Development of an esterase-responsive near-infrared fluorescent probe for rapid antibiotic susceptibility testing of...

Rapid antimicrobial susceptibility testing (AST) is critical for guiding anti-infective therapy, yet the development of simple and rapid phenotypic methods remains challenging. Herein, we report a novel fluorescence sensing strategy that dramatically accelerates AST by leveraging a near-infrared (NIR) turn-on probe, IR-A. This probe features a hemicyanine scaffold functionalized with an esterase-responsive acetate group, serving as a direct biochemical reporter for bacterial metabolic activity. Upon hydrolysis by intracellular esterases in viable bacteria, IR-A yields a strong NIR fluorescence signal, enabling wash-free, high signal-to-noise monitoring. By employing a four-parameter logistic (4PL) model to analyze the dose-response curve, we achieve quantitative pharmacodynamic readouts, such as IC₅₀. The entire AST protocol leveraging this probe can be completed within 3 h, providing reliable minimum inhibitory concentration (MIC) values and category. This work establishes a new strategy for rapid phenotypic AST by integrating target-activated molecular sensing with robust quantitative analysis, offering a powerful tool for both clinical diagnostics and basic antimicrobial research.