Epitope-Resolved Digital SERS Profiling of Structurally Dynamic Antigens via a Multi-Epitope Bispecific Antibody Framework.

Digital surface-enhanced Raman scattering (SERS) immunoassays digitize epitope binding events to achieve ultrasensitive protein detection. However, existing implementations predominantly rely on single-epitope recognition, yielding 1D molecular view of antigen structure and obscuring how conformational heterogeneity or mutation-induced changes affect epitopes. This limitation cannot be resolved simply by combining multiple monoclonal antibodies, as heterogeneous conjugation and uncontrolled binding collapse epitope-specific responses into pooled, non-assignable signals. Here, we introduce EpiCount-SERS (Epitope-Resolved Digital Counting by SERS), a multi-epitope digital SERS framework that enables epitope-resolved immunochemical profiling. The platform employs nanobody-based bispecific antibody fragments that pair epitope-specific nanobodies with a unified anti-methoxy polyethylene glycol conjugation domain, enabling orientation-controlled attachment to spectrally encoded SERS nanotags. Epitope-specific binding events are discretized into independent digital channels, allowing distinct epitopes on the SARS-CoV-2 receptor-binding domain to be interrogated in parallel. Digital enumeration across these channels generates epitope-resolved molecular fingerprints that capture information beyond single-epitope assays. EpiCount-SERS achieves sub-ng mL- 1 sensitivity for recombinant protein, detects inactivated virus at approximately 102 copies µL- 1, and classifies clinical nasopharyngeal swab samples with accuracy of 88.3% (area under the curve = 0.9467). Because epitope binders can be exchanged without altering the unified conjugation strategy, EpiCount-SERS provides a scalable framework for digital immunochemical profiling of structurally dynamic protein targets.