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A Heating-Superfusion Platform Technology for the Investigation of Protein Function in Single Cells

Shijun Xu (Institutionen för kemi och kemiteknik, Fysikalisk kemi) ; Alar Ainla ; Kent Jardemark ; Aldo Jesorka (Institutionen för kemi och kemiteknik, Fysikalisk kemi) ; Gavin Jeffries (Institutionen för kemi och kemiteknik, Fysikalisk kemi)
Analytical Chemistry (0003-2700). Vol. 87 (2015), 1, p. 381-387.
[Artikel, refereegranskad vetenskaplig]

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.

Nyckelord: Chemicals and CAS Registry Numbers: Hemolysin Proteins



Denna post skapades 2015-03-03. Senast ändrad 2016-07-12.
CPL Pubid: 213326

 

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Institutioner (Chalmers)

Institutionen för kemi och kemiteknik, Fysikalisk kemi

Ämnesområden

Analytisk kemi

Chalmers infrastruktur