Intracellular protein determination using droplet-based immunoassays

Martino, C., Zagnoni, M., Sandison, M., Chanasakulniyom, M., Pitt, A.R. and Cooper, J.M. (2011) Intracellular protein determination using droplet-based immunoassays. Analytical Chemistry, 83(13), pp. 5361-5368. (doi:10.1021/ac200876q)

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Publisher's URL: http://dx.doi.org/10.1021/ac200876q

Abstract

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins developed using microdroplet technology The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from similar to 50 pM to 1 mu M. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Sandison, Dr Mairi and Pitt, Dr Andrew and Chanasakulniyom, Miss Mayuree and Cooper, Professor Jonathan
Authors: Martino, C., Zagnoni, M., Sandison, M., Chanasakulniyom, M., Pitt, A.R., and Cooper, J.M.
College/School:College of Science and Engineering > School of Engineering > Biomedical Engineering
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Analytical Chemistry
ISSN:0003-2700

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
460401Droplet-based microfluidics for single cell-omics.Jonathan CooperBiotechnology and Biological Sciences Research Council (BBSRC)BB/F005024/1Biomedical Engineering