To address the persistent challenge of biofouling on aluminum alloys in marine antifouling applications, the work aims to investigate the effect of antimicrobial peptide (AMP) architecture, specifically its branching degree, on the resultant antibacterial and antifouling efficacy of functionalized surfaces. The core objective is to establish a clear structure-performance relationship to guide the rational design of high-performance, environmentally benign coatings, moving beyond traditional biocidal agents that pose ecological risks. To this end, three AMP variants with distinct topological structures of a linear peptide (branching degree 1), a two-branched hyperbranched peptide (branching degree 2), and a four-branched hyperbranched peptide (branching degree 4), were covalently immobilized onto 5083 aluminum alloy substrates. The fabrication process involved a multi-step surface engineering strategy. Initially, the aluminum substrates underwent a boiling-water pretreatment to generate a hydroxyl-rich surface, enhancing its reactivity. Subsequently, a polyimide-based interfacial layer (PMPI) was grafted onto this activated surface, providing a stable foundation with reactive maleimide groups. Finally, the AMPs, pre-modified with a cysteine coupling agent, were immobilized via a Michael addition reaction between the thiol groups of cysteine and the maleimide groups on the PMPI layer, ensuring stable and oriented attachment. The successful functionalization and surface properties of the resulting samples denoted as Al-Peptide 1 (linear), Al-Peptide 2 (2-branched), and Al-Peptide 4 (4-branched) were comprehensively characterized. Water contact angle (WCA) measurements revealed a significant increase in surface hydrophilicity with higher branching degrees, with contact angles of 42.4°, 34.7°, and 28.3° for the linear, 2-branched, and 4-branched surfaces, respectively. This trend was attributed to the higher surface density of hydrophilic groups presented by the hyperbranched architectures. Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analyses confirmed the successful grafting of both the PMPI layer and the AMPs, with characteristic peaks for amide bonds and the presence of sulfur (from cysteine) providing definitive evidence. The antibacterial and antifouling performance was rigorously evaluated against two representative marine bacteria: Vibrio natriegens (Gram-negative) and Bacillus. (Gram-positive). Quantitative assays demonstrated that all AMP-functionalized surfaces exhibited markedly superior performance compared to the untreated and PMPI-only controls. Notably, a clear dependency on branching degree was observed. The 2-branched hyperbranched surface (Al-Peptide 2) delivered the optimal performance, achieving antibacterial rates of 77.4% against Vibrio natriegens and 68.2% against Bacillus. Correspondingly, its antifouling rates reached 89.3% and 88.7%, respectively. While the 4-branched surface also outperformed the linear one, its efficacy was slightly lower than that of the 2-branched variant. Confocal laser scanning microscopy (CLSM) visualization corroborated these findings, showing substantially less bacterial adhesion on the hyperbranched surfaces, with the 2-branched sample exhibiting the sparsest biofilm coverage. Scanning electron microscopy (SEM) further revealed that bacteria attached to the AMP-modified surfaces displayed compromised membrane integrity, confirming a membrane-disruptive mechanism of action. In conclusion, this work systematically demonstrates that the branching degree of AMPs is a critical determinant for the antibacterial and antifouling performance of functionalized aluminum surfaces. The 2-branched hyperbranched architecture emerges as the optimal configuration, striking a delicate balance that maximizes functional benefits. Its branched structure enhances the local density of active cationic groups, improves contact with negatively charged bacterial membranes, and increases the peptide's stability on the surface. Concurrently, the heightened hydrophilicity contributes to fouling resistance. However, excessive branching, as in the 4-branched peptide, can introduce steric hindrance and potentially lead to suboptimal molecular orientation or self-aggregation, which may marginally reduce the accessibility of active sites, thereby explaining its slightly inferior performance compared to the 2-branched analogue. Therefore, the principle of "optimal branching" rather than "maximum branching" is paramount. These findings provide a fundamental mechanistic insight into peptide-based surface design and offer a practical, eco-friendly strategy for engineering advanced antifouling aluminum surfaces. The identified optimal structure holds significant promise for application in marine antifouling, where durable and non-toxic fouling control is essential.
