Digital SERS Nanostructured Platform for Amplification-free Single-Molecule DNA Detection and Pre-Symptomatic Diagnosis of Kiwifruit Soft Rot

We present a digital surface-enhanced Raman spectroscopy (SERS) platform enabling enzyme- and amplification-free single-molecule DNA detection through Poisson distribution-driven quantification and geometrically optimized plasmonic nanostructures. Utilizing high-throughput UV lithography, we fabricated large-area nanopillar arrays (1.5 × 1.5 cm) with tunable heights (500–1300 nm) and periodicities (4–10 μm), addressing key limitations in conventional SERS methods via three innovations: (1) dynamic hotspot generation: Target DNA induces plasmonic dimerization between functionalized gold nanoprobes and Au-capped nanopillars, creating spatially resolved SERS enhancement (1326 cm–1 DTNB signal), (2) digital counting algorithms: Compensate Raman intensity variations by quantifying activated pillars as discrete molecular events, and (3) geometric confinement: Spatial confinement effects enabling statistically robust single-molecule occupancy. This platform achieved 10 fM sensitivity for Diaporthe spp. DNA detection, outperforming PCR-electrophoresis (1 pM) by 2 orders of magnitude. Applied to presymptomatic kiwifruit diagnosis, the system identified pathogen DNA in asymptomatic samples prior to visual symptom development, validated by characteristic DTNB peaks (1326 cm–1) absent in negative controls. The modular design permits multiplexed detection through probe diversification, while the absence of enzymatic steps enables field-deployable operation. By integrating single-molecule statistics with plasmonic signal amplification, this work establishes a robust framework for precision molecular diagnostics, demonstrating transformative potential for agricultural pathogen surveillance and low-abundance biomarker analysis. The technology’s batch-producible nanopillar architecture and digital quantification strategy overcome longstanding reproducibility challenges in SERS-based detection, positioning it as a versatile tool for next-generation biosensing applications.