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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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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 |
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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 |
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