The preparation of high quantum yield, stable, and multifunctional fluorescent probes is of great significance in the fields of biomedicine and photoelectric sensing. Here, a triphenylamine-based D-π-A fluorescent molecule (TPA-CN) was designed and prepared, demonstrating a fluorescence quantum yield of 88.84%. With a polystyrene nanosphere as the carrier, TPA-CN was encapsulated inside the nanosphere to form intra-nanosphere confining domains. These nanodomain-enhanced fluorescent nanospheres exhibited a fluorescence quantum yield of 98.21%. Using antigen-antibody specificity and the selective catalytic activity of a bioenzyme, with chloramphenicol as a model target, a dual-signal readout biosensor (in fluorescence and colorimetric modes) was designed for ultrasensitive and instrument-free determination. The detection limit was 24 pg/mL within 30 min in fluorescence mode, 38-fold more sensitive and 10-fold faster than that of enzyme linked immunosorbent assays. The nanodomain-enhanced fluorescent probes and dynamic biosensor provide a robust and versatile solution for public health and environmental monitoring needs.
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