Xu Shijun, Ainla Alar, Jardemark Kent, Jesorka Aldo, Jeffries Gavin D M
Department of Chemical and Biological Engineering, Chalmers University of Technology Kemivägen 10, SE-412 96 Gothenburg, Sweden.
Anal Chem. 2015 Jan 6;87(1):381-7. doi: 10.1021/ac5031418. Epub 2014 Dec 18.
Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.
在此，我们报告了一种研究不同温度下单细胞内酶活性的新方法，该方法利用局部激光加热探针与可自由定位的微流控灌注装置相结合。通过将单个细胞定向暴露于成孔剂α-溶血素，我们控制了膜通透性，实现了底物的靶向递送。轻度通透化的细胞暴露于荧光底物以监测细胞内酶的活性，同时使用红外激光加热系统调节靶细胞周围的局部温度。我们在不同细胞系中的五个不同温度下对细胞内碱性磷酸酶活性进行了定量估计，构建了单细胞水平上酶活性的温度响应曲线。细胞通透化后迅速测定酶活性，仅在200秒内生成五点温度响应曲线。
