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

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Abstract

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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Notes

  1. GE FF60 has been replaced in the product line with updated FF80HP from GE Healthcare.

References

  1. Tonkinson JL, Stillman BA (2002) Nitrocellulose: a tried and true polymer finds utility as a post-genomic substrate. Front Biosci 7:c1–c12

    Article  CAS  Google Scholar 

  2. Fridley GE, Holstein CA, Oza SB, Yager P (2013) The evolution of nitrocellulose as a material for bioassays. MRS Bull 38:326–330. doi:10.1557/mrs.2013.60

    Article  CAS  Google Scholar 

  3. Gubala V, Harris LF, Ricco AJ, Tan MX, Williams DE (2012) Point of care diagnostics: status and future. Anal Chem 84:487–515. doi:10.1021/ac2030199

    Article  CAS  Google Scholar 

  4. Mansfield M (2009) Nitrocellulose membranes for lateral flow immunoassays: a technical treatise. In: Wong RC, Tse HY (eds) Lateral flow immunoass. Humana Press, New York, pp 95–113

    Google Scholar 

  5. O’Farrell B (2009) Evolution of lateral flow-based immunoassay systems. In: Wong RC, Tse HY (eds) Lateral flow immunoass. Humana Press, New York, pp 1–33

    Chapter  Google Scholar 

  6. Vermasvuori R (2009) Production of recombinant proteins and monoclonal antibodies—techno-economical evaluation of the production methods. Helsinki University of Technology. http://lib.tkk.fi/Lic/2009/urn100121.pdf

  7. Fleishman SJ, Whitehead TA, Ekiert DC, Dreyfus C, Corn JE, Strauch E-M, Wilson IA, Baker D (2011) Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science 332:816–821. doi:10.1126/science.1202617

    Article  CAS  Google Scholar 

  8. Whitehead TA, Chevalier A, Song Y, Dreyfus C, Fleishman SJ, De Mattos C, Myers CA, Kamisetty H, Blair P, Wilson IA, Baker D (2012) Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing. Nat Biotechnol 30:543–548. doi:10.1038/nbt.2214

    Article  CAS  Google Scholar 

  9. Wu P, Castner DG, Grainger DW (2008) Diagnostic devices as biomaterials: a review of nucleic acid and protein microarray surface performance issues. J Biomater Sci Polym Ed 19:725–753. doi:10.1163/156856208784522092

    Article  CAS  Google Scholar 

  10. Rusmini F, Zhong Z, Feijen J (2007) Protein immobilization strategies for protein biochips. Biomacromolecules 8:1775–1789. doi:10.1021/bm061197b

    Article  CAS  Google Scholar 

  11. Jonkheijm P, Weinrich D, Schröder H, Niemeyer CM, Waldmann H (2008) Chemical strategies for generating protein biochips. Angew Chem Int Ed 47:9618–9647. doi:10.1002/anie.200801711

    Article  CAS  Google Scholar 

  12. Pelton R (2009) Bioactive paper provides a low-cost platform for diagnostics. TrAC Trends Anal Chem 28:925–942. doi:10.1016/j.trac.2009.05.005

    Article  CAS  Google Scholar 

  13. Sicard C, Brennan JD (2013) Bioactive paper: biomolecule immobilization methods and applications in environmental monitoring. MRS Bull 38:331–334. doi:10.1557/mrs.2013.61

    Article  CAS  Google Scholar 

  14. Yu A, Shang J, Cheng F, Paik BA, Kaplan JM, Andrade RB, Ratner DM (2012) Biofunctional Paper via the Covalent Modification of Cellulose. Langmuir 28:11265–11273. doi:10.1021/la301661x

    Article  CAS  Google Scholar 

  15. Yetisen AK, Akram MS, Lowe CR (2013) Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 13:2210. doi:10.1039/c3lc50169h

    Article  CAS  Google Scholar 

  16. Holstein CA, Fridley GE, Adcock CA, Yager P (2015) Methods and Models to Examine Protein Adsorption to Nitrocellulose. Anal Chem (in review)

  17. Metkar SS, Mahajan SK, Sainis JK (1995) Modified procedure for nonspecific protein staining on nitrocellulose paper using Coomassie brilliant blue R-250. Anal Biochem 227:389–391. doi:10.1006/abio.1995.1297

    Article  CAS  Google Scholar 

  18. Li B, Olsen CE, Moore DR (2011) Porous membranes having a polymeric coating and methods for their preparation and use. US patent application, US20130171618 A1

  19. Li B, Olsen CE, Moore DR (2011) Porous membranes having a polymeric coating and methods for their preparation and use. US patent application, US20130171368 A1

  20. Li B, Olsen CE, Moore DR (2011) Porous membranes having a polymeric coating and methods for their preparation and use. US patent application, US20130171026 A1

  21. Koga N, Tatsumi-Koga R, Liu G, Xiao R, Acton TB, Montelione GT, Baker D (2012) Principles for designing ideal protein structures. Nature 491:222–227. doi:10.1038/nature11600

    Article  CAS  Google Scholar 

  22. Huang P-S, Oberdorfer G, Xu C, Pei XY, Nannenga BL, Rogers JM, DiMaio F, Gonen T, Luisi B, Baker D (2014) High thermodynamic stability of parametrically designed helical bundles. Science 346:481–485. doi:10.1126/science.1257481

    Article  CAS  Google Scholar 

  23. Currie LA (1995) Nomenclature in evaluation of analytical methods including detection and quantification capabilities (IUPAC Recommendations 1995). Pure Appl Chem 67:1699–1723. doi:10.1351/pac199567101699

    Article  CAS  Google Scholar 

  24. Holstein CA, Griffin M, Hong J, Sampson PD (2015) A statistical method for determining and comparing limits of detection of bioassays. Anal Chem. doi:10.1021/acs.analchem.5b02082

  25. Van Oss CJ, Good RJ, Chaudhury MK (1987) Mechanism of DNA (Southern) and protein (Western) blotting on cellulose nitrate and other membranes. J Chromatogr 391:53–65

    Article  Google Scholar 

  26. American Chemical Society, American Chemical Society (1995) Proteins at interfaces II: fundamentals and applications. American Chemical Society, Washington, DC

    Google Scholar 

  27. EMD Millipore Corporation (2008) Rapid lateral flow test strips: consideration for product development. EMD Millipore Corporation, Billerica, MA

  28. Přistoupil TI, Kramlová M, Štěrbíková J (1969) On the mechanism of adsorption of proteins to nitrocellulose in membrane chromatography. J Chromatogr A 42:367–375. doi:10.1016/S0021-9673(01)80636-1

    Article  Google Scholar 

Download references

Acknowledgments

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.

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Authors and Affiliations

  1. Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98105, USA

    Carly A. Holstein, Aaron Chevalier, Steven Bennett, Caitlin E. Anderson, Karen Keniston & Paul Yager

  2. Department of Biochemistry, University of Washington, 1705 NE Pacific St., Seattle, WA, 98195-7350, USA

    Aaron Chevalier & David Baker

  3. General Electric Global Research Center, 1 Research Cir, Niskayuna, NY, 12309, USA

    Cathryn Olsen, Bing Li, Brian Bales & David R. Moore

  4. School of Chemical, Biological, and Environmental Engineering, Oregon State University, 103 Gleeson Hall, Corvallis, OR, 97331, USA

    Elain Fu

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  1. Carly A. Holstein
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  11. David Baker
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Corresponding author

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). https://doi.org/10.1007/s00216-015-9052-0

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  • DOI: https://doi.org/10.1007/s00216-015-9052-0

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