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

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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.

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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.

Author information

Correspondence to Carly A. Holstein.

Additional information

Published in the topical collection Fiber-based Platforms for Bioanalytics with guest editors Antje J. Baeumner and R. Kenneth Marcus.

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Holstein, C.A., Chevalier, A., Bennett, S. et al. Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers. Anal Bioanal Chem 408, 1335–1346 (2016).

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  • Paper-based assays
  • Protein immobilization
  • Protein engineering
  • Nitrocellulose