Programmable Dual‐Targeting Nano Bioconjugates: a Universal Bioconjugation Strategy to Enhance Single Molecule Proteomic Assays

Abstract Bioconjugation is crucial for functionalizing nanomaterials in biomedical applications, yet existing methods often struggle with site specificity, controlled orientation, stability and biofouling. Here, a universal bioconjugation strategy is presented for functionalizing nanoparticles with bispecific antibody fragments (BsAb‐Fs) to enable robust functionalization with any protein binders for precision biomedicine. Controlled bioconjugation relies on high‐affinity BsAb‐Fs (Kd≈nM range), site‐specifically conjugated to methoxy‐polyethylene glycol (mPEG)‐coated nanoparticles, preserving programmable target‐binding site for optimal orientation. The bioconjugation of BsAb‐Fs to nanoparticles optimizes surface presentation and spatial orientation, enhancing target binding. The robustness of this strategy is demonstrated for single‐cytokine detection in complex biological environments with surface‐enhanced Raman scattering (SERS) using active single‐particle SERS nanotags. The BsAb‐Fs based bioconjugation of SERS nanotags reduces batch‐to‐batch variability and improves colloidal stability, which improves SERS‐based immunoassay performance compared to covalent bioconjugation methods. This work applies this strategy for the simultaneous detection of three key cytokines regulating inflammatory response (human TNF‐α, IL‐12, IL‐1β) with exceptional specificity and sensitivity, achieving attomolar level detection of cytokines. Clinical evaluation is conducted by measuring cytokine levels in patients with major depressive disorder, highlighting the potential of this novel bioconjugation approach for diagnostics and personalized medicine.