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 washfree 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.

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards

“…69B). 436 Higher-order multiplexing via incorporation of more A dyes, more separations distances, or more lanthanide complexes, quantification of RNA biomarkers (e.g., micro-RNA), 419 or implementation into wash-free imaging or standard benchtop fluorescence plate readers, 449,450 are amongst the versatile extensions of this multiplexed FRET-DNA scaffold biosensor design demonstrated to date.…”

Mapping the Distribution of Electronic States within the 5D4 and 7F6 Levels of Tb3+ Complexes with Optical Spectroscopy

“…Lanthanide luminescence has found widespread use in anticounterfeiting agents, 1 sensing, 2-3 bioassays, [4][5][6][7] and display technology. 8 Terbium(III) ions in particular, has emerged on the forefront of a number of applications such as FRET based biosensing, [9][10][11][12][13][14][15][16] , luminescent bioprobes, [17][18] and singe-molecule magnets. [19][20][21][22] In addition, the high energy gap between the emitting 5 D4 level and the lower lying 7 F6-0 manifold makes Tb(III) luminescence less susceptible to quenching by the chemical environment than other luminescent lanthanides.…”