Precise tuning of microstructure for surface bacteriostasis using two-photon polymerization 3D printing technology

In nature, many biological surfaces exhibit inherent bacteriostatic property due to the existence of special microstructures. However, the key factors and underlying mechanisms driving this property remain unclear. A significant challenge lies in the lack of proper techniques for precisely fabricating such microstructures as well as finely tuning their morphological parameters. In this study, we adopted a two-photon 3D printing-based approach to fabricate microstructures on specified surfaces with accurate control over their morphology, enabling the investigation of structural bacteriostasis. Through abstracting the subtle morphology on shark skin, we replicated their bacteriostatic microstructures and were able to regulate their morphology at the micron scale. By culturing Streptococcus mutans on the surface of these microstructures, we validated their bacteriostatic performance and demonstrated that morphological parameters significantly influenced the efficacy of structural bacteriostasis. Other kinds of microstructures such as micro-holes with bacteriostatic property could also be fabricated and investigated utilizing this two-photon polymerization technology. We believe this strategy offers a powerful tool for researching bacteriostatic mechanisms of various microstructures and will inspire their broad applications in both daily and industrial settings.

- Otto M. Physical stress and bacterial colonization. FEMS Microbiol Rev. 2014;38(6):1250-1270. doi: 10.1111/1574-6976.12088
- Byrd AL, Belkaid Y, Segre JA. The human skin microbiome. Nat Rev Microbiol. 2018;16(3):143-155. doi: 10.1038/nrmicro.2017.157
- Muhammad MH, Idris AL, Fan X, et al. Beyond risk: bacterial biofilms and their regulating approaches. Front Microbiol. 2020;11:928. doi: 10.3389/fmicb.2020.00928
- Nobbs AH, Lamont RJ, Jenkinson HF. Streptococcus adherence and colonization. Microbiol Mol Biol Rev. 2009;73(3):407-450. doi: 10.1128/mmbr.00014-09
- Akcalı A, Lang NP. Dental calculus: the calcified biofilm and its role in disease development. Periodontol 2000. 2018;76(1):109-115. doi: 10.1111/prd.12151
- Karygianni L, Ren Z, Koo H, Thurnheer T. Biofilm matrixome: extracellular components in structured microbial communities. Trends Microbiol. 2020;28(8):668-681. doi: 10.1016/j.tim.2020.03.016
- Arciola CR, Campoccia D, Montanaro L. Implant infections: adhesion, biofilm formation and immune evasion. Nat Rev Microbiol. 2018;16(7):397-409. doi: 10.1038/s41579-018-0019-y
- Sharma D, Misba L, Khan AU. Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob Resist Infect Control. 2019;8:76. doi: 10.1186/s13756-019-0533-3
- Roy R, Tiwari M, Donelli G, Tiwari V. Strategies for combating bacterial biofilms: a focus on anti-biofilm agents and their mechanisms of action. Virulence. 2018; 9(1):522-554. doi: 10.1080/21505594.2017.1313372
- Wu H, Moser C, Wang HZ, Høiby N, Song ZJ. Strategies for combating bacterial biofilm infections. Int J Oral Sci. 2015;7(1):1-7. doi: 10.1038/ijos.2014.65
- Nwabuife JC, Omolo CA, Govender T. Nano delivery systems to the rescue of ciprofloxacin against resistant bacteria “E. coli; P. aeruginosa; Saureus; and MRSA” and their infections. J Control Release. 2022;349:338-353. doi: 10.1016/j.jconrel.2022.07.003
- Fu X, Rehman U, Wei L, et al. Silver-dendrimer nanocomposite as emerging therapeutics in anti-bacteria and beyond. Drug Resist Updat. 2023;68:100935. doi: 10.1016/j.drup.2023.100935
- Kalghatgi S, Spina CS, Costello JC, et al. Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in Mammalian cells. Sci Transl Med. 2013;5(192):192ra85. doi: 10.1126/scitranslmed.3006055
- Jaggessar A, Shahali H, Mathew A, Yarlagadda P. Bio-mimicking nano and micro-structured surface fabrication for antibacterial properties in medical implants. J Nanobiotechnology. 2017;15(1):64. doi: 10.1186/s12951-017-0306-1
- Shahali H, Hasan J, Cheng HH, Ramarishna S, Yarlagadda PK. A systematic approach towards biomimicry of nanopatterned cicada wings on titanium using electron beam lithography. Nanotechnology. 2021;32(6):065301. doi: 10.1088/1361-6528/abbeaa
