Controlled self-assembly of plant proteins into high-performance multifunctional nanostructured films

This study addresses how to process abundant, low-cost, plant-derived proteins—typically poorly water-soluble—into functional materials with controlled micro/nanostructure and high performance. The research question is whether an environmentally friendly, scalable processing route can induce and control nanoscale self-assembly in water-insoluble plant proteins to yield robust, transparent films with tunable functionalities. The context is the replacement of petroleum-derived plastics with biodegradable protein-based materials; prior work focused on animal proteins or engineered proteins faces cost, allergenicity, and sustainability challenges. The purpose is to develop a solvent and processing strategy to dissolve, denature, and direct plant protein assembly into β-sheet-rich networks, enabling strong, transparent films and micro/nano-patterning for added functions. The significance lies in overcoming solubility limitations and eliminating harsh chaotropes or intensive purification, unlocking scalable plant-protein materials for coatings, packaging, and photonic applications.

Previous protein-based films have primarily used synthetic peptides and animal-derived proteins (e.g., silk, β-lactoglobulin, lysozyme) or engineered proteins, achieving controlled self-assembly but with drawbacks of high cost, potential allergenicity, and environmental concerns. Plant proteins are sustainable and abundant, including as industrial by-products, but are typically poorly soluble in water, impeding controlled self-assembly. Prior progress often relied on complex purification to isolate water-soluble fractions, limiting scalability, or used nonvolatile chaotropic agents for denaturation, which can remain in products and affect properties. Ultrasonication and solvent systems have been studied to improve dispersion and functional properties of soy proteins, and fibril formation has been reported for some plant proteins; however, a scalable, green method to induce nanoscale order and translate it into high-performance films has remained elusive.

- Solvent system and dissolution: Soy protein isolate (SPI, 92% purity) dispersed at 10 w/v% in aqueous 30 v/v% acetic acid to form a colloidal slurry. Ultrasonication (Bandelin HD2070, 70 W; 40% amplitude; 0.7 s on/0.3 s off) at elevated temperature (90 °C) for 30 min applied to disrupt aggregates, unfold proteins, and enhance solvation; facilitates formation of small soluble aggregates. - Self-assembly by cooling: Upon controlled cooling from 90 °C to ~20 °C, the SPI solution undergoes sol–gel transition to a self-standing hydrogel. TEM/AFM on diluted solutions (2 w/v% and 0.1 w/v% in 30% acetic acid) confirm fibrillar aggregates (length 100–200 nm, diameter 5–10 nm). Hydrogel network observed by SEM/cryo-SEM as fine-stranded, densely packed aggregates. - Film formation: Glycerol added as plasticizer at 20–40 w/w% relative to dry mass (typical 30%). Hot solution cast onto pre-heated (90 °C) glass Petri dish; upon cooling, a thin hydrogel forms, then air-dried at room temperature for 3 days to yield free-standing films. TEM reveals β-sheet nanocrystals (~5–10 nm; 3.5 Å spacing). A large-scale film (30 × 40 cm) prepared by casting 60 mL of 10% SPI on a 30 × 40 cm tray pre-heated at 110 °C; dried 3 days. - Controls: Nonstructured films prepared by dispersing 10 w/v% SPI in alkaline solution (pH 10), heating at 95 °C for 30 min, adding 30% glycerol, casting and drying. - Coating process: Dip-coating paperboard (0.75 mm) in hot SPI solution (10 w/v% in 30% acetic acid, no plasticizer) immediately post-sonication; withdraw slowly; air-dry 3 days to form coating. Variant includes addition of hydrophobic zein (0.5 w/v%) in 60% acetic acid with 10% SPI. - Micro/nano patterning (soft lithography): PDMS negative templates prepared from SU-8 microfabricated masters (micropillars) and from a DVD for nanogratings (~740–750 nm pitch). Sonicated SPI solution with 30% glycerol cast on degassed PDMS templates and dried to form patterned films. - Characterization: Turbidity (A600), solubility by centrifugation and gravimetry, DLS for particle size, zeta potential; TEM (Talos F200X), AFM (non-contact, PPP-NCHR tips); SEM (Verios 460) including critical point drying and cryo-SEM; FTIR-ATR (Bruker VERTEX 70) with second derivative analysis of Amide I to quantify secondary structure; XRD (CuKα) to assess β-sheet reflections; Tensile testing (Tinius Olsen H25KS, 250 N, 2 mm/min) on 5-mm strips; UV–vis transmittance (Cary 500); Oxygen permeability by Oxtran 2/21 at 23 °C, 50% RH; Contact angle (FTA1000B).

