Bioinspired silk–catechol–enzyme co-assemblies as jigsaw-designed conformation-locking bioactive-biocatalytic platforms

Abstract

Immobilized enzymes offer a sustainable, cost-effective alternative to soluble catalysis, but an adaptable platform preserving native conformation, stability, and broad enzyme/material compatibility remains elusive. We report a facile, general, jigsaw-designed platform via co-assembling silk fibroin (SF), protocatechualdehyde (PA), and enzymes. Mussel-inspired PA remodels SF into β-sheet-rich “nano-pockets” to conformationally lock bioenzymes, while its aldehyde and catechol moieties anchor enzymes and surfaces, yielding structurally and functionally adaptable, bioactive-biocatalytic self-assemblies. The platform conferred exceptional stability: enzymes withstood 20 cycles at 50 °C and retained 47.7% activity after one-year ambient storage. The assemblies accommodated diverse enzymes (ALP, GOx, HRP, SOD, CAT), adhered universally to solid surfaces, MOFs, and liquid metal microspheres, and were programmable into coatings, films, or hydrogels. Rational enzyme selection enabled biomimetic mineralization (ALP), antioxidation (SOD/CAT), and antibacterial activity (GOx). Spatial organization of cascade enzymes in core–shell nanoparticles enabled gated catalysis, while segregation on conductive supports facilitated enzyme-mediated electrochemical corrosion control. Moreover, these biointerfacing assemblies elicited favorable cell adhesion, biocompatibility, and anti-inflammatory/-infective effects in vitro/vivo. This modular platform provides a versatile blueprint for robust, catalytically bioactive materials tailored to advanced bioapplications and readily extendable to immobilize other functional biomolecules like peptides.

Graphical abstract

A silk-catechol co-assembly platform conformationally locks enzymes for adaptable robust biocatalysis and enables versatile bioactive functions for advanced biomedical applications.

Highlights

• •Silk-catechol co-assembly platform confers enzyme immobilization and protection with additional bioactivity.
• •β-sheet nano-pockets lock enzyme conformation, boosting stability and activity.
• •Encapsulated enzymes withstand 20 cycles at 50 °C and remain active after one-year ambient storage.
• •Versatile enzyme/substrate compatibility, multi-enzyme cascades, and spatial programmability.
• •Demonstrated in vitro/vivo efficacy in mineralization, anti-inflammation/-infection, and tissue repair, underscoring its bioapplication versatility.

1. Introduction

Enzymes, nature's exquisite catalytic masterpiece, enable highly selective and sustainable chemical transformations under mild conditions, underpinning a wide array of biopharmaceutical and biomedical applications [[1], [2], [3]]. Yet, their practical utility remains critically hampered by conformational fragility, high production costs, and inefficient recyclability, deficits that inflate operational expenses and contradict circular economy principles [4]. Immobilizing enzymes to solid supports addresses these barriers by transforming soluble catalysts into reusable heterogeneous systems, yet current strategies suffer from inherent trade-offs: weak physical interactions lead to leaching, covalent conjugation risks conformational distortion, and conventional porous carriers impose mass-transfer limitations and poor substrate versatility [[5], [6], [7]]. Consequently, a simple adaptable bioactive entrapment system that simultaneously preserves enzyme conformation, ensures stability, and accommodates diverse enzymes and material interfaces remains an unmet need, driving the rational design of advanced immobilization matrices and strategies.

Preserving native enzyme conformation is paramount for maintaining catalytic activity, as denaturation constitutes the primary cause of functional loss [1,7]. Bearing this in mind, a rational strategy is to encapsulate such fragile proteins within polymeric matrices possessing well-defined, rigid polymeric secondary structures (e.g., β-sheets) to restrict deleterious conformational fluctuations [[8], [9], [10]]. Silk fibroin (SF), a biocompatible protein derived from Bombyx mori cocoons, provides an exemplary natural scaffold for this purpose. Conformationally, SF assembles into extensive, mechanically robust β-sheet crystalline domains, driven by hydrogen bonding and hydrophobic packing of its repetitive peptide motifs [[10], [11], [12]]. Notably, these domains can organize into nanoscale “pocket-like” architectures that entrap and shield biomolecules [8,13,14], offering a protective yet dynamic microenvironment ideal for enzyme stabilization [15].

Aside from conformational stabilization, a practical immobilization platform must also achieve strong and versatile adhesion to diverse material surfaces to accommodate utilities such as biosensors and medical implants [16,17]. Here, nature again provides inspiration to solving this. Marine mussels adhere tenaciously to solid surfaces via catechol-rich proteins, wherein the catechol moiety of 3,4-dihydroxyphenylalanine enables robust interfacial adhesion through covalent or metal coordination, hydrogen bonding, and π-interactions, and other interplays [17]. This property has inspired the incorporation of catechol and derived polyphenols into diverse material systems to enhance cohesion or substrate adhesion. Critically, beyond serving as a molecular bridge for surface anchoring, catechols are documented to actively induce and enhance β-sheet formation in SF [18,19], a feature that can be readily harnessed to amplify the generation of protective molecular pockets as described above. Expanding beyond conventional polyphenols, we selected protocatechualdehyde (PA) for its distinctive molecular architecture and reactivity [20]: it uniquely combines a catechol group for universal surface anchoring with an aldehyde function capable of forming dynamic Schiff base linkages with amino groups on SF or enzymes. Moreover, PA also possesses inherent anti-inflammatory and antioxidant properties [21,22], further broadening the platform's functional scope.

Integrating SF and PA yields a synergistic, “jigsaw-inspired” co-assembly immobilization platform that concurrently resolves conformational stability and interfacial adhesion (Fig. 1). Its adaptable, modular architecture enables on-demand encapsulation of diverse enzymes (including multi-enzyme cascades) within the same β-sheet-rich matrix, while PA-mediated dynamic covalent anchoring ensures robust immobilization across virtually any material surface. Compared to existing biocatalytic hybrids such as enzyme–metal/covalent organic framework composites [[23], [24], [25]], where the biocatalyst is confined within rigid, often diffusion-limited architectures, our SFPA system provides a hydrated, flexible matrix that could better preserves native catalytic conformation and facilitate mass transfer. Furthermore, unlike conventional carrier-dependent systems that suffer from limited substrate compatibility and require complex, multistep synthesis or pre-treatment [23], the SFPA platform enables one-pot, substrate-independent adhesion across metals, polymers, inorganic non-metals, and semiconductors without specialized chemistry or elaborate processing. Its morphological programmability enables fabrication into coatings, films, microspheres, or hydrogels by simply tuning parameters such as the SF/PA ratio, additives, and assembly templates, offering exceptional processability and flexibility to suit diverse applications. Moreover, PA's inherent antimicrobial [26] and antioxidant/anti-inflammatory [27,28] potencies could expand the platform's utility beyond immobilization toward therapeutic applications.

Fig. 1.

Bioinspired silk–catechol–enzyme co-assemblies as jigsaw-designed conformation-locking bioactive-catalytic platforms. (a) Overall concept and merits of the platform. (b) PA-mediated remodeling of SF into β-sheet-rich networks. (c) Enzymes conformationally locked within β-sheet-protective nanoscale pockets. (d) Platform versatility: diverse enzymes, broad substrate compatibility across scales, and morphological programmability. (e) Representative bioactive-catalytic functions enabled by the platform.

In this study, we first investigated the SF–PA self-assembly behaviors through integrated experimental and computational approaches, elucidating the molecular interplay between SF and PA and the mechanism of PA-mediated β-sheet remodeling with dynamic covalent anchoring. Unlike conventional polyphenol–silk systems that rely primarily on hydrogen bonding [18,19], the bifunctionality of PA, fusing a catechol group for universal adhesion and an aldehyde group for dynamic Schiff base formation, enables a synergistic covalent–noncovalent crosslinking network. This unique feature leads to the formation of β-sheet “nano-pockets” that conformationally lock enzymes, a concept distinct from mere encapsulation. We then demonstrated exceptional enzymatic stability, substrate versatility, and morphological programmability of the resulting SFPA platform for modular enzyme immobilization. Rational selection of enzymes could impart advanced biofunctionalities, including biomimetic mineralization, antioxidative defense, and antibacterial activity. Notably, compartmentalizing cascade enzymes within acid-labile core–shell nanoparticles could enable gated, stimuli-responsive catalysis, while spatial segregation on conductive supports facilitates enzyme-mediated charge transfer for localized electrochemical corrosion control. Importantly, these biointerfacing assemblies promoted cell adhesion and exhibited potent anti-inflammatory and anti-infective efficacies in mouse subcutaneous implantation models, boding well for bioapplications. By addressing the classic activity–stability trade-off while enabling broad substrate compatibility and programmable biofunctionalities, this work presents silk–catechol self-assemblies as a robust, versatile, and bioactive foundation for next-generation biocatalytic interfaces.

