Flexible gold nanoparticle SERS tape for rapid, label-free and ultrasensitive detection and differentiation of Shiga toxin variants

Surface-Enhanced Raman Spectroscopy (SERS) is a highly sensitive technique that enhances Raman signals using plasmonic nanomaterials, enabling the detection of trace biomolecules. Flexible SERS substrates offer advantages such as low cost, adaptability to various surfaces, and reliable performance in complex environments, making them ideal for real-time applications in food safety, environmental monitoring, and diagnostics. In this work, we present a flexible SERS sensor based on Kapton film coated with gold nanoparticles, fabricated through a simple bottom-up method. As a case study, we evaluated, for the first time to the best of our knowledge, its performance in detecting Shiga toxins (Stxs), which are produced by Shiga toxin-producing Escherichia coli (STEC) and are responsible for severe illnesses such as hemorrhagic colitis and hemolytic uremic syndrome. Early and accurate detection of Stxs is critical for outbreak control and timely treatment. We focused on three variants: Shiga toxin 1 (Stx1), Shiga toxin 2 (Stx2), and cleaved Stx2, which are structurally similar but differ in pathogenicity. The SERS spectra collected from each toxin revealed subtle but consistent differences, and Principal Component Analysis (PCA) successfully discriminated between them. Notably, our sensor achieved an outstanding enhancement factor of 7 × 10 6 and reached an ultra-low limit of detection (LOD) of 15 pM for Stx2 representing, to our knowledge, the lowest LOD reported to date for this toxin using a flexible SERS platform. We also evaluated the LOD in clinically relevant concentrations measured in sera, demonstrating that our system provides a sensitivity suitable for application in real diagnostic settings in the earlier stages of the disease. These results demonstrate the high sensitivity and discriminative power of our sensor, highlighting its strong potential for real-time, on-site monitoring of hazardous biomolecules in fields such as environmental surveillance, food safety, and clinical diagnostics. • Novel flexible SERS platform achieves ultra-low LOD (15 pM) for Stx2, lowest reported for flexible SERS to date. • First-time spectral discrimination of Stx1, Stx2, and cleaved Stx2 via PCA on flexible SERS tape. • High enhancement factor (7 × 10 6 ). • Bottom-up fabrication yields low-cost, high-performance plasmonic biosensors. • Ideal for real-time biosensing in food safety, diagnostics, and environment.