In Situ-Imaging Detection Of Celluar Cholesterol And Tissue Transglutaminase

Cell is the basic unit of the organism structure and function.Cholesterol is abundant in the cytoplasmic membrane and stored intracelluar in the form of cholesterol ester to maintain the dynamic equilibrium of cholesterol in the cell.If the equilibrium is disturbed,the metabolism and physiological activities will be disturbed,resulting in atherosclerosis and fatty liver as well as other diseases.Besides cholesterol,various enzymes are also necessary for cell metabolism and regulation.Tissue transglutaminase?TG2?is a kind of enzyme possessing allosteric effect and able to crosslink proteins.It is riched in tissues and many types of cells.The conformation and activity of TG2 are influenced by Ca²⁺and GTP.The expression of TG2 is not only participated in many cell physiology activities,including cell differentiation and apoptosis as well as signal transmission,but also related to cardiovascular disease,celiaca disease and neurological disorders.Hence it is significant to develop the strategy to detect cholesterol and tissue transglutaminase respectively,especially in cells,for many human diseases diagnosis and therapy.The proposed strategys include the following parts:?1?An effective membrane-to-intracellular cholesterol detection strategy was designed based on cascade reactions.A biochip array was firstly fabricated by consecutively immobilizing luminol modified gold nanoparticles?Au@luminol?,soybean peroxidase?SBP?and cholesterol oxidase?ChoX?on the cellulose acetate?CA?membrane functionalized home-made micropore array.When cholesterol existed,it was oxidized by ChoX generating H₂O₂,which further triggered the CL reaction under the SBP catalysis.The proposed strategy exhibited a wide linear range from 0.12?M to 1000?M and relatively low detection limit?LOD?of 0.08?M.The strategy has good stability and selectivity as well as high sensitivity and accuracy.Furthermore,it could be used to serum sample detection and in-situ detect membrane cholesterol and intracelluar esterified cholesterol in cells.Additionally,the cholesterol content in cells was changed after stimulated by drugs,such as apolipoprotein A-I?ApoA-I?,pitavastatin or probucol,which opened a new door for studying clinic treatment of the cholesterol-related diseases.?2?A fluorescent probe based on the aggregation induced luminescence?AIE?principle was designed to detect the activity of transglutaminase?TG2?.The probe was obtained by the linkage between the aggregation induced luminescent molecule tetraprene?TPE?and the TG2-specific recognized peptide through the click reaction.In the presence of Ca²⁺,the TG2 presented stretching conformation with activity.The lysine?K?and glutamine?Q?in two peptides of the probes was coupling by TG2,making the TPE molecular aggregation and the molecular rotation limited.Fluorescence emission phenomenon was occurred under the 315 nm excitation irradiation,realizing the sensitive detection of TG2.The detected concentration ranged from 0.1 nM-2.5 nM and the detection limit was 0.02 nM.This probe had good stability and selectivity and was successfully applied to detect TG2 in cells by targeted fluorescence imaging.In addition,the inhibitor cysteamine had obvious inhibitory effect on TG2 activity in cells,which proved that the proposed probe had good effectiveness and reliability in the detection of celluar TG2 activity.