Analytical and Bioanalytical Chemistry

, Volume 408, Issue 5, pp 1335–1346 | Cite as

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers

  • Carly A. HolsteinEmail author
  • Aaron Chevalier
  • Steven Bennett
  • Caitlin E. Anderson
  • Karen Keniston
  • Cathryn Olsen
  • Bing Li
  • Brian Bales
  • David R. Moore
  • Elain Fu
  • David Baker
  • Paul Yager
Research Paper
Part of the following topical collections:
  1. Fiber-based Platforms for Bioanalytics


To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant “flu binders” for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.


Paper-based assays Protein immobilization Protein engineering Nitrocellulose 



The authors thank colleagues in the Yager, Baker, and GE Global Research groups for helpful discussions regarding this work. The authors particularly acknowledge Rashmi Ravichandran for her help in producing many of the recombinant flu binder proteins. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE – 0718124, by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI096184, and by the University of Washington Department of Bioengineering. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2015_9052_MOESM1_ESM.pdf (574 kb)
ESM 1 (PDF 573 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Carly A. Holstein
    • 1
    Email author
  • Aaron Chevalier
    • 1
    • 2
  • Steven Bennett
    • 1
  • Caitlin E. Anderson
    • 1
  • Karen Keniston
    • 1
  • Cathryn Olsen
    • 3
  • Bing Li
    • 3
  • Brian Bales
    • 3
  • David R. Moore
    • 3
  • Elain Fu
    • 4
  • David Baker
    • 2
  • Paul Yager
    • 1
  1. 1.Department of BioengineeringUniversity of WashingtonSeattleUSA
  2. 2.Department of BiochemistryUniversity of WashingtonSeattleUSA
  3. 3.General Electric Global Research CenterNiskayunaUSA
  4. 4.School of Chemical, Biological, and Environmental EngineeringOregon State UniversityCorvallisUSA

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