Radical‐Mediated In Situ Fluorescence Dye Deposition: A Simple Interfacial Signal Amplification Reaction for Ultrasensitive Immunoassay on Barcode Beads

ABSTRACT Ultrasensitive detection of low‐abundance protein biomarkers is crucial for early disease diagnosis but remains challenging for the conventional barcode beads‐based suspension chip platform due to the limited detection sensitivity. Here, we report a conceptually novel reaction termed “radical‐mediated in situ fluorescence dye deposition” (RIFD) as a simple interfacial signal amplification strategy to overcome the limitation. This first discovered RIFD follows a universal “three‐element principle”, where the coexistence of beads, free radicals, and dyes suffices for fluorescence labeling, eliminating the pre‐conjugation of dyes to detection probes required in traditional methods. Mechanistic studies reveal that this radical‐triggered RIFD possibly follows three distinct pathways, including amide condensation on lysine residues, covalent biphenyl formation on tyrosine residues, or radical‐dye co‐deposition. It facilitates local and ultrafast (within 5 min) dye‐trapping specifically on target‐positive barcode beads. Consequently, the established RIFD‐based immunoassay achieves a limit of detection of 12 fg/mL for IL‐10, a 100‐fold improvement over the conventional suspension chip method, and also successfully differentiates Alzheimer's disease patients from healthy controls by quantifying low‐abundance plasma p‐Tau217. Multiplexed detection is further validated with a three‐plex cytokines panel. Our reported RIFD represents a powerful in situ fluorescence labeling tool, advancing protein biomarker detection toward the sub‐pg/mL level with broad implications for clinical diagnostics.