Intracellular protein determination using droplet-based immunoassays

Chiara Martino, Michele Zagnoni, Mairi E. Sandison, Mayuree Chanasakulniyom, Andrew Pitt, Jonathan M. Cooper

Research output: Contribution to journalArticle

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 ~50 pM to 1 µM. 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.
Original languageEnglish
Pages (from-to)5361-5368
Number of pages8
JournalAnalytical Chemistry
Volume83
Issue number13
DOIs
Publication statusPublished - 16 May 2011

Bibliographical note

Copyright © 2011 American Chemical Society

Keywords

  • Western blotting
  • calibration
  • cell line
  • humans
  • immunoassay
  • proteins

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