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Focal molography is a new method for the in situ analysis of molecular interactions in biological samples

Nature Nanotechnology volume 12, pages 1089–1095 (2017)Cite this article

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An Author Correction to this article was published on 11 February 2021

This article has been updated

Abstract

Focal molography is a next-generation biosensor that visualizes specific biomolecular interactions in real time. It transduces affinity modulation on the sensor surface into refractive index modulation caused by target molecules that are bound to a precisely assembled nanopattern of molecular recognition sites, termed the ‘mologram’. The mologram is designed so that laser light is scattered at specifically bound molecules, generating a strong signal in the focus of the mologram via constructive interference, while scattering at nonspecifically bound molecules does not contribute to the effect. We present the realization of molograms on a chip by submicrometre near-field reactive immersion lithography on a light-sensitive monolithic graft copolymer layer. We demonstrate the selective and sensitive detection of biomolecules, which bind to the recognition sites of the mologram in various complex biological samples. This allows the label-free analysis of non-covalent interactions in complex biological samples, without a need for extensive sample preparation, and enables novel time- and cost-saving ways of performing and developing immunoassays for diagnostic tests.

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Figure 1: Schematic illustration of the fundamental components of focal molography and the submicrometre near-field lithographic process used in molography.
Figure 2: Comparison of sandwich immunoassay performed by molography and OWLS (diffractometric versus refractometric biosensor).
Figure 3: Determination of assay specific affinity for antibody binding to β-amyloid peptide.
Figure 4: Real-time detection of a therapeutic antibody in human plasma.
Figure 5: In situ measurement of secreted immunoglobulins in a hybridoma cell culture.

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Change history

  • 25 October 2017

    In the version of this Article originally published, the illumination pattern below the phase mask was incorrectly positioned in Fig. 1b (ii) and Zeptosens was misspelled in two instances in Methods. These errors have been corrected in all versions of the Article.

  • 11 February 2021

    A Correction to this paper has been published: https://doi.org/10.1038/s41565-021-00863-x

References

  1. Engvall, E. & Perlmann, P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8, 871–874 (1971).

    Article  CAS  Google Scholar 

  2. Love, J. C., Estroff, L. A., Kriebel, J. K., Nuzzo, R. G. & Whitesides, G. M. Self-assembled monolayers of thiolates on metals as a form of nanotechnology. Chem. Rev. 105, 1103–1169 (2005).

    Article  CAS  Google Scholar 

  3. Shimomura, O. The discovery of aequorin and green fluorescent protein. J. Microsc. 217, 3–15 (2005).

    Article  CAS  Google Scholar 

  4. McMeekin, T. L., Groves, M. L. & Hipp, N. J. in Amino Acids and Serum Proteins Vol. 44 (ed. Stekol, J. A.) 54–66 (American Chemical Society, 1964).

    Book  Google Scholar 

  5. Vörös, J. The density and refractive index of adsorbing protein layers. Biophys. J. 87, 553–561 (2004).

    Article  CAS  Google Scholar 

  6. Homola, J. Present and future of surface plasmon resonance biosensors. Anal. Bioanal. Chem. 377, 528–539 (2003).

    Article  CAS  Google Scholar 

  7. Brolo, A. G. Plasmonics for future biosensors. Nat. Photon. 6, 709–713 (2012).

    Article  CAS  Google Scholar 

  8. Wilson, R. Sensitivity and specificity: twin goals of proteomics assays. Can they be combined? Expert Rev. Proteomics 10, 135–149 (2013).

    Article  CAS  Google Scholar 

  9. Lee, G. U., Metzger, S., Natesan, M., Yanavich, C. & Dufrene, Y. F. Implementation of force differentiation in the immunoassay. Anal. Biochem. 287, 261–271 (2000).

    Article  CAS  Google Scholar 

  10. Mulvaney, S. P. et al. Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics. Biosens. Bioelectron. 23, 191–200 (2007).

    Article  CAS  Google Scholar 

  11. Myszyka, D. G. Improving biosensor analysis. J. Mol. Recognit. 12, 279–284 (1999).

    Article  Google Scholar 

  12. Kozma, P., Kehl, F., Ehrentreich-Förster, E., Stamm, C. & Bier, F. F. Integrated planar optical waveguide interferometer biosensors: a comparative review. Biosens. Bioelectron. 58, 287–307 (2014).

    Article  CAS  Google Scholar 

  13. Zhang, J. et al. Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA. Nat. Nanotech. 1, 214–220 (2006).

    Article  CAS  Google Scholar 

  14. Stern, E. et al. Label-free biomarker detection from whole blood. Nat. Nanotech. 5, 138–142 (2010).

    Article  CAS  Google Scholar 

  15. Cornell, B. A. et al. A biosensor that uses ion-channel switches. Nature 387, 580–583 (1997).

    Article  CAS  Google Scholar 

  16. Tsay, Y. G. et al. Optical biosensor assay (OBA). Clin. Chem. 37, 1502–1505 (1991).

    Article  CAS  Google Scholar 

  17. Cleverley, S., Chen, I. & Houle, J. F. Label-free and amplified quantitation of proteins in complex mixtures using diffractive optics technology. J. Chromatogr. 878, 264–270 (2010).

