Experimental Optimization of a Plasmonic Surface Biofunctionalization, toward the Bimodal Biosensing and Kinetic Characterization of sPD1

Soluble PD1 (sPD1) plays a complex role in cancer pathophysiology, reportedly dependent on its interactions with immune checkpoint proteins and therapeutic monoclonal antibodies. Yet, no biosensor platform currently affords simultaneous quantification and kinetic profiling of sPD1-antibody interactions. Here, we introduce a surface plasmon resonance (SPR) refractometric biosensor setup functionalized with nivolumab that integrates direct, label-free quantification and real-time functional analysis of sPD1 in a buffer and human serum. Sensor functionalization strategies and robust regeneration protocols were investigated and optimized. The biosensor achieved a limit of detection of 5 ng/mL (limit of quantification (LOQ) 8.7 ng/mL; dynamic range 8.7 ng/mL to 376 μg/mL) and quantified sPD1 with 93 ± 5% recovery in 1% serum and 62 ± 30% in 10% serum. Kinetic constants (<i>k</i> _a ≈ 2.32 × 10⁵ M^-1 s^-1; <i>k</i> _d ≈ 1.03 × 10^-3 s^-1; <i>K</i> _D ≈ 4.66 nM) match literature values for the nivolumab-PD1 interaction. This dual-mode SPR platform represents the first attempt to achieve two distinct analytical functions: (i) quantitative detection of soluble PD-1 (sPD1) and (ii) kinetic characterization of the sPD1-antibody interaction, in a single platform, within biological media. The emergent significance of sPD1 as a liquid biopsy biomarker in immuno-oncologic profiling positions this biosensor setup as a powerful tool for research and potential clinical monitoring of immune checkpoint dynamics.