Electrochemical detection of miRNA-21 based on molecular beacon formed by thymine-mercury(II)-thymine base pairing

Abstract

We present a molecular beacon-based electrochemical biosensor with high sensitivity and specificity for the detection of microRNA-21. A special oligonucleotide probe was prepared containing a nucleotide sequence complementary to miR-21 and consecutively linking eight and six thymines to the 3 ' and 5 ' ends, respectively, to allow the formation of a T-Hg2+-T complex-based molecular beacon on the electrode surface by the selective binding of Hg2+ ions. The introduction of multiple thymines at the end of the probe avoids base overlapping between the miRNA sequence and the molecular beacon formation sequence, enabling a universal probe design that can detect all types of miRNAs. A ferrocene moiety was attached to the 5 '-end of the specially designed probe as an electrochemical signal indicator. The molecular beacons are formed by six consecutive T-Hg2+-T pairs by Hg2+ addition, and the molecular beacons are destroyed by perfect hybridization between 22 bases as a result of miR-21 addition. Based on this detection mechanism, we were able to detect miR-21 with LODs of 0.64 pM and 1.08 pM in buffer solution and human serum, respectively. In addition, the specifically designed oligonucleotide probe showed perfect specificity in detecting only miR-21 without binding to other miRNAs. Finally, the sensor showed excellent miR-21 recovery ability from samples spiked into serum, indicating that the method described in this study worked perfectly, even in a turbid complex matrix such as human serum.