An Fluorescent Probe For Sensing Different Concentration Ranges Of Glutathione Based On Diketopyrrolopyrrole Base Nanoaggregates

Intracellular thiol compounds such as cysteine?Cys?,homocysteine?Hcy?and glutathione?GSH?play a crucial role in cell regulation.GSH is the most abundant type of biothiol in animals.The different levels of GSH are directly related to people's pathological features,including liver damage,cancer,AIDS and cardiovascular diseases.GSH content is increased in tumor cells,so it is possible to know whether a cell has a lesion according to the change in the content of GSH.Therefore,it is necessary to detect different levels of GSH determine whether the cells have lesions.The three amino acids of cysteine?Cys?,homocysteine?Hcy?and glutathione?GSH?have very similar structure,contain amine groups and sulfhydryl groups.Now a lot of amino acids of fluorescent probes are reacted with amino groups or sulfhydryl groups in amino acids.The detection of the three amino acids is often disturbed.Therefore,it is important to design and synthesize fluorescent probes that can detect an amino acid from these three amino acids.GSH is the most difficult to selectively detect in these three amino acids due to its large molecule and large steric hindrance.It is necessary to detect different concentrations of GSH.To detect GSH in different concentration ranges,two reaction sites are needed and experience complex synthesis.However,fluorescent probes with different concentrations of GSH detected by one reaction site have not been reported.Therefore,we designed fluorescent probe TPE-DPP of the Schiff base structure have only one reaction.TPE-DPP is a Schiff base structure formed by the reaction of DPP-CHO and TPE-NH₂,and can realize the ratiometric fluorescence detection of GSH in different concentration ranges.The probe TPE-DPP can form stable nanoparticles in a solution of?THF:H₂O=4:6,v/v?.Due to the particle size of the nanoparticles,it can react differently with the three amino acids,GSH can be detected alone in the three amino acids.GSH??-glutamyl-cysteinyl-glycine?is a ubiquitous tripeptide.Due to the presence of two carboxylic acid groups,thiol group and amino group,when it is a low concentration of GSH,the carboxyl group in GSH is easily ionized to form GSH^?,hydrolyzing the C=N double bond of TPE-DPP to form DPP-CHO and TPE-NH₂.DPP-CHO emits strong orange fluorescence when it is in solution.When it is a high concentration of GSH,the C=N double bond of GSH and TPE-DPP undergoes nucleophilic addition,the fluorescence emission spectrum of TPE-NH₂ overlaps with the ultraviolet absorption spectrum of DPP-CHO.When a nucleophilic addition reaction occurs,the FRET effect of the electron-donating TPE to the electron-withdrawing DPP can occur,induce obvious fluorescence,enabling detection of different concentration ranges of GSH.Moreover,the selectivity of the probe TPE-DPP is also very high.The fluorescence emission peak of the probe TPE-DPP is at 578 nm,when the cysteine?Cys?and homocysteine?Hcy?is added,the emission peak intensity was no significant change,and when GSH was added at a low concentration?0-0.1 mM?,the fluorescence emission peak at 578 nm gradually increased,and a new fluorescence emission peak appeared at 411 nm,with the concentration of GSH increase,the fluorescence intensity of these two fluorescent emission peaks gradually increases.In the presence of high concentration?0.1-1.0 mM?GSH,the fluorescence intensity of the emission peak at 578 nm continues to increase,while the fluorescence intensity at 411nm gradually decreases.The specificity is also important for potential applications.The capability of TPE-DPP nanoaggregates to detect other biothiols was evaluated.Only GSH could induce obvious fluorescence whereas Cys and Hcy can hardly turn on the fluorescence of TPE-DPP.At the same time,other amino acids also show no fluorescence response to TPE-DPP which has wonderfully potential applications in biological fields.The detection limit of GSH can reach 0.05?M.Because TPE-DPP nanoparticles have good selectivity and good sensitivity,cell experiments were carried out to investigate the permeability of GSH in HeLa cells.Most organic solvents have some damage to cells.Therefore,the water-soluble fluorescent probe can reduce the damage to the cells and adapt to the cell environment well,and minimize the damage to the cells during the fluorescence imaging process.Therefore,we encapsulated TPE-DPP with amphiphilic polymer F127 to prepare water-soluble TPE-DPP-F127nanoparticles for cell imaging.We used GSH inhibitors as a control experiment to prove that TPE-DPP can selective detection of GSH and good imaging results in cells.