Dual DNAzyme-MoS₂/GDY Catalytic Assembly Enables Smartphone-Based Multiplex Detection of Sugarcane Pokkah Boeng Pathogens at Sub-Femtomolar Levels

Rapid on-site detection of sugarcane pokkah boeng disease caused by Fusarium pathogens remains challenging due to the lack of portable platforms combining high sensitivity and multiplexing capability. Here, we present a self-powered biosensor integrating a dual DNAzyme-driven catalytic system with a MoS₂/graphdiyne (GDY) nanohybrid-modified biofuel cell (EBFC) for simultaneous detection of <i>Fusarium sacchari</i> and <i>Fusarium verticillioides</i>. The key innovation lies in the windmill-shaped dual DNAzyme structure that enables Mn²⁺/Mg²⁺-dependent target recycling, synergistically coupled with the hybridization chain reaction (HCR) and triplex catalytic hairpin assembly (TCHA) for exponential signal amplification. The MoS₂/GDY nanohybrid provides an ideal conductive substrate with 3.8-fold higher DNA loading capacity than pristine MoS₂, while the integration of a charge-storage capacitor boosts detection sensitivity by 10.4- and 9.8-fold compared with conventional EBFCs through transient current amplification. The smartphone-coupled system achieves unprecedented detection limits of 21.3 aM (<i>F. sacchari</i>) and 54.3 aM (<i>F. verticillioides</i>) with a dynamic range spanning 5 orders of magnitude (0.1 fM-10 nM), demonstrating excellent specificity against non-target pathogens (more than 95% signal discrimination). This smartphone-integrated biosensor represents a field-ready diagnostic tool for rapid on-site screening of sugarcane fungal pathogens, offering a transformative approach to mitigate crop losses through early disease intervention and precision agriculture management.