Key words
biofouling /
bio-peptide /
hyperbranched /
antibacterial property /
antifouling performance
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References
[1] 白秀琴, 贺小燕, 危卫, 等. 船体表面防污减阻技术[M]. 武汉: 武汉理工大学出版社, 2020.
BAI X Q, HE X Y, WEI W, et al.Technologies of Antifouling and Drag Reduction for Ship Hull Surface[M]. Wuhan: Wuhan University of Technology Press, 2020.
[2] 贺小燕. 海洋生物污损条件膜和生物膜的形成及调控机制研究[D]. 宁波: 中国科学院大学(中国科学院宁波材料技术与工程研究所), 2017.
HE X Y.Formation and regulation mechanisms of conditioning layer and biofilm during marine biofouling[D]. Ningbo: Ningbo Institute of Material Technology, Chinese Academy of Sciences, 2017.
[3] WANG G S, LI X, WANG Z.APD3: The Antimicrobial Peptide Database as a Tool for Research and Education[J]. Nucleic Acids Research, 2016, 44(D1): D1087-D1093.
[4] BRADY D, GRAPPUTO A, ROMOLI O, et al.Insect Cecropins, Antimicrobial Peptides with Potential Therapeutic Applications[J]. International Journal of Molecular Sciences, 2019, 20(23): 5862.
[5] ZHANG X G, FENG X B, MA L, et al.A Sonosensitive Diphenylalanine-Based Broad-Spectrum Antimicrobial Peptide[J]. Nature Biomedical Engineering, 2025, 9(8): 1349-1365.
[6] LOHNER K.New Strategies for Novel Antibiotics: Peptides Targeting Bacterial Cell Membranes[J]. General Physiology and Biophysics, 2009, 28(2): 105-116.
[7] 张良, 潘滢浩, 袁瑜, 等. 基于细胞膜损伤机制的抗菌肽结构优化研究进展[J]. 中国抗生素杂志, 2023, 48(7): 721-727.
ZHANG L, PAN Y H, YUAN Y, et al.Research Progress on Structure Modification of Antimicrobial Peptides Targeting Membrane Disruption[J]. Chinese Journal of Antibiotics, 2023, 48(7): 721-727.
[8] SENGUPTA D, LEONTIADOU H, MARK A E, et al.Toroidal Pores Formed by Antimicrobial Peptides Show Significant Disorder[J]. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2008, 1778(10): 2308-2317.
[9] TIELEMAN D P, SANSOM M S P. Molecular Dynamics Simulations of Antimicrobial Peptides: From Membrane Binding to Trans-Membrane Channels[J]. International Journal of Quantum Chemistry, 2001, 83(3/4): 166-179.
[10] GIFFORD J L, HUNTER H N, VOGEL H J.Lactoferricin: A Lactoferrin-Derived Peptide with Antimicrobial, Antiviral, Antitumor and Immunological Properties[J]. Cellular and Molecular Life Sciences, 2005, 62(22): 2588-2598.
[11] WANG J J, DOU X J, SONG J, et al.Antimicrobial Peptides: Promising Alternatives in the Post Feeding Antibiotic Era[J]. Medicinal Research Reviews, 2019, 39(3): 831-859.
[12] BROGDEN K A.Antimicrobial Peptides: Pore Formers or Metabolic Inhibitors in Bacteria[J]. Nature Reviews Microbiology, 2005, 3(3): 238-250.
[13] NGUYEN L T, HANEY E F, VOGEL H J.The Expanding Scope of Antimicrobial Peptide Structures and Their Modes of Action[J]. Trends in Biotechnology, 2011, 29(9): 464-472.
[14] ZHANG L, WANG H, LI X, et al.Hyperbranched Antimicrobial Peptides: Design, Synthesis, and Mechanism of Action[J]. Biomaterials, 2021, 275: 120856.
[15] WANG Q, ZHANG R, LI M, et al.Self-Assembled Hyperbranched Peptides for Biofilm Disruption and Bacterial Killing[J]. Nano Letters, 2023, 23(10): 4567-4575.
