Label-free Microarray-based Binding Affinity Constant Measurement with Modified Fluidic Arrangement


The oblique-incidence reflectivity difference (OI-RD) scanning microscopy has the capability of simultaneously measuring binding curves of a protein probe with tens of thousands molecular targets in a microarray and yielding reaction rate constants. However, the quality of reaction rate constants is influenced by the fluidic system. To improve the quality of reaction rate constant measurement over the entire microarray, we demonstrate a fluidic chamber that allows the fluid to flow from the bottom to the top uniformly across the microarray and thus provides more uniform and accurate measurements of reaction rate constants with simplified fluidic design.

This is a preview of subscription content, access via your institution.


  1. 1.

    Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. Quantitative monitoring of gene-expression patterns with a complementary-DNA micro. Science 270, 467–470 (1995).

    CAS  Article  Google Scholar 

  2. 2.

    Stoughton, R.B. Applications of DNA microarrays in biology. Annu. Rev. Biochem. 74, 53–82 (2005).

    CAS  Article  Google Scholar 

  3. 3.

    MacBeath, G. Protein microarrays and proteomics. Nat. Genet. 32, 526–532 (2002).

    CAS  Article  Google Scholar 

  4. 4.

    Stoevesandt, O., Taussig, M.J. & He, M.Y. Protein microarrays: high-throughput tools for proteomics. Expert Rev. Proteomics 6, 145–157 (2009).

    CAS  Article  Google Scholar 

  5. 5.

    Feizi, T., Fazio, F., Chai, W.C. & Wong, C.H. Carbohydrate microarrays -a new set of technologies at the frontiers of glycomics. Curr. Opin. Struct. Biol. 13, 637–645 (2003).

    CAS  Article  Google Scholar 

  6. 6.

    Song, X.Z. et al. Shotgun glycomics: a microarray strategy for functional glycomics. Nat. Methods 8, 85-U125 (2011).

    CAS  Article  Google Scholar 

  7. 7.

    Andersen, M.R. et al. A trispecies aspergillus microarray: comparative transcriptomics of three aspergillus species. Proc. Natl. Acad. Sci. U. S. A. 105, 4387–4392 (2008).

    CAS  Article  Google Scholar 

  8. 8.

    Gobert, G.N. et al. Transcriptomics tool for the human Schistosoma blood flukes using microarray gene expression profiling. Exp. Parasitol. 114, 160–172 (2006).

    CAS  Article  Google Scholar 

  9. 9.

    Phelps, T.J., Palumbo, A.V. & Beliaev, A.S. Metabolomics and microarrays for improved understanding of phenotypic characteristics controlled by both genomics and environmental constraints. Curr. Opin. Biotechnol. 13, 20–24 (2002).

    CAS  Article  Google Scholar 

  10. 10.

    Soanes, K.H. et al. Molecular characterization of zebrafish embryogenesis via DNA microarrays and multiplatform time course metabolomics studies. J. Proteome Res. 10, 5102–5117 (2011).

    CAS  Article  Google Scholar 

  11. 11.

    Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 293, 2101–2105 (2001).

    CAS  Article  Google Scholar 

  12. 12.

    Sun, Y.S., Landry, J.P., Fei, Y.Y. & Zhu, X.D. Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions. Langmuir 24, 13399–13405 (2008).

    CAS  Article  Google Scholar 

  13. 13.

    Rich, R.L. & Myszka, D.G. Survey of the year 2007 commercial optical biosensor literature. J. Mol. Recognit. 21, 355–400 (2008).

    CAS  Article  Google Scholar 

  14. 14.

    Singh, B.K. & Hillier, A.C. Surface plasmon resonance imaging of biomolecular interactions on a gratingbased sensor array. Anal. Chem. 78, 2009–2018 (2006).

    CAS  Article  Google Scholar 

  15. 15.

    Hanel, C. & Gauglitz, G. Comparison of reflectometric interference spectroscopy with other instruments for label-free optical detection. Anal. Bioanal. Chem. 372, 91–100 (2002).

    CAS  Article  Google Scholar 

  16. 16.

    Ozkumur, E. et al. Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications. Proc. Natl. Acad. Sci. U. S. A. 105, 7988–7992 (2008).

    CAS  Article  Google Scholar 

  17. 17.

    Cross, G.H. et al. The metrics of surface adsorbed small molecules on the Young’s fringe dual-slab waveguide interferometer. J. Phys. D: Appl. Phys. 37, 74–80 (2004).

    CAS  Article  Google Scholar 

  18. 18.

    Wang, Z.H. & Jin, G. A label-free multisensing immunosensor based on imaging ellipsometry. Anal. Chem. 75, 6119–6123 (2003).

    CAS  Article  Google Scholar 

  19. 19.

    Arwin, H. Ellipsometry on thin organic layers of biological interest: characterization and applications. Thin Solid Films 377, 48–56 (2000).

    Article  Google Scholar 

  20. 20.

    Nand, A. et al. In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging. Anal. Biochem. 464, 30–35 (2014).

