DNA-Directed Antibody Immobilization for Robust Protein Microarrays: Application to Single Particle Detection ‘DNA-Directed Antibody Immobilization

  • Nese Lortlar Ünlü
  • Fulya Ekiz Kanik
  • Elif Seymour
  • John H. Connor
  • M. Selim ÜnlüEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1571)


Protein microarrays are emerging tools which have become very powerful in multiplexed detection technologies. A variety of proteins can be immobilized on a sensor chip allowing for multiplexed diagnostics. Therefore, various types of analyte in a small volume of sample can be detected simultaneously. Protein immobilization is a crucial step for creating a robust and sensitive protein microarray-based detection system. In order to achieve a successful protein immobilization and preserve the activity of the proteins after immobilization, DNA-directed immobilization is a promising technique. Here, we present the design and the use of DNA-directed immobilized (DDI) antibodies in fabrication of robust protein microarrays. We focus on application of protein microarrays for capturing and detecting nanoparticles such as intact viruses. Experimental results on Single-particle interferometric reflectance imaging sensor (SP-IRIS) are used to validate the advantages of the DDI method.

Key words

Protein microarrays DNA-directed antibody immobilization Label free detection Single particle detection SP-IRIS 


  1. 1.
    MacBeath G, Schreiber SL (2000) Printing proteins as microarrays for high-throughput function determination. Science 289:1760–1763Google Scholar
  2. 2.
    Sun H, Chen GYJ, Yao SQ (2013) Recent advances in microarray technologies for proteomics. Chem Biol 20:685–699CrossRefGoogle Scholar
  3. 3.
    Hall DA, Tacek J, Snyder M (2007) Protein microarray technology. Mech Ageing Dev 128(1):161–167CrossRefGoogle Scholar
  4. 4.
    Avci O, Lortlar ÜN, Yalcin A et al (2015) Interferometric Reflectance imaging sensor (IRIS)-a platform technology for multiplexed diagnostics and digital detection. Sensors 15(7):17649–17665CrossRefGoogle Scholar
  5. 5.
    Schwenk JM, Lindberg J, Sundberg M et al (2007) Determination of binding specificities in highly multiplexed bead-based antibody assays for antibody proteomics. Mol Cell Proteomics 6:125–132CrossRefGoogle Scholar
  6. 6.
    Cretich M, Daaboul GG, Sola L et al (2015) Digital detection of biomarkers assisted by nanoparticles: application to diagnostics. Trends Biotechnol 33(6):343–351CrossRefGoogle Scholar
  7. 7.
    Yurt A, Daaboul GG, Connor JH et al (2012) Single nanoparticle detectors for biological applications. Nanoscale 4(3):715–726CrossRefGoogle Scholar
  8. 8.
    Walt D (2013) Optical methods for single molecule detection and analysis. Anal Chem 85(3):1258–1263CrossRefGoogle Scholar
  9. 9.
    Daaboul GG, Lopez CA, Chinnala J et al (2014) Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for ebola and marburg detection. ACS Nano 8(6):6047–6055CrossRefGoogle Scholar
  10. 10.
    Monroe MR, Daaboul GG, Tuysuzoglu A et al (2013) Single nanoparticle detection for multiplexed protein diagnostics with attomolar sensitivity in serum and unprocessed whole blood. Anal Chem 85(7):3698–3706CrossRefGoogle Scholar
  11. 11.
    Sevenler D, Lortlar ÜN, Ünlü MS (2015) Nanoparticle biosensing with interferometric reflectance imaging. In: Vestergaard MC, Kerman K, Hsing I-M, Tamiya E (eds) Nanobiosensors and nanobioanalyses. Springer, Tokyo, pp 81–95Google Scholar
  12. 12.
    Monroe MR, Reddington A, Collins AD et al (2011) Multiplexed method to calibrate and quantitate fluorescence signal for allergen-specific IgE. Anal Chem 83(24):9485–9491CrossRefGoogle Scholar
  13. 13.
    Niemeyer CM, Boldt L, Ceyhan B et al (1999) DNA-directed immobilization: efficient, reversible, and site-selective surface binding of proteins by means of covalent DNA-streptavidin conjugates. Anal Biochem 268:54–63CrossRefGoogle Scholar
  14. 14.
    Ladd J, Boozer C, Yu Q et al (2004) DNA-directed protein immobilization on mixed self-assembled monolayers via a streptavidin bridge. Langmuir 20:8090–8095CrossRefGoogle Scholar
  15. 15.
    Schroeder H, Adler M, Gergk K et al (2009) User configurable microfluidic device for multiplexed immunoassays based on DNA-directed assembly. Anal Chem 81:1275–1279CrossRefGoogle Scholar
  16. 16.
    Washburn AL, Gomez J, Bailey RC (2011) DNA-encoding to improve performance and allow parallel evaluation of the binding characteristics of multiple antibodies in a surface-bound immunoassay format. Anal Chem 83:3572–3580CrossRefGoogle Scholar
  17. 17.
    Wacker R, Niemeyer CM (2004) DNA- μFIA-a readily configurable microarray-fluorescence immunoassay based on DNA-directed immobilization of proteins. Chem Bio Chem 5:453–459CrossRefGoogle Scholar
  18. 18.
    Wacker R, Schroder H, Niemeyer CM (2004) Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study. Anal Biochem 330:281–287CrossRefGoogle Scholar
  19. 19.
    Seymour E, Daaboul GG, Zhang X et al (2015) DNA-directed antibody immobilization for enhanced detection of single viral pathogens. Anal Chem 87(20):10505–10512CrossRefGoogle Scholar
  20. 20.
    Avci O, Adato R, Yalcin OA et al (2016) Physical modeling of interference enhanced imaging and characterization of single nanoparticles. Opt Express 24(6):6094–6114CrossRefGoogle Scholar
  21. 21.
    Pirri G, Damin F, Chiari M et al (2004) Characterization of the polymeric adsorbed coating for DNA microarray glass slides. Anal Chem 76(4):1352–1358CrossRefGoogle Scholar
  22. 22.
    Yalçin A, Damin F, Özkumur E et al (2009) Direct observation of conformation of a polymeric coating with implications in microarray applications. Anal Chem 81(2):625–630CrossRefGoogle Scholar
  23. 23.
    Romanov V, Davido SN, Miles AR et al (2014) A critical comparison of protein microarray fabrication technologies. Analyst 139(6):1303–1326CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Nese Lortlar Ünlü
    • 1
    • 2
  • Fulya Ekiz Kanik
    • 3
  • Elif Seymour
    • 4
  • John H. Connor
    • 5
  • M. Selim Ünlü
    • 1
    • 3
    • 5
    Email author
  1. 1.Biomedical Engineering DepartmentBoston UniversityBostonUSA
  2. 2.Faculty of MedicineBahcesehir UniversityIstanbulTurkey
  3. 3.Electrical and Computer Engineering DepartmentBoston UniversityBostonUSA
  4. 4.Biotechnology Research Program DepartmentASELSAN Research CenterAnkaraTurkey
  5. 5.Microbiology DepartmentBoston University School of MedicineBostonUSA

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