title: Single cell multiplexed assay for proteolytic activity using droplet microfluidics.
abstract: Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology.
authors.affiliation: Department of Biomedical Engineering, National University of Singapore, Block E4, #04-08, 4 Engineering Drive 3, Singapore 117583, Singapore.
authors.forename: Ee Xien
authors.initials: EX
authors.lastname: Ng
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Miles A
authors.initials: MA
authors.lastname: Miller
authors.affiliation: Department of Biomedical Engineering, National University of Singapore, Block E4, #04-08, 4 Engineering Drive 3, Singapore 117583, Singapore; Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore 1385602, Singapore.
authors.forename: Tengyang
authors.initials: T
authors.lastname: Jing
authors.affiliation: Department of Biomedical Engineering, National University of Singapore, Block E4, #04-08, 4 Engineering Drive 3, Singapore 117583, Singapore; Singapore Institute for Neurotechnology, 28 Medical Dr. #05-COR, Singapore 117456, Singapore. Electronic address: biecch@nus.edu.sg.
authors.forename: Chia-Hung
authors.initials: CH
authors.lastname: Chen
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation; D045744:Cell Line, Tumor; D057075:Enzyme Assays / Q000295:instrumentation; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D010447:Peptide Hydrolases / Q000378:metabolism*; D059748:Proteolysis; D059010:Single-Cell Analysis / Q000295:instrumentation*
chemical_list: D010447:Peptide Hydrolases
keywords: Droplet microfluidics; FRET-sensors; Proteolytic activity matrix analysis; Single cell assay