High-throughput single-cell analysis for enzyme activity without cytolysis

Analytical Chemistry
Ning GaoZhao-Lun Fang

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.

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Citations

Sep 7, 2006·Analytical and Bioanalytical Chemistry·Glen K Shoemaker, Monica M Palcic
Mar 13, 2008·Analytical and Bioanalytical Chemistry·Ning BaoChang Lu
Apr 21, 2007·Analytical Chemistry·Aparna AgarwalStephen G Weber
Feb 7, 2009·Analytical Chemistry·Jessica OlofssonOwe Orwar
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Dec 30, 2014·Analytical and Bioanalytical Chemistry·Shijun XuAldo Jesorka
Sep 6, 2013·Analytical Chemistry·Jessica OlofssonOwe Orwar

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