Surfactant-free polymer microspheres with molecular confinement-enabled optical encoding for dual-color immunochromatographic assays.

A surfactant-free, confinement-assisted optical encoding strategy is presented for preparing color-encoded poly(styrene-co-methacrylic acid) microspheres as polymeric chromogenic probes for dual-color immunochromatographic assays. Hydrophobic dyes were immobilized within styrene-rich polymer domains, while the carboxyl-enriched shell remained accessible for antibody conjugation. The obtained microspheres showed uniform morphology, stable optical properties, and low nonspecific interference. Loading of five hydrophobic chromophores confirmed the versatility of this confinement-assisted encoding platform for constructing distinguishable color probes. Red- and blue-encoded microspheres were further employed as dual-color labels in a representative MxA/CRP lateral-flow immunoassay for the simultaneous detection of myxovirus resistance protein A (MxA) and C-reactive protein (CRP). The dual-color immunochromatographic assay enabled visual detection within 15 min, with naked-eye visual detection thresholds of 50 ng mL- 1 for MxA and 5 µg mL- 1 for CRP, providing distinguishable dual-color readouts and good reproducibility under the tested conditions. This study provides a simple and cost-effective approach for preparing surfactant-free color-encoded polymer probes and highlights their applicability as a material platform in multiplex immunochromatographic analysis.