Rapid identification of multiplexed pathogens via a two-step dual-channel fluorescence turn-on array

Bacterial infections have been an increasingly serious threat to human health. However, the rapid identification of multiplexed bacteria remains challenging due to their intricate composition. Herein, we developed a two-step, dual-channel fluorescence "turn-on" sensor array that sequentially amplifies signals via Indicator Displacement Analysis (IDA) and Aggregation-Induced Emission (AIE). Three weakly fluorescent, positively charged conjugated fluorophores (A1-A3) with AIE properties were designed to form electrostatic complexes (C1-C3) with negatively charged graphene oxide (GO). Upon addition of bacteria, fluorophores were released from the electrostatic complexes via IDA, resulting in fluorescence turn-on. These fluorophores then aggregated on the bacterial surface, further enhancing fluorescence. This array accurately differentiated among 10 distinct bacterial strains, achieving 98.3 % classification accuracy within 30 s. Finally, the approach facilitated semi-quantitative bacterial analysis, multiplex identification, and robust differentiation in artificial urine samples, presenting a promising method for early infectious disease diagnosis.