Direct Access and Control of the Intracellular Solution Environment in Single Cells
Artikel i vetenskaplig tidskrift, 2009

Methods that can control and vary the solution environment around single cells are abundant. In contrast, methods that offer direct access to the intracellular proteome and genome in single cells with the control, flexibility, and convenience given by microfluidic methods are both scarce and in great demand. Here, we present such a method based on using a microfluidic device mounted on a programmable scanning stage and cells on-chip permeabilized by the pore-forming glycoside digitonin. We characterized the on-chip digitonin poration, as well as the solution exchange within cells. Intracellular solution exchange times vary with the dose of exposure to digitonin from less than a second to tens of seconds. Also, the degree of permeabilization obtained for cells treated with the same dose varies considerably, especially for low doses of digitonin exposure and low permeabilities. With the use of the presented setup, the degree of permeabilization can be measured during the permeabilization process, which allows for "on-line" optimization of the digitonin exposure time. Using this calibrated permeabilization method, we demonstrate the generation of intracellular oscillations, intracellular gradients, and the delivery of substrate to initiate enzymatic reactions in situ. This method holds the potential to screen and titrate intracellular receptors or enzymes or to generate intracellular oscillations, useful in the study of signaling pathways and oscillation decoding among other applications.

lymphocytes

fluorescein

protein-kinase

adrenal chromaffin cells

electroporation

permeabilized mammalian-cells

microfluidics

saccharomyces-cerevisiae

digitonin

membranes

Författare

Jessica Olofsson

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Helen Bridle

Chalmers

Aldo Jesorka

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Ida Isaksson

Chalmers

Sebastian Weber

University of Pittsburgh

Owe Orwar

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Analytical Chemistry

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

Vol. 81 5 1810-1818

Ämneskategorier

Fysikalisk kemi

Kemi

DOI

10.1021/ac802081m