Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

High-throughput fabrication of antimicrobial phage microgels and example applications in food decontamination

Nature Protocols (2024)Cite this article

Subjects

Abstract

Engineered by nature, biological entities are exceptional building blocks for biomaterials. These entities can impart enhanced functionalities on the final material that are otherwise unattainable. However, preserving the bioactive functionalities of these building blocks during the material fabrication process remains a challenge. We describe a high-throughput protocol for the bottom-up self-assembly of highly concentrated phages into microgels while preserving and amplifying their inherent antimicrobial activity and biofunctionality. Each microgel is comprised of half a million cross-linked phages as the sole structural component, self-organized in aligned bundles. We discuss common pitfalls in the preparation procedure and describe optimization processes to ensure the preservation of the biofunctionality of the phage building blocks. This protocol enables the production of an antimicrobial spray containing the manufactured phage microgels, loaded with potent virulent phages that effectively reduced high loads of multidrug-resistant Escherichia coli O157:H7 on red meat and fresh produce. Compared with other microgel preparation methods, our protocol is particularly well suited to biological materials because it is free of organic solvents and heat. Bench-scale preparation of base materials, namely microporous films (the template for casting microgels) and pure concentrated phage suspension, requires 3.5 h and 5 d, respectively. A single production run, that yields over 1,750,000 microgels, ranges from 2 h to 2 d depending on the rate of cross-linking chemistry. We expect that this platform will address bottlenecks associated with shelf-stability, preservation and delivery of phage for antimicrobial applications, expanding the use of phage for prevention and control of bacterial infections and contaminants.

Key points

  • The authors detail how to prepare phage microgels while preserving phage bioactivity using peelable microporous templates, and employ the microgels as an antimicrobial spray to reduce multidrug-resistant bacteria contamination of food products, one example of their potential use.

  • Phage microgels are more stable than free phages for storage and phage delivery, and have the advantage over other antimicrobials of being able to selectively target specific bacteria at the strain level.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of the preparation and applications of phage-built microgels.
Fig. 2: Preparation of PS honeycomb templates.
Fig. 3: Preparation of phage-built microgels.
Fig. 4: Characterization of phage-built microgels.
Fig. 5: Evaluation of bacteria-killing ability of phage microgels.
Fig. 6: Food decontaminating application of phage microgel spray.

Similar content being viewed by others

Data availability

The data supporting the findings of this study are available in the supporting primary research paper25.

References

  1. The promise of phages. Nat. Biotechnol. 41, 583–583 (2023).

  2. Souza, G. R. et al. Three-dimensional tissue culture based on magnetic cell levitation. Nat. Nanotechnol. 5, 291–296 (2010).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  3. Chiang, C. Y. et al. Weaving genetically engineered functionality into mechanically robust virus fibers. Adv. Mater. 19, 826–832 (2007).

    Article  CAS  Google Scholar 

  4. Chung, W. J. et al. Biomimetic self-templating supramolecular structures. Nature 478, 364–368 (2011).

    Article  CAS  PubMed  ADS  Google Scholar 

  5. Chen, S. et al. Identification of highly selective covalent inhibitors by phage display. Nat. Biotechnol. 39, 490–498 (2021).

    Article  CAS  PubMed  Google Scholar 

  6. Tian, L. et al. Bacteriophage‐built gels as platforms for biomedical applications. Can. J. Chem. Eng. 100, 2191–2203 (2022).

    Article  CAS  Google Scholar 

  7. Jackson, K., Peivandi, A., Fogal, M., Tian, L. & Hosseinidoust, Z. Filamentous phages as building blocks for bioactive hydrogels. ACS Appl. Bio. Mater. 4, 2262–2273 (2021).

    Article  CAS  PubMed  Google Scholar 

  8. Salmond, G. P. C. & Fineran, P. C. A century of the phage: past, present and future. Nat. Rev. Microbiol. 13, 777–786 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Antimicrobial resistanceWorld Health Organization http://www.who.int/en/news-room/fact-sheets/detail/antimicrobial-resistance (2021).

  10. Smith, G. P. & Petrenko, V. A. Phage display. Chem. Rev. 97, 391–410 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. Smith, G. P., Petrenko, V. A. & Matthews, L. J. Cross-linked filamentous phage as an affinity matrix. J. Immunol. Methods 215, 151–161 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Jung, S. M., Qi, J., Oh, D., Belcher, A. & Kong, J. M13 virus aerogels as a scaffold for functional inorganic materials. Adv. Funct. Mater. 27, 1603203 (2017).

    Article  Google Scholar 

  13. Peivandi, A., Tian, L., Mahabir, R. & Hosseinidoust, Z. Hierarchically structured, self-healing, fluorescent, bioactive hydrogels with self-organizing bundles of phage nanofilaments. Chem. Mater. 31, 5442–5449 (2019).

