Electrochemical biosensor for microRNA detection based on hybridization protection against nuclease S1 digestion

Nuclease S1 can catalyze the nonspecific endo- and exonucleolytic cleavage of single-stranded DNA and RNA to yield nucleoside 5′-phosphates and 5′-phosphooligonucleotides. However, it cannot hydrolyze double-stranded DNA, double-stranded RNA, or DNA-RNA hybrid. Inspired by this specific property, a simple electrochemical method was developed for microRNA detection based on hybridization protection against nuclease S1 digestion. In the absence of hybridization process, the assembled probe DNA on the electrode surface can be easily digested by nuclease S1 and a strong electrochemical signal can be generated due to the decreased repulsive force towards the redox probe. However, after hybridization with target microRNA, the digestion activity of nuclease S1 is inhibited, which can lead to a weak electrochemical signal. Based on the change of the electrochemical signal, the detection of target microRNA-319a can be achieved. Under optimal experiment conditions, the electrochemical signal was proportional to microRNA-319a concentration from 1000 to 5 pM and the detection limit was 1.8 pM (S/N = 3). The developed method also showed high detection selectivity and reproducibility. Furthermore, the proposed method was successfully applied to assay the expression level of microRNA-319a in the leaves of rice seedlings after being incubated with different concentrations of 6-benzylaminopurine.