Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

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 cell distribution 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 cell surface 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.

Real-time measurement of thrombin generation using continuous droplet microfluidics

“…By controlling these two parameters, biomarkers, such as thrombin, were selected from the fluid flow in the sample solutions based on their size. HFE-7500 fluorocarbon oil (3M Novec TM , Singapore) with 2% Krytox (modified with a PEG head) surfactant 21,22 was used as the continuous phase. Syringes supplied by Terumo were connected to the microfluidic device with 0.38 mm (ID) medical grade polyethylene microtubing and were operated using Harvard Apparatus syringe pumps.…”

High spatial and temporal resolution cell manipulation techniques in microchannels

“…Then we will present the latest developments of microfluidic methods for fast cell stimulation and quenching reactions. Further examples such as for inertial, 17 droplet, 18,19 pneumatic, 20,21 filter-based, 22,23 electroosmotic, 24 immunoaffinity 25 and digital 26 microfluidics, cell traps 27 and optical tweezers, 28 are not covered here, but we recommend corresponding in-depth literature. [29][30][31][32][33][34][35]…”

Microfluidics in systems biology — hype or truly useful?

“…Over the last decade, many refinements of droplet techniques on-chip have led to simplification of operation of these devices, and an enlarged repertoire of applications. Some examples include systems for studying bacterial population dynamics [32], studying kinase signaling [33], performing directed evolution in yeasts [34][35], screening C. elegans [36], and performing mammalian embryo vitrification [37]. Furthermore, parallel development of biologically compatible surfactant and oil systems have also contributed to the rapid growth of the field [38][39].…”