Preparation And Application Of ROS Recognition And Phosphorylation/Glycosylation Nanofluorescent Probes

Protein and bioactive molecules,as important life substances,play an important role in the whole process of biological development and the maintenance of human normal life activities.Protein post-translational modification can coordinate and control the activity of most proteins,among which protein phosphorylation and glycosylation play an important role in almost every aspect of every organism.Such as gene transcription,signal transduction,tumorigenesis and so on.The level of bioactive molecules such as glucose and sialoglycoside also plays a very important role in the occurrence and development of diseases,among which hyperglycemia is an independent risk factor for cardiovascular death.Glucose may accelerate the foaming of macrophages by affecting the flow of cholesterol in macrophages,thus inducing the occurrence of atherosclerotic lesions.Peroxynitrite（ONOO^-）is a highly active reactive oxygen species,which is an important biological oxidant and can generate various toxic substances in the body,thus causing DNA damage and cell death in the body.Therefore,simultaneous identification and detection of multiple active species and understanding of the interaction between bioactive molecules are crucial for exploring the pathogenesis of diseases.Based on this,two kinds of nano-fluorescent probes（I₃^--Rh B@PCN-224 and BSA-AB）were designed using the I₃^--Rh B association modified by metal-organic framework PCN-224and BSA encapsulated small molecule fluorescent probes,respectively.The simultaneous detection and imaging analysis of protein phosphorylation and glucose levels and sialic acid and ONOO^-levels were performed,respectively,to explore the changes in serum levels of bioactive molecules in early atherosclerosis（AS）mice.Specific work contents are as follows:1.Using the electrostatic adsorption between the metallic organic framework PCN-224and I₃^--Rh B association,we constructed a nano-fluorescent probe I₃^--Rh B@PCN-224 that can simultaneously identify glucose and phosphorylation sites,and thoroughly studied the changes of protein phosphorylation and glucose level in the early stage of atherosclerosis without plaque formation.In vitro simultaneous detection of glucose and phosphorylation sites was realized by fluorescence spectrum detection,and it was proved that the probe had no cytotoxicity and realized imaging analysis in cells.The advantage of two-photon characteristics of porphyrins and glucose recognition units could effectively reduce background fluorescence and improve imaging sensitivity.Finally,the AS rat model was constructed by feeding a high-fat diet.The changes of protein phosphorylation and glucose levels in the early AS rat model were observed by two-photon fluorescence imaging.The results showed that the protein phosphorylation and glucose levels in the early AS mice were higher than those in normal mice.This study provides a suitable fluorescence tool for further exploring the pathogenesis and early diagnosis of atherosclerosis.2.Nano-composite probe BSA-AB was prepared by using two small molecule fluorescent probes encapsulated by BSA.Fluorescence probe FBA with 2.7-diaminofluorene as fluorophore and boric acid as active center and fluorescence probe FIB with luciferin as fluorophore and boric acid as identifying group were studied and prepared respectively,and then wrapped with bovine serum albumin（BSA）.The synthesized nanocomposite probe BSA-AB achieves simultaneous fluorescence detection of sialic acid and ONOO^-in vitro.Moreover,it was proved that the probe had no cytotoxicity and could be used for in vivo imaging analysis.The changes of SA and ONOO^-levels in the blood and tissues of early AS mice were detected,and the results showed that the levels of SA and ONOO^-in early AS mice were higher than those in normal mice.This work provides a new analytical method for the study of SA and ONOO^-related signaling channels to reveal the disease mechanism.