    CAS  Google Scholar 

  18. Johnson-Buck, A. et al. Kinetic fingerprinting to identify and count single nucleic acids. Nat. Biotechnol. 33, 730–732 (2015).

    Article  CAS  Google Scholar 

  19. Gunnarsson, A. et al. Drug discovery at the single molecule level: inhibition-in-solution assay of membrane-reconstituted β-secretase using single-molecule imaging. Anal. Chem. 87, 4100–4103 (2015).

    Article  CAS  Google Scholar 

  20. Fattinger, C. Focal molography: coherent microscopic detection of biomolecular interaction. Phys. Rev. X 4, 031024 (2014).

    Google Scholar 

  21. Gedda, L., Björkelund, H. & Andersson, K. Real-time immunohistochemistry analysis of embedded tissue. Appl. Radiat. Isot. 68, 2372–2376 (2010).

    Article  CAS  Google Scholar 

  22. Bondza, S., Stenberg, J., Nestor, M., Andersson, K. & Bjorkelund, H. Conjugation effects on antibody–drug conjugates: evaluation of interaction kinetics in real time on living cells. Mol. Pharm. 11, 4154–4163 (2014).

    Article  CAS  Google Scholar 

  23. Binnig, G. Coherent signal picks out biomolecular interactions. Physics 7, 84 (2014).

    Article  Google Scholar 

  24. Woolley, C. F., Hayes, M. A., Mahanti, P., Douglass Gilman, S. & Taylor, T. Theoretical limitations of quantification for noncompetitive sandwich immunoassays. Anal. Bioanal. Chem. 407, 8605–8615 (2015).

    Article  CAS  Google Scholar 

  25. Homola, J. Surface plasmon resonance sensors for detection of chemical and biological species. Chem. Rev. 108, 462–493 (2008).

    Article  CAS  Google Scholar 

  26. Ashkenazi, A. & Dixit, V. M. Death receptors: signaling and modulation. Science 281, 1305–1308 (1998).

    Article  CAS  Google Scholar 

  27. Jemmerson, R., LaPlante, B. & Treeful, A. Release of intact, monomeric cytochrome c from apoptotic and necrotic cells. Cell Death Differ. 9, 538–548 (2002).

    Article  CAS  Google Scholar 

  28. Jonkheijm, P., Weinrich, D., Schröder, H., Niemeyer, C. M. & Waldmann, H. Chemical strategies for generating protein biochips. Angew. Chem. Int. Ed. 47, 9618–9647 (2008).

    Article  CAS  Google Scholar 

  29. Pirrung, M. C. & Huang, C. Y. A general method for the spatially defined immobilization of biomolecules on glass surfaces using ‘caged’ biotin. Bioconjug. Chem. 7, 317–321 (1996).

    Article  CAS  Google Scholar 

  30. Sundberg, S. A. et al. Spatially-addressable immobilization of macromolecules on solid supports. J. Am. Chem. Soc. 117, 12050–12057 (1995).

    Article  CAS  Google Scholar 

  31. Vossmeyer, T., DeIonno, E. & Heath, J. R. Light-directed assembly of nanoparticles. Angew. Chem. Int. Ed. 36, 1080–1083 (1997).

    Article  CAS  Google Scholar 

  32. Fodor, S. P. et al. Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767–773 (1991).

    Article  CAS  Google Scholar 

  33. Serrano, Â., Zürcher, S., Tosatti, S. & Spencer, N. D. Imparting nonfouling properties to chemically distinct surfaces with a single adsorbing polymer: a multimodal binding approach. Macromol. Rapid Commun. 37, 622–629 (2016).

    Article  CAS  Google Scholar 

  34. Schmitt, K., Oehse, K., Sulz, G. & Hoffmann, C. Evanescent field sensors based on tantalum pentoxide waveguides—a review. Sensors 8, 711–738 (2008).

    Article  CAS  Google Scholar 

  35. Pasche, S., De Paul, S. M., Vörös, J., Spencer, N. D. & Textor, M. Poly(L-lysine)-graft-poly(ethylene glycol) assembled monolayers on niobium oxide surfaces: a quantitative study of the influence of polymer interfacial architecture on resistance to protein adsorption by ToF-SIMS and in situ OWLS. Langmuir 19, 9216–9225 (2003).

    Article  CAS  Google Scholar 

  36. Aizenberg, J., Rogers, J. A., Paul, K. E. & Whitesides, G. M. Imaging profiles of light intensity in the near field: applications to phase-shift photolithography. Appl. Opt. 37, 2145–2152 (1998).

    Article  CAS  Google Scholar 

  37. Tanaka, T. et al. A novel optical lithography technique using the phase-shifter fringe. Jpn J. Appl. Phys. 30, 1131–1136 (1991).

