High-throughput single-cell analysis for enzyme activity without cytolysis.
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.
Gao
Ning
N
School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China.
Wang
Wenlei
W
Zhang
Xiaoli
X
Jin
Wenrui
W
Yin
Xuefeng
X
Fang
Zhaolun
Z
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
D006873:Hydroquinones; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D009195:Peroxidase; C031927:hydroquinone