Selection of phage-displayed peptides on live adherent cells in microfluidic channels

Abstract

Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phagedisplayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.

Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

“…Our chip design permits end-to-end experimentation, which mitigates manual errors typically associated with conventional assays. The integration of all sample preparation – including cell fixation, membrane permeabilization, immunostaining, on-chip imaging and flow cytometry – into a closed monolithic automated platform increases reproducibility and minimizes labor, contamination, loss of cells and reagents, and variations in cell microenvironment [8], [25], [26]. Individual time courses reported below were generated using a single device, with data collected in real time.…”

Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides

“…75 Recently, a phage-displayed peptide has been selected on live cells in a microfluidic device. 76 This approach uses fewer cells (10 2 –10 4 ), and the flow of fluids through the chamber is more efficient in removing nonbinding phage. Additionally, fewer cells are lost in the wash process, thus minimizing the risk of losing phage clones.…”

Micro Total Analysis Systems: Fundamental Advances and Applications in the Laboratory, Clinic, and Field

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