A digital SERS platform powered by bispecific antibody fragments for SARS-CoV-2 diagnostics.

Surface-enhanced Raman scattering (SERS) immunoassays are powerful analytical tools for protein detection while typically rely on the availability of suitable antibodies. Compared to full-length antibodies, antibody fragments provide advantages such as rapid and cost-effective production. However, oriented conjugation of antibody fragments to SERS nanotags, essential for maintaining their functionality, remains relatively underexplored. Here, we introduce a bispecific antibody (BsAb)-programmable digital immunochemistry detection regime implemented on an advanced digital SERS platform ("DigibiSERS"), enabling deterministic molecular orientation, variance-robust single-particle event calling, and kinetic regularization within one unified platform. Specifically, we fuse an anti-nucleocapsid nanobody with a single-chain variable fragment targeting methoxy polyethylene glycol (mPEG) grafted onto the SERS nanotag surfaces. This design facilitates straightforward, oriented BsAb conjugation, preserving its functionality. The DigibiSERS platform, incorporating single-particle active SERS nanotags, nanomixing-enhanced microchips, and digital readouts, demonstrates high sensitivity and specificity, achieving detection limits of 2.01 ng/mL for nucleocapsid protein and 2.7 copies/mL for virus. An area under the curve (AUC) of 0.8783 highlights the potential of engineered antibody fragments in enhancing the clinical sensitivity and practicality of SERS-based immunoassays for infectious disease diagnostics.