A Distance-Adjustable 3D DNA Scaffold Accelerated Quadruped DNA Walker for the Photoelectrochemical Biosensing of RNA in Phytophthora Infestans

Phytophthora infestans, the causative agent of potato late blight, poses a destructive threat to global agricultural production. Herein, a 3D DNA scaffold is elaborately designed as DNA tracks for accelerating the walking process of the quadruped walker, further accompanied by the perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA)/CdS/TiO₂ photoelectrode for the detection of RNA in Phytophthora infestans. Upon interaction with the target RNA, the quadruped walker was released to expeditiously walk along the 3D DNA scaffold and initiate the unfolding of hairpin 1 (H1), where H1 could completely pair with the alkaline phosphatase-modified hairpin 2 (AP-H2). Impressively, compared to traditional DNA track, the unique structure of the 3D DNA scaffold with equidistant binding sites and high continuity endowed the quadruped walker with a faster walking rate and better reaction efficiency. Consequently, a substantial quantity of AP-H2 was anchored, causing the hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA) by the immobilized AP. AA, as an active electron donor, can significantly increase the photocurrent signal. Thus, the proposed biosensor presents remarkable sensitivity in RNA detection with a detection limit down to 3.3 fM. This strategy of designing a 3D DNA scaffold as a DNA track provides a high-efficiency sensing platform and a valuable potential application for other biomarker detection.