Single-step, homogeneous and sensitive detection for microRNAs with dual-recognition steps based on luminescence resonance energy transfer (LRET) using upconversion nanoparticles

A single-step, homogeneous and sensitive LRET assay is presented for the detection of miRNAs. The amplification-free assay provides a unique combination of high specificity with dual-recognition approach of different hybridization and ligation steps and preventing background auto-fluorescence in biological samples using upconversion nanoparticles (UCNPs) as signal-producing nanoprobes. The assay probe is composed of signal-producing unit (a pair of homogeneous upconversion luminescence resonance energy transfer (UC-LRET)-based oligonucleotides) and recognition unit (two adaptor oligonucleotides). In the presence of target miRNAs, the probe and target miRNAs leads to the formation of stable double-strands and semi-stable adaptor-miRNAs complexes with an adaptor nick. Ligation of the nick using ligase cause the formation of stable double-strands, resulting in UCNPs-to-dye UC-LRET for detection of the miRNAs with near-infrared radiation (980 nm). Sensitive detection of miRNA-21 at concentrations of 200 pM to 1.4 nM and detection limits of 0.095 nM with good precision of 3.9% (RSD) for seven repeated measurements of 500 pM miRNAs demonstrate the feasibility of both high throughput and point-of-care clinical diagnostics. The homogeneous UC-LRET assay without any washing can be extended to the application in other important types of nucleic acid analysis.