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Synthesis and functionalization of scalable and versatile 2D protein films via amyloid-like aggregation

Nature Protocols volume 19, pages 539–564 (2024)Cite this article

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Abstract

Two-dimensional (2D) protein films can be used to modify the properties of surfaces, and find applications predominantly in the fields of biomaterials, lithography, optics and electronics. However, it is difficult to produce scalable homogeneous and robust protein films with an easy, low-cost, green and efficient method. Further challenges include encapsulating and releasing functional building blocks in the film without inactivating them, and maintaining or improving the bioactivities of proteins used for the formation of the films. Here we detail the process to prepare large 2D protein films with user-defined features and structures via the amyloid-like aggregation of commonly synthesized proteins. These films can be synthesized at meter scales, have high interface adhesion, high functional expansibility and tunable functional properties, obtained by controlling the position of the disulfide bond breakage. For example, we can retain or even enhance the natural antibacterial, biomineralization and antifouling activity of proteins involved in film formation, and the properties can also be expanded through the physical blending or chemical grafting of additional functional blocks on the surface of the film. A 2D protein film can be prepared in ~3 h using four alternative coating techniques: immersion, transfer, hydrogel stamping and spraying. The characterization process of the film requires ~5 d. The procedure can be carried out by users with basic expertise in materials science.

Key points

  • Amyloid-like aggregation of native globular proteins leverages internal hydrophilic and external hydrophobic bonds to unfold the protein structures, which aggregate into oligomers, and then oligomerizes them as β-sheet structures to form two-dimensional protein films at the interface of the solution.

  • Alternative methods to prepare two-dimensional protein films include casting, vacuum filtration and interface collection. The advantages of using amyloid-like aggregation include ease of use, biocompatibility and high levels of flexibility and functionalization.

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Fig. 1: Schematic preparation process of the 2D protein film based on amyloid-like aggregation.
Fig. 2: Four strategies to prepare lysozyme 2D protein film.
Fig. 3: The preparation of 2D protein film with TCEP by amyloid-like aggregation.
Fig. 4: The characteristic of 2D protein film prepared with TCEP.
Fig. 5: The characterization of chemical groups on 2D lysozyme films.
Fig. 6: Adhesion performant of 2D protein film.
Fig. 7: Bioactive 2D protein film prepared by thiol–disulfide exchange.
Fig. 8: Synthesis and characterization of the PEG-grafted 2D lysozyme film.

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Data availability

Source data for the figures in this study are available in figshare (https://doi.org/10.6084/m9.figshare.23937909). Source data are provided with this paper.

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Acknowledgements

We are grateful for funding from the National Science Fund for Distinguished Young Scholars (no. 52225301 to P.Y.), the National Key R&D Program of China (nos. 2020YFA0710400 and 2020YFA0710402 to P.Y.), the National Natural Science Foundation of China (no. 51903147 to Y.L., no. 21905166 to H.R.), the 111 Project (no. B14041 to P.Y.), the International Science and Technology Cooperation Program of Shaanxi Province (no. 2022KWZ-24 to P.Y.), the Fundamental Research Funds for the Central Universities (no. GK202305001 to P.Y.) and the Postdoctoral Research Foundation of China (no. 2019M653864 to Y.L.).

Author information

Author notes

  1. These authors contributed equally: Yongchun Liu, Shuting Miao, Hao Ren.

Authors and Affiliations

  1. Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, China

    Yongchun Liu, Shuting Miao, Hao Ren, Lihua Tian, Jian Zhao & Peng Yang

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  1. Yongchun Liu
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Contributions

P.Y. conceived this project and supervised the writing. Y.L. and S.M. developed the protocol and codrafted the manuscript, with the assistance of H.R., L.T. and J.Z., who contributed to the discussion and manuscript modification.

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Correspondence to Peng Yang.

