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

This is the authors' abstract. We don't add key points for this paper.

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. • Novel probe design: To our knowledge, this is the first demonstration of BsAb-functionalized SERS nanotags , enabling noncovalent and orientation-controlled conjugation for improved assay fidelity. • Integrated digital immunoassay: Combining BsAb nanotags with nanomixing-enhanced microfluidics and digital readout improved sensitivity, signal uniformity, and reproducibility. • Clinical validation: We demonstrate accurate discrimination between SARS-CoV-2-positive and healthy individuals using clinical swab samples, underscoring the translational relevance of this approach.