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Microfluidic Molecular Trap: Probing Extracellular Signaling by Selectively Blocking Exchange of Specific Molecules in Cell-Cell Interactions

[+] Author Affiliations
Bryson M. Brewer, Rebecca M. Sappington, Deyu Li

Vanderbilt University, Nashville, TN

Yandong Gao

University of California-Davis, Davis, CA

Paper No. IMECE2013-64489, pp. V010T11A005; 2 pages
doi:10.1115/IMECE2013-64489
From:
  • ASME 2013 International Mechanical Engineering Congress and Exposition
  • Volume 10: Micro- and Nano-Systems Engineering and Packaging
  • San Diego, California, USA, November 15–21, 2013
  • Conference Sponsors: ASME
  • ISBN: 978-0-7918-5639-0
  • Copyright © 2013 by ASME

abstract

Communication among cell populations is achieved via a wide variety of soluble, extracellular signaling molecules [1]. In order to investigate the role of specific molecules in a cellular process, researchers often utilize in vitro cell culture techniques in which the molecule under question has been removed from the signaling pathway. Traditionally, this has been accomplished by eliminating the gene in the cell that is responsible for coding the targeted ligand/receptor by using modern DNA technology such as gene knockout; however, this process is expensive, time-consuming, and labor intensive. Previously, we have demonstrated a microfluidic platform that uses a semi-permeable barrier with embedded receptor-coated nanoparticles to selectively remove a specific molecule or ligand from the extracellular signaling pathway in a cell co-culture environment [2]. This initial proof-of-principle was conducted using biotinylated nanoparticles and fluorescently tagged avidin molecules, as the avidin/biotin complex is the strongest known non-covalent interaction between a protein and a ligand (Dissociation constant kd = 10−15 M). Also, the trap was only effective for short time periods (<15 min) because the high concentration of fluorescently tagged avidin molecules required for visualization quickly saturated the barrier. However, nearly all biologically relevant ligand-receptor interactions have lower binding affinities than the avidin-biotin complex, with dissociation constants that are larger by several orders of magnitude. In addition, many in vitro cell culture experiments are conducted over multiple hours or days. Thus, a practically useful molecular trap device must be able to operate in a lower binding affinity regime while also lasting for extended time periods. Here we present results in which a biotinylated-particle barrier was used to successfully block lower concentrations of fluorescently tagged avidin for multiple days, showcasing the applicability of the device for long term experiments. In addition, we introduce a modified molecular trap in which the protein A/goat IgG complex was used to demonstrate the effectiveness of the platform for lower binding affinity protein-ligand interactions. These results indicate the potential usefulness of the microfluidic molecular trap platform for probing extracellular signaling pathways.

Copyright © 2013 by ASME
Topics: Microfluidics

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