    Article  CAS  Google Scholar 

  38. Hill, K. O., Malo, B., Bilodeau, F., Johnson, D. C. & Albert, J. Bragg gratings fabricated in monomode photosensitive optical fiber by UV exposure through a phase mask. Appl. Phys. Lett. 62, 1035 (1993).

    Article  CAS  Google Scholar 

  39. Vörös, J. et al. Optical grating coupler biosensors. Biomaterials 23, 3699–3710 (2002).

    Article  Google Scholar 

  40. Panza, F. et al. Amyloid-based immunotherapy for Alzheimer's disease in the time of prevention trials: the way forward. Expert Rev. Clin. Immunol. 10, 405–419 (2014).

    Article  CAS  Google Scholar 

  41. Vaisocherová, H. et al. Ultralow fouling and functionalizable surface chemistry based on a zwitterionic polymer enabling sensitive and specific protein detection in undiluted blood plasma. Anal. Chem. 80, 7894–7901 (2008).

    Article  CAS  Google Scholar 

  42. Riedel, T. et al. Hepatitis B plasmonic biosensor for the analysis of clinical serum samples. Biosens. Bioelectron. 85, 272–279 (2016).

    Article  CAS  Google Scholar 

  43. Piliarik, M., Bocková, M. & Homola, J. Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma. Biosens. Bioelectron. 26, 1656–1661 (2010).

    Article  CAS  Google Scholar 

  44. MacDougall, D. & Crummett, W. B. Guidelines for data acquisition and data quality evaluation in environmental chemistry. Anal. Chem. 52, 2242–2249 (1980).

    Article  CAS  Google Scholar 

  45. Armbruster, D. A. & Pry, T. Limit of blank, limit of detection and limit of quantitation. Clin. Biochem. Rev. 29(Suppl. 1), S49–S52 (2008).

    Google Scholar 

  46. Fan, X. et al. Sensitive optical biosensors for unlabeled targets: a review. Anal. Chim. Acta 620, 8–26 (2008).

    Article  CAS  Google Scholar 

  47. Kolomenskii, A. A., Gershon, P. D. & Schuessler, H. A. Sensitivity and detection limit of concentration and adsorption measurements by laser-induced surface-plasmon resonance. Appl. Opt. 36, 6539–6547 (1997).

    Article  CAS  Google Scholar 

  48. Horvath, R., Pedersen, H. C., Skivesen, N., Selmeczi, D. & Larsen, N. B. Monitoring of living cell attachment and spreading using reverse symmetry waveguide sensing. Appl. Phys. Lett. 86, 071101 (2005).

    Article  CAS  Google Scholar 

  49. Pawlak, M. et al. Zeptosens’ protein microarrays: a novel high performance microarray platform for low abundance protein analysis. Proteomics 2, 383–393 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank V. Guzenko (ETH) and W. Arens (IMT) for technical support in fabrication of the phase mask, S. Tosatti (SuSoS) and S. Zürcher (SuSoS) for consulting regarding surface science questions and production of the copolymer, A. Nichtl (Roche Diagnostics) for support with various reagents, H.P. Herzig (EPFL) and A. Naqavi (EPFL) for support with numerical simulations and J. Hehl and T. Schwarz (ScopeM/ETH) for STED support. For designing and fabricating numerous hardware components, the authors thank T. Kissling, R. Rietmann (Roche) and S. Wheeler (ETH). The authors also thank A. Lieb for support with ZeptoReader-related issues. The authors thank the following for discussions on various aspects of the project: M. Hennig, K. Mueller, M. Lauer, A. Rufer, G. Dernick, M. Marcinowski, M. Essenpreis, M. Hein, O. Gutmann, A. Drechsler, M. Glauser, N. Milicevic, J. Spinke, A. Maurer, C. Patsch, C. Cusulin, J. Fingerle, R. Staack and A. Poehler. The authors acknowledge the Roche Postdoc Fellowship (RPF) Program, ETH Zurich and the NCCR Molecular Systems Engineering for funding.

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Author notes

  1. Volker Gatterdam and Andreas Frutiger: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH Zurich, 8092 Zurich, Switzerland

    Volker Gatterdam, Andreas Frutiger & Janos Vörös

  2. Roche Diagnostics GmbH, 82377 Penzberg, Germany

    Klaus-Peter Stengele & Dieter Heindl

  3. Roche Pharma Research and Early Development, Roche Innovation Center Basel, 4070 Basel, Switzerland

    Thomas Lübbers & Christof Fattinger

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Contributions

Experiments were designed by V.G., K.-P.S., D.H., T.L., J.V. and C.F. C.F. performed the calculations for the phase mask and all other optical components. Molographic experiments were performed by V.G. A.F. performed the numerical simulations and wrote the evaluation software with support from J.V. and C.F. All authors read and approved the manuscript for submission.

Corresponding authors

Correspondence to Janos Vörös or Christof Fattinger.

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The authors declare no competing financial interests.

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Gatterdam, V., Frutiger, A., Stengele, KP. et al. Focal molography is a new method for the in situ analysis of molecular interactions in biological samples. Nature Nanotech 12, 1089–1095 (2017). https://doi.org/10.1038/nnano.2017.168

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