Photoacoustic-fluorescence Dual-modal Imaging Probes Located In The Golgi Apparatus Reveal Changes In PH And Cysteine ??during Inflammation

Golgi apparatus is an important organelle in eukaryotic cells.Golgi apparatus is mainly involved in modification and classified transportation of protein and lipid.In this process,the pH of Golgi plays a crucial role.When the pH homeostasis of Golgi apparatus is destroyed,it will lead to the dysfunction of cells,abnormal glycosylation,and even lead to the disorganization of the structure of Golgi apparatus and further cleavage.Golgi pH will deviate from the normal range in many major diseases.Therefore,the real-time,in-situ and accurate detection of intracellular Golgi pH is of great significance.However,there are few reports on the pH detection methods of Golgi apparatus.Recent studies have speculated that transmembrane protein 165?TMEM165?is a reverse ion channel for Ca²⁺/H⁺of Golgi,but due to the lack of effective pH detection tools for Golgi,this speculation has not been directly proved.Therefore,we hope to develop a small molecule fluorescent probe that can detect the pH change of Golgi in high-efficiency target,in-situ and real-time,which provide direct evidence for the hydrogen ion transport function of TMEM165 and further validates pH changes in inflammation and tumor sites.In addition,since a large amount of protein is processed and modified in the Golgi apparatus,the level of cysteine also directly affects the normal function of Golgi apparatus.However,as far as we know,small molecule fluorescent probe has been reported to detect cysteine in Golgi.Therefore,it is necessary to develop a fluorescent probe that can detect cysteine in Golgi in real time and with specificity.The main contents of this paper are as follows:1.We designed and synthesized a fluorescent and photoacoustic dual-mode probe CPH,which can efficiently monitor the Golgi pH change in real time.CPH uses p-aminobenzene sulfonamide as the localization group,and uses a near-infrared dye tricarbocyanine?Cy7?as a fluorophore,which also has photoacoustic properties.CPH probe has the ability to quantitatively detect pH in cells and in vivo.Using the probe CPH,we also further verified the function of TMEM165 as a Ca²⁺/H⁺reverse ion channel in the Golgi apparatus.2.We aim to design and synthesize a fluorescent-photoacoustic dual-mode imaging probe that locates Golgi apparatus and detects cysteine in situ.Cysteine fluorescent probe HC-Cys was synthesized by using p-aminobenzene sulfonamide as Golgi localization group,benzohydroxyl hemicyenine as fluorophore,and acrylate group as cysteine identification group.The fluorescence of the probe HC-Cys is very weak.When cysteine is present,the ester group is broken and the fluorescence is significantly enhanced.In addition,due to the near-infrared and photoacoustic properties of benzohydroxyl hemicyenine,the probe can also be used for dual-mode imaging of photoacoustic and fluorescent in vivo.