2. Results and discussion

2.1. SF–PA interactions and self-assembly: experimental and simulation insights

The assembly of our platform originates from molecular interactions between SF and PA. Structurally, SF from Bombyx mori contains abundant amino acid residues (e.g., tyrosine, alanine, serine, glycine) [11,12,29], while PA uniquely integrates a catechol moiety for universal interfacial adhesion and an aldehyde moiety capable of forming dynamic Schiff base linkages with amino groups [20]. Previous studies have demonstrated that polyphenolic molecules, including tannic acid (TA), epigallocatechin-gallate (EGCG), and related catechol-rich compounds, can induce β-sheet conformational transitions in SF chains [18,19]. This regulatory capacity of phenolics toward protein secondary structure arises from their ability to assume multiple non-covalent interplays (e.g., hydrogen bonds, electrostatic/hydrophobic associations, and π-π stacking) with protein chains [18]. Given these precedents, we hypothesized that PA, as a bifunctional catechol-aldehyde molecule, would similarly engage SF through synergistic non-covalent and covalent interplays (Fig. S1), thus altering its conformation and possibly driving visible assembly phenomena in solution.

To investigate this hypothesis, we first examined the mixing behavior of SF with PA at varying concentrations. Aqueous PA solutions (0.5–2 M, 60 °C) remained clear and transparent, though noticeable darkening occurred at higher concentrations (a phenomenon correlating to the oxidation of catechol moieties under ambient oxygen [30]) (Fig. 2a). Upon mixing with equivalent volumes of SF solution (30 mg/mL), all PA-containing mixtures immediately turned turbid, forming milky emulsions, whereas SF alone in water stayed transparent. This rapid turbidity development indicates that PA-induced aggregation of SF, likely driven by the multiple weak interactions discussed above between the catechol moieties and SF chain segments [18,19]. Optical microscopy further revealed PA concentration-dependent microphase separation: numerous spherical microdroplets emerged in the 2 M PA mixture, few were observed at 1 M PA, and none at 0.5 M PA or in the SF control (Fig. 2b andS2).

Fig. 2.

Molecular insights into SF–PA interaction via experimental observation and MD simulation analysis. (a) Digital images showing appearance evolution of SF–PA mixtures at indicated concentrations and time points; SF or PA alone served as controls. (b) Optical micrograph of SF–PA mixture (2 M PA). (c–h) MD simulation results: (c) total, Coulombic (Coul), and Lennard-Jones (LJ) energies for SF + PA and SF + PAH systems; (d) RMSD analysis; (e) time-dependent hydrogen bond counts; (f) initial molecular structures of SF, PA, and PAH; (g) binding modes of PA/PAH with SF; (h) structural fluctuations over 100 ns simulation.

To gain insight into the morphological evolution of these assemblies, we air-dried and freeze-dried the mixtures and examined them by SEM (Fig. S3). Air-dried pure SF lacked regular structures, whereas SF mixed with 0.5 M or 1 M PA produced microparticles whose sizes increased with PA concentrations. At 1 M PA, plate-like features appeared alongside particles. Freeze-drying, which could better preserve hydrated assembly states, revealed more distinct morphologies: SF alone showed only a film with uneven surface, while PA-containing samples developed needle-like structures at 0.5 M PA, which grew into larger plate-like forms at higher concentrations. This concentration-dependent morphological progression suggests that PA not only triggers SF aggregation but also actively modulates the microscopic packing and organization of SF chains, although the precise molecular mechanism (e.g., whether it involves β-sheet formation) requires further investigation. PA appears to function as a structural regulator, potentially shifting the balance between hydrophobic interactions and electrostatic repulsion, thereby directing the assembly pathway [31].

The specificity of SF–phenolics interactions was further probed by comparing PA with tannic acid (TA) and gallic acid (GA), both of which feature multiple phenolic hydroxyl groups but no aldehyde moiety (Fig. S4). At equivalent concentrations, TA and GA induced immediate gelation of SF, whereas PA produced only slight turbidity. The rapid gelation in the former case can be attributed to the abundant phenolic hydroxyls in TA and GA, which enable extensive hydrogen-bond crosslinking with SF, as previously documented [32,33]. In contrast, PA possesses fewer hydroxyl groups but introduces an aldehyde moiety capable of forming dynamic Schiff base linkages with amino residues on SF. This distinction implies a fundamentally different interaction balance: while TA and GA rely predominantly on non-covalent associations [32,33], PA likely engage SF through a synergistic combination of dynamic covalent and non-covalent interactions, establishing a competitive equilibrium that prevents excessive cross-linking, thereby guiding controlled β-sheet rearrangement to form protective conformation-locking nano-pockets.

Given that the catechol moiety of PA trends to undergo protonation in aqueous environments, we further modeled its interactions with SF in both neutral (PA) and protonated (PAH) states by molecular dynamics (MD) simulations. This comparative approach is designed to particularly elucidate the role of electrostatic forces in the assembly process. The simulation systems, comprising an SF molecule surrounded by multiple PA or PAH molecules in explicit water, were analyzed over a 100 ns production run to evaluate interaction energies and conformational dynamics.

The analysis of non-bonded interaction energies revealed distinct profiles for the two systems (Fig. 2c). A more negative total energy value indicates a more stable system. Both SF–PA and SF–PAH complexes exhibited negative values for total energy, Coulombic (Coul, electrostatic), and Lennard-Jones (LJ, van der Waals) components, confirming overall attractive interactions. However, a critical difference emerged: the SF–PAH system displayed significantly more negative total and Coulombic energies compared to SF-PA. This indicates that protonation substantially strengthens the electrostatic attraction between PA and SF, constituting the primary contributor to the enhanced stability of the SF–PAH complex. Correspondingly, the root-mean-square deviation (RMSD) of the protein backbone reached a stable plateau more rapidly in the SF-PAH system, further corroborating its superior conformational stability (Fig. 2d). Analysis of hydrogen bonding provided additional nuance (Fig. 2e). The number of hydrogen bonds formed was similar in both systems, with a marginally lower count in the SF–PAH complex. This suggests that while hydrogen bonding contributes to the interaction, the dramatic stability boost from protonation is not primarily mediated by an increase in these bonds. Instead, the dominant mechanism is the enhanced net electrostatic complementarity between the positively charged PAH and the negatively charged patches on the SF surface.

The structural consequences of these interactions were visualized from the simulation trajectories (Fig. 2g and h). Over the simulation period, the SF chain in the presence of PAH adopted a more compact conformation. Notably, the formation of a hollow space within the SF structure was observed, resulting from the inward pulling of one polypeptide segment. This visualization directly demonstrates that protonated PAH can engage with multiple segments of the SF chain, effectively acting as a molecular crosslinker that compacts the protein architecture. We monitored secondary structure transitions in each system over 100 ns MD simulations, focusing on residues 1–10, which showed marked differences across conditions (Fig. S5a–c). In the SF alone system, residues 1–10 remained predominantly coil during the first 20 ns, with only half ultimately transitioning to bend. In SF + PA, coil and bend appeared within 5 ns. Notably, SF + PAH exhibited minimal coil from the start, and coil residues progressively decreased, converting nearly entirely to bend, turn, or helix. These observations indicate that PA effectively drives SF from random coil to ordered conformations. Quantitative time-evolution of secondary structure content (Fig. S5d–f) showed that both SF + PA and SF + PAH reduced coil proportion and increased α-helix and β-sheet compared to SF alone, confirming PA-promoted transition toward ordered structures. We further examined the effect of SF:PA ratio (Fig. S6). At low PA concentrations, PA molecules effectively interacted with multiple SF chain segments, promoting local ordering and β-sheet formation. At high concentrations, excessive PA molecules surrounded the SF chain, engaging in non-specific interactions that saturated hydrogen-bonding sites and physically obstructed regular interchain packing required for β-sheet propagation. These simulations thus provide a partial molecular rationale for the experimental observations, revealing that the enhanced electrostatic interactions from PAH promote SF aggregation and microphase separation, leading to the microdroplets (Fig. 2b) and nanostructures (Fig. S3).

