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

ABSTRACT 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 10 2 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.