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.