2.2. Preparation and characterization of model SFPA–enzyme interfacial assemblies

Building upon the molecular understanding of SF–PA interactions, we proceeded to construct a bioactive-biocatalytic interface for materials characterization and preliminary functional validation. Titanium was selected as a model substrate given its widespread use in biomedical implants [34], where the inherent bioinertness of this metal necessitates surface functionalization to achieve desired biological performance [30,35]. Here, alkaline phosphatase (ALP) was selected as a model enzyme due to its well-characterized structure and catalytic mechanism, well-established activity assays, biological relevance, and cost-effectiveness [23]. Through simple mixing of SF, PA, and ALP on titanium surfaces, where PA's adhesive catechol moieties and β-sheet conformational transition synergistically favor the formation of high-quality, substrate-adherent enzyme-immobilized coatings, the resulting assemblies were designated Ti-SFPA_ALP (Fig. 3a), with unmodified Ti, Ti-SF, Ti-SFPA, Ti-ALP and Ti-SF_ALP serving as controls for comparative analysis.

Fig. 3.

Characterization and mineralization performance of SFPA_ALP assemblies on titanium. (a, b) Schematic illustration of Ti-SFPA_ALP fabrication (a) and ALP-catalyzed CaGP hydrolysis for surface mineralization (b). (c) Water contact angle measurements: representative images (left) and quantification (right). (d) FTIR spectra. (e) Deconvoluted FTIR Amide I bands showing secondary structure composition of each coating. (f) SEM images of samples after mineralization in CaGP for 3 d.

The successful deposition and chemical modification were first corroborated by water contact angle measurements (Fig. 3c). Bare titanium exhibited a moderately hydrophilic surface with contact angles of approximately 53.8°. Coating with SF alone increased this value to around 76.8°, reflecting the hydrophobic character imparted by SF's β-sheet-rich domains [23]. Incorporation of PA into the coating reduced the contact angles slightly to approximately 66.5°, mirroring the introduction of polar catechol and aldehyde groups. Encapsulating ALP within the SFPA matrix (Ti-SFPA_ALP) resulted in a further increase to about 76.5°, likely attributable to the exposure of the enzyme's hydrophobic domains.

Fourier transform infrared (FTIR) spectroscopy was conducted to provide direct evidence for the chemical and structural composition evolution within the assemblies (Fig. 3d). Increased intensity was observed at 1513 cm⁻¹ (indicative of β-sheet configuration [36]), while the random coil-associated band at 1233 cm⁻¹ diminished. These spectral changes indicated a reduction in random coil content alongside an increase in β-sheet structure within the PA-containing coating. Successful PA loading was further evidenced by emerging vibrational signals at 1290 cm⁻¹ (C–O), 1590 cm⁻¹ (aromatic C=C), 1118 cm⁻¹ (C=O), 876 cm⁻¹ (C–H), and 816 cm⁻¹ (O–H) [[36], [37], [38]], all of which exhibited increased intensity. Additional spectral changes confirmed ALP encapsulation [[39], [40], [41]]: peaks at 1401 cm⁻¹ (COO⁻) and 1063 cm⁻¹ (C–O–C) both intensified following enzyme addition, demonstrating successful loading of ALP into the SFPA matrix.

To quantitatively assess the secondary structural changes induced by PA, deconvolution of the Amide I band was performed (Fig. 3e), revealing the distribution of β-sheet, random coil, α-helix, and turn conformations within the coatings [42]. Upon transitioning from the SF to the SFPA coating, the analysis revealed a marked decrease in random coil content from 33.87% to 10.71% and a significant increase in β-sheet content from 26.83% to 50.48%. The secondary structure distribution in the final SFPA_ALP coating (47.69% β-sheet content) remained largely similar to that of SFPA, confirming that enzyme encapsulation occurs without disrupting the PA-induced β-sheet-rich architecture. This structural reinforcement was further supported by circular dichroism (CD) spectroscopy of coating solutions (Fig. S7). The SF solution exhibited a pronounced negative minimum near 197 nm, characteristic of random coil conformation [18]. Upon PA addition, this minimum diminished while the negative band emerged around 218 nm strengthened (corresponding to the β-sheet conformation [18]). This solution-phase evidence confirmed that PA actively directs SF conformational rearrangement prior to any drying or assembly process, consistent with the MD simulations showing PA-mediated chain compaction and secondary structure remodeling (Fig. 2h).

To quantify Schiff base bond density, ¹H NMR was performed on SF, PA, and the mixture of SF and PA using sodium 3-(trimethylsilyl)propionate as an internal standard (Fig. S8). A new imine proton signal at ∼8.2 ppm confirmed Schiff base formation [43,44]. Interestingly, the aldehyde proton signal of PA at 9.672 ppm decreased by 32.6% in the mixture of SF and PA, corresponding to Schiff base bond densities of 8.15 × 10⁻⁵ mol/g [45]. Upon varying the ratio of SF/PA in the mixed solution, the proportion of PA participated in the Schiff base reaction also changed, demonstrating that covalent crosslinking density in the SFPA system can be tuned.

Beyond compositional and conformational effects, the interfacial stability of coatings proved crucial for practical application. PA incorporation qualitatively improved coating robustness. After aqueous rinsing, Ti-SF samples exhibited visible edge wrinkling and partial delamination, whereas Ti-SFPA coatings remained intact and uniformly adhered (Fig. S9). Cross-cut adhesion testing confirmed that both coatings maintained good adhesion (Fig. S10), but the absence of edge defects in PA-containing samples suggested enhanced interfacial bonding, likely arising from PA's catechol-mediated substrate anchoring combined with the mechanically reinforced β-sheet network. Furthermore, these nano-assemblies demonstrated favorable optical properties owing to their ultrathin nature. Transparency measurements showed that the SFPA coating possessed comparable or superior light transmittance relative to the SF coating (Figs. S11 and S12), an advantageous feature for applications requiring optical clarity, visual monitoring, or optical interfacing [12].

The ultimate validation of our platform lies in its biological functionality. To evaluate the biofunctionality of the self-assembled enzyme constructs, Ti-SFPA_ALP coatings were incubated at 37 °C in calcium glycerophosphate (CaGP) solution, a soluble organic phosphate ester serving as both calcium source and phosphatase substrate [23]. ALP-catalyzed CaGP hydrolysis generates orthophosphate and Ca²⁺ ions for biomimetic apatite deposition, a process recapitulating bone formation. Bare Ti, Ti-ALP (ALP absorbed on Ti), Ti-SFPA (assemblies without ALP), Ti-SF (assemblies without ALP and PA) and Ti-SF_ALP (ALP-containing SF coating) served as controls. After 3 d, SEM revealed abundant spherical mineral deposits exclusively on ALP-containing coatings (Fig. 3f). Notably, Ti-SFPA_ALP surfaces exhibited denser and more numerous mineral particles compared to Ti-ALP and Ti-SF_ALP, despite identical enzyme loading. Energy-dispersive X-ray spectroscopy (EDS) substantiated these deposits as bony calcium phosphate minerals (Fig. S13). The superior mineralization of Ti-SFPA_ALP over Ti-SF_ALP arises primarily from high ALP activity (see Section 2.3 below) conferred by the SFPA immobilization platform. Beyond this enzymatic contribution, the SFPA matrix itself likely plays an auxiliary role based on literature: catechol moieties may locally concentrate Ca²⁺ via coordination to facilitate nucleation [23,46]. while the PA-induced β-sheet domains provide ordered templates for oriented hydroxyapatite growth [47], analogous to collagen in natural bone. To dissect the contributions of enzymatic catalysis and matrix templating, we set three sample groups: Ti-SFPA (SFPA matrix alone, without ALP), Ti-SFPA + free ALP (same ALP activity as the coating, but added freely in solution), and Ti-SFPA_ALP (ALP immobilized within the SFPA coating). These samples were immersed in CaGP solution for 3 d, followed by alizarin red S staining to quantify mineral deposition (Fig. S14). The relative mineralization degrees (normalized to Ti-SFPA_ALP as 100%) were approximately 9.98% for Ti-SFPA and 16.35% for Ti-SFPA + free ALP. In certain sense, the SFPA matrix alone contributed about 10% to the overall mineralization. These results confirm that the excellent mineralization performance of Ti-SFPA_ALP is primarily due to the immobilized ALP, with the SFPA matrix playing a supporting role.