    Article  CAS  Google Scholar 

  14. Peivandi, A. et al. Inducing microscale structural order in phage nanofilament hydrogels with globular proteins. ACS Biomater. Sci. Eng. 8, 340–347 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Chen, P. Y. et al. Assembly of viral hydrogels for three-dimensional conducting nanocomposites. Adv. Mater. 26, 5101–5107 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Oh, J. W. et al. Biomimetic virus-based colourimetric sensors. Nat. Commun. 5, 3043 (2014).

    Article  PubMed  ADS  Google Scholar 

  17. Ohmura, J. F. et al. Highly adjustable 3D nano-architectures and chemistries: via assembled 1D biological templates. Nanoscale 11, 1091–1101 (2019).

    Article  CAS  PubMed  Google Scholar 

  18. Lee, J. H. et al. Production of tunable nanomaterials using hierarchically assembled bacteriophages. Nat. Protoc. 12, 1999–2013 (2017).

    Article  CAS  PubMed  Google Scholar 

  19. Lee, J. H. et al. Phage-based structural color sensors and their pattern recognition sensing system. ACS Nano 11, 3632–3641 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Chen, L., Zhao, X., Lin, Y., Su, Z. & Wang, Q. Dual stimuli-responsive supramolecular hydrogel of bionanoparticles and hyaluronan. Polym. Chem. 5, 6754–6760 (2014).

    Article  CAS  Google Scholar 

  21. Sawada, T., Yanagimachi, M. & Serizawa, T. Controlled release of antibody proteins from liquid crystalline hydrogels composed of genetically engineered filamentous viruses. Mater. Chem. Front. 1, 146–151 (2017).

    Article  CAS  Google Scholar 

  22. Fernandez-nieves, A., Wyss, H. M., Mattsson, J. & Weitz, D. A. Microgel Suspensions https://doi.org/10.1002/9783527632992 (Wiley, 2011).

  23. Li, Y. et al. Selectively suppressing tumor angiogenesis for targeted breast cancer therapy by genetically engineered phage. Adv. Mater. 32, e2001260 (2020).

    Article  PubMed  Google Scholar 

  24. Yun, Y. H., Goetz, D. J., Yellen, P. & Chen, W. Hyaluronan microspheres for sustained gene delivery and site-specific targeting. Biomaterials 25, 147–157 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Tian, L. et al. Self-assembling nanofibrous bacteriophage microgels as sprayable antimicrobials targeting multidrug-resistant bacteria. Nat. Commun. 13, 7158 (2022).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  26. Mao, A. S. et al. Deterministic encapsulation of single cells in thin tunable microgels for niche modelling and therapeutic delivery. Nat. Mater. 16, 236–243 (2017).

    Article  MathSciNet  CAS  PubMed  ADS  Google Scholar 

  27. Zhu, C., Tian, L., Liao, J., Zhang, X. & Gu, Z. Fabrication of bioinspired hierarchical functional structures by using honeycomb films as templates. Adv. Funct. Mater. 28, 1–8 (2018).

    Article  Google Scholar 

  28. Årdal, C. et al. Antibiotic development—economic, regulatory and societal challenges. Nat. Rev. Microbiol. 18, 267–274 (2020).

    Article  PubMed  Google Scholar 

  29. Emergency cases treated with investigational phage bank. Adaptive Phage Therapeutics https://aphage.com/science/case-studies/ (2023).

  30. Maitz, J., Merlino, J., Rizzo, S., McKew, G. & Maitz, P. Burn wound infections microbiome and novel approaches using therapeutic microorganisms in burn wound infection control. Adv. Drug Deliv. Rev. 196, 114769 (2023).

    Article  CAS  PubMed  Google Scholar 

  31. Awwad, S. et al. Principles of pharmacology in the eye. Br. J. Pharmacol. 174, 4205–4223 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Jóhannesson, G., Stefánsson, E. & Loftsson, T. Microspheres and nanotechnology for drug delivery. Dev. Ophthalmol. 55, 93–103 (2016).

    Article  PubMed  Google Scholar 

  33. Rahman, R., Scharff, R. L. & Wu, F. Foodborne disease outbreaks in flour and flour-based food products from microbial pathogens in the United States, and their health economic burden. Risk Anal. https://doi.org/10.1111/risa.14132 (2023).

  34. Prasad, A. et al. Advancing in situ food monitoring through a smart lab‐in‐a‐package system demonstrated by the detection of salmonella in whole chicken. Adv. Mater. https://doi.org/10.1002/adma.202302641 (2023).

