Multiplexed protein analysis using encoded antibody-conjugated microbeads

Nora Pisanic, Eric E. Roller, Kristopher D. Barbee, Matthias Franzreb, Xiaohua Huang

Research output: Contribution to journalArticle

Abstract

We describe a method for multiplexed analysis of proteins using fluorescently encoded microbeads. The sensitivity of our method is comparable to the sensitivity obtained by enzyme-linked immunosorbent assay while only 5 μl sample volumes are needed. Streptavidin-coated, 1 μm beads are encoded with a combination of fluorophores at different intensity levels. As a proof of concept, we demonstrate that 27 microbead populations can be readily encoded by affinity conjugation using three intensity levels for each of three different biotinylated fluorescent dyes. Four populations of encoded microbeads are further conjugated with biotinylated capture antibodies and then combined and immobilized in a microfluidic flow cell for multiplexed protein analysis. Using four uniquely encoded microbead populations, we show that a cancer biomarker and three cytokine proteins can be analysed quantitatively in the picogram per millilitre range by fluorescence microscopy in a single assay. Our method will allow for the fabrication of high density, bead-based antibody arrays for multiplexed protein analysis using integrated microfluidic devices and automated sample processing.

Original languageEnglish (US)
Pages (from-to)1104-1113
Number of pages10
JournalJournal of the Royal Society Interface
Volume8
Issue number61
DOIs
StatePublished - Aug 7 2011
Externally publishedYes

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Microspheres
Antibodies
Proteins
Lab-On-A-Chip Devices
Microfluidics
Assays
Population
Protein Array Analysis
Immunosorbents
Streptavidin
Fluorophores
Fluorescence microscopy
Tumor Biomarkers
Fluorescent Dyes
Fluorescence Microscopy
Enzymes
Dyes
Enzyme-Linked Immunosorbent Assay
Cytokines
Fabrication

Keywords

  • Combinatorial encoding
  • Microbeads
  • Multiplexed protein immunoassays

ASJC Scopus subject areas

  • Biophysics
  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Biomaterials
  • Biochemistry

Cite this

Multiplexed protein analysis using encoded antibody-conjugated microbeads. / Pisanic, Nora; Roller, Eric E.; Barbee, Kristopher D.; Franzreb, Matthias; Huang, Xiaohua.

In: Journal of the Royal Society Interface, Vol. 8, No. 61, 07.08.2011, p. 1104-1113.

Research output: Contribution to journalArticle

Pisanic, Nora ; Roller, Eric E. ; Barbee, Kristopher D. ; Franzreb, Matthias ; Huang, Xiaohua. / Multiplexed protein analysis using encoded antibody-conjugated microbeads. In: Journal of the Royal Society Interface. 2011 ; Vol. 8, No. 61. pp. 1104-1113.
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