Cargando…

Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching

Label-free homogeneous optical detection of low concentration of oligonucleotides using graphene oxide in complex solutions containing proteins remains difficult. We used a colloidal graphene oxide (GO) as a fluorescent probe quencher to detect microRNA-21 spiked-in cell culture medium, overcoming p...

Descripción completa

Detalles Bibliográficos
Autores principales: Nitu, Florentin R., Savu, Lorand, Muraru, Sorin, Stoian, Ioan, Ionită, Mariana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912907/
https://www.ncbi.nlm.nih.gov/pubmed/33540562
http://dx.doi.org/10.3390/nano11020368
_version_ 1783656683812159488
author Nitu, Florentin R.
Savu, Lorand
Muraru, Sorin
Stoian, Ioan
Ionită, Mariana
author_facet Nitu, Florentin R.
Savu, Lorand
Muraru, Sorin
Stoian, Ioan
Ionită, Mariana
author_sort Nitu, Florentin R.
collection PubMed
description Label-free homogeneous optical detection of low concentration of oligonucleotides using graphene oxide in complex solutions containing proteins remains difficult. We used a colloidal graphene oxide (GO) as a fluorescent probe quencher to detect microRNA-21 spiked-in cell culture medium, overcoming previously reported problematic aspects of protein interference with graphene oxide. We used a “turn off” assay for specific quenching-based detection of oligo DNA-microRNA hybridization in solution. A fluorescein conjugated 30-mer single-stranded DNA (ssDNA) probe was combined with a complementary synthetic microRNA (18 nucleotides) target. The probe-target hybridization was detected by specific quenching due to photoinduced electron transfer (PET). On the next step, GO captures and quenches the unhybridized probe by fluorescence resonance energy transfer (FRET) in the presence of cell culture medium supplemented with platelet lysate, 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton X-100 and 50% formamide. This resulted in sensitive measurement of the specific probe-target complexes remaining in solution. The detection is linear in the range of 1 nM and 8 nM in a single 100 μL total volume assay sample containing 25% cell culture medium supplemented with platelet lysate. We highlight a general approach that may be adopted for microRNA target detection within complex physiological media.
format Online
Article
Text
id pubmed-7912907
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-79129072021-02-28 Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching Nitu, Florentin R. Savu, Lorand Muraru, Sorin Stoian, Ioan Ionită, Mariana Nanomaterials (Basel) Article Label-free homogeneous optical detection of low concentration of oligonucleotides using graphene oxide in complex solutions containing proteins remains difficult. We used a colloidal graphene oxide (GO) as a fluorescent probe quencher to detect microRNA-21 spiked-in cell culture medium, overcoming previously reported problematic aspects of protein interference with graphene oxide. We used a “turn off” assay for specific quenching-based detection of oligo DNA-microRNA hybridization in solution. A fluorescein conjugated 30-mer single-stranded DNA (ssDNA) probe was combined with a complementary synthetic microRNA (18 nucleotides) target. The probe-target hybridization was detected by specific quenching due to photoinduced electron transfer (PET). On the next step, GO captures and quenches the unhybridized probe by fluorescence resonance energy transfer (FRET) in the presence of cell culture medium supplemented with platelet lysate, 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton X-100 and 50% formamide. This resulted in sensitive measurement of the specific probe-target complexes remaining in solution. The detection is linear in the range of 1 nM and 8 nM in a single 100 μL total volume assay sample containing 25% cell culture medium supplemented with platelet lysate. We highlight a general approach that may be adopted for microRNA target detection within complex physiological media. MDPI 2021-02-02 /pmc/articles/PMC7912907/ /pubmed/33540562 http://dx.doi.org/10.3390/nano11020368 Text en © 2021 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
Nitu, Florentin R.
Savu, Lorand
Muraru, Sorin
Stoian, Ioan
Ionită, Mariana
Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching
title Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching
title_full Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching
title_fullStr Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching
title_full_unstemmed Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching
title_short Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching
title_sort label-free homogeneous microrna detection in cell culture medium based on graphene oxide and specific fluorescence quenching
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912907/
https://www.ncbi.nlm.nih.gov/pubmed/33540562
http://dx.doi.org/10.3390/nano11020368
work_keys_str_mv AT nituflorentinr labelfreehomogeneousmicrornadetectionincellculturemediumbasedongrapheneoxideandspecificfluorescencequenching
AT savulorand labelfreehomogeneousmicrornadetectionincellculturemediumbasedongrapheneoxideandspecificfluorescencequenching
AT murarusorin labelfreehomogeneousmicrornadetectionincellculturemediumbasedongrapheneoxideandspecificfluorescencequenching
AT stoianioan labelfreehomogeneousmicrornadetectionincellculturemediumbasedongrapheneoxideandspecificfluorescencequenching
AT ionitamariana labelfreehomogeneousmicrornadetectionincellculturemediumbasedongrapheneoxideandspecificfluorescencequenching