Cytometric Bead Arrays


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.


van Andel E, de Bus I, Tijhaar EJ, et al. (2017) Highly specific binding on antifouling zwitterionic polymer‐coated microbeads as measured by flow cytometry. ACS Applied Materials & Interfaces 9: 38211–38221.

Armstrong B, Stewart M and Mazumder A (2000) Suspension arrays for high throughput multiplexed single nucleotide polymorphism genotyping. Cytometry 40: 102–108.

Bellisario R, Colinas RJ and Pass KA (2000) Simultaneous measurement of thyroxine and thyrotropin from newborn dried blood‐spot specimens using a multiplexed fluorescent microsphere immunoassay. Clinical Chemistry 46: 1422–1424.

Berger SS, Lauritsen KT, Boas U, Lind P and Andresen LO (2017) Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead‐based multiplex analysis. Journal of Veterinary Diagnostic Investigation 29: 797–804.

Binder SR (2006) Autoantibody detection using multiplex technologies. Lupus 15: 412–421.

Blazer LL, Roman DL, Muxlow MR and Neubig RR (2010) Use of flow cytometric methods to quantify protein–protein interactions. Current Protocols in Cytometry 51: 1–15.

Brenner S, Williams SR, Vermaas EH, et al. (2000) In vitro cloning of complex mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs. Proceedings of the National Academy of Sciences of the United States of America 97: 1665–1670.

Buranda T, Wu Y and Sklar LA (2009) Subsecond analyses of G‐protein coupled‐receptor ternary complex dynamics by rapid mix flow cytometry. Methods in Enzymology 461: 227–247.

Cai H, White PS, Torney D, et al. (2000) Flow cytometry‐based minisequencing: a new platform for high‐throughput single‐nucleotide polymorphism scoring. Genomics 66: 135–143.

Chan BM, Badh A, Berry KA, Grauer SA and King CT (2018) Flow cytometry‐based epitope binning using competitive binding profiles for the characterization of monoclonal antibodies against cellular and soluble protein targets. SLAS Discovery 23: 613–623.

Christopoulou S, Karaiskou S and Kalogianni DP (2018) Microbead‐based simultaneous fluorometric detection of three nut allergens. Microchimica Acta 185: 13.

Cretich M, Sola L and Gagni P (2013) Novel fluorescent microarray platforms: a case study in neurodegenerative disorders. Expert Review of Molecular Diagnostics 13: 863–873.

Dekking E, Velden VHJ, Van Der Böttcher S, et al. (2010) Detection of fusion genes at the protein level in leukemia patients via the flow cytometric immunobead assay. Best Practice & Research Clinical Haematology 23: 333–345.

Diaz MR and Fell JW (2004) High‐throughput detection of pathogenic yeasts of the genus Trichosporon. Journal of Clinical Microbiology 42: 3696–3706.

Edelmann L, Hashmi G, Song YH, et al. (2004) Cystic fibrosis carrier screening: validation of a novel method using bead‐chip technology. Genetics in Medicine 6: 431–438.

Edwards BS, Oprea T, Prossnitz ER and Sklar LA (2004) Flow cytometry for high‐throughput, high‐content screening. Current Opinion in Chemical Biology 8: 392–398.

Feldhaus MJ, Lualhati M, Cardon K, Roth B and Kamb A (2000) Oligonucleotide‐conjugated beads for transdominant genetic experiments. Nucleic Acids Research 28: 534–543.

Gao M, Lian H, Yu L, et al. (2019) Rolling circle amplification integrated with suspension bead array for ultrasensitive multiplex immunodetection of tumor markers. Analytica Chimica Acta 1048: 75–84.

Givan AL (1992) Flow Cytometry, First Principles, 2nd edn. Wiley‐Liss: New York.

González‐Buitrago JM (2006) Multiplexed testing in the autoimmunity laboratory. Clinical Chemistry and Laboratory Medicine 44: 1169–1174.

Hurley JD, Engle LJ, Davis JT, Welsh AM and Landers JE (2004) A simple bead‐based approach for multi‐SNP molecular haplotyping. Nucleic Acids Research 32: e186.

Iannone MA, Taylor JD, Chen J, et al. (2000) Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry. Cytometry 39: 131–140.

Kalogianni DP, Elenis DS, Christopoulos TK and Ioannou PC (2007) Multiplex quantitative competitive polymerase chain reaction based on a multianalyte hybridization assay performed on spectrally encoded microspheres. Analytical Chemistry 79: 6655–6661.

Kalogianni DP, Bazakos C, Boutsika LM, et al. (2015) Olive oil DNA fingerprinting by multiplex SNP genotyping on fluorescent microspheres. Journal of Agricultural Food Chemistry 63: 3121–3128.

Kounelli ML and Kalogianni DP (2017) A sensitive DNA‐based fluorometric method for milk authenticity of dairy products based on spectrally distinct microspheres. European Food Research and Technology 243: 1773–1781.

Lan W‐J, Lin Y‐M, Men Z‐H and Yan L (2017) Surface‐decorated S. cerevisiae for flow cytometric array immunoassay. Analytical and Bioanalytical Chemistry 409: 5259–5267.

Luo Y (2005) Selectivity assessment of kinase inhibitors: strategies and challenges. Current Opinion in Molecular Therapeutics 7: 251–255.

Mahony J, Chong S, Merante F, et al. (2007) Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead‐based assay. Journal of Clinical Microbiology 45: 2965–2970.

