A Novel Ratiometric Electrochemical Biosensor Using\nOnly One Signal Tag for Highly Reliable and Ultrasensitive Detection\nof miRNA-21

Abstract

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