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

## TL;DR

A new biosensor was developed to detect and analyze interactions of soluble PD1 with antibodies in biological samples.

## Contribution

The first biosensor platform combining quantitative detection and kinetic analysis of sPD1–antibody interactions in biological media.

## Key findings

- The biosensor achieved a detection limit of 5 ng/mL and quantification in serum with 62-93% recovery.
- Kinetic constants for sPD1–nivolumab interaction matched literature values (K_D ≈ 4.66 nM).
- The platform enables dual-mode analysis in buffer and human serum.

## Abstract

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
(k
a ≈ 2.32 × 105 M–1 s–1; k
d ≈ 1.03 × 10–3 s–1; K
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.