Ingeniería de una sonda dúplex de tres hebras de disposición plana para un circuito de ADN catalítico hacia una...

Nucleic acid probe design and immobilization are fundamental to the high performance of biosensors. However, the dependence on elaborate fabrication processes or meticulous optimization of probe immobilization poses challenges to achieving reliable bioanalysis and high sensing efficiency. Herein, we report an effective strategy for constructing electrochemical DNA biosensor that leverages a catalytic DNA reaction on a uniquely designed three-stranded duplex (TSD) probe. The TSD probe is engineered with an internally positioned thiol group to adopt a favorable flat-lying immobilization orientation. Compared to conventional upright TSD probe, the flat-lying design facilitates a more accessible interface and more stable assembly density with reduced dependence on immobilization concentration. This flat-lying system demonstrated superior sensing performance, including a faster reaction rate (completed within 1.5 h vs. 2.5 h for upright probes) and a lower detection limit of 244 fM for target, which is about 40-fold better than the upright configuration. The sensor also demonstrated excellent selectivity against mismatched sequences, better reproducibility and was successfully applied for target detection in diluted serum. This work presents a novel and facile probe design and immobilization paradigm that eliminates the traditional need for complex density optimization, offering a robust sensing platform for highly sensitive and efficient DNA detection.