Target-triggered triple isothermal cascade amplification strategy for ultrasensitive microRNA-21 detection at sub-attomole level

MicroRNA-21 (miR-21) is a promising diagnostic biomarker for breast cancer screening and disease progression, thus the method for the sensitive and selective detection of miR-21 is vital to its clinical diagnosis. Herein, we develop a novel method to quantify miR-21 levels as low as attomolar sensitivity by a target-triggered triple isothermal cascade amplification (3TICA) strategy. An ingenious unimolecular DNA template with three functional parts has been designed: 5'-fragment as the miR-21 recognition unit, middle fragment as the miR-21 analogue amplification unit, and 3'-fragment as the 8-17 DNAzyme production unit. Triggered by miR-21 and accompanied by polymerase-nicking enzyme cascade, new miR-21 analogues autonomously generated for the successive re-triggering and cleavage process. Simultaneously, the 8-17 DNAzyme-contained sequence could be exponentially released and activated for the second cyclic cleavage toward a specific ribonucleotide (rA)-contained substrate, inducing a remarkably amplified generation of HRP-mimicking DNAzyme in the presence of hemin. Finally, the amperometric technique was used to record the catalytic reduction current of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H₂O₂. The increase in the steady-state current was proportional with the increase of the miR-21 concentration from 1 aM to 100 pM. An ultra-low detection limit of 0.5 aM with an excellent selectivity for even discriminating differences between 1-base mismatched target and miR-21 was achieved. This simple and cost-effective 3TICA strategy is promising for the detection of any short oligonucleotides, simply by altering the target recognition unit in the template sequence.