title: C3N4Nanosheet Modified Microwell Array with Enhanced Electrochemiluminescence for Total Analysis of Cholesterol at Single Cells.
abstract: Here, a g-C3N4nanosheet modified microwell array providing enhanced electrochemiluminescence (ECL) and better visible sensitivity was prepared to simultaneously analyze total (membrane and intracellular) cholesterol at single cells. The detection limit for ECL visualization of hydrogen peroxide at microwell array was improved to be 500 nM that guaranteed the detection of low concentration cholesterol at single cells in parallel. To achieve single cell cholesterol analysis, the individual cells cultured at the microwell array were exposed to cholesterol oxidase generating hydrogen peroxide for luminescence analysis of membrane cholesterol, and then treated with triton X-100, cholesterol esterase, and cholesterol oxidase to produce hydrogen peroxide from intracellular cholesterol for luminescence determination. The observation of the luminescence spots at microwells in these two steps confirmed the codetection of membrane and intracellular cholesterol at single cells. The inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) resulted in less intracellular cholesterol storage (less luminescence) and more membrane cholesterol (more luminescence). The correlation of the luminescence intensity with the amount of cholesterol confirmed that our assay could simultaneously monitor membrane and intracellular cholesterol pools at different cellular states, which should offer more information for the study of cholesterol-related pathways at single cells.
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing 210093, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University , Chongqing 400010, China.
authors.forename: Depeng
authors.initials: D
authors.lastname: Jiang
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University , Chongqing 400010, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Qin
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University , Chongqing 400010, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Xia
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing 210093, China.
authors.forename: Dechen
authors.identifier: 0000-0002-2845-3621
authors.initials: D
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing 210093, China.
authors.forename: Hong-Yuan
authors.initials: HY
authors.lastname: Chen
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000032:analysis* / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000378:metabolism; D055664:Electrochemical Techniques; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis; D057230:Limit of Detection; D008163:Luminescent Measurements / Q000379:methods*; D046228:Microarray Analysis; D049329:Nanostructures / Q000737:chemistry*; D009570:Nitriles / Q000737:chemistry*; D017830:Octoxynol / Q000737:chemistry; D059010:Single-Cell Analysis; D002787:Sterol Esterase / Q000378:metabolism; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D009570:Nitriles; C011206:cyanogen; D017830:Octoxynol; D002784:Cholesterol; D006861:Hydrogen Peroxide; D002789:Cholesterol Oxidase; D002785:Sterol O-Acyltransferase; D002787:Sterol Esterase