Quantitation of superoxide radical (O 2–·) production at the site of radical generation remains challenging. A simple method to detect nanomolar to micromolar levels of superoxide radical in aqueous solution has been developed and optimized. This method is based on the efficient trapping of O2–· using a novel fluorescent probe (2‐chloro‐1,3‐dibenzothiazolinecyclohexene), coupled with a spectra character‐signaling increase event. A high‐specificity and high‐sensitivity fluorescent probe was synthesized in‐house and used to image O2–· in living cells. Better selectivity for O2–· over competing cellular reactive oxygen species and some biological compounds illustrates the advantages of our method. Under optimal conditions, the linear calibration range for superoxide anion radicals was 5.03 × 10−9−3.33 × 10−6 m. The detection limit was 1.68 × 10−9 m. Fluorescence images of probe‐stained macrophages stimulated with 4β‐phorbol 12‐myristate 13‐acetate were obtained successfully using a confocal laser scanning microscope.
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