Ultrasensitive quantitation of circulating miR-195–5p with triple strand displacement amplification cascade

Circulating miR-195-5p has been proposed as a promising peripheral biomarker for the diagnosis, prognosis and severity assessment of various diseases. However, the demand for its sensitive and convenient quantification has not been met yet. Herein, we proposed a one-pot isothermal approach, in which the target signal acquisition, amplification and conversion (fluorescence read-out) system was integrated by a triple strand displacement amplification (SDA) cascade. Using this triple SDA strategy, miR-195-5p can be at least detected at 1 aM, and the linear dynamic range (from 100 aM to 1 pM) is wide enough to meet the detection needs of clinical miRNA level. A proof-of-principle study, using this novel methodology to directly analyze the spiking serum samples with different levels of miR-195-5p, demonstrated the potential of circulating miR-195-5p detection for clinical point-of-care assay. This one-pot isothermal triple SDA approach, we believe, will be a simple and feasible tool for ultrasensitive quantification of circulating miR-195-5p, and may promote the wide application of this potential biomarker in non-invasive clinical diagnosis.