2.3. Immobilization efficiency and interfacial biocatalytic activity with exceptional thermal, operational, and storage stability

A primary objective of enzyme immobilization is to confer enhanced stability against operational and environmental stresses, thereby extending functional lifespan and enabling reuse [[1], [2], [3]]. We next evaluated the robustness and efficacy of the SFPA platform for enzyme immobilization and activity preservation. This evaluation is grounded in the platform's design principle (Fig. 1a–d): PA covalently tethers enzymes via dynamic Schiff base linkages to prevent leaching, while the PA-induced, β-sheet-rich SF network is hypothesized to create protective nanoscale pockets that conformationally lock the encapsulated enzyme, shielding it from denaturation.

Firstly, we quantified the loading amount and immobilization efficiency using ALP (4 KU/mL) and horseradish peroxidase (HRP) (300 U/mL) as model enzymes in SFPA versus SF matrices on titanium (Fig. S15). For ALP, the total protein leakage (including SF and enzyme) was 51.4% for Ti-SF_ALP and 33.4% for Ti-SFPA_ALP; for HRP, the protein leakage was 57.1% for Ti-SF_HRP and 39.7% for Ti-SFPA_HRP. Enzyme leakage decreased from 62.2% (Ti-SF_ALP) to 36.7% (Ti-SFPA_ALP) for ALP, and from 65.2% (Ti-SF_HRP) to 35.6% (Ti-SFPA_HRP) for HRP. The loading amount for ALP was 0.39 μg/mg in Ti-SF_ALP and 0.48 μg/mg in Ti-SFPA_ALP; for HRP, it was 0.41 μg/mg in SF-HRP and 0.53 μg/mg in Ti-SFPA_HRP. The immobilization efficiency for ALP decreased from 8.4% (Ti-SF_ALP) to 7.9% (Ti-SFPA_ALP), and for HRP increased from 1.1% (Ti-SF_HRP) to 2.3% (Ti-SFPA_HRP). Taking ALP as an example, Ti-SFPA_ALP also showed decreased specific activity (from 337.57 U/mg in Ti-SF_ALP to 316.59 U/mg) and greater retained activity per coating mass (from 63.8 mU in Ti-SF_ALP to 100.2 mU), supporting the protective and stabilizing effect of the β-sheet-enriched nanopockets.

Additionally, we investigated the enzyme retention capacity of the SFPA platform, employing a hydrogel-based diffusion assay (Fig. 4a). Hydrogels, with their more open and hydrated network compared to dried coatings, facilitate the diffusion of unbound enzymes and thus provide a more sensitive assessment of matrix retention capability [48]. The assay relies on spectrophotometric detection of p-nitrophenol (p-NP, OD405) generated by ALP-catalyzed hydrolysis of p-nitrophenyl phosphate disodium salt hexahydrate (p-NPP) in alkaline buffer at 37 °C [23. To this end, SF_ALP/glycerol and SFPA_ALP/glycerol hydrogels were prepared by incorporating glycerol into the SF-based assembly solutions to induce gelation. The resulting hydrogels (Fig. S16) were then incubated in deionized water at 37 °C under shaking, with the medium replaced every 12 h. Leached enzyme activity in the collected supernatant was quantified over 48 h (Fig. 4b–e). Both hydrogels released a fraction of loosely associated enzyme within the first 24 h, but critically, cumulative leakage from SFPA_ALP was substantially lower than from SF-ALP at all time points. These results demonstrate that the PA-modified SF network possesses superior capacity to retain encapsulated enzymes, an effect attributable to enhanced β-sheet physical crosslinking and dynamic covalent anchoring that together create a tighter enzyme-retaining matrix.

Fig. 4.

Enzyme retention capacity and reusability and stability of SFPA_ALP self-assembly systems. (a) Schematic of hydrogel-based enzyme release study to assess retention capacity. (b–e) ALP activity of leached enzymes over 0–12 h (b), 12–24 h (c), 24–36 h (d), and 36–48 h (e) from SFPA_ALP versus SF_ALP hydrogels. (f) Schematic of the cyclic thermal stability test. (g–i) Cyclic ALP activity of Ti-SFPA_ALP over 20 cycles at 25 °C (g), 37 °C (h), and 50 °C (i), with Ti-ALP and Ti-SF_ALP as controls. (j) Enzyme storage stability of various samples after ambient storage for up to 12 months. (k) Effect of PA concentration on the enzymatic activity of Ti-SFPA_ALP.

We then assessed the thermal and operational resilience of immobilized ALP on titanium under simulated conditions. To this end, Ti-SFPA_ALP coatings were subjected to 20 consecutive cycles of dry incubation at 25, 37, and 50 °C spanning storage, physiological, and accelerated stress conditions, with ALP activity measured after each cycle using the standard p-NPP assay (Fig. 4f–i). Ti-ALP (directly adsorbed enzyme) and Ti-SF_ALP (ALP entrapped in SF without PA) served as controls. Across all temperatures, a progressive decline in activity was observed for all groups, but with striking differences in magnitude. Ti-ALP lost nearly all detectable activity within the first few cycles, reflecting the vulnerability of surface-adsorbed enzymes to thermal denaturation and leaching. Ti-SF_ALP exhibited intermediate stability, retaining moderate activity over multiple cycles, consistent with a stabilizing effect from SF encapsulation solely [8]. Remarkably, Ti-SFPA_ALP consistently maintained the highest residual activity throughout 20 cycles at all temperatures tested. Even at the challenging temperature of 50 °C, where free enzymes typically undergo rapid inactivation, the SFPA-immobilized ALP retained appreciable activity throughout the cycling, demonstrating that PA incorporation confers exceptional thermal resilience. The superior stability of the Ti-SFPA_ALP system is attributed to the synergistic effects of the SFPA matrix. Combining our simulation results with experimental characterizations (Fig. 2, Fig. 3), we envision that PA actively remodels SF into a more compact, β-sheet-rich conformation, which safeguards encapsulated enzymes. Similar phenomenon was also observed by others [15]. For instance, Kaplan and colleagues demonstrated that SF films undergoing β-sheet transition can form pocket-type microdomains capable of stabilizing entrapped enzymes such as glucose oxidase (GOx), lipase, and HRP during ultralong-term storage (10 months at 37 °C) [8]. This structural rearrangement likely affords a protective, hydrophobic microenvironment around the enzyme molecules, restricting large-scale conformational fluctuations that provoke denaturation under thermal stress.

To verify whether the β-sheet nanopockets enhance enzyme thermotolerance, free ALP (at the same concentration as in the coating solution) was subjected to repeated temperature cycling at 25, 37, and 50 °C. After three cycles, the enzymatic activity of free ALP decreased to 51.9%, 41.1%, and 35.3% of its initial value, respectively. Under identical conditions, Ti-SFPA_ALP retained 60.5%, 61.5%, and 60.7% of its initial activity, respectively (Fig. S17). These results demonstrate that immobilization within the SFPA matrix significantly enhances ALP thermotolerance, particularly at elevated temperatures, which we attribute to the protective effect of the β-sheet-enriched nanopockets. To further validate this protection, green fluorescent protein (GFP) was used as a folding reporter, as its fluorescence depends on proper folding of its β-barrel structure [49]. After 24 h of incubation at room temperature, free GFP retained only 4.2% of its initial fluorescence intensity, whereas Ti-SFPA_GFP retained 70.9% under the same conditions (Fig. S18). These results demonstrate that immobilization within the β-sheet nanopockets stabilizes the three-dimensional structure of GFP, preventing unfolding and loss of function.

To assess whether the enzyme active sites are spatially confined by the β-sheet nanopockets, we compared the catalytic activity of free and immobilized ALP using two substrates of different sizes: p-NPP (smaller) and 4-methylumbelliferyl phosphate (4-MUP) (larger). Standard curves were established with free ALP and 1 mM substrate, and the activities of Ti-SF_ALP and Ti-SFPA_ALP were expressed as equivalent free ALP units (mU). For Ti-SF_ALP, the activity decreased by 58.2% when switching from p-NPP (63.8 mU) to 4-MUP (26.65 mU); for Ti-SFPA_ALP, the decrease was 61.3% (from 100.2 to 38.76 mU). In both cases, activity toward the larger substrate was significantly lower, indicating steric hindrance imposed by the nanopockets (Fig. S19). Additionally, the K_m increased from 512.18 μmol/L (free ALP) to 807.14 μmol/L for Ti-SFPA_ALP, consistent with reduced substrate accessibility due to spatial confinement effect (Fig. S20). These results attest to the conclusion that the β-sheet-enriched nanopockets restrict access of substrates, especially bulky ones, to the enzyme active sites.

