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Automated microfluidic phosphoprofiling
GREGG ANDRESKI
extracellular signal-regulated kinase and p38. membranes with methanol to allow the When biologists requested a microfluidic fluorescently labeled phosphospecific antiThe group monitored the phosphorylation platform to study pathogenic bacteria, a bodies to enter the cell nuclei. Fluoresof these proteins through automated workteam of engineers at Sandia National cence was detected only inside the cell Laboratories in California rose to the chal- flows for adherent and nonadherent cells. nuclei that were exposed to LPS. lenge. To study potential bioterror After fluorescence imaging of the agents, the biologists required a stained cells, a trypsin-containing contained system that would minibuffer was introduced at a high mize the aerosolized spread of bacflow rate to detach the cells from teria. In a recent AC paper (DOI the incubation chamber and 10.1021/ac8024224), Nimisha sweep them downstream. A Srivastava (now at the University of sheath liquid provided hydrodyCalifornia Santa Barbara) and her namic focusing of the cell suscolleagues at Sandia present the pension before point detection. design and validation for the phosFor validation, the cells were colphoFlow Chip (pFC), an autolected and analyzed off-line by mated microfluidic platform for conventional flow cytometry. phosphoprofiling adherent and To simulate nonadherent nonadherent cells. “It’s completely cells, the researchers dynamically automated, which is a huge concoated the channels with polyvenience, because these assays can ethylene glycol to prevent the run over many hours and days,” An artistic representation of pathogenic bacteria (blue) interactmacrophages from sticking. A says Srivastava. porous filter in the side channel The established benchtop phos- ing with a macrophage (red) in a microfluidic environment. retained the free-floating cells phoprofiling method combines Working with cells was a challenge for without impeding the pressure-driven phosphostaining and flow cytometry. the engineers, who were accustomed to flow. The group detected increased levels of Washing steps in this traditional benchtop working with beads. The group had to phosphorylation after the LPS challenge, as workflow involve centrifugation, which figure out how to attach and detach the expected. In a separate experiment, the incan lead to both aggregation and loss of cells without damaging or killing them. vestigators confirmed that the flow itself did cells. The authors circumvented these And getting the adherent cells to stick not activate the macrophages. problems by retaining the cells in the exclusively in a designated location was no Srivastava says that noteworthy advanchannels during washing, either by adhesimple matter. “We used this tubing to tages of the pFC assay over the benchtop sion to the channel floor or by inserting a deliver cells to the actual glass device, and method include minimal cell loss and the monolithic porous polymer filter (for adthat tubing, we realized, was extremely ability to take measurements rapidly after herent and nonadherent cells, respeccrucial,” says Srivastava. “We had to coat the challenge compound is introduced. tively). Fast mixing occurred in narrow the tubing so cells wouldn’t stick to the One drawback is that the pFC has to be spiral channels, and incubation took place inside, because even if one cell stuck to reoptimized for each new cell type. in wide spiral channels. The pFC design The group is currently involved in disallowed control and experimental assays to one part of the tubing, other cells would just pile up on it. It’s sort of [like] buildcussions with companies about licensing be run simultaneously on the same chip. ing up a plaque inside an artery and suffer- the technology to produce pFCs as To mimic the interaction between ing from a heart attack.” add-on modules for existing commercial pathogenic bacteria and the primary imIn the adherent workflow, cells were flow cytometry instruments. Next, the mune system, the researchers incubated brought into the incubation chamber, engineers will be installing the pFC platmacrophages with lipopolysaccharide where they settled and attached to the form in the Biosafety Level 4 lab of their (LPS), a major component of the outer bottom after the pressure-driven flow was collaborators. Srivastava expects that this membrane of Gram-negative bacteria. stopped. Then, LPS was introduced into will bring a new set of challenges. “That’s Why work with adherent macrophages? the experimental incubation chamber (but when the rubber hits the road, and we’ll “They are the ones that actually recognize not the control chamber) to “challenge” probably have to go in and reengineer the bacteria once it enters the body,” the macrophages for a determined period things,” she says. “But I’m really looking Srivastava explains. LPS activates macof time. After chemically fixing the cells, forward to these next steps.” rophages through a signaling cascade that the researchers permeabilized the cell —Christine Piggee involves phosphorylation of proteins such as 3204
ANALYTICAL CHEMISTRY /
MAY 1, 2009
10.1021/AC9005167 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 03/26/2009