  35. GRAS notice 755, Preparation containing two bacterial phages specific to Escherichia coli O157Food and Drug Administration https://www.fda.gov/media/117249/download (2018).

  36. GRAS Notice 834, preparation containing bacterial phages specific to shiga-toxin producing Escherichia coliFood and Drug Administration https://www.fda.gov/media/133519/download (2019).

  37. Chen, Y., Wang, Y., Paez-Espino, D., Polz, M. F. & Zhang, T. Prokaryotic viruses impact functional microorganisms in nutrient removal and carbon cycle in wastewater treatment plants. Nat. Commun. 12, 5398 (2021).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  38. Bayat, F., Didar, T. F. & Hosseinidoust, Z. Emerging investigator series: bacteriophages as nano engineering tools for quality monitoring and pathogen detection in water and wastewater. Environ. Sci. Nano 8, 367–389 (2021).

    Article  CAS  Google Scholar 

  39. Lewis, J. M. & Sagona, A. P. Armed phages are heading for clinical trials. Nat. Microbiol. https://doi.org/10.1038/s41564-023-01415-w (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Headen, D. M., Aubry, G., Lu, H. & García, A. J. Microfluidic-based generation of size-controlled, biofunctionalized synthetic polymer microgels for cell encapsulation. Adv. Mater. 26, 3003–3008 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Eydelnant, I. A., Betty Li, B. & Wheeler, A. R. Microgels on-demand. Nat. Commun. 5, 3355 (2014).

    Article  PubMed  ADS  Google Scholar 

  42. Brugger, B. & Richtering, W. Magnetic, thermosensitive microgels as stimuli-responsive emulsifiers allowing for remote control of separability and stability of oil in water-emulsions. Adv. Mater. 19, 2973–2978 (2007).

    Article  CAS  Google Scholar 

  43. An, H. Z., Helgeson, M. E. & Doyle, P. S. Nanoemulsion composite microgels for orthogonal encapsulation and release. Adv. Mater. 24, 3838–3844 (2012).

    Article  CAS  PubMed  Google Scholar 

  44. Schmitt, C. et al. Internal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment. Soft Matter 6, 4876–4884 (2010).

    Article  CAS  ADS  Google Scholar 

  45. Nicolai, T. Formation and functionality of self-assembled whey protein microgels. Colloids Surf. B Biointerfaces 137, 32–38 (2016).

    Article  CAS  PubMed  Google Scholar 

  46. Phan-Xuan, T. et al. On the crucial importance of the ph for the formation and self-stabilization of protein microgels and strands. Langmuir 27, 15092–15101 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Li, Y. et al. Rapid assembly of heterogeneous 3D cell microenvironments in a microgel array. Adv. Mater. 28, 3543–3548 (2016).

    Article  CAS  PubMed  ADS  Google Scholar 

  48. Yeh, J. et al. Micromolding of shape-controlled, harvestable cell-laden hydrogels. Biomaterials 27, 5391–5398 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Wang, J. et al. Antimicrobial peptides: promising alternatives in the post feeding antibiotic era. Med. Res. Rev. 39, 831–859 (2019).

    Article  CAS  PubMed  Google Scholar 

  50. Hou, K. et al. Microbiota in health and diseases. Signal Transduct. Target. Ther. 7, 135 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Burmeister, A. R. & Turner, P. E. Trading-off and trading-up in the world of bacteria–phage evolution. Curr. Biol. 30, R1120–R1124 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Daly, R., Sader, J. E. & Boland, J. J. Taming self-organization dynamics to dramatically control porous architectures. ACS Nano 10, 3087–3092 (2016).

    Article  CAS  PubMed  Google Scholar 

  53. Takehiro Nishikawa et al. Fabrication of honeycomb film of an amphiphilic copolymer at the air−water interface. Langmuir 18, 5734–5740 (2002).

    Article  Google Scholar 

  54. Wang, W. et al. Deterministic reshaping of breath figure arrays by directional photomanipulation. ACS Appl. Mater. Interfaces 9, 4223–4230 (2017).

    Article  CAS  PubMed  Google Scholar 

  55. Zhang, A., Bai, H. & Li, L. Breath figure: a nature-inspired preparation method for ordered porous films. Chem. Rev. 115, 9801–9868 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Wang, X., Bukusoglu, E. & Abbott, N. L. A practical guide to the preparation of liquid crystal-templated microparticles. Chem. Mater. 29, 53–61 (2017).

    Article  CAS  Google Scholar 

  57. Widawski, G. & Rawiso, M. Self-organized honeycomb morphology of star-polymer polystyrene films. Nature 369, 387–389 (1994).

