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Isothermal Rolling Circle Amplification and Lanthanide‐Based FRET for Femtomolar Quantification of MicroRNA

Abstract : 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.
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https://hal.archives-ouvertes.fr/hal-03747854
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Submitted on : Monday, August 8, 2022 - 6:04:32 PM
Last modification on : Wednesday, August 10, 2022 - 3:22:32 AM

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Analysis Sensing - 2022 - De...
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Mariia Dekaliuk, Pierre Busson, Niko Hildebrandt. Isothermal Rolling Circle Amplification and Lanthanide‐Based FRET for Femtomolar Quantification of MicroRNA. Analysis & Sensing, Wiley, inPress, ⟨10.1002/anse.202200049⟩. ⟨hal-03747854⟩

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