Mitigating Interfacial Displacement via Abundance-Dependent Antibody Modification for SeNP-Based Multiple-Biomarker Biosensing

Multiplex-antibody co-immobilization constitutes a critical bottleneck for nanoparticle-enabled multiple-biomarker lateral-flow analytical platforms. Competitive adsorption induces severe interfacial displacement, in which high-abundance antibodies displace pre-adsorbed counterparts, degrading antibody bioactivity and overall assay performance. Here, we report an abundance-dependent antibody coating strategy to suppress such displacement via prioritized low-abundance antibody immobilization for rational surface-occupancy modulation on selenium nanoparticles (SeNPs). Validated by western blot and fluorescence assays, this strategy boosts labeling efficiency by 51.92% over conventional simultaneous co-labeling. As a proof-of-concept assay, the SeNP-enabled system achieves concurrent detection of AFP and CEA, delivering lower visual LODs (2.5 ng/mL for AFP and 0.5 ng/mL for CEA) than the simultaneous-labeling counterpart. When benchmarked against chemiluminescence using 315 clinical serum samples, the system attains 97.14% diagnostic coincidence. This work delivers a rational antibody-anchoring guideline for high-performance POCT screening of multiple biomarkers.