An ingenious electrochemical aptasensor for detection of CYFRA 21–1 based on dual-output toehold mediated strand displacement reaction.

• A high-efficiency electrochemical biosensor was proposed based on dual-output toehold mediated strand displacement reaction (TMSDR). • The whole sensing process was completely enzyme-free. • The homogeneous approach improved the efficiency of TMSDR. Herein, a "signal-on" electrochemical aptasensor based on dual-output toehold mediated strand displacement reaction (TMSDR) was developed to detect cytokeratin fragment antigen 21–1 (CYFRA 21–1). First, the methylene blue (MB) labeled signal probe (SP) was combined with a long template strand (TS) to form the three-strands duplex, which was immobilized on magnetic beads. The sequence of TS was innovatively designed with two toehold zones. Thus, with the presence of target DNA and Fuel strand (F), double SP and target were released through TMSDR. Besides, the released target could participate in the next cycle, achieving the further amplification. The released SP was selected by magnetic separation, then added to the gold electrode (GE) to hybridize with the hairpin DNA. Ultimately, the target can be detected by recording the current response of the MB. Under optimized conditions, the response peak current was linear with the concentration of CYFRA 21–1 in range from 10 pM to 1 μM with detection limit of 8.079 pM. The proposed signal amplification strategy was enzyme-free in homogenous solution and the using of magnetic beads improved the recognition efficiency and simplified the experimental steps, which endowed the sensor with super-sensitivity. [ABSTRACT FROM AUTHOR]