Development Of Ratiometric Fluorescence Probes For Biosensing And Imaging Of Nitric Oxide

Nitric oxide(NO)is an important physiological activity regulator in the body.It participates in various life activities of the body.It plays a vital role in the protection of the cardio-cerebrovascular system,the signal transduction of the nervous system,and the immune system.The destruction of NO homeostasis will cause the internal environment of the organism to be disordered,which will lead to a series of diseases.However,NO has a short half-life and it is easily diffused into other tissues and systems to interact with reactive oxygen species and other related substances.Therefore,it is very important to achieve rapid response and highly selective detection of NO,which is helpful to understand and analyze the pathology of diseases closely related to NO.At present,a variety of methods have been reported for the detection and research of NO.Among them,fluorescence analysis methods have been widely used in the detection of NO because it can realize in-situ detection and bioimaging sensing.However,there are still certain difficulties and challenges in realizing real-time detection and imaging analysis with high selectivity and high sensitivity for NO: the internal environment of the organism is complex,and there are many similar substances.The analysis and detection of related substances at the cell,tissue or living body level requires highly selective fluorescent probes;living tissues are generally deep(> 100 μm)so in order to achieve real-time detection and biosensing of NO in living tissues and zebrafish require fluorescent probes that have strong penetrating ability and are not interfered by the environment.This paper has carried out the following research on the above scientific issues:In the first work,a ratio probe(CN-NO)based on fluorescence resonance energy transfer(FRET)was synthesized to achieve high selectivity and sensitivity for real-time detection of NO levels in neurons.After the probe CN-NO reacted with NO,the FRET system was formed between the energy donor and the energy acceptor in the CN-NO molecule of the probe,thereby realizing the ratiometric detection of NO and improving the detection of the probe.The accuracy of the experiments successfully explored the changes of NO in neurons,and found that the ischemic state may lead to a significant increase in the level of NO concentration in the cell.In the second work,a highly selective and highly stable two-photon ratiometric fluorescent probe(RBD@Au NCs)based on organic-inorganic nanomaterials was constructed to realize the quantitative analysis and real-time imaging of NO in neurons,brain tissue and zebrafish.The probe also had the advantages of high accuracy,rapid response(55 s),good two-photon performance,little tissue damage,strong penetrating ability,and good water solubility.The two-photon fluorescent probe was composed of gold nanoclusters(Au NCs)modified with NO recognition molecule RBD.Through the special design of the molecule,the alkynyl group was introduced into the structure of the NO recognition molecule RBD,thereby increasing the stability of the two-photon fluorescent probe,which was more conducive to the probe’s good detection effect in the complex biological environment.The introduction of Au NCs into the probe RBD@Au NCs as an internal reference increased the water solubility of the probe,and realized the positioning of the probe and the quantitative detection of NO.Through the imaging analysis of NO in biological samples using this probe,it was found that the neuronal death induced under hypoxia was regulated by the internal NO level.In addition,it was also found that the NO content in different brain regions of mice increased differently during hypoxia,and the burst of NO would cause the death of live zebrafish under hypoxia.These findings provided new tools for understanding pathologies related to NO.