Donor-Acceptor Organic Fluorophores Encapsulated in Polystyrene Nanoparticles as High-Brightness Reporters for Fluorescent Lateral Flow Immunoassay of C-Reactive Protein.

Fluorescent lateral flow immunoassays (LFAs) offer improved sensitivity, quantitative readout, and multiplexing over conventional colorimetric formats. However, individual organic fluorophores often provide insufficient signal for highly sensitive strip assays; although dye-loaded polymer nanoparticles can amplify fluorescence by delivering many fluorophores per particle, their performance remains limited by dye absorptivity and emissivity, self-quenching under dense loading, encapsulation efficiency, and optical background from nitrocellulose membranes. Here, we report two rationally designed donor-acceptor fluorophores, QNN and B_PEG, and encapsulate them into carboxylated polystyrene nanoparticles (PSNPs) as fluorescent reporters for C-reactive protein (CRP) LFAs. Their extended donor-acceptor structures and PEG-like side chains were designed to enhance fluorescence, reduce aggregation-related quenching, and support swelling-diffusion loading in mixed aqueous/organic media. After normalization of the commercial PS control using the PS-dominated UV absorption at 235 nm, QNN_PSNP and B_PEG_PSNP generated approximately 2- and 13-fold stronger fluorescence, respectively, than a size-comparable commercial fluorescent PS control, while preserving their optical properties after antibody conjugation and remaining photostable under continuous UV irradiation. In CRP LFAs, the QNN-based assay extended the working range to 0.1-100 ng mL⁻¹ with robust visual discrimination at the lower end, whereas the B_PEG-based assay operated over 1-100 ng mL⁻¹ as a spectrally distinct blue-emitting counterpart. The superior strip-level performance of QNN, despite its lower intrinsic quantum yield, highlights the importance of emission wavelength, optical filtering, and membrane-derived background in practical LFA sensitivity. These results identify QNN and B_PEG as complementary fluorophore candidates for PSNP-based fluorescent LFA reporters.