title: High-throughput single-cell analysis for enzyme activity without cytolysis.
abstract: A novel high-throughput method without cytolysis for determination of enzyme activity inside single cells was developed by a combination of chemical cell perforation and an intracellular enzyme-catalyzed reaction. Peroxidase (PO) inside human neutrophils was chosen as the model system. Cells were perforated with digitonin to form micropores on the cell membrane. The perforated cells, with physiological buffer saline of pH 7.4 containing hydroquinone (H2Q) and H2O2, were continuously propelled by pressure through a capillary as the microsampler and microreactor. Small molecules H2Q and H2O2 could diffuse into the cell interior through the micropores on the cell membrane, and the large molecule PO remained in the cell interior. Intracellular PO converted H2Q into benzoquinone (BQ). BQ diffused out from the cell interior to the cell surface through the micropores and formed a BQ zone around the cell. The process proceeded in the capillary during cell movement. The BQ zones around every moving perforated cell were continuously delivered to the capillary outlet by hydraulic flow and detected. An average detection rate of >1 cell/min was obtained.
authors.affiliation: School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.forename: Wenlei
authors.initials: W
authors.lastname: Wang
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Zhang
authors.forename: Wenrui
authors.initials: W
authors.lastname: Jin
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Yin
authors.forename: Zhaolun
authors.initials: Z
authors.lastname: Fang
mesh_terms: D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D004563:Electrochemistry; D004566:Electrodes; D019075:Electrophoresis, Capillary / Q000379:methods; D004789:Enzyme Activation; D006735:Horseradish Peroxidase / Q000032:analysis; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D006873:Hydroquinones / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000379:methods; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology*; D009195:Peroxidase / Q000032:analysis*; D016062:Porosity; D012680:Sensitivity and Specificity; D013499:Surface Properties; D013997:Time Factors
chemical_list: D006873:Hydroquinones; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D009195:Peroxidase; C031927:hydroquinone