    CAS  Article  Google Scholar 

  21. 21.

    Boozer, C., Kim, G., Cong, S.X., Guan, H.W. & Londergan, T. Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologies. Curr. Opin. Biotechnol. 17, 400–405 (2006).

    CAS  Article  Google Scholar 

  22. 22.

    Landry, J.P., Fei, Y.Y. & Zhu, X.D. Simultaneous Measurement of 10,000 Protein-Ligand Affinity Constants Using Microarray-Based Kinetic Constant Assays. Assay Drug Dev. Technol. 10, 250–259 (2012).

    CAS  Article  Google Scholar 

  23. 23.

    Landry, J.P., Proudian, A.P., Malovichko, G. & Zhu, X.D. Kinetic identification of protein ligands in a 51,200 small-molecule library using microarrays and a label-free ellipsometric scanner. In Proceedings of SPIE BIOS 2013, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI, 8587 IV. SPIEInt Soc. Opt. Eng. (2013).

    Google Scholar 

  24. 24.

    Zhu, C.G. et al. Calibration of oblique-incidence reflectivity difference for label-free detection of a molecular layer. Appl. Opt. 55, 9459–9466 (2016).

    CAS  Article  Google Scholar 

  25. 25.

    Nabighian, E., Bartelt, M.C. & Zhu, X.D. Kinetic roughening during rare-gas homoepitaxy. Phys. Rev. B. 62, 1619–1622 (2000).

    CAS  Article  Google Scholar 

  26. 26.

    Thomas, P., Nabighian, E., Bartelt, M.C., Fong, C.Y. & Zhu, X.D. An oblique-incidence optical reflectivity difference and LEED study of rare-gas growth on a lattice-mismatched metal substrate. Appl. Phys. A. 79, 131–137 (2003).

    Article  Google Scholar 

  27. 27.

    Zhu, X.D., Wicklein, S., Gunkel, F., Xiao, R. & Dittmann, R. In situ optical characterization of LaAlO3 epitaxy on SrTiO3(001). Europhys. Lett. 109, 37006 (2015).

    Article  Google Scholar 

  28. 28.

    Schwarzacher, W., Gray, J.W. & Zhu, X.D. Oblique incidence reflectivity difference as an in situ probe of Co electrodeposition on polycrystalline Au. Electrochem. Solid-State Lett. 6, C73–C76 (2003).

    CAS  Article  Google Scholar 

  29. 29.

    Wu, G.Y. et al. Pb electrodeposition on polycrystalline Cu in the presence and absence of Cl-: A combined oblique incidence reflectivity difference and in situ AFM study. Surf. Sci. 601, 1886–1891 (2007).

    CAS  Article  Google Scholar 

  30. 30.

    Fei, Y.Y. et al. Characterization of receptor binding profiles of influenza A viruses using an ellipsometrybased label-free glycan microarray assay platform. Biomolecules 5, 1480–1489 (2015).

    CAS  Article  Google Scholar 

  31. 31.

    Fei, Y.Y. et al. Use of real-time, label-free analysis in revealing low-affinity binding to blood group antigens by Helicobacter pylori. Anal. Chem. 83, 6336–6341 (2011).

    CAS  Article  Google Scholar 

  32. 32.

    Landry, J.P. et al. Discovering Small Molecule Ligands of Vascular Endothelial Growth Factor That Block VEGF-KDR Binding Using Label-Free Microarray-Based Assays. Assay Drug Dev. Technol. 11, 326–332 (2013).

    CAS  Article  Google Scholar 

  33. 33.

    Fei, Y.Y. et al. A novel high-throughput scanning microscope for label-free detection of protein and smallmolecule chemical microarrays. Rev. Sci. Instrum. 79, 013708 (2008).

    CAS  Article  Google Scholar 

  34. 34.

    Guo, X.X. et al. Characterization of protein expression levels with label-free detected reverse phase protein arrays. Anal. Biochem. 509, 67–72 (2016).

    CAS  Article  Google Scholar 

  35. 35.

    Wang, J.J. et al. Epigallocatechin-3-gallate (EGCG) enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity. Cell Death Discov. 3, e17034 (2017).

    Article  Google Scholar 

  36. 36.

    Zhu, C.G. et al. Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry. Sensors 16, 378 (2016).

    Article  Google Scholar 

  37. 37.

    Jung, H.S. et al. Impact of hapten presentation on antibody binding at lipid membrane interfaces. Biophys. J. 94, 3094–3103 (2008).

    CAS  Article  Google Scholar 

  38. 38.

    Zhang, N. et al. Plasmonic metal nanostructure array by glancing angle deposition for biosensing application. Sens. Actuators B-Chem. 183, 310–318 (2013).

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding authors

Correspondence to Hao Chen or Yiyan Fei.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hu, J., Chen, R., Zhu, C. et al. Label-free Microarray-based Binding Affinity Constant Measurement with Modified Fluidic Arrangement. BioChip J 12, 11–17 (2018).

Download citation


  • Optical biosensors
  • Protein microarray
  • Kinetics measurement
  • Affinity constant