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

Abstract

AbstractWe 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.