Beyond thermal cycling, long-term storage stability represents a critical benchmark for practical utility. It is well established that soluble enzymes are notoriously labile under ambient conditions, typically losing activity within days to weeks, with preservation over months achievable only under frozen storage (−20 °C or −80 °C) [8,15]. We challenged our coatings by storing them at room temperature for 12 months. It is notable that, after this ultralong ambient storage, Ti-SFPA_ALP samples still retained 47.7% of their initial catalytic activity (Fig. 4j). To further corroborate that this retained activity translates into sustained biological function, the 12-month aged samples were subjected to the CaGP mineralization assay. Both Ti-SF_ALP and Ti-SFPA_ALP surfaces still supported mineral deposition, albeit to a lesser extent than fresh samples (Fig. S21). This long-term preservation again implicates the enzyme conformation protection by PA-induced β-sheet pockets over extended periods.

To elucidate the role of PA concentration, we fabricated assemblies with varying SF/PA ratios. The resulting enzymatic activity profile followed a distinct volcano-shaped trend (Fig. 4k). Activity increased with PA concentration up to an optimal value (125 mM), then declined at a further higher concentration. This non-monotonic relationship reveals PA's dualistic function. At optimal concentrations, PA effectively induces β-sheet formation and provides sufficient dynamic covalent sites via Schiff base linkages for stable enzyme immobilization [50,51], maximizing activity retention. At excessive concentrations, however, PA may oversaturate the system, competing with enzyme–SF binding/interactions or disrupting the formation of a coherent β-sheet network. This interpretation aligns with the MD simulations disclosing that PAH can engage multiple SF chain segments; at high density, such multivalent interactions may over-constrain the matrix, reducing the flexibility needed for optimal catalytic motion. To investigate whether the PA concentration-dependent enzyme activity correlates with β-sheet content, we quantified the β-sheet fraction of SFPA_ALP coatings prepared at varying PA concentrations (0, 0.5, 1.25, 12.5, 125, and 250 mM) by FTIR amide I deconvolution. The β-sheet contents were 26.83%, 35.68%, 43.88%, 59.55%, 58.36%, and 54.06%, respectively (Fig. S22). The β-sheet content increased with PA concentration up to 12.5 mM, indicating PA-induced SF conformational transition, but decreased at higher concentrations (125 and 250 mM), suggesting that excessive PA may have disrupted ordered β-sheet formation.

To highlight the merits of the SF-based system as an enzyme immobilization matrix, we selected gelatin (Gel, a non-β-sheet protein) and hyaluronic acid (HA, a polysaccharide) as controls. Under identical conditions, both control coatings either dissolved or detached after washing (Fig. S23a), while SF_ALP and SFPA_ALP remained adhered and intact (with SF_ALP showing slight edge shrinkage). Enzyme activity assays showed that SFPA_ALP exhibited the highest ALP activity, whereas the controls elicited negligible activity (Fig. S23b). These results confirm that the β-sheet network and PA-mediated adhesion are critical for maintaining coating integrity and enzyme activity.

2.4. Universal substrate adhesion and biological affinity of SFPA assemblies

A key challenge for biofunctionalization strategies is achieving compatibility with both biological systems and diverse material substrates without complex pre-treatment protocols, a prerequisite for broad translational impact [16,20]. We hypothesized that the SFPA assemblies' design inherently confers this adaptability: the catechol moieties of PA mediate robust adhesion to diverse surfaces through multiple interactions (e.g., coordination, hydrogen bonding, and π-interactions), while SF contributes additional physical bonding and biological affinity via its amino acid side chains [14]. We therefore systematically evaluated the platform's ability to form uniform coatings on diverse solid materials and to present bioactive cues for interfacing with biological systems.

We first assessed coating formation on a panel of technologically and biomedically relevant substrates, including polymers (PTFE, PEEK), metals (titanium), inorganic non-metals (glass), and semiconductors (silicon). Visual inspection, aided by a co-dissolved dye, confirmed the formation of continuous, adherent layers on all substrates tested (Fig. 5a). X-ray photoelectron spectroscopy (XPS) provided definitive chemical evidence: following coating application, the survey spectra of all substrates exhibited prominent peaks for C, N, and O characteristic of the organic SFPA matrix, while substrate-specific signals were substantially attenuated (Fig. 5b). Water contact angles of the coated surfaces converged to a moderately hydrophilic range (66–75°) regardless of the native wettability of the bare substrates (Fig. 5c), indicating a conformal coating that masked the original surface and created a chemically uniform biointerface. We further evaluated the adhesion strength of SFPA coatings on different materials (Ti, PTFE, glass) via micro-scratch testing by applying a linearly increasing normal force over 5 mm. The critical loads (Lc2, coating failure threshold) were 30.83 N on Ti, 17.26 N on PTFE, and 9.98 N on glass (Fig. S24), indicating substrate-dependent adhesion behaviors. The highest adhesion on Ti likely arises from strong interfacial interactions such as metal-catechol coordination and hydrogen bonding [17]. For PTFE, despite its lack of adhesion-favoring reactive groups, has a hydrophobic rough surface in our study, which could lead to hydrophobic interactions, van der Waals forces, and chain entanglement with SF/PA (Fig. S25) [17,52]. In contrast, the smooth glass surface minimizes mechanical interlocking, and relies primarily on weak non-covalent interactions, resulting in the lowest adhesion. Apart from 2D planar substrates, we also tested the adaptability of the SFPA platform for uniformly modifying 3D complex medical devices, including porous titanium-based scaffolds and PEEK screws (Fig. S26), confirming its ability to conform to complex 3D geometries and topographies for practical applications.

Fig. 5.

Material-independent SFPA coatings with tunable surface chemistry, wettability, and enhanced cell adhesion. (a) Photographs of uncoated (left), SFPA-coated (middle), and dye-visualized SFPA-coated (right) PTFE, PEEK, glass, and Si substrates. (b) XPS spectra confirming surface chemistry alteration. (c) Water contact angle characterization of SFPA coatings: representative images (left) and quantitative data (right) before and after coating. (d) Calcein AM fluorescence images of MC3T3-E1 cells adherent on uncoated and coated substrates. (e) Schematic of SFPA-enabled substrate-independent cell adhesion. (f) Quantification of cell metabolic activity by resazurin assay (ns, non-significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Tailored surface functionalization ultimately aims to create a biointerfacing platform that proactively interacts with living systems [34,53]. With this goal in mind, we evaluated the ability of SFPA-assembled material substrates to universally support mammalian cell adhesion, a fundamental prerequisite for tissue integration [16]. To this end, MC3T3-E1 pre-osteoblasts were cultured on uncoated and coated substrates (PTFE, PEEK, glass, and Si) for 6 h, followed by Calcein AM staining and fluorescence imaging. A striking difference was observed (Fig. 5d and e): cells on uncoated surfaces remained largely rounded with limited attachment, whereas SF- and particularly SFPA-coated surfaces supported significantly enhanced adhesion and spreading. Notably, SFPA-coated substrates consistently exhibited the highest cell density and most extensive spreading, with cells displaying well-defined pseudopodia. In addition, quantitative resazurin assays corroborated these observations: metabolically active cells reduced the blue, non-fluorescent resazurin dye to fluorescent resorufin (ex/em 560/590 nm) [23], revealing a statistically significant increase in cell metabolic activity on SFPA-coated surfaces compared to both SF-coated and uncoated controls (Fig. 5f). The SFPA coating is supposed to synergistically assist cell adhesion and functionality through a dual mechanism: its moderately hydrophilic, aldehyde-functionalized surface promotes protein adsorption [16,20], while SF's inherent biocompatibility and cell-interactive motifs facilitate integrin-mediated attachment and spreading [23]. To further evaluate biocompatibility, MC3T3-E1 cells were cultured on SF and SFPA coatings, followed by live/dead staining and CCK-8 assays (Fig. S27). Both the SF and SFPA groups exhibited sound cell spreading and normal proliferation over 5 d. CCK-8 quantification showed no significant difference between the coated groups and the negative control. These results confirm the favorable biocompatibility of both SF and SFPA coatings, supporting their suitability for cell adhesion and proliferation.

