| Homocysteine(Hcy)is internationally considered to be an independent risk factor for vascular disease.According to recent studies,the concentration of cysteine(Cys),which is only one methane less structurally than Hcy and can be produced by Hcy metabolism,is also significantly associated with the incidence of cardiovascular disease.In addition,glutathione(GSH),which can be converted by Cys metabolism,has been reported to protect endothelial cells from oxidative stress.In summary,current studies have shown that Cys and GSH are also associated with the regulation of endosthal cell function in blood vessels,but fewer studies have been conducted on specific mechanisms.Fluorescent probe detection technology has been widely used in labeling nucleic acids,proteins and some cellular immune molecules,and has promoted the research of relevant basic mechanisms.Among them,near-infrared fluorescence detection has the least damage to tissue and has a certain clinical application prospect.Melamine-based isofluoroflolone derivative is a near-infrared fluorescence(NIR)dye that has appeared in recent years.However,there is less research on biomolecular detection at present based on melamine-based isofluorolketone as the basic unit.In this paper,three near-infrared small molecule fluorescent probes are designed and synthesized based on melamine-based isofluorolketone,which can not only achieve quantitative detection of Cys or GSH,but also locate Cys or GSH for imaging and dynamic tracing.It provides a powerful means to explore the mechanism of Cys and GSH and vascular endothelial cells,and has certain clinical application prospects.In this paper,combined with modern fluorescent probe detection technology,three near-infrared small molecule fluorescence probes are designed and synthesized to not only achieve quantitative detection of Cys or GSH,but also to locate cys or GSH for imaging and dynamic specting.It provides a powerful means to explore the mechanism of Cys and GSH and vascular endosthal cells,and has a certain clinical application prospect.This thesis mainly includes three parts as following:1.Based on melamine-based isofluorolone and paraben formaldehyde,a new"Turn-off"type fluorescent probe—IMA is designed and synthesized for detection of Cys,which identifies Cys by aldehyde group.The probe has near-infrared fluorescence at an emission wavelength of 652 nm but quenched after reaction with Cys.And the fluorescence intensity has a good linear relationship with Cys concentration,the detection limit is 1.06×10-8 mol/L.The probe has successfully completed the detection of Cys concentration in plasma and also successfully used for fluorescence imaging in living cells.However,because of"Turn-off"type,IMA fluorescent probe is limited in application of dynamic tracing in vivo.2.In order to overcome the above defects,we have introduced a melamine isofluorosterone at the aldehyde base and an acrylamide group at the hydroxyl base based on the IMA fluorescent probe to synthesize a new"Turn-off"type fluorescent probe named IMT.And the the introduction of acrylamide group,which can specifically identify Cys,can make the probe fluorescence quenched and specificity detecte Cys.After reaction with Cys,the acrylamide group leaved,and the fluorescence recovered at an emission wavelength of 595 nm.And the fluorescence intensity has a good linear relationship with Cys concentration,the detection limit is 1.20×10-8 mol/L,which is much lower than the normal Cys concentrations in the body.In addition,IMT fluorescent probe was successfully used in Cys imaging in living cells.Therefore,IMT fluorescent probe can not only be applied to quantitative detection of Cys concentration in plasma,but also has the potential for Cys tracer imaging in the body.3.On the basis of IMA fluorescent probe,the acrylic group is introduced directly at the hydroxyl base position and a new"Turn-on"type fluorescent probe was synthesized and named IMC,which can recognize GSH by acrylate group and aldehyde group together.The probe itself fluorescence is very weak,after reaction with GSH,the generation of hydrophobic sixteen-membered ring derivatives,and released fluorescence at an emission wavelength of 650 nm.The IMC fluorescent probe also showed excellent sensitivity and selectivity,which can resist interference from common amino acid interference,including Cys.The detection limit is 1.27×10-8 mol/L.Next,we investigated the fluorescence imaging performance of the IMC probe and found that it achieved satisfied imaging results in both living cells and in mice.In summary,the detection limits of the three fluorescent probes above are lower than the physiological concentration minimums of Cys and GSH.And it has the application prospect of detecting Cys and GSH concentration in plasma.In addition,we carried out cytotoxicity tests on all three fluorescent probes,and found that after 24hours of incubation with one of the three fluorescent probes above at a concentration of100?mol/L,the HUVEC cell survival rate was still as high as 90%,indicating that all the three fluorescent probes have low cytotoxicity.At the same time,all the three fluorescent probes were successfully used in living cell imaging and mice,and showed good application prospect of molecular imaging in living body. |
PDF Full Text Request