Cytometric Bead Arrays

Abstract

Flow cytometry in combination with microspheres (beads) in a suspension have gained increasing interest by the research community. Various types of such beads are commercially available or self‐prepared by researchers, differing in size or the fluorophores that they contain, producing different sets of beads. They also contain functional groups that enable their conjugation with specific biomolecules, mainly oligonucleotides, antibodies, protein and other molecules. Recognition reactions of several analytes, such as immunoassays or hybridisation assays, take place on the surface of the beads, while a reporter fluorophore is used for the detection of the interaction between the recognition molecules and the analyte. The characterisation and classification of beads in different groups, as well as the detection of the fluorescence emitted from the reporter molecules is accomplished by a flow cytometer, as the beads pass one by one in front of one or two laser beams.

Key Concepts

  • Flow cytometry is applied for the detection of various analytes.
  • Flow cytometry offers high multiplexing potential and a high‐throughput format.
  • There are available up to 500 different sets of distinct beads.
  • Beads can be distinguished by their size, their shape or the fluorophores that they contain.
  • Beads carry on their surface several functional groups that allow easy coupling to recognition molecules such as oligonucleotides, proteins and biomolecules.
  • The panel of analytes can be easily changed, offering a great flexibility of bead‐based systems.

Keywords: beads; flow cytometry; microparticles; microspheres; fluorescence; array; DNA; protein

Figure 1. Principle of the analysis of beads by flow cytometry.
Figure 2. The three parts of a flow cytometer: fluidics, optics and electronics.
Figure 3. Schematic illustration of (a) an immunoassay and (b) a hybridisation assay on the bead's surface.
Figure 4. (a) SNP detection by a ligation reaction in combination with hybridisation of the ligated products to oligonucleotide‐functionalised beads, while a fluorophore attached to the one probe is used for the detection. (b) Schematic illustration of the general approach for the detection of interactions between two molecules using streptavidin beads, while the second molecule is labelled with phycoerythrin (PE). (c) SNP detection using allele‐specific hybridisation reaction on the surface of oligonucleotide‐functionalised Qbeads that is obtained when two different quantum dots are incorporated into the beads. The detection is accomplished by a streptavidin–phycoerythrin–Cy5 (SA–PE–Cy5) conjugate. B: biotin, F: fluorophore.
Figure 5. (a) Detection of a protein biomarker (P) by a bead‐based immunoassay. One specific antibody is attached on a bead, while the second antibody is coupled to an RCA primer. After rolling circle amplification (RCA), a probe labelled with a fluorophore (F) is repeatedly hybridised to the single‐stranded RCA, resulting in signal enhancement. (b) A hybridisation chain reaction (HCR) on streptavidin beads through a biotinylated‐probe and two complementary hairpins carrying a fluorophore at one end is used for signal enhancement for the detection of target DNA. B: biotin, F: fluorophore.
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Darzynkiewicz Z and Zhao H (2014) Cell cycle analysis by flow cytometry. In: Encyclopedia of Life Sciences. Wiley Online Library.

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Kalogianni, Despina P(Jun 2019) Cytometric Bead Arrays. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0028611]