Physicochemical Tuning of Bispecific Antibody Fragment Immobilization for Robust Digital SERS Biosensing.

Surface-enhanced Raman scattering (SERS) biosensing platforms offer exceptional sensitivity and multiplexing capabilities for biomarker detection, but their performance is often limited by uncontrolled ligand orientation on nanotags. To address this, we developed a SERS nanotag incorporating a bispecific antibody fragment (BsAb) engineered to bind the SARS-CoV-2 receptor-binding domain (RBD) and methoxy polyethylene glycol (mPEG) simultaneously, enabling orientation-controlled immobilization onto mPEG-grafted plasmonic nanostructures. We systematically evaluated how immobilization conditions─particularly pH relative to the BsAb's isoelectric point (pI)─influence surface density, antigen accessibility, and overall assay performance using a combination of orthogonal physicochemical and functional assays. Our results show that immobilization at pH above the BsAb's pI improves antigen accessibility and nanotag stability, despite reduced immobilization density. When integrated into a digital SERS biosensing platform, the BsAb-functionalized SERS nanotags enabled highly specific digital detection of RBD in plasma, achieving a low limit of detection (3.97 ng/mL) and outperforming enzyme-linked immunosorbent assay in assay time, reagent consumption, and multiplexing potential. This study establishes a robust strategy for designing and optimizing antibody fragment-based SERS nanotags, advancing their application in next-generation diagnostic platforms.