Rapid Identification of Multiplexed Pathogens via a Two-Step Dual-Channel Fluorescence Turn-on Array

Abstract

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 seconds. 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.