Development and application of multivalent nanobody-functionalized plasmonic probes in SERS sensing platforms.

Surface-enhanced Raman scattering (SERS) immunoassays have emerged as highly sensitive, multiplexed analytical techniques for detecting protein biomarkers. Traditional SERS immunoassays typically rely on antibody-based SERS probes for target protein detection; however, it is challenging to obtain antibodies that are both highly effective at identifying natural proteins and suitable for SERS probe conjugation. Herein, we engineer a MultiValent Probe (MVP), consisting of multivalent nanobodies as the protein-targeting ligand to provide improved binding avidity and Raman reporter-coated gold-silver alloy nanoboxes for single-particle signal readouts. The multivalent nanobodies exhibit precise antigen recognition and exceptional affinity, and are expressed in a mammalian system for cost-effective and large-scale production. We thoroughly characterize the MVP via nanoparticle tracking analysis, nanoflow cytometry, and differential centrifugal sedimentation. To further enhance assay performance, we integrate MVP with a nanomixing-enhanced microfluidic chip to develop an MVP-based SERS-microfluidic immunoassay. As a proof of concept, we demonstrate the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins and virions from multiple strains in clinical nasopharyngeal samples (39 healthy and 39 infected), showing 84.6% concordance with RT-qPCR. This work highlights the potential of MVP-incorporated SERS-microfluidic immunoassays for diagnostics of pandemic diseases and broader applications in detecting a wide range of viral pathogens.