Challenges and Solutions in Developing Label-Free Optical Chip Based on Surface Plasmon Resonance Imaging (SPRi) for Multiprotein Assembly Sensing

Abstract Understanding biomolecular interactions is essential for disease diagnostics and therapeutic development, yet practical implementation is hampered by under-reported failure modes and technical constraints in the literature. Although commercial surface plasmon resonance (SPR) systems have advanced the field, real-world laboratories still face recurring pitfalls in data interpretation, surface chemistry, and microfluidic design that are often far from idealized reports. To address these practical obstacles, this study employs a null-ellipsometry phase-interrogation SPR imaging (SPRi) technique, which delivers high sensitivity and stability by extracting phase information rather than relying on intensity or simple angular-dip tracking. We show that conventional gold substrates limit both imaging and kinetic measurements, and that surface chemistry critically determines nonspecific binding. To overcome these limitations, we optimized surface functionalization via microcontact printing to produce protein-repellent and functionalized regions for immunoassays, revealing how nonspecific adsorption depends on coating chemistry. We also developed a custom microfluidic cell to accelerate solution exchange and reduce mass-transport artifacts that distort kinetic constants in commercial setups. Finally, we introduce a multilayer dielectric sensor chip that extends the detectable thickness range of protein complexes at a fixed angle of incidence around 60° and 658 nm illuminations, offering a scalable platform for analyzing complex multiprotein assemblies.