Louis
author_sort Ben Aissa, Sondes
collection PubMed
description In this work, we describe the use of a new truncated aptamer for the determination of ofloxacin (OFL), being a principal quinolone commonly used in both human and animal healthcare. Since the affinity of a 72-mer ssDNA sequence has been previously described without further investigations, this paper demonstrates the first computational prediction of the binding motif between this aptamer and OFL through in silico molecular docking studies. Besides, we suggest the application of the characterized recognition mechanism in a simple FRET (Förster Resonance Energy Transfer) pattern for the rapid aptasensing of the quinolone of interest. Accordingly, our approach harnesses the fluorescence quenching of the fluorescein-tagged aptamer (FAM-APT) induced by its partial hybridization to a tetramethyl rhodamine-labelled complementary ssDNA (TAMRA-cDNA). In such a structure, dye labels brought into close proximity act as a FRET pair. Upon ofloxacin addition, an affinity competition occurs to form a more stable FAM-APT/OFL complex, thus unquenching the FAM-APT signal. Interestingly, the recovered fluorescence intensity was found to correlate well with the antibiotic’s concentrations in the range of 0.2–200 μM in HEPES buffer, with a linear response that ranged between 0.2 and 20 μM. The rapid apta-assay achieved limits of detection and quantification of 0.12 and 0.40 μM, respectively. The truncated aptamer has also shown an improved specificity toward OFL than other quinolones, compared to the original full-length aptamer described in previous works. Finally, the practical application of the developed apta-assay was successfully confirmed to detect OFL quinolone in spiked milk samples, with satisfactory recoveries ranging between 97.4% and 111.4%.
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spelling pubmed-77617772020-12-26 Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay Ben Aissa, Sondes Mastouri, Mohamed Catanante, Gaëlle Raouafi, Noureddine Marty, Jean Louis Antibiotics (Basel) Article In this work, we describe the use of a new truncated aptamer for the determination of ofloxacin (OFL), being a principal quinolone commonly used in both human and animal healthcare. Since the affinity of a 72-mer ssDNA sequence has been previously described without further investigations, this paper demonstrates the first computational prediction of the binding motif between this aptamer and OFL through in silico molecular docking studies. Besides, we suggest the application of the characterized recognition mechanism in a simple FRET (Förster Resonance Energy Transfer) pattern for the rapid aptasensing of the quinolone of interest. Accordingly, our approach harnesses the fluorescence quenching of the fluorescein-tagged aptamer (FAM-APT) induced by its partial hybridization to a tetramethyl rhodamine-labelled complementary ssDNA (TAMRA-cDNA). In such a structure, dye labels brought into close proximity act as a FRET pair. Upon ofloxacin addition, an affinity competition occurs to form a more stable FAM-APT/OFL complex, thus unquenching the FAM-APT signal. Interestingly, the recovered fluorescence intensity was found to correlate well with the antibiotic’s concentrations in the range of 0.2–200 μM in HEPES buffer, with a linear response that ranged between 0.2 and 20 μM. The rapid apta-assay achieved limits of detection and quantification of 0.12 and 0.40 μM, respectively. The truncated aptamer has also shown an improved specificity toward OFL than other quinolones, compared to the original full-length aptamer described in previous works. Finally, the practical application of the developed apta-assay was successfully confirmed to detect OFL quinolone in spiked milk samples, with satisfactory recoveries ranging between 97.4% and 111.4%. MDPI 2020-12-03 /pmc/articles/PMC7761777/ /pubmed/33287135 http://dx.doi.org/10.3390/antibiotics9120860 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ben Aissa, Sondes
Mastouri, Mohamed
Catanante, Gaëlle
Raouafi, Noureddine
Marty, Jean Louis
Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay
title Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay
title_full Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay
title_fullStr Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay
title_full_unstemmed Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay
title_short Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay
title_sort investigation of a truncated aptamer for ofloxacin detection using a rapid fret-based apta-assay
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761777/
https://www.ncbi.nlm.nih.gov/pubmed/33287135
http://dx.doi.org/10.3390/antibiotics9120860
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