Construction And Application Of Fluorescent Probes For Identifying Biological Active Substances

Biological active substances play an important role in regulating cell functions and their metabolic imbalance will cause a series of pathological changes.Therefore,the detection and imaging of related substances are of great significance in the diagnosis and treatment of diseases.Fluorescent probes are sensitive means to detect various ions,enzymes and other biomarkers in organisms.They can rapidly realize analysis and detection of related active molecules and real-time distribution imaging,which are practical tools for life science research.In this paper,three fluorescent probes were designed and synthesized,which can be applied to specific and high-sensitivity detection of zinc ions and receptor tyrosine kinases and cellular bioimaging.Zinc plays a key role in a variety of biological processes and its dysfunction is associated with many diseases.The probe CANQ was synthesized from coumarin aldehyde derivatives and quinoxaline-hydrazide derivatives by schiff base reaction.The probe is a hydrazone compound with many active ligands and has unique optical selectivity for Zn²⁺ions,resulting in enhanced fluorescence.When Zn²⁺ions were added into the solution system,the fluorescence signal was significantly enhanced at 522 nm,and the binding constant is8.28×10⁴M^-1.High resolution mass spectrometry and Job’s plot curves showed that probe CANQ formed a 1:1 complex with Zn²⁺ion.The probe CANQ has good biocompatibility and can be used for fluorescence imaging of Zn²⁺ions in MCF-7 cells,which provide reference experience for monitoring Zn²⁺ions in biological systems.Receptor tyrosine kinases are transmembrane protein kinases that can catalyze thephosphorylation of tyrosine residues and activate intracellular signaling pathway transduction,which are closely related to tumor growth and metastasis.The fluorescent probe SP1 is composed of a pyrene fluorophore connected to the molecular backbone of sunitinib through6-aminocaproic acid,which can form a dimer in solution,resulting in fluorescence quenching.When probe interacts with receptor tyrosine kinase,conformational changes lead to fluorescence recovery.The probe SP1 can effectively inhibit the activity of receptor tyrosine kinases and adding receptor tyrosine kinase to the probe solution system the emission peak significantly enhanced at 545 nm,which has good selectivity and sensitivity for the detection of kinases.Probe SP1 has low biological toxicity and can be applied to fluorescence imaging of tumor cells overexpressing tyrosine kinase receptors,which can achieve cell membrane imaging and tumor recognition by interacting with receptor tyrosine kinases accumulated on cancer cell membranes.At the same time the probe SP1 can be applied to the rapid fluorescence imaging of new blood vessels in the chick embryo allantoic membrane tissue,and show excellent tumor fluorescence imaging in the HT-29 xenograft nude mouse model.The long wavelength emitting fluorescent dye has deeper tissue penetration ability and lower background interference.The Nile blue fluorescent dye is used to construct the fluorescent probe SNB by linking the long flexible chain to the sunitinib molecular backbone.Probes were obtained by modifying Nile blue derivatives with receptor tyrosine kinase inhibitors.By interacting with the specific domain of the kinase,it shows fluorescence enhancement at 635 nm in a solution system and can selectively and rapidly identify the receptor tyrosine kinase.The probe has no obvious effect on cell proliferation and growth and can accurately locate receptor tyrosine kinase overexpressed HT-29,A549 and HUVEC cell membranes.The probe SNB can be applied to cell fluorescence imaging and cell sorting by flow cytometry.At the same time,it can effectively distinguish tumor tissues in the mouse HT-29 tumor model and can monitor the changes of receptor tyrosine kinases in complex biological systems.Probes can be used as molecular tools for tumor imaging and have potential applications in the early diagnosis of tumors.