Chronofluorometry Based on a Facile Fluorescent Probe for Rapid Detection of Fractional Exhaled Nitric Oxide and Bioimaging in Live Cells

Fractional exhaled nitric oxide (FeNO) is an established biomarker of type 2 airway inflammation in asthma. The advancement of rapid and sensitive FeNO detection technology is crucial for improving the diagnosis and treatment of patients with asthma or airway inflammation. In this study, we introduce chronofluorometry as a novel method for quantitative analysis using specifically designed and synthesized fluorescent probes. This approach enables the successive, rapid, sensitive, and simple determination of the NO levels. We have designed and synthesized several facile fluorescent probes centered around 7-methyl-2-phenylimidazo[1,2-<i>a</i>]pyridine (ImPy) as the core structure, incorporating different functional groups for the detection of FeNO. Notably, the ImPy-Ph-CF₃ probe exhibited exceptionally high selectivity and an exceptionally rapid response time of less than 35 s. The reaction mechanism and fluorescence quenching process were thoroughly investigated, revealing that NO specifically binds to the C3 site of ImPy-Ph-CF₃, disrupting the original energy transfer mechanism and resulting in significant fluorescence quenching. Utilizing chronofluorometry based on the sensing platform, a low limit of detection (LOD) of 0.061 μM was achieved within a NO concentration range from 0.01 to 33.0 μM. Additionally, the platform has been successfully applied to detect FeNO in patients with asthma or airway inflammation and for bioimaging of NO in live cells. Thus, the proposed chronofluorometry provides a new quantitative method for NO detection, showing great potential for clinical applications.