Isothermal Rolling Circle Amplification and Lanthanide‐Based FRET for Femtomolar Quantification of MicroRNA
Quantification des microARN au niveau femtomolaire par amplification par cercle roulant et FRET à base de lanthanide
Résumé
Quantification of DNA and RNA biomarkers via isothermal amplification technologies can significantly simplify biosensing and clinical diagnostics. However, highly sensitive and wash-free assay formats based on time-resolved Förster resonance energy transfer (TR-FRET) have been limited to terbium (Tb) based FRET pairs and their application on specialized clinical instruments. Here, we present the implementation of rolling circle amplification (RCA)-FRET to europium-dye (Eu-ATTO620) FRET pairs and its application on standard benchtop fluorescence plate readers. Eu-ATTO620 RCA-FRET was used to quantify microRNA-21 (miR-21) in a 1 to 100 pM concentration range on a SPARK multimode plate reader system. Detection limits down to 120±20 fM (18±3 attomol of miR-21) and quantification of endogenous miR-21 from the plasma of ovarian cancer patients using a KRYPTOR clinical plate reader demonstrated the broad applicability and clinical relevance of Eu-dye-based RCA-FRET. The extension to both Eu and Tb based TR-FRET and to standard bioanalytical laboratory instruments has the potential to significantly broaden the application and versatility of RCA-FRET for simple, highly-sensitive, and multiplexed bioanalysis.
Domaines
Biochimie, Biologie Moléculaire
Fichier principal
Analysis Sensing - 2022 - Dekaliuk - Isothermal Rolling Circle Amplification and Lanthanide‐Based FRET for Femtomolar.pdf (1.48 Mo)
Télécharger le fichier
Origine | Publication financée par une institution |
---|