Triggering dual-suppression and single-enhancement signal pattern via bipedal DNA walker and CHA for ratiometric...

In this study, an ultra-sensitive and specific ratiometric electrochemical aptasensor for the detection of alpha-fetoprotein (AFP) based on synergistic signal amplification mechanism of catalytic hairpin assembly (CHA) and bipedal DNA walker was proposed. We synthesized two novel nanocomposites for aptasensor: UiO-66@NG@Fc serves as a stable reference signal, while UiO-66-NH₂@BTC@MB functions as the signal source for the nanoprobe (Nanopro). This study innovatively introduces a single-step dual-amplification strategy that achieves a "dual-decrease and single-increase" signal pattern for methylene blue (MB) and ferrocene (Fc). By synergistically integrating two common electrochemical techniques, the detection mechanism operates as follows: First, high-specificity binding between AFP and its aptamer (AP273) triggers the initial attenuation of the MB signal (I_MB); subsequently, a CHA-based bipedal DNA walker cleaves the substrate strand (c-DNA), causing secondary I_MB attenuation. This design enables massive detachment of Nanopro from the electrode surface, thereby enhancing the Fc signal (I_Fc). Compared to individual detection of I_MB or I_Fc, the ratiometric approach using I_MB/I_Fc as the quantitative signal significantly improves sensitivity. This electrochemical ratiometric response (I_MB/I_Fc) for AFP demonstrated an excellent linear response within a range of 1.00 × 10^-2 to 1.00 × 10³ pg/mL, with an ultra-low detection limit of 4.93 fg/mL. Additionally, this sensor demonstrated good specificity, reproducibility, long-term stability, and achieved high recoveries (99.92%-106.01%) in human serum samples, representing that it had enormous potential in the clinical application.