Layer-by-layer characterizations of protein complexes by surface plasmon resonance enhanced raman system - detection of heparin-platelet factor IV complexes as an example

Although nanostructure-based strategies for Raman signal enhancement can effectively enhance the signal, they typically rely on complex material synthesis and result in uneven structural distribution on the substrate, affecting system stability and practical applicability. To address these limitations, this study presents a surface plasmon resonance (SPR) enhanced Raman detection platform based on the Kretschmann configuration. By exciting surface plasmon polaritons (SPPs) through attenuated total reflection (ATR) and integrating the advantages of SPR, such as real-time detection, label-free operation, and molecular fingerprint recognition, this approach enhances both the Raman signal and overall detection sensitivity. The system's feasibility was validated using the heparin–platelet factor 4 (PF4) complex as the target analyte. The results revealed a significant improvement in sensitivity, with the system capable of detecting concentrations as low as 1 ng/mL, compared to the previous detection limit of 50 ng/mL. Furthermore, a detailed analysis of the incident angle and polarization conditions revealed that the Raman signal enhancement was most pronounced at the SPR resonance angle under p-polarized illumination, thereby validating the optical coupling enhancement mechanism. Moreover, multilayered analysis associated with specific Raman molecular features enabled the successful identification of the fabrication processes. Ultimately, this system is anticipated to serve as an analytical tool for traditional immunoassays by allowing the identification of molecular binding interactions through enhanced Raman spectral signals. This not only provides a technical foundation for high-sensitivity biosensing but also enhances detection accuracy and specificity.