A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

Because nitric oxide (NO) plays important roles in nerve conduction, signal regulation, and immune protection, analysis of NO is of great significance for understanding the physiological and pathological processes related to neurological diseases. Herein, a highly stable and selective two-photon ratiometric fluorescent probe was developed for real-time sensing and imaging of NO in neurons, brain tissues, and larval zebrafish, in which a Rhodamine B derivative (RBD) was designed for specific recognition of NO and gold nanoclusters (AuNCs) were synthesized as reference element. The developed organic–inorganic nanoprobe exhibited high stability against biological thiol compounds and high selectivity against other reactive oxygen and nitrogen species, metal ions, and acids. In addition, the response time of the present nanoprobe was less than ∼55 s. By using the developed nanoprobe, we proved that hypoxia-induced neuronal death was regulated by NO. Moreover, it was found that the hypoxia-induced NO increase in different brain regions was various and that the NO burst contributed to hypoxia-induced death of zebrafish.