Engineered self-driven intelligent nanomachine induced by target-mediated knock-on effect to determine attomolar...

There remain great challenges in improving amplification efficiency and detection specificity of enzymatic biosensing platforms for genetic disease diagnosis. Herein, we constructed a single probe-based intelligent nanomachine (ESINM) to amplify cancer-related nucleic acids through engineered self-driven multiple cycles based on target-mediated knock-on effect. Strategically, to make the most of each base, the well-designed versatile ESINM probe is made up of eight functional regions to implement target and enzyme recognition, intramolecular configuration transformation, signal amplification. The free ESINM probe, like a dormant seed, remains a stable quenching hairpin structure with recognition function and allosteric activity restrained. Once the nicking-mediated cyclical strand-displacement polymerization between the target and ESINM probe is carried out to generate two types of key nicking fragments (NFs), a series of knock-on intermolecular and intramolecular priming-directed strand replication/nicking/displacement circuits spontaneously occur among NFs, ESINM probe, and all other intermediates, in which a dramatic stream of NFs of interest flow out instead of depleting. As a result, fluorescent DNA dendrimer like shining vine is formed. In such a high-efficient magnification way, the target can be tested down to 1 × 10^-6 nM within 80 min with a detection limit of 479 × 10^-9 nM. This one-step, mix-to-detection, one probe-involved biosensor without any wasted species possesses outstanding point mutation recognition ability and practical availability in biological samples, exhibiting great potentials in biochemical analysis and early diagnosis of genetic diseases.