Organic Fluorophores with Large Stokes Shift for the Visualization of Rapid Protein and Nucleic Acid Assays

Abstract Organic small‐molecule fluorophores, characterized by flexible chemical structure and adjustable optical performance, have shown tremendous potential in biosensing. However, classical organic fluorophore motifs feature large overlap between excitation and emission spectra, leading to the requirement of advanced optical set up to filter desired signal, which limits their application in scenarios with simple settings. Here, a series of wavelength‐tunable small‐molecule fluorescent dyes ( PTs ) bearing simple organic moieties have been developed, which exhibit Stokes shift up to 262 nm, molar extinction coefficients ranged 30,000–100,000 M −1 cm −1 , with quantum yields up to 54.8 %. Furthermore, these dyes were formulated into fluorescent nanoparticles ( PT‐NPs ), and applied in lateral flow assay (LFA). Consequently, limit of detection for SARS‐CoV‐2 nucleocapsid protein reached 20 fM with naked eye, a 100‐fold improvement in sensitivity compared to the pM detection level for colloidal gold‐based LFA. Besides, combined with loop‐mediated isothermal amplification (LAMP), the LFA system achieved the visualization of single copy level nucleic acid detection for monkeypox (Mpox).