- Xu M, Wang X, Wang B, et al. Carbonized lotus leaf/ZnO/ Au for enhanced synergistic mechanical and photocatalytic bactericidal activity under visible light irradiation. Colloids Surf B Biointerfaces. 2022;215:112468. doi: 10.1016/j.colsurfb.2022.112468
- Satheesh S, Ba-akdah MA, Al-Sofyani AA. Natural antifouling compound production by microbes associated with marine macroorganisms — a review. Electron J Biotechnol. 2016;21:26-35. doi: 10.1016/j.ejbt.2016.02.002
- Ge X, Ren C, Ding Y, et al. Micro/nano-structured TiO(2) surface with dual-functional antibacterial effects for biomedical applications. Bioact Mater. 2019;4:346-357. doi: 10.1016/j.bioactmat.2019.10.006
- Wang S, Deng Y, Yang L, Shi X, Yang W, Chen ZG. Enhanced antibacterial property and osteo-differentiation activity on plasma treated porous polyetheretherketone with hierarchical micro/nano-topography. J Biomater Sci Polym Ed. 2018;29(5):520-542. doi: 10.1080/09205063.2018.1425181
- Gao A, Yan Y, Li T, Liu F. Biomimetic urchin-like surface based on poly (lactic acid) membrane for robust anti-wetting and anti-bacteria properties. Mater Lett. 2019;237:240-244. doi: 10.1016/j.matlet.2018.11.063
- Li Y-N, Zhao Y, Liu J-J. Precisely engineered bionic Cu-doped titanium dioxide nanoarrays/fluorine-doped tin oxide with excellent antibacterial and antifouling properties. Tungsten. 2024;6(3):522-528. doi: 10.1007/s42864-023-00255-9
- Zhang Y, Zhao W, Chen Z, et al. Influence of biomimetic boundary structure on the antifouling performances of siloxane modified resin coatings. Colloids Surf A: Physicochem Eng Aspects. 2017;528:57-64. doi: 10.1016/j.colsurfa.2017.05.044
- Liu M, Wang S, Wei Z, Song Y, Jiang L. Bioinspired design of a superoleophobic and low adhesive water/solid interface. Adv Mater. 2009;21(6):665-669. doi: 10.1002/adma.200801782
- Cormican CM, Bektaş S, Martin-Martinez FJ, Alexander S. Emerging trends in bioinspired superhydrophobic and superoleophobic sustainable surfaces. Adv Mater. 2025;37(12):2415961. doi: 10.1002/adma.202415961
- Xu Y, Luan X, He P, et al. Fabrication and functional regulation of biomimetic interfaces and their antifouling and antibacterial applications: a review. Small. 2024; 20(21):e2308091. doi: 10.1002/smll.202308091
- Shao Z, Shen R, Gui Z, et al. Ethyl cellulose/gelatin/β- cyclodextrin/curcumin nanofibrous membrane with antibacterial and formaldehyde adsorbable capabilities for lightweight and high-performance air filtration. Int J Biol Macromol. 2024;254(Pt 2):127862. doi: 10.1016/j.ijbiomac.2023.127862
- Wu W, Han C, Liang R, et al. Fabrication and performance of graphene flexible pressure sensor with micro/nano structure. Sensors (Basel). 2021;21(21):7022. doi: 10.3390/s21217022
- Gao Y, Ding Q, Li W, Gu R, Zhang P, Zhang L. Role and mechanism of a micro-/nano-structured porous zirconia surface in regulating the biological behavior of bone marrow mesenchymal stem cells. ACS Appl Mater Interfaces. 2023;15(11):14019-14032. doi: 10.1021/acsami.2c22736
- Chen Y, Wang C, Zhang Z, et al. 3D-printed piezocatalytic hydrogels for effective antibacterial treatment of infected wounds. Int J Biol Macromol. 2024;268(Pt 2):131637. doi: 10.1016/j.ijbiomac.2024.131637
- Jing X, Fu H, Yu B, Sun M, Wang L. Two-photon polymerization for 3D biomedical scaffolds: Overview and updates. Front Bioeng Biotechnol. 2022;10:994355. doi: 10.3389/fbioe.2022.994355
- Pao YH, Rentzepis PM. Laser‐induced production of free radicals in organic compounds. Appl Phys Lett. 1965;6(5):93-95. doi: 10.1063/1.1754182
- Zhou X, Liu X, Gu Z. Photoresist development for 3D printing of conductive microstructures via two-photon polymerization. Adv Mater. 2024;36(48):2409326. doi: 10.1002/adma.202409326
- O’Halloran S, Pandit A, Heise A, Kellett A. Two-photon polymerization: fundamentals, materials, and chemical modification strategies. Adv Sci (Weinh). 2023;10(7):e2204072. doi: 10.1002/advs.202204072
- Sanders ME, Akkermans LM, Haller D, et al. Safety assessment of probiotics for human use. Gut Microbes. 2010;1(3):164-185. doi: 10.4161/gmic.1.3.12127
