A Novel Ratiometric Electrochemical Biosensor Using Only One Signal Tag for Highly Reliable and Ultrasensitive Detection of miRNA-21.

Herein, a novel ratiometric electrochemical biosensor with methylene blue (MB) as the only one signal tag was proposed for highly reliable and ultrasensitive detection of microRNA-21 (miRNA-21) under the assistance of an intelligent target-induced dual signal amplification (T-DSA). First, a small amount of target miRNA-21 could produce abundant mimic targets DNA S1 and Zn2+ through target-induced recycle and acid dissolution, respectively. Then, S1 triggered rolling circle amplification (RCA) to generate functional DNA nanospheres (DSP) encoded by DNAzyme and substrate sequence for loading numerous signal tag MB with a remarkable electrochemical signal (signal on), and the Zn2+ cofactor mediated the nonviolent DNAzyme-catalyzed cleavage of DSP to sharply release MB with obviously reduced electrochemical responses (signal off). Impressively, our strategy could controllably load and release the only signal tag MB through the well-designed DSP to effectively avoid the false positive responses caused by the non-ideal upright state of DNA probes connected to electrodes in traditional distance-dependent signal adjustment ratiometric strategies with two different signal tags. Meanwhile, with the aid of innovative T-DSA recycle and RCA-produced functional DSP, the detection sensitivity of this sensing platform was significantly improved. As a result, the proposed biosensor successfully realized highly reliable and ultrasensitive detection of miRNA-21 with a detection limit down to 26.7 aM, which shows exceptional promise in biological analysis and medical diagnosis.