McBride MT, Gammon S, Pitesky M, et al. (2003) Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents. Analytical Chemistry 75: 1924–1930.

Meijgaarden KE, van Khatri B, Smith SG, et al. (2018) Cross‐laboratory evaluation of multiplex bead assays including independent common reference standards for immunological monitoring of observational and interventional human studies. PLoS ONE 13: 1–17.

Mizrahi O, Shalom EI, Baniyash M and Klieger Y (2018) Quantitative flow cytometry: concerns and recommendations in clinic and research. Cytometry Part B (Clinical Cytometry) 94B: 211–218.

Morgan E, Varro R, Sepulveda H, et al. (2004) Cytometric bead array: a multiplexed assay platform with applications in various areas of biology. Clinical Immunology 110: 252–266.

Pan Y, Wei X, Liang T, et al. (2018) A magnetic beads‐based portable flow cytometry immunosensor for in‐situ detection of marine biotoxin. Biomedical Microdevices 20: 60–68.

Pataki J, Szabó M, Lantos E, et al. (2005) Biological microbeads for flow‐cytometric immunoassays, enzyme titrations, and quantitative PCR. Cytometry Part A 68A: 45–52.

Pedersen RO, Nowatzke WL, Cho CY, Oliver KG and Garber EAE (2018) Cross‐reactivity by botanicals used in dietary supplements and spices using the multiplex xMAP food allergen detection assay. Analytical and Bioanalytical Chemistry 410: 5791–5806.

Rho J, Jang W, Hwang I, Lee D and Heon C (2018) Multiplex immunoassays using virus‐tethered gold microspheres by DC impedance‐based flow cytometry. Biosensors and Bioelectronics 102: 121–128.

Schneiderhan‐Marra N, Kirn A, Döttinger A, et al. (2005) Protein microarrays – a promising tool for cancer diagnosis. Cancer Genomics & Proteomics 2: 37–42.

Shapiro HM (1995) Practical Flow Cytometry, 3rd edn. Wiley‐Liss: New York.

Sklar LA, Edwards BS, Graves SW, Nolan JP and Prossnitz ER (2002) Flow cytometric analysis of ligand–receptor interactions and molecular assemblies. Annual Review of Biophysics and Biomolecular Structure 31: 97–119.

Sledz W, Los E and Lojkowska E (2010) Technology Luminex xMAP? A new tool in the diagnostics of plant diseases. Biotechnologica 1: 97–108.

Spiro A, Lowe M and Brown D (2000) A bead‐based method for multiplexed identification and quantitation of DNA sequences using flow cytometry. Applied and Environmental Microbiology 66: 4258–4265.

Stuchlý J and Kalina T (2014) Analyses of large flow cytometry datasets. Cytometry Part A 85A: 203–205.

Suárez H, Gámez‐Valero A, Reyes R, et al. (2017) A bead‐assisted flow cytometry method for the semi‐quantitative analysis of extracellular vesicles. Scientific Reports 7: 1–11.

Sukhanova A, Susha AS, Bek A, et al. (2007) Nanocrystal‐encoded fluorescent microbeads for proteomics: antibody profiling and diagnostics of autoimmune diseases. Nano Letters 7: 2322–2327.

Sukhanova A and Nabiev I (2008) Fluorescent nanocrystal‐encoded microbeads for multiplexed cancer imaging and diagnosis. Critical Reviews in Oncology/Hematology 68: 39–59.

Summerbell RC, Lévesque CA, Seifert KA, et al. (2005) Microcoding: the second step in DNA barcoding. Philosophical Transactions of the Royal Society B 360: 1897–1903.

Tang Y‐W and Stratton CW (2013) Advanced Techniques in Diagnostic Microbiology, 2nd edn. Springer: New York.

Van der Pol E, Sturk A, Leeuwen v, et al. (2018) Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation. Journal of Thrombosis and Haemostasis 16: 1236–1245.

Vignali DAA (2000) Multiplexed particle‐based flow cytometric assays. Journal of Immunological Methods 243: 243–255.

Wedemeyer N and Pötter T (2001) Flow cytometry: an ‘old’ tool for novel applications in medical genetics. Clinical Genetics 60: 1–8.

Whitehead GS, Walker JKL, Berman KG, Foster WM and Schwartz DA (2003) Allergen‐induced airway disease is mouse strain dependent. American Journal of Physiology‐Lung Cellular and Molecular Physiology 285: 32–42.

Xu H, Sha MY, Wong EY, et al. (2003) Multiplexed SNP genotyping using the Qbead TM system: a quantum dot‐encoded microsphere‐based assay. Nucleic Acids Research 31: 1–10.

Yan X, Zhong W, Tang A, et al. (2005) Multiplexed flow cytometric immunoassay for influenza virus detection and differentiation. Analytical Chemistry 77: 7673–7678.

Yu B, Li F, Zhao T, et al. (2018) Hybridization chain reaction‐based flow cytometric bead sensor for the detection of emetic Bacillus cereus in milk. Sensors and Actuators B: Chemical 256: 624–631.

Further Reading

Darzynkiewicz Z and Zhao H (2014) Cell cycle analysis by flow cytometry. In: Encyclopedia of Life Sciences. Wiley Online Library.

Hunt SV (2001) Cell separation techniques used in immunology. In: Encyclopedia of Life Sciences. Wiley Online Library.

Steinkamp JA (2002) Flow cytometers. 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. [doi: 10.1002/9780470015902.a0028611]