    Article  CAS  ADS  Google Scholar 

  58. Zhang, A. et al. Formation of breath figure arrays in methanol vapor assisted by surface active agents. ACS Appl. Mater. Interfaces 6, 8921–8927 (2014).

    Article  CAS  PubMed  Google Scholar 

  59. Zhao, B., Zhang, J., Wang, X. & Li, C. Water-assisted fabrication of honeycomb structure porous film from poly (l-lactide). J. Mater. Chem. 16, 509–513 (2006).

    Article  CAS  Google Scholar 

  60. Liu, C., Lang, W., Shi, B. & Guo, Y. Fabrication of ordered honeycomb porous polyvinyl chloride (PVC) films by breath figures method. Mater. Lett. 107, 53–55 (2013).

    Article  CAS  Google Scholar 

  61. Yabu, H., Tanaka, M., Ijiro, K. & Shimomura, M. Preparation of honeycomb-patterned polyimide films by self-organization. Langmuir 19, 6297–6300 (2003).

    Article  CAS  Google Scholar 

  62. Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual 3rd edn https://doi.org/10.1177/0261018311403863 (Cold Spring Harbour Laboratory Press, 2001).

  63. Chung, W. J., Lee, D. Y. & Yoo, S. Y. Chemical modulation of M13 bacteriophage and its functional opportunities for nanomedicine. Int. J. Nanomed. 9, 5825–5836 (2014).

    Google Scholar 

  64. Courchesne, N. M. D. et al. Assembly of a bacteriophage-based template for the organization of materials into nanoporous networks. Adv. Mater. 26, 3398–3404 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Tian, L., He, L., Jackson, K., Mahabir, R. & Hosseinidoust, Z. Bacteria repellent protein hydrogel decorated with tunable, isotropic, nano-on-micro hierarchical microbump array. Chem. Commun. 57, 10883–10886 (2021).

    Article  CAS  Google Scholar 

  66. Migneault, I., Dartiguenave, C., Bertrand, M. J. & Waldron, K. C. Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques 37, 790–802 (2004).

    Article  CAS  PubMed  Google Scholar 

  67. Jones, C. G. in Forensic Microscopy for Skeletal Tissues (ed. Bell, L. S.) 1–20 (Springer, 2012).

Download references

Acknowledgements

This research was partially supported by the Canada Research Chairs Program (T.F.D. and Z.H.) and Ontario Early Researcher Award (T.F.D.). Z.H. and T.F.D. acknowledge support from Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants Program. S.K. and K.J. are funded by Vanier Canada Graduate Scholarships awarded by the Natural Sciences and Engineering Research Council and Canadian Institutes of Health Research, respectively.

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, McMaster University, Hamilton, Ontario, Canada

    Lei Tian, Kyle Jackson, Leon He, Mathura Thirugnanasampanthar, Mellissa Gomez & Zeinab Hosseinidoust

  2. Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Ontario, Canada

    Kyle Jackson & Zeinab Hosseinidoust

  3. School of Biomedical Engineering, McMaster University, Hamilton, Ontario, Canada

    Shadman Khan, Fereshteh Bayat, Tohid F. Didar & Zeinab Hosseinidoust

  4. Department of Mechanical Engineering, McMaster University, Hamilton, Ontario, Canada

    Tohid F. Didar

  5. Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

    Tohid F. Didar & Zeinab Hosseinidoust

  6. Brockhouse Institute for Materials Research, McMaster University, Hamilton, Ontario, Canada

    Tohid F. Didar & Zeinab Hosseinidoust

Authors
  1. Lei Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kyle Jackson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leon He
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shadman Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mathura Thirugnanasampanthar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mellissa Gomez
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fereshteh Bayat
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tohid F. Didar
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zeinab Hosseinidoust
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.T. designed the protocol of microgel preparation, led the experiments, prepared all figures and contributed to the manuscript writing. K.J. participated in the experiments and made significant contributions to the manuscript writing. L.H. made significant contributions to the experiments. S.K. and T.F.D. made significant contributions to the protocol for the food decontamination test. M.T., M.G. and F.B. contributed to the manuscript writing. Z.H. led the team, supervised the experimental design and guided the manuscript writing.

Corresponding authors

Correspondence to Tohid F. Didar or Zeinab Hosseinidoust.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Longzu Cui and David H. Kohn for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Tian, L. et al. Nat. Commun. 13, 7158 (2022): https://doi.org/10.1038/s41467-022-34803-7

Peivandi, A. et al. Chem. Mater. 31, 5442–5449 (2019): https://doi.org/10.1021/acs.chemmater.9b00720

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tian, L., Jackson, K., He, L. et al. High-throughput fabrication of antimicrobial phage microgels and example applications in food decontamination. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00964-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41596-024-00964-6

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing