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Cell Ordering Using Pinch Flow Microchannel for Signle Cell Kinase Assay

Abstract

Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform celldistribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cellsurface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

image of FIG. 1.

 

 Fig 1. Schematic representation of the microchannel design with pinched flow structures and droplet encapsulation of cells

Inertial focusing channel: length of 5 mm and height of 50  m (Fig.1 (a)). These structures consist of contracting and expanding chambers. Cells traveling through the cell-focusing channel with chambers (90  m wide) and orifices (30  m wide) are focused along the center of the channel (Fig. 1(b) ). Cell clumps are separated into individual cells due to the shear forces exerted on them during their transit through the pinching regions of the microchannel. Subsequently, the separated cells move in groups through a cell pinching channel with chambers (90  m wide)/orifices (12  m wide) in a uniform spacing (Fig. 1(c) ). The cells are ordered along the short straight channel before entering a laminar stream of assay reagents (Fig. 1(d) ), which are encapsulated along with the cells downstream in the continuous oil phase droplet-forming region (Fig. 1(e) ).

 Source:  Biomicrofluidics 8, 034104 (2014); http://dx.doi.org/10.1063/1.4878635

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