A Rapid, Amplification‐Free, and Sensitive Diagnostic Assay for Single‐Step Multiplexed Fluorescence Detection of MicroRNA

Abstract

The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short-sequence nucleic acids next-generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single-step, single-temperature, and amplification-free assay provides a unique combination of performance parameters compared to state-of-the-art miRNA detection technologies. Precise multiplexed quantification of miRNA-20a, -20b, and -21 at concentrations between 0.05 and 0.5 nM in a single 150 μL sample and detection limits between 0.2 and 0.9 nM in 7.5 μL serum samples demonstrate the feasibility of both high-throughput and point-of-care clinical diagnostics.

Quantitative and Specific Detection of Exosomal miRNAs for Accurate Diagnosis of Breast Cancer Using a Surface‐Enhanced Raman Scattering Sensor Based on Plasmonic Head‐Flocked Gold Nanopillars

“…MicroRNAs (miRNAs) are recognized as critical regulators of multiple biological processes in cancer, breast cancer in particular, including tumorigenesis, tumor progression, stress response, and drug resistance . Since miRNA discovery, increasing evidence has indicated their essential roles in tumor progression and metastasis; therefore, miRNAs have emerged as potential biomarkers for cancer diagnosis, prognosis, and/or treatment efficacy . Thus, it was shown that miRNA expression patterns correlate with breast cancer subtype and the hormone receptor status …”

miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects

“…Recently, a plethora of novel assay concepts for measurement of miRNAs has been published, including formats based on multiplexed Förster or flourescence resonance energy transfer (FRET) and quantum dot assay designs [116][117][118], and ligation-mediated hybridization to microarrays [119], respectively. It remains to be elucidated if any of these methods can take the next step to move into the clinical routine.…”

Production of a fluorescence resonance energy transfer (FRET) biosensor membrane for microRNA detection

“…[13][14][15] Fluorescence resonance energy transfer (FRET), in which the electronic excitation energy of an energy donor chromophore is transferred to an energy acceptor in the presence of a target, has been used previously in miRNA detection. [16][17][18] Traditional fluorophores used in FRET-based detection include Cy5, FAM and Texas red. However, these are limited by intrinsic drawbacks including broad emission bands and photobleaching effects.…”