title: Microfluidics-Enabled Enzyme Activity Measurement in Single Cells.
abstract: Cellular heterogeneity has presented a significant challenge in the studies of biology. While most of our understanding is based on the analysis of ensemble average, individual cells may process information and respond to perturbations very differently. Presented here is a highly sensitive platform capable of measuring enzymatic activity at the single-cell level. The strategy innovatively combines a rolling circle-enhanced enzyme activity detection (REEAD) assay with droplet microfluidics. The single-molecule sensitivity of REEAD allows highly sensitive detection of enzymatic activities, i.e. at the single catalytic event level, whereas the microfluidics enables isolation of single cells. Further, confined reactions in picoliter-sized droplets significantly improve enzyme extraction from human cells or microorganisms and result in faster reaction kinetics. Taken together, the described protocol is expected to open up new possibilities in the single-cell research, particularly for the elucidation of heterogeneity in a population of cells.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Mollers Alle 3, Building 1131, locale 328, 8000, Aarhus C, Denmark.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Tesauro
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Mollers Alle 3, Building 1131, locale 328, 8000, Aarhus C, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Frøhlich
authors.affiliation: Department of Pathology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Magnus
authors.initials: M
authors.lastname: Stougaard
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Ho
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Mollers Alle 3, Building 1131, locale 328, 8000, Aarhus C, Denmark. brk@mb.au.dk.
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
mesh_terms: D004264:DNA Topoisomerases, Type I / Q000032:analysis / Q000378:metabolism*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D004264:DNA Topoisomerases, Type I
keywords: Enzyme activity; Human topoisomerase I; Microfluidics; Rolling circle amplification; Single-cell analysis; Single-molecule detection