- Chowdhury MAH, Ashrafudoulla M, Mevo SIU, Mizan MFR, Park SH, Ha SD. Current and future interventions for improving poultry health and poultry food safety and security: a comprehensive review. Compr Rev Food Sci Food Saf. 2023;22(3):1555-1596. doi: 10.1111/1541-4337.13121
- Chitrakar B, Zhang M, Adhikari B. Dehydrated foods: are they microbiologically safe? Crit Rev Food Sci Nutr. 2019;59(17):2734-2745. doi: 10.1080/10408398.2018.1466265
- Xu D, Gu T, Lovley DR. Microbially mediated metal corrosion. Nat Rev Microbiol. 2023;21(11):705-718. doi: 10.1038/s41579-023-00920-3
- Battaglia TW, Mimpen IL, Traets JJH, et al. A pan-cancer analysis of the microbiome in metastatic cancer. Cell. 2024;187(9):2324-2335.e19. doi: 10.1016/j.cell.2024.03.021
- Yong J, Chew KW, Khoo KS, Show PL, Chang JS. Prospects and development of algal-bacterial biotechnology in environmental management and protection. Biotechnol Adv. 2021;47:107684. doi: 10.1016/j.biotechadv.2020.107684
- Roager HM, Licht TR. Microbial tryptophan catabolites in health and disease. Nat Commun. 2018;9(1):3294. doi: 10.1038/s41467-018-05470-4
- Skoulas E, Manousaki A, Fotakis C, Stratakis E. Biomimetic surface structuring using cylindrical vector femtosecond laser beams. Sci Rep. 2017;7:45114. doi: 10.1038/srep45114
- Li Y, Zhang LY, Zhang C, Zhang ZR, Liu L. Bioinspired antifouling Fe-based amorphous coating via killing-resisting dual surface modifications. Sci Rep. 2022;12(1):819. doi: 10.1038/s41598-021-04746-y
- Zhu W, Ma X, Gou M, Mei D, Zhang K, Chen S. 3D printing of functional biomaterials for tissue engineering. Curr Opin Biotechnol. 2016;40:103-112. doi: 10.1016/j.copbio.2016.03.014
- Su R, Chen J, Zhang X, et al. 3D-printed micro/nano-scaled mechanical metamaterials: fundamentals, technologies, progress, applications, and challenges. Small. 2023;19(29):e2206391. doi: 10.1002/smll.202206391
- Zhang Y, Su Y, Zhao Y, Wang Z, Wang C. Two-photon 3D printing in metal-organic framework single crystals. Small. 2022;18(18):e2200514. doi: 10.1002/smll.202200514
- Ivanova EP, Hasan J, Webb HK, et al. Bactericidal activity of black silicon. Nat Commun. 2013;4:2838. doi: 10.1038/ncomms3838
- Ivanova EP, Hasan J, Webb HK, et al. Natural bactericidal surfaces: mechanical rupture of Pseudomonas aeruginosa cells by Cicada wings. Small. 2012;8(16):2489-2494. doi: 10.1002/smll.201200528
- Linklater DP, Nguyen HKD, Bhadra CM, Juodkazis S, Ivanova EP. Influence of nanoscale topology on bactericidal efficiency of black silicon surfaces. Nanotechnology. 2017;28(24):245301. doi: 10.1088/1361-6528/aa700e
- Bandara CD, Singh S, Afara IO, et al. Bactericidal effects of natural nanotopography of dragonfly wing on Escherichia coli. ACS Appl Mater Interfaces. 2017;9(8):6746-6760. doi: 10.1021/acsami.6b13666
- Linklater DP, Juodkazis S, Rubanov S, Ivanova EP. Comment on “Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on Escherichia coli”. ACS Appl Mater Interfaces. 2017;9(35):29387-29393. doi: 10.1021/acsami.7b05707
- Wang X, Bhadra CM, Yen Dang TH, et al. A bactericidal microfluidic device constructed using nano-textured black silicon. RSC Adv. 2016;6(31):26300. doi: 10.1039/C6RA03864F
- Zhu WT, Huo FY, Cao LM, et al. Two-photon polymerization 3D printing of biomimetic microstructures for functionalizing surfaces to inhibit bacterial growth. Chem Eng J. 2025;511:161907. doi: 10.1016/j.cej.2025.161907
- Li B, Tan H, Anastasova S, Power M, Seichepine F, Yang GZ. A bio-inspired 3D micro-structure for graphene-based bacteria sensing. Biosens Bioelectron. 2019;123:77-84. doi: 10.1016/j.bios.2018.09.087
- Akbari E, Buntat Z, Afroozeh A, Zeinalinezhad A, Nikoukar A. Escherichia coli bacteria detection by using graphene-based biosensor. IET Nanobiotechnol. 2015;9(5):273-279. doi: 10.1049/iet-nbt.2015.0010
- Krishnamurthi VR, Harris N, Rogers A, Zou M, Wang Y. Interactions of E. coli with cylindrical micro-pillars of different geometric modifications. Colloids Surf B Biointerfaces. 2022;209(Pt 2):112190. doi: 10.1016/j.colsurfb.2021.112190