2.5. Morphological programmability and materials/enzymatic versatility for adaptable biocatalysis

Moving beyond the universal adherence and bio-affinity of the SFPA assemblies, we further explored their inherent adaptability across two additional, crucial dimensions: morphological programmability and enzymatic versatility. A practical enzyme immobilization platform should not be confined to planar coatings but should adapt to diverse physical forms, such as films, hydrogels, and micro/nanoscale carriers, to suit varied application scenarios [15]. Concurrently, it must accommodate a broad spectrum of biocatalysts beyond a single model enzyme. The modular “jigsaw” design of the SFPA co-assembly, wherein abundant PA molecules provide dynamic interfacial anchoring and multiple enzyme-binding sites while SF offers a structurally tunable, conformationally protective, and inherently bioactive matrix, inherently confers such flexibility. In the following, we shall demonstrate the platform's versatility to assume multiple morphologies, stabilize diverse enzymes on varying substrates, and enable complex multi-enzyme cascades (Fig. 6a).

Fig. 6.

Versatility of SFPA-based enzyme assemblies across substrates and enzymes. (a) Schematic illustrating platform generality. (b–d) Cyclic activity of ALP-immobilized assemblies on PEEK, Si, and mixed cellulose ester (MCE) substrates. (e–g) Enzymatic activity of GOx-, HRP-, and SOD-immobilized SFPA assemblies on Ti. (h) SEM images of GOx/HRP-co-immobilized liquid metal (LM) microspheres. (i) UV-vis detection of the GOx/HRP cascade reaction on LM microspheres.

By modulating assembly conditions such as PA concentration and environmental humidity, we readily obtained self-supporting macroscopic films that could be peeled intact from substrates (Fig. S28). This processability is attributed to PA's role in directing β-sheet formation; higher humidity during drying mimics “water-annealing”, a well-established method to enhance β-sheet content in SF [54]. Films prepared with an optimal PA concentration (20 mM) exhibited notable mechanical resilience (detailed in Fig. 8c later). Beyond self-supporting films, the platform's morphological programmability extends to other forms. As described in Section 2.3, enzyme-loaded hydrogels were readily fabricated by incorporating glycerol into the assembly system (Fig. S16). Furthermore, as shall be demonstrated later in this section and the next, the SFPA_Enzyme modification strategy can be applied to liquid metal (LM) microspheres and metal-organic framework (MOF) nanoparticles while preserving enzymatic function. These examples underline the inherent morphological programmability of the SFPA platform, positioning it as a versatile toolbox for designing application-tailored biocatalytic materials across length scales.

Fig. 8.

In vivo anti-inflammatory and biocompatibility evaluation of SOD/CAT-immobilized SFPA films after subcutaneous implantation. (a) Schematic of the subcutaneous implantation procedure. (b) Schematic illustrating SFPA film preparation. (c) Macroscopic appearance and mechanical resilience of the SFPA film. (d) H&E staining of peri-implant tissue sections, with arrows highlighting neutrophil infiltration. (e) Masson's trichrome staining of peri-implant tissues. (f) Quantification of inflammatory cell infiltration areas (ns, non-significant, ∗p < 0.05, ∗∗∗p < 0.001). (g) Fibrous capsule thickness measurements (ns, non-significant, ∗p < 0.05).

To verify that the enzyme-stabilizing capability of the SFPA matrix is independent of the underlying substrate chemistry, we evaluated the activity of ALP immobilized on a range of macroscopic materials. In addition to titanium discussed in Section 2.3, substrates included PEEK, silicon, and mixed cellulose ester (MCE) filter membranes, representing high-performance polymers, semiconductors, and soft, porous architectures, respectively. Across multiple assay cycles, a consistent performance order was witnessed on all solid substrates: SFPA_ALP > SF_ALP > directly adsorbed ALP (Fig. 6b–d). In other words, the SFPA-immobilized ALP consistently exhibited the highest and most stable activity, demonstrating the platform's key merit: decoupling enzyme stabilization from substrate-specific surface chemistry to enable versatile heterogeneous biocatalyst design.

The generality of the stabilization strategy was further tested with enzymes possessing distinct structures and catalytic mechanisms: glucose oxidase (GOx, catalyzing glucose oxidation to gluconic acid and H₂O₂ [8]), horseradish peroxidase (HRP, catalyzing H₂O₂-mediated oxidation to yield •OH [20]), and superoxide dismutase (SOD, dismutating superoxide anions to H₂O₂ and O₂ [35]). Each enzyme was immobilized on titanium via the same one-pot co-assembly protocol and exhibited a performance hierarchy similar to that observed for ALP (Section 2.3) across multiple test cycles (Fig. 6e–g). Notably, the SFPA_GOx system maintained pronounced activity over 3 days' continuous operation (Fig. 6e), demonstrating resilience under self-generated harsh conditions (low pH and oxidative stress from accumulated reaction products). This retained activity translated into functional relevance, as Ti-SFPA_GOx coatings elicited distinct inhibition zones against Staphylococcus aureus (S. aureus) microbes via localized antibacterial H₂O₂ production (Fig. S29). Similarly, HRP and SOD immobilized via SFPA showed notably higher activity compared to SF-only or directly adsorbed controls (Fig. 6f and g). The consistent success across oxidases, peroxidases, and dismutases validates the broad, enzyme-independent applicability of the SFPA encapsulation strategy, wherein dynamic covalent anchoring and β-sheet pocket confinement function as generic mechanisms for enzyme immobilization and protection.

To transcend the limitation of single-enzyme systems, we co-immobilized GOx and HRP on GaInSn-based LM microcarriers to afford a cascade catalytic microsystem (Fig. S30). The resulting enzyme-modified microspheres successfully mediated the GOx/HRP tandem reaction, wherein GOx catalyzed glucose oxidation to generate H₂O₂, which then served as the substrate for HRP to oxidize the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB, yielding a blue product upon HRP-mediated oxidation in the presence of H₂O₂) [55]). The successful modification of GOx and HRP on the LM nanoparticles was verified by the colorimetric signal with characteristic absorbance peak at 652 nm upon glucose addition (Fig. 6i). This result proves the platform's applicability to non-planar, microscopic carriers and, more importantly, demonstrates that the SFPA matrix can co-localize multiple enzymes while maintaining their individual activities and facilitating efficient intermediate transfer, enabling sophisticated cascade reactions within a confined microscale volume.

2.6. Spatially programmed cascade enzymes in macro-/nanoreactors for designer catalysis

Building upon the demonstrated versatility of the SFPA platform in enzyme immobilization and substrate compatibility, we next explored its capacity to orchestrate more sophisticated, application-driven biocatalytic systems. The precise spatial organization of multiple enzymes is a cornerstone of advanced biocatalysis, enabling controlled reaction sequences, logic-gated responses, and the transduction of biochemical signals into advanced material/device functions [56,57]. Leveraging the SFPA platform's capacity for substrate-independent spatial nanoassembly, we engineered two distinct proof-of-concept systems: (i) nanoscale, stimulus-responsive core–shell MOF particles for spatiotemporally controlled cascade reactions (Fig. 7a), and (ii) macroscopic U-shaped conductive metal staple-type devices wherein spatially segregated cascade enzymes at the two ends drive a self-powered electrochemical process (Fig. 7b).

Fig. 7.

Demonstrative applications of the SFPA enzyme immobilization platform. (a) Schematic of cascade enzyme activity in the MOF_HRP/SFPA_GOx core–shell system with internally encapsulated HRP and externally immobilized GOx. (b) Schematic of galvanic current generation from a metal staple bearing spatially-segregated GOx and HRP assemblies at opposite ends upon insertion into agar medium. (c) SEM images showing morphology evolution of MOF particles after sequential surface modifications. (d) Enzymatic activity of MOF-based composites with and without shell degradation. (e) Visual detection of hydroxyl radicals via TMB colorimetric reaction using intact and cut stainless steel staples with spatially segregated GOx (left)/HRP (right) assemblies embedded in agarose gel; the cut staple (marked with ×) represents an open-circuit configuration. (f) TMB chromogenic reaction using intact and cut zinc wires with spatially segregated enzyme assemblies (left: GOx, right: HRP); the cut wire (marked with ×) represents an open-circuit configuration. (g) Quantification of cascade reaction products from the zinc wire system under closed- and open-circuit conditions.

