title: 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.
authors.affiliation: Department of Biomedical Engineering, National University of Singapore, Singapore 117575.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Ramji
authors.affiliation: Department of Biomedical Engineering, National University of Singapore, Singapore 117575.
authors.forename: Ming
authors.initials: M
authors.lastname: Wang
authors.affiliation: Clearbridge Biomedics Pte. Ltd., Singapore 118257.
authors.forename: Ali Asgar S
authors.initials: AA
authors.lastname: Bhagat
authors.affiliation: National Cancer Centre Singapore, Singapore 169610.
authors.forename: Daniel
authors.initials: D
authors.lastname: Tan Shao Weng
authors.affiliation: Department of Biomedical Engineering, National University of Singapore, Singapore 117575 ; Singapore Institute for Neurotechnology, Singapore 117456.
authors.forename: Nitish V
authors.initials: NV
authors.lastname: Thakor
authors.affiliation: Department of Biomedical Engineering, National University of Singapore, Singapore 117575 ; Department of Mechanical Engineering, National University of Singapore, Singapore 117575.
authors.forename: Chwee
authors.initials: C
authors.lastname: Teck Lim
authors.affiliation: Department of Biomedical Engineering, National University of Singapore, Singapore 117575 ; Singapore Institute for Neurotechnology, Singapore 117456.
authors.forename: Chia-Hung
authors.initials: CH
authors.lastname: Chen