Isothermal circular strand displacement-based assay for microRNA detection in liquid biopsy

Extracellular miRNAs are promising targets for developing new assays for the early diagnosis and prognosis of diseases based on liquid biopsy. The detection of miRNAs in liquid biopsies is challenged by their short sequence length, low concentration, and interferences with bodily fluid components. Isothermal circular strand displacement polymerization has emerged as a convenient method for nucleic acid amplification and detection. Herein, we describe an innovative strategy for microRNA detection directly from biological fluids based on hairpin probe-assisted isothermal amplification reaction. We designed and optimized the assay to detect target analytes in 1 µL of the complex media's biological matrix using a microfluidic device for the straightforward analysis of multiple samples. We validated the assay to detect circulating miR-127-5p in synovial fluid, recently indicated as a predictive biomarker for osteoarthritis disease. The combined use of a mutant polymerase operating with high yield and a primer incorporating locked nucleic acid nucleosides allowed detection of miR-127-5p with 34 fmol L^-1 LOD. We quantified circulating miR-127-5p directly in synovial fluid, thus demonstrating that the assay may be employed for the convenient detection of 4.3 ± 0.5 pmol L^-1 concentrated miRNAs in liquid biopsy samples.