title: A heating-superfusion platform technology for the investigation of protein function in single cells.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology Kemivägen 10, SE-412 96 Gothenburg, Sweden.
authors.forename: Shijun
authors.initials: S
authors.lastname: Xu
authors.forename: Alar
authors.initials: A
authors.lastname: Ainla
authors.forename: Kent
authors.initials: K
authors.lastname: Jardemark
authors.forename: Aldo
authors.initials: A
authors.lastname: Jesorka
authors.forename: Gavin D M
authors.initials: GD
authors.lastname: Jeffries
mesh_terms: D002463:Cell Membrane Permeability; D057809:HEK293 Cells; D006361:Heating; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D018613:Microscopy, Confocal; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D006460:Hemolysin Proteins