Ultrasensitive electrochemical detection of microRNA with star trigon structure and endonuclease mediated signal amplification

MicroRNAs play important roles in gene regulation. They can be used as effective biomarkers for diagnosis and prognosis of diseases like cancers. Due to their intrinsic properties of short length, low abundance and sequence homology among family members, it is difficult to realize sensitive and selective detection with economical use of time and cost. Herein, we report an ultrasensitive electrochemical method for microRNA analysis employing two oligonucleotides and one endonuclease. Generally, a glassy carbon electrode is first covered with gold nanoparticles (AuNPs) mediated by poly(diallyldimethylammonium chloride) (PDDA). Then, thiolated capture probe (CP) with methylene blue (MB) labeled at 5′ end is modified on the pretreated electrode. Hybridization occurs among target microRNA, CP and auxiliary probe (AP), forming a star trigon structure on the electrode surface. Subsequently, endonuclease recognizes and cleaves CP on CP/AP duplex, releasing microRNA and AP back to the solution. The two regenerated elements can then form another star trigon with other CP molecules, initiating cycles of CP cleavage and MB departure. Significant decrease of electrochemical signals is thus observed, which can be used to reflect the concentration of microRNA. This proposed method has a linear response to microRNA in a wide range from 100 aM to 1 nM and the sensitivity of attomolar level can be achieved. Moreover, it has high selectivity against single-base mismatch sequences and can be used directly in serum samples. Therefore, this method shows great feasibility for the detection of microRNA and may have potential applications in cancer diagnosis and prognosis.