[16] ROSSI L, BIANCHI E, PINI A, et al.Hyperbranched Peptides with Dual Antibacterial and Anti-Inflammatory Properties[J]. Advanced Science, 2025, 12(5): 2304567.
[17] 楼彤, 白秀琴, 贺小燕, 等. 抗菌肽在海洋防污领域的研究进展[J]. 中国表面工程, 2022, 35(2): 1-11.
LOU T, BAI X Q, HE X Y, et al.Research Progress of Antimicrobial Peptides in the Field of Marine Antifouling[J]. China Surface Engineering, 2022, 35(2): 1-11.
[18] FENG L B, ZHANG H X, WANG Z L, et al.Superhydrophobic Aluminum Alloy Surface: Fabrication, Structure, and Corrosion Resistance[J]. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2014, 441: 319-325.
[19] LIU W C, LOU T, BAI X Q, et al.Experimental and Molecular Dynamics Simulation Study on Antifouling Performance of Antimicrobial Peptide-Modified Aluminum Alloy Surfaces[J]. Surface and Interface Analysis, 2024, 56(8): 532-543.
[20] AMMA S I, LUO J W, PANTANO C G, et al.Specular Reflectance (SR) and Attenuated Total Reflectance (ATR) Infrared (IR) Spectroscopy of Transparent Flat Glass Surfaces: A Case Study for Soda Lime Float Glass[J]. Journal of Non-Crystalline Solids, 2015, 428: 189-196.
[21] KIM S H, PANTANO C G, AMMA S I.Infrared Reflection Absorption Spectroscopy (IRRAS) for Surface Analysis of Dielectrics: Principles and Applications[J]. Review of Scientific Instruments, 2024, 95(6): 065106.
[22] ETC N L I L J H. Frontiers in Infrared Spectroscopy at Surfaces and Interfaces[J]. Surface Science Reports, 41(1-8): 1-120.
[23] ZHANG Y, LIU X, WANG L.Infrared-Spectroscopic Single-Shot Laser Mapping Ellipsometry: Fast Characterization of Structured Surfaces and Molecular Interactions[J]. Surface and Coatings Technology, 2017, 332: 1-8.
[24] MARTINEZ G, GARCIA R, SANCHEZ J.Analysis of XPS Peaks for Identifying Carbon Nanotube Oxidation States: A Comparative Study with FTIR[J]. Carbon, 2025, 210: 118765.
[25] KIM S H, LEE J H, PARK J W.Advanced Peak Fitting Methods for XPS Analysis of Metal-Organic Frameworks[J]. Microporous and Mesoporous Materials, 2025, 368: 112345.
[26] LEE J H, PARK J W, KIM S H.Frontiers in XPS Peak Interpretation: From Basic Principles to Machine Learning Applications[J]. Surface and Interface Analysis, 2025, 57(3): 289-301.
[27] ZHANG Y, LIU X, WANG L.XPS Peak Shape Analysis of SiO2-Coated Silicon Wafers: Effects of Surface Roughness[J]. Applied Surface Science, 2025, 667: 161234.
[28] WANG H, ZHANG Y, LIU Z.FTIR and XPS Combined Analysis of Polymer Degradation: Peak Assignment and Correlation[J]. Polymer Degradation and Stability, 2025, 212: 113501.
[29] FERNANDEZ J E, MARTINEZ G, GARCIA R.Machine Learning-Assisted Peak Fitting for Complex {XPS} Spectra of Transition Metal Oxides[J]. Journal of Cheminformatics, 2025, 17(1): 56.
[30] KIM S H, LEE J H, PARK J W.XPS Peak Intensity Correction for Surface Roughness: A Case Study of Titanium Nitride Films[J]. Thin Solid Films, 2025, 758: 140234.
[31] ALFA CHEMISTRY.XPS Peak Analysis for Surface Chemical State Identification: A Practical Guide[J]. Surface Coating Technology, 2025, 428: 128901.
[32] ROUST E, HECKER C, SCHODLOK M.Rock Sample Surface Preparation Effects on XPS Peak Intensity: Implications for Geological Analysis[J]. Minerals, 2025, 15(6): 789.
Funding
National Natural Science Foundation of China (52475212, 52501426, 52505185)
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