5-Carboxyfluorescein

5-Carboxyfluorescein (CAS No.: 76823-03-5) is a well-characterized green fluorescent probe belonging to the fluorescein family, widely employed in biomedical research for the labeling and tracking of biomolecules. As a single isomer derivative of carboxyfluorescein, it exhibits excitation and emission maxima near 490–492 nm and 518–520 nm, enabling sensitive detection in fluorescence-based assays. The molecule contains a reactive carboxylic acid group that can be activated, commonly via carbodiimide chemistry, to form stable conjugates with primary amines on peptides, proteins, and nucleic acids, facilitating site-specific fluorescent labeling. It functions as a non-targeted reporter probe rather than engaging specific signaling pathways, with its utility arising from its high fluorescence quantum yield, photostability, and favorable aqueous solubility. In vitro, 5-carboxyfluorescein is extensively used in fluorescence microscopy, flow cytometry, and biochemical assays to monitor molecular interactions, intracellular trafficking, and dynamic biological processes, with effective concentrations typically spanning the low micromolar range depending on labeling efficiency and assay configuration. It is also broadly applied in cell-based imaging, tracer studies, and high-throughput screening platforms in drug discovery, where it supports the quantitative analysis of binding events, enzymatic activity, and transport mechanisms without directly modulating biological targets.