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Here, a universal bioconjugation strategy is presented for functionalizing nanoparticles with bispecific antibody fragments (BsAb\u2010Fs) to enable robust functionalization with any protein binders for precision biomedicine. Controlled bioconjugation relies on high\u2010affinity BsAb\u2010Fs (Kd\u2248nM range), site\u2010specifically conjugated to methoxy\u2010polyethylene glycol (mPEG)\u2010coated nanoparticles, preserving programmable target\u2010binding site for optimal orientation. The bioconjugation of BsAb\u2010Fs to nanoparticles optimizes surface presentation and spatial orientation, enhancing target binding. The robustness of this strategy is demonstrated for single\u2010cytokine detection in complex biological environments with surface\u2010enhanced Raman scattering (SERS) using active single\u2010particle SERS nanotags. The BsAb\u2010Fs based bioconjugation of SERS nanotags reduces batch\u2010to\u2010batch variability and improves colloidal stability, which improves SERS\u2010based 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\u2010\u03b1, IL\u201012, IL\u20101\u03b2) 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.<\/jats:p>","DOI":"10.1002\/smtd.202501235","type":"journal-article","created":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T06:42:26Z","timestamp":1763966546000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Programmable Dual\u2010Targeting Nano Bioconjugates: a Universal Bioconjugation Strategy to Enhance Single Molecule Proteomic Assays"],"prefix":"10.1002","volume":"10","author":[{"given":"Fahimeh","family":"Farokhinejad","sequence":"first","affiliation":[{"name":"Centre of Personalized Nanomedicine Australian Institute for Bioengineering and Nanotechnology (AIBN) The University of Queensland  Brisbane QLD 4072 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