Twinkle Light‐Inspired Aggregation‐Induced Emission “Lighting‐Up” Bacteriophages to Enhance Immunoassays via Spontaneous Amino‐Yne Click Reaction

ABSTRACT Traditional fluorescence immunoassays are often hindered by false negatives or quantification inaccuracies, especially at high target concentrations, due to the aggregation‐caused quenching effect of fluorescent indicators. This study introduces a novel fluorescence immunoassay strategy that leverages the spontaneous amino‐yne click reaction to covalently assemble activated alkyne‐based luminogens with aggregation‐induced emission characteristics (AIEgens) onto the surface of bifunctional M13 bacteriophages, thereby facilitating efficient “lighting‐up” fluorescence signal output in conjunction with magnetic‐mediated immunorecognition. To further enhance the load of activated alkyne‐based AIEgens and improve the fluorescence “lighting‐up” efficiency, M13 bacteriophages were engineered to display varying numbers of surface‐exposed lysine residues. This was achieved by inserting different quantities of lysines between the signal peptide and the amino acid sequence of the pVIII protein via a point mutation strategy. Benefiting from the synergy of AIEgen stacking‐enhanced fluorescence output and M13 bacteriophage‐driven signal amplification, the developed “lighting‐up” immunoassay enabled highly sensitive and rapid detection of targets, from small molecules to pathogenic microorganisms. This work provides valuable insights into the design of “lighting‐up” AIEgens for enhancing fluorescence immunoassays. Moreover, the proposed strategy offers great versatility, allowing it to be readily adapted to detect other targets simply by pairing the target with the M13 bacteriophages.