Single cell kinase signaling assay using pinched flow coupled droplet microfluidics |
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| Authors: | Ramesh Ramji Ming Wang Ali Asgar S Bhagat Daniel Tan Shao Weng Nitish V Thakor Chwee Teck Lim Chia-Hung Chen |
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| Institution: | 1.Department of Biomedical Engineering, National University of Singapore, Singapore 117575;2.Clearbridge Biomedics Pte. Ltd., Singapore 118257;3.National Cancer Centre Singapore, Singapore 169610;4.Singapore Institute for Neurotechnology, Singapore 117456;5.Department of Mechanical Engineering, National University of Singapore, Singapore 117575 |
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| 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. |
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