Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification
Aptamer-based assays and sensors are garnering increasing interest as alternatives to antibodies, particularly due to their increased flexibility for implementation in alternative assay formats, as they can be employed in assays designed for nucleic acids, such as molecular aptamer beacons or aptame...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-11-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/12/11/972 |
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| author | Miriam Jauset-Rubio Mayreli Ortiz Ciara K. O’Sullivan |
| author_facet | Miriam Jauset-Rubio Mayreli Ortiz Ciara K. O’Sullivan |
| author_sort | Miriam Jauset-Rubio |
| collection | DOAJ |
| description | Aptamer-based assays and sensors are garnering increasing interest as alternatives to antibodies, particularly due to their increased flexibility for implementation in alternative assay formats, as they can be employed in assays designed for nucleic acids, such as molecular aptamer beacons or aptamer detection combined with amplification. In this work, we took advantage of the inherent nucleic acid nature of aptamers to enhance sensitivity in a rapid and facile assay format. An aptamer selected against the anaphylactic allergen β-conglutin was used to demonstrate the proof of concept. The aptamer was generated by using biotinylated dUTPs, and the affinity of the modified aptamer as compared to the unmodified aptamer was determined by using surface plasmon resonance to calculate the dissociation constant (K<sub>D</sub>), and no significant improvement in affinity due to the incorporation of the hydrophobic biotin was observed. The modified aptamer was then applied in a colorimetric competitive enzyme-linked oligonucleotide assay, where β-conglutin was immobilized on the wells of a microtiter plate, competing with β-conglutin free in solution for the binding to the aptamer. The limit of detection achieved was 68 pM, demonstrating an improvement in detection limit of three orders of magnitude as compared with the aptamer simply modified with a terminal biotin label. The concept was then exploited by using electrochemical detection and screen-printed electrodes where detection limits of 326 fM and 7.89 fM were obtained with carbon and gold electrodes, respectively. The assay format is generic in nature and can be applied to all aptamers, facilitating an easy and cost-effective means to achieve lower detection limits. |
| first_indexed | 2024-03-09T19:14:35Z |
| format | Article |
| id | doaj.art-ce3548608dc54e87ac04f03d7000068e |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-09T19:14:35Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-ce3548608dc54e87ac04f03d7000068e2023-11-24T03:55:13ZengMDPI AGBiosensors2079-63742022-11-01121197210.3390/bios12110972Exploiting the Nucleic Acid Nature of Aptamers for Signal AmplificationMiriam Jauset-Rubio0Mayreli Ortiz1Ciara K. O’Sullivan2Interfibio Consolidated Research Group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007 Tarragona, SpainInterfibio Consolidated Research Group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007 Tarragona, SpainInterfibio Consolidated Research Group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007 Tarragona, SpainAptamer-based assays and sensors are garnering increasing interest as alternatives to antibodies, particularly due to their increased flexibility for implementation in alternative assay formats, as they can be employed in assays designed for nucleic acids, such as molecular aptamer beacons or aptamer detection combined with amplification. In this work, we took advantage of the inherent nucleic acid nature of aptamers to enhance sensitivity in a rapid and facile assay format. An aptamer selected against the anaphylactic allergen β-conglutin was used to demonstrate the proof of concept. The aptamer was generated by using biotinylated dUTPs, and the affinity of the modified aptamer as compared to the unmodified aptamer was determined by using surface plasmon resonance to calculate the dissociation constant (K<sub>D</sub>), and no significant improvement in affinity due to the incorporation of the hydrophobic biotin was observed. The modified aptamer was then applied in a colorimetric competitive enzyme-linked oligonucleotide assay, where β-conglutin was immobilized on the wells of a microtiter plate, competing with β-conglutin free in solution for the binding to the aptamer. The limit of detection achieved was 68 pM, demonstrating an improvement in detection limit of three orders of magnitude as compared with the aptamer simply modified with a terminal biotin label. The concept was then exploited by using electrochemical detection and screen-printed electrodes where detection limits of 326 fM and 7.89 fM were obtained with carbon and gold electrodes, respectively. The assay format is generic in nature and can be applied to all aptamers, facilitating an easy and cost-effective means to achieve lower detection limits.https://www.mdpi.com/2079-6374/12/11/972aptameraptasensorsbiotinylated dNTPsenzyme-linked oligonucleotide assay (ELONA)chronoamperometryelectrochemical detection |
| spellingShingle | Miriam Jauset-Rubio Mayreli Ortiz Ciara K. O’Sullivan Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification Biosensors aptamer aptasensors biotinylated dNTPs enzyme-linked oligonucleotide assay (ELONA) chronoamperometry electrochemical detection |
| title | Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification |
| title_full | Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification |
| title_fullStr | Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification |
| title_full_unstemmed | Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification |
| title_short | Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification |
| title_sort | exploiting the nucleic acid nature of aptamers for signal amplification |
| topic | aptamer aptasensors biotinylated dNTPs enzyme-linked oligonucleotide assay (ELONA) chronoamperometry electrochemical detection |
| url | https://www.mdpi.com/2079-6374/12/11/972 |
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