An enzyme-free and label-free signal-on aptasensor based on DNAzyme-driven DNA walker strategy

Herein, a signal-on electrochemical aptasensor for highly sensitive detection of thrombin (TB) was constructed based on the DNAzyme-driven DNA walker strategy. We developed a new dual functional hairpin DNA (HP) containing a substrate sequence of the Mg²⁺-dependent DNAzyme (in the loop region) and the G-quadruplex forming segment (in the stem region). The DNA walker (TBA₂-DWs), containing a TB aptamer and an enzymatic sequence, was introduced onto gold electrode (GE) by aptamers-target specific recognition, and thus initiated the enzymatic sequences to hybridize with the substrate sequence. Then, the DNA walker could repeatedly bind and cleave HP in the assistance of Mg²⁺, unlocking many active G-quadruplex forming sequences. Finally, hemin can further bind the G-quadruplex to form G-quadruplex/hemin complexes and generate enhanced current output. The aptasensor for TB assay showed a linear detection range from 1 pM to 60000 pM with a lower detection limit of 0.58 pM. And more, the proposed detection strategy was enzyme-free and label-free.