A Heating-Superfusion Platform Technology for the Investigation of Protein Function in Single Cells
Artikel i vetenskaplig tidskrift, 2015

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 a-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.

Chemicals and CAS Registry Numbers: Hemolysin Proteins

Författare

Xu Shijun

Chalmers, Kemi och kemiteknik, Kemi och biokemi, Fysikalisk kemi

Alar Ainla

Harvard University

Kent Eric Jardemark

Karolinska Institutet

Aldo Jesorka

Chalmers, Kemi och kemiteknik, Kemi och biokemi, Fysikalisk kemi

Gavin Jeffries

Chalmers, Kemi och kemiteknik, Kemi och biokemi, Fysikalisk kemi

Analytical Chemistry

0003-2700 (ISSN) 1520-6882 (eISSN)

Vol. 87 381-387

Ämneskategorier

Analytisk kemi

DOI

10.1021/ac5031418