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Related links

Key reference using this protocol

Yang, F. et al. Nat. Commun. 9, 5443 (2018): https://doi.org/10.1038/s41467-018-07888-2

Key data used in this protocol

Wang, D. et al. Adv. Mater. 28, 7414–7423 (2016): https://doi.org/10.1002/adma.201506476

Li, C. et al. Adv. Mater. 31, e1903973 (2019): https://doi.org/10.1002/adma.201903973

Xu, Y. et al. Angew. Chem. Int. Ed. Engl. 59, 2850–2859 (2020): https://doi.org/10.1002/anie.201912848

Saif, B. et al. ACS Nano. 13, 7736–7749 (2019): https://doi.org/10.1021/acsnano.9b01392

Extended data

Extended Data Fig. 1 2D lysozyme film prepared with Sn2+.

a-c, Survey (a) and high-resolution XPS spectra of Sn 3d (b) and S 2p (c) of the film. d, Raman spectra of native lysozyme and film, with the inset showing the characteristic peaks for the clusters of -S-(Sn)x-S- at 309 cm-1 and reduction of the disulfide bond at 505 cm-1, respectively. e, Evolution of ANS fluorescence to monitor the exposition of hydrophobic domain during amyloid-like aggregation. f, The fluorescence intensity of the ThT-stained reaction solution at different aggregation time. Figure adapted with permission from ref. 31, ACS.

Extended Data Fig. 2 2D lysozyme film prepared with cysteine.

a, The NPM assay for native lysozyme and partially unfolded lysozyme. The inset shows the reaction between NPM and free thiol to enhance the fluorescence emission. b, Typical MALDI-TOF-MS spectra of native lysozyme and cysteine-reduced lysozyme. c, The SDS-PAGE of native lysozyme (lane 1) and partially unfolded lysozyme (lane 2). d,The activity assay of native lysozyme, cysteine and partially unfolded lysozyme. e, ANS assay for native lysozyme, cysteine, and partially unfolded lysozyme. f, Intrinsic tryptophan fluorescence assay for native lysozyme and partially unfolded lysozyme. Figure adapted with permission from ref. 23, Wiley.

Source data

Extended Data Fig. 3 Characterization of the 2D lysozyme film prepared with Sn2+.

a, Digital photograph showing a large-area protein film at the air/water interface. b, Scanning transmission electron microscopy (STEM) image of the protein film. c, Corresponding energy-dispersive X-ray spectroscopy (EDS) elemental mapping of Sn in the protein film. d, Water contact angle measurements of the protein film on different substrates. e, Transmittance of the protein film recorded by UV/vis spectrometer. f, Fluorescence spectra of the protein film stained with ThT. g, CLSM image of the ThT-dyed protein film. h,Digital photograph of the protein film dyed with Congo red. i, Film thickness as a function of lysozyme concentration. Figure adapted with permission from ref. 31, ACS.

Extended Data Fig. 4 Characterization of the 2D lysozyme film prepared with cysteine.

a, Digital photograph of a large-area protein film at the air/water interface. b,c, AFM (b) and TEM (c) images of the protein film surface. d, UV/vis absorption spectrum of the protein film in the range of 200-800 nm, along with a digital photograph showing the film-coated quartz with a leaf as a background. e, Water contact angle of the protein film coated on different substrates. f, CD spectra of the protein film stained with ThT. g, Digital photograph of the protein film dyed by Congo Red and an CLSM image of the ThT-dyed protein film. h, The thickness of the film as a function of lysozyme concentration. i, The effect of repetitive coating times on the thickness of the nanofilm. Figure adapted with permission from ref. 23, Wiley.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Tables 1 and 2.

Reporting Summary

Supplementary Data 1

Statistical source data for Supplementary Fig. 5b–d.

Source data

Source Data Fig. 8

Statistical source data for Fig. 8d.

Source Data Extended Data Fig. 2

Unprocessed sodium dodecyl sulfate polyacrylamide gel electrophoresis.

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Liu, Y., Miao, S., Ren, H. et al. Synthesis and functionalization of scalable and versatile 2D protein films via amyloid-like aggregation. Nat Protoc 19, 539–564 (2024). https://doi.org/10.1038/s41596-023-00918-4

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