Polymer Microspheres Copolymerized with Deep Red Fluorescent Molecules as a Label for Lateral Flow Immunochromatography

The development of fluorescently labeled microspheres is a critical aspect of advancing the technology of lateral flow immunochromatography (LFIA) for biological detection. Nevertheless, potential interference posed by the background fluorescence originating from the nitrocellulose (NC) membrane would significantly impact the sensitivity and accuracy of microsphere-based detection in LFIA. In this work, an attempt was made to extend the π-conjugated system and asymmetric structure of rhodamine fluorophore, resulting in the synthesis of dye molecules (RB2) incorporating double bonds, which can reach an absolute photoluminescence quantum yield (PLQY) of 30.01% in EtOH. Subsequently, carboxyl group functionalized fluorescent microspheres were prepared in a two-step copolymerization via soap-free emulsion polymerization. The obtained microspheres were characterized by scanning electron microscopy, transmission electron microscopy, DLS, Fourier transform infrared spectroscopy, ultraviolet spectrophotometry, and fluorescence spectrophotometry. The results showed that RB2 was successfully copolymerized into the microspheres, and the resulting microspheres had good dispersion and stability with high red fluorescence intensity (λabs ∼ 610 nm, λem ∼ 660 nm). Utilizing these microspheres, the resulting lateral flow immunoassay was successfully found to detect SARS-CoV-2 N protein with a detection limit of 2.5 pg/mL and the linear concentration spanning from 2.5 pg/mL to 10 ng/mL. The results confirm the effectiveness of the synthetic fluorescent microspheres as the label for LFIA.