Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 408, Issue 5, Pages 1335-1346Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-015-9052-0
Keywords
Paper-based assays; Protein immobilization; Protein engineering; Nitrocellulose
Funding
- National Science Foundation Graduate Research Fellowship Program [DGE - 0718124]
- National Institute of Allergy and Infectious Diseases of National Institutes of Health [R01AI096184]
- University of Washington Department of Bioengineering
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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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