We first designed a core–shell nanosystem wherein HRP encapsulated within an acid-labile ZIF-8 core and GOx incorporated in the SFPA shell are spatially compartmentalized, with their interaction gated by an acidic trigger that can be externally applied or self-generated via GOx activity (Fig. 7a). ZIF-8 was selected for its well-established combination of biocompatibility, facile one-pot enzyme encapsulation during rapid synthesis, and its characteristic decomposition under mildly acidic conditions [23,56]. This design creates a spatiotemporal barrier: the outer GOx remains accessible to environmental glucose, while the inner HRP is sequestered within the initially stable MOF core. GOx oxidizes glucose to gluconolactone and H₂O₂, concurrently generating gluconic acid that lowers the local pH. This self-generated acidic microenvironment triggers dissolution of the ZIF-8 core, releasing HRP to utilize the co-produced H₂O₂ for generating hydroxyl radicals, thereby producing an amplified colorimetric signal with substrates such as TMB.

SEM confirmed successful hierarchical assembly: pristine ZIF-8 crystals evolved from faceted, smooth-surfaced particles to MOF_HRP with retained crystallinity and marginally rougher surfaces, and finally to core-shell MOF_HRP/SFPA_GOx structures wherein the original facets were obscured by a conformal, rough polymeric overlayer (Fig. 7c). TEM imaging further confirmed the core–shell architecture, revealing a uniform polymeric shell of approximately 65 nm (Fig. S31). Crucially, a TMB-based assay verified the triggerability and effectiveness of this compartmentalized catalysis. Intact core–shell particles produced a minimal signal, indicating successful spatial segregation that prevented HRP from accessing GOx-generated H₂O₂ (Fig. 7d). Only upon deliberate partial degradation of the MOF structure, which can enable more efficient spatial enzyme/substrate diffusion and cascade reaction, was a significant catalytic signal detected. This establishes a gate-keeping, feedback-driven biocatalytic system wherein cascade activity remains locked until triggered by a specific microenvironmental cue such as local acidosis, a design concept directly relevant for targeted therapeutic activation in pathological microenvironments including bacterial biofilms and tumors [58].

The GOx/HRP cascade reaction is inherently redox-active, involving not only substrate conversion but also electron transfer processes that, if properly harnessed, can be coupled to electrode surfaces to generate self-powered electrical signals or modulate interfacial electrochemistry [56]. To exploit this interplay between spatially organized biocatalysis and electrochemistry, we regionally immobilized GOx and HRP on opposite ends of U-shaped conductive substrates (first a commercial steel staple, then a pure zinc wire), creating simple “galvanic” systems (Fig. 7e and f). A striking observation emerged when the enzyme-functionalized ends were vertically embedded in glucose-containing agarose gel with TMB: the intact staple (closed circuit) developed color at the GOx-coated end substantially faster and more intensely than its cut counterpart (open circuit), indicating that electron flow through the conductive substrate influences the cascade reaction kinetics. This aligns with recent reports demonstrating that GOx/HRP cascade reactions spatially integrated into conductive microneedle platforms can generate microcurrents with pro-healing therapeutic effects [56,57].

We recognized that commercial staples may release impurity ions like Fe²⁺ upon corrosion, which could participate in Fenton chemistry with GOx-generated H₂O₂ and generate hydroxyl radicals independently of HRP [59], complicating interpretation. We therefore turned to custom-bent U-shaped pure zinc wires, which are less prone to such interference, to isolate the coupling effect while guiding a more evident interfacial electrochemical corrosion behavior. While no distinct TMB color change was observed around the samples, an unexpected reversal in the wire's electrochemical degradation pattern emerged (Fig. 7f). In the open-circuit configuration, corrosion was primarily localized to the GOx-coated end, likely due to local acidification from gluconic acid production. Remarkably, closed-circuit conditions redirected pronounced corrosion to the HRP-coated end (circled region, Fig. 7f), implying that the enzyme-generated micro-current modulates local electrochemistry to control anodic corrosion site. UV-vis analysis of the reaction solution further indicated that the cascade reaction kinetics were suppressed under closed-circuit conditions (Fig. 7g), linking the electrochemical state directly to the biocatalytic output. This presents a novel paradigm for designing self-regulating systems, such as biodegradable implants whose corrosion profile can be programmed by engineered enzyme pairs.

2.7. In vivo biocompatibility and anti-inflammatory/anti-infective effects and mechanisms of SFPA-based functional assemblies

The bioactive-biocatalytic nature of our platform motivated its evaluation for advanced biomedical applications, specifically in scenarios where implantable materials must actively manage host immune reactions and/or combat infections [53]. The inherent morphological programmability, facile processability, and biomolecule adaptability of the SFPA system enabled the fabrication of two model sample forms for these in vivo validations: (i) robust, freestanding films functionalized with antioxidant cascade enzymes for subcutaneous implantation to assess anti-inflammatory functionality and tissue biocompatibility (Fig. 8), and (ii) antimicrobial peptide-functionalized titanium implants to assess antibacterial efficacy against infections (Fig. 10).

Fig. 10.

In vivo antibacterial evaluation of HHC36-immobilized SFPA assemblies on Ti implants. (a) Schematic of subcutaneous implantation for antibacterial assessment. (b) SEM image of retrieved implants, with bacteria false-colored for clarity. (c, d) Bacterial counts on implant surfaces determined by dilution plating (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (e) Giemsa staining of peri-implant tissue, with red arrows indicating bacterial/biofilm clusters. (f) Quantification of immune cell infiltration from H&E staining. (g) H&E staining of peri-implant tissues, with black arrows indicating neutrophil infiltration (∗p < 0.05, ∗∗∗p < 0.001).

To fabricate macroscopic self-supporting films, SFPA solutions were cast on hydrophobic PTFE substrates under optimized conditions, dried, and subsequently peeled to obtain freestanding films (Fig. 8b) [9]. Films prepared with an optimal PA concentration (20 mM) exhibited remarkable mechanical resilience, maintaining structural integrity after repeated folding, crumpling, and smoothing (Fig. 8c). This robust performance likely arises from the PA-induced, high β-sheet content within the SF matrix, which forms a network of physical crosslinks that transduces molecular-scale reinforcement into macroscopic toughness essential for applications such as flexible bioelectronics [18,60]. To impart proactive anti-inflammatory functionality, we encapsulated tandem antioxidant enzymes SOD and CAT within the SFPA film matrix (SFPA_SOD/CAT). The SOD/CAT cascade represents natural cellular antioxidant defense mechanism, sequentially neutralizing superoxide anions and H₂O₂ (Fig. 8a), key inflammatory mediators [35]. These films, alongside control SF and plain SFPA films, were implanted subcutaneously in mice (Fig. 8a).

Uncontrolled inflammation and fibrous encapsulation of implanted materials can compromise their function, longevity, and tissue regeneration [53]. Histological evaluation by H&E staining revealed distinct host responses over 14 d (Fig. 8d, f). All groups exhibited acute inflammation with neutrophil infiltration at day 3. By day 7, clear differences emerged: the SF film group sustained the most extensive inflammation, while the SFPA group exhibited a moderate reduction, and the SFPA_SOD/CAT group demonstrated the most significant and sustained attenuation. The anti-inflammatory effect is believed to be synergistic: (i) PA itself possesses intrinsic antioxidant activity, reported to modulate endogenous antioxidant enzymes (e.g., by elevating GSH-PX and SOD activity, decreasing xanthine oxidase (XOD) and NADPH oxidase (NOX) activity, and lowering malondialdehyde (MDA) levels) [27] and directly scavenge radicals [28]; (ii) The co-immobilized SOD/CAT cascade affords sustained, localized enzymatic scavenging of •O₂⁻ and H₂O₂ [35], thus counteracting ROS-mediated pro-inflammatory signaling. Consistent with the inflammatory profile, analysis of fibrotic encapsulation via Masson's trichrome staining and capsule thickness measurement showed that the SF film induced the thickest, most disorganized fibrous capsules (Fig. 8e, g), likely exacerbated by its fragmentation in vivo (Fig. S32). In contrast, both SFPA and SFPA_SOD/CAT films elicited significantly thinner and more organized fibrous layers (Fig. 8e, g), confirming the anti-inflammatory and anti-fibrotic roles of PA and the co-immobilized enzymes.