- Dissolution and aggregate control: Processing SPI in 30% v/v acetic acid with ultrasonication at 90 °C yields translucent solutions with smaller particle sizes (29 ± 9.1 nm) versus water-dispersed control (148 ± 68 nm), increased transparency and solubility. - Fibrillar self-assembly: Cooling induces self-assembly into fibrillar aggregates (100–200 nm long, 5–10 nm diameter) and a fine-stranded hydrogel network; not observed when diluted prior to cooling, confirming temperature-triggered assembly. - β-sheet nanocrystallinity: Dried films contain β-sheet nanocrystals (~5–10 nm) with ~3.5 Å spacing. FTIR during cooling shows ~25% increase in intermolecular β-sheet content and Amide I shift to lower wavenumbers (stronger H-bonding). Final films exhibit higher intermolecular β-sheet content (65%) than original SPI powder (49%) and nonstructured films (46%). XRD shows stronger β-sheet reflections (~10 Å d-spacing) versus powder. - Mechanical performance: Self-assembled SPI films (30 wt% glycerol) show tensile strength 15.6 ± 2.07 MPa and Young’s modulus 209 ± 39.1 MPa, outperforming nonstructured SPI films with 30% glycerol (strength 9.30 ± 1.53 MPa; modulus 131 ± 22.6 MPa). Tuning glycerol (20–40 wt%) adjusts modulus from 483 ± 58.4 to 92.7 ± 25.3 MPa and strength from 25.0 ± 3.49 to 6.18 ± 0.98 MPa. Properties comparable to some engineering plastics (e.g., LDPE, PTFE), achieved without reinforcers. - Optical transparency and scalability: High transparency with transmittance at 550 nm of 93.7%, surpassing nonstructured films (77.4%). Fabrication demonstrated at 30 × 40 cm scale; films can be thermally welded to form 3D items (e.g., a bag). - Coating/barrier function: Dip-coated SPI layers on paperboard reduce water uptake significantly; cobalt chloride humidity sensor confirms slower water diffusion. Incorporation of 0.5 w/v% zein further reduces water uptake. Oxygen permeability measured at 2012 ± 820 ccum/m²-day-atm at 23 °C, 50% RH, comparable to PVC and OPET. - Surface patterning and function: Micropillar patterning (~10 µm diameter, ~15 µm height) increases water contact angle from 45° to 99°, imparting hydrophobicity. Nanopatterning using DVD gratings (~750 nm pitch) produces iridescent, structurally colored films.

The findings demonstrate that an aqueous acetic acid/ultrasonication approach overcomes the poor water solubility of plant proteins, enabling controlled thermal self-assembly into β-sheet-rich fibrillar networks that translate into robust, transparent films. The increase in intermolecular β-sheet content underpins enhanced mechanical properties without additives or cross-linkers, approaching those of certain plastics. The sol–gel transition allows simple dip-coating to impart barrier layers with reduced water uptake and competitive oxygen permeability, and the process is compatible with lithographic templating for micro/nano-structured surfaces, enabling hydrophobicity and photonic effects. The method uses low-toxicity, low-cost solvents and scalable operations (ultrasonication, casting), addressing cost and sustainability issues of animal or engineered protein systems and suggesting applicability across different plant protein feedstocks (e.g., pea protein).

This work introduces a scalable, green processing route to dissolve and direct the self-assembly of plant proteins into β-sheet-rich nanostructured films with high transparency, tunable mechanics, and multifunctionality. The approach leverages aqueous acetic acid, ultrasonication, and temperature-controlled assembly, followed by solvent evaporation, to yield robust films and coatings, and is compatible with micro/nano-patterning to add hydrophobic and photonic functions. These advances open opportunities for sustainable, plant-protein-based materials to replace petroleum-derived plastics in applications such as packaging, coatings, and photonics. Future work could explore broader plant protein feedstocks, optimize solvent recovery and continuous processing, integrate additional functional fillers or motifs, and scale industrially relevant forming methods (e.g., extrusion, roll-to-roll, 3D shaping).