Jinai Chen,Yujiao Wei,Liming Wang,Leyi Chen,Shengnan He,Honglin Liu
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.
Yuzhu Li,Zhongxiang Ding,Hongyan Wang,Cheng Qu,Guangping Li,Honglin Liu
In clinical diagnostics, human breath presents an alternative and more convenient sample than biofluids for detecting the ingestion of nonvolatile drugs. Surface-enhanced Raman spectroscopy (SERS) is a powerful vibrational spectroscopy technique with high sensitivity based on molecular fingerprinting. However, the low affinity of traditional SERS substrates for aerosols and the stochastic fluctuation of the SERS signal at low concentrations limit their application in breath aerosol analysis. In this study, we synthesized hydrogel microsphere SERS substrates with highly reversible swelling/shrinking properties that enhance target analyte accumulation in breath aerosols and promote plasmonic nanoparticle aggregation for intense Raman hotspot formation. Furthermore, these hydrogel microsphere SERS substrates function as a three-in-one system, enabling multilevel selectivity based on size, charge, and hydrophilicity for target molecules simultaneously without pretreatment. Notably, by “digitizing” the SERS signal of each individual hydrogel microsphere and calculating the proportion of positive microspheres, the hydrogel microspheres can serve as a digital SERS platform that circumvents the low stability issues resulting from fluctuations in SERS signal intensity. Consequently, the digital SERS platform achieved a detection limit of 0.5 ppm for fentanyl in simulated breath aerosols. This innovative sensing strategy not only demonstrates a promising approach for screening nonvolatile drugs but also simplifies the sampling process, holding great potential for clinical diagnosis of breath aerosols.
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