We consider the SOD/CAT-immobilized SFPA films to have favorable stability based on several observations. First, immobilized enzymes withstood repeated thermal cycling up to 50 °C (Fig. 4i), indicating that β-sheet nanopockets could restrict conformational flexibility and protect against denaturation. This property would likely enhance resistance to physiological stressors such as proteases and temperature shifts in vivo. Second, the SFPA_SOD/CAT group exhibited the most sustained anti-inflammatory effect over 14 d (Fig. 8d–g), implying that SOD and CAT remained active to resist inflammation progression. Third, the SFPA_SOD/CAT film remained largely intact after retrieval, whereas SF film without PA fragmented (Fig. S32). This difference is attributed to higher β-sheet content (Fig. 3e) and PA-mediated crosslinking, which should play a role to strengthen the network and slow hydrolytic degradation.

Noting that SFPA films showed significantly stronger anti-inflammatory activity than SF films, we considered that these functions might be linked to how SFPA interacts with immune cells. To this end, we performed transcriptomic analysis on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages cultured on SF and SFPA films to explore the possible cellular mechanisms behind these effects (Fig. 9). Compared with the SF group, the SFPA group exhibited reduced expression of pro-inflammatory genes (IL-1a, IL-1b, and Ccl2) [61]; Relative to the Control group, SF upregulated Stabilin-1 (Stab-1) and Spexin (Spx), both associated with anti-inflammatory and reparative functions [62,63] (Fig. 9b, Fig. S33). These suggest intrinsic immunomodulatory activity of SF and SFPA. Gene Ontology (GO) and KEGG analyses further delineated group-specific biological processes and pathways. GO enrichment revealed that SFPA, compared with SF, was associated with differential molecular functions and biological processes related to inflammation and tissue repair (Fig. 9d), while SF versus Control primarily affected repair-related processes (Fig. S34a). KEGG analysis identified significant modulation of inflammation-related pathways, including the chemokine signaling pathway and C-type lectin receptor signaling pathway (Fig. 9e; Fig. S34b) [64,65]. Consistently, GSEA demonstrated enhanced anti-inflammatory capacity in the SFPA group relative to SF (Fig. 9f), characterized by suppression of pro-inflammatory pathways (e.g., “chemokine signaling” and “C-type lectin receptor signaling”) [66] and activation of the mRNA surveillance pathway that contributes to immune homeostasis. In comparison, SF alone upregulated the anti-inflammatory PPAR signaling pathway versus Control (Fig. S34c) [67]. The enhanced anti-inflammatory and pro-reparative effects of SFPA are attributable to PA's dual role in modulating the structural properties of SF and imparting additional ROS-regulating bioactivity.

Fig. 9.

Mechanisms underlying the anti-inflammatory and repair-promoting effects of SFPA. (a) Principal component analysis (PCA) of RAW264.7 cells from SFPA and SF groups. (b) Volcano plot of differentially expressed genes. (c) Heatmap clustering of gene expression. (d) GO and (e) KEGG enrichment analyses. Pathways related to inflammation and tissue repair are highlighted in red and green boxes, respectively. (f) Gene set enrichment analysis (GSEA) results. (g) Schematic of the experimental workflow.

Beyond modulating inflammation, preventing infection is paramount for implant success, particularly for percutaneous or transcutaneous devices where the skin barrier is breached [53,59]. To demonstrate the platform's capacity for diverse bioactive cargoes, we further functionalized Ti implants with SFPA encapsulating an established broad-spectrum antimicrobial peptide HHC36 (Ti-SFPA_HHC36). Following contamination with S. aureus and subcutaneous implantation for 3 d, retrieved implants were analyzed (Fig. 10a). SEM and quantitative plating revealed a stark contrast (Fig. 10b–d). Uncoated Ti and Ti-SFPA surfaces harbored numerous viable bacteria, with a modest reduction on Ti-SFPA attributable to PA's intrinsic antibacterial properties [26]. In contrast, the Ti-SFPA_HHC36 surface showed a drastic reduction in adherent bacteria, with many exhibiting membrane damages. Histological analysis of peri-implant tissues using Giemsa and H&E staining confirmed the functional efficacy (Fig. 10e–g). Tissues surrounding Ti-SFPA_HHC36 implants had minimal bacterial clusters and significantly reduced inflammatory cell infiltration compared to controls. These results hint at a dual mechanism of action: immediate contact-mediated killing at the surface, coupled with sustained release of HHC36 during coating degradation to eliminate planktonic bacteria in surrounding tissues and potentially recruit host antimicrobial immune responses [68,69]. Given the potential hemolytic risk of antimicrobial peptides [70], we evaluated the hemocompatibility of free and immobilized HHC36 using mouse red blood cells. Free HHC36 exhibited a hemolysis ratio of 4.08% (approaching the 5% ISO10993-4 threshold), whereas SFPA-immobilized HHC36 showed a lower ratio of 3.19%, indicating improved hemocompatibility, likely due to charge shielding and reduced direct cellular contact (Fig. S35).

No systemic toxicity was detected in major organs (heart, liver, spleen, lungs, kidneys) in either implantation model (Figs. S36 and S37). These studies validate the SFPA platform as a biocompatible, adaptable toolkit for engineering biofunctional interfaces. The platform's capability to actively tailor host responses is demonstrated simply by exchanging the encapsulated bioactive cargo, for instance, from antioxidant enzymes to antimicrobial peptides. This modular design, coupled with the straightforward assembly process and the inherent bioactivity of the SFPA matrix, highlights its immense potential for the development of next-generation, multifunctional smart biomaterials and regenerative medical devices.

3. Conclusions

We have demonstrated that β-sheet-rich silk–catechol–enzyme self-assemblies constitute a facile, bioinspired, jigsaw-designed platform that reconciles catalytic activity preservation with interfacial adaptability crucial for enzyme immobilization. The resulting biocatalytic constructs exhibited enhanced tolerance to harsh environments, including elevated temperatures, and maintained exceptional one-year-long storage stability. The platform accommodated a broad spectrum of solid and soft substrates across multiple length scales and was morphologically programmable into coatings, films, or hydrogels. Rational enzyme integration endowed on-demand biofunctionalities, including biomimetic mineralization, antioxidative defense, and antibacterial effects. Beyond simple immobilization, spatial regional organization of cascade enzymes within acid-labile MOF core–shell nanoparticles enabled gated, stimuli-responsive catalysis, while segregation on conductive supports facilitated enzyme-mediated charge transfer that directed localized electrochemical corrosion. Biologically, these assemblies elicited universal cell adhesion and exhibited favorable biocompatibility and anti-infective/-inflammatory properties in vitro or vivo.

Beyond these, our study presents a versatile bioactive blueprint for advanced heterogeneous biocatalysis, with potential applications ranging from implant coatings and wound dressings to sustainable manufacturing and smart biosensors. The inherent conformational programmability of SF and the structural diversity and chemical reactivity of catechols allow this jigsaw-inspired strategy to be adaptable to various material formulations and readily extrapolatable to other biomolecule immobilization, including peptides, growth factors (e.g., BMP-2), and antibiotics [[71], [72], [73]], for activity retention or controlled delivery. Despite the promise, further evaluation is needed under pathological conditions (e.g., pH fluctuations, protease activity, immune infiltration) that may affect coating stability and long-term enzyme activity, along with systematic assessment of degradation product biodistribution, immunogenicity, and long-term biosafety and effectiveness, which are essential for clinical translation.

4. Experimental section

The detailed experimental methods were described in the Supporting Information. All animal experiments were performed in accordance with the guideline approved by Institutional Animal Ethical and Welfare Committee of Sun Yat-sen University.

Ethics approval and consent to participate

The animal experiments were carried out in accordance with protocols approved by the Institutional Animal Ethical and Welfare Committee of Sun Yat-sen University (Approval No. SYSU-IACUC-2026-000281). This study did not involve any human participants, human data, or human tissue; therefore, consent to participate is not applicable.

CRediT authorship contribution statement

Jiahui Hao: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Yuling Zhang: Investigation, Software, Visualization, Writing – original draft. Haiying Luo: Investigation. Xinyang Wang: Formal analysis, Methodology, Software. Min Wen: Project administration, Visualization, Writing – review & editing. Jiang Qian: Formal analysis, Resources. Zhaojun Jia: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

Declaration of competing interest

Zhaojun Jia and Jiahui Hao are inventors of a Chinese patent on the subject of this work. The other authors declare that they have no competing financial interests.

Appendix A. Supplementary data

The following is the Supplementary data to this article: