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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-02-01
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| Series: | Nanomaterials |
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| Online Access: | https://www.mdpi.com/2079-4991/11/2/368 |
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| author | Florentin R. Nitu Lorand Savu Sorin Muraru Ioan Stoian Mariana Ionită |
| author_facet | Florentin R. Nitu Lorand Savu Sorin Muraru Ioan Stoian Mariana Ionită |
| author_sort | Florentin R. Nitu |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-09T06:07:37Z |
| format | Article |
| id | doaj.art-c6b1a6887d8645069e855579b1fc4746 |
| institution | Directory Open Access Journal |
| issn | 2079-4991 |
| language | English |
| last_indexed | 2024-03-09T06:07:37Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Nanomaterials |
| spelling | doaj.art-c6b1a6887d8645069e855579b1fc47462023-12-03T12:02:33ZengMDPI AGNanomaterials2079-49912021-02-0111236810.3390/nano11020368Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence QuenchingFlorentin R. Nitu0Lorand Savu1Sorin Muraru2Ioan Stoian3Mariana Ionită4Faculty of Medical Engineering, University Politehnica of Bucharest, Gh. Polizu St., No. 1-7, 011061 Bucharest, RomaniaMolecular Biology Department, Genetic Lab, Milcov Street, No. 5, Sector 1, 012244 Bucharest, RomaniaFaculty of Medical Engineering, University Politehnica of Bucharest, Gh. Polizu St., No. 1-7, 011061 Bucharest, RomaniaRoyal Hospital, Splaiul Unirii Street, No. 313A, Sector 3, 030138 Bucharest, RomaniaFaculty of Medical Engineering, University Politehnica of Bucharest, Gh. Polizu St., No. 1-7, 011061 Bucharest, RomaniaLabel-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.https://www.mdpi.com/2079-4991/11/2/368graphene oxidesurfactantsfluorescence quenchingFREToptical microRNA sensor |
| spellingShingle | Florentin R. Nitu Lorand Savu Sorin Muraru Ioan Stoian Mariana Ionită Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching Nanomaterials graphene oxide surfactants fluorescence quenching FRET optical microRNA sensor |
| 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 | graphene oxide surfactants fluorescence quenching FRET optical microRNA sensor |
| url | https://www.mdpi.